CANCER ETIOLOGY, DIAGNOSIS AND TREATMENTS TUMOR SUPPRESSOR GENES FUNCTIONS, REGULATION AND HEALTH EFFECTS. Nova Science Publishers, Inc.

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1 CANCER ETIOLOGY, DIAGNOSIS AND TREATMENTS TUMOR SUPPRESSOR GENES FUNCTIONS, REGULATION AND HEALTH EFFECTS

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3 CANCER ETIOLOGY, DIAGNOSIS AND TREATMENTS TUMOR SUPPRESSOR GENES FUNCTIONS, REGULATION AND HEALTH EFFECTS MEHMET GUNDUZ AND ESRA GUNDUZ EDITORS New York

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5 Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Contents Physico-Chemical Properties of the Putative Tumor Suppressor Protein, 101F6 1 Alajos Bérczi and Motonari Tsubaki The Inhibitor of Growth (ING) Gene Family: Potential Use in Cancer Diagnostics and Therapy 17 Mehmet Gunduz, Eyyup Uctepe, Catherine Moroski Erkul, Senol Dane and Esra Gunduz Multifunction New Tumor Suppressor Gene Family from Cancer to Metastasis: A Disintegrin and Metalloproteinases with Thrombospondin Motifs (ADAMTS) 45 Kadir Demircan, Yunus Emre Bilgen, Tugrul Celik, Yudum Yaral, Birsen Dogan, Zisan Akcaaga, Zahide Nur Unal and Mehmet Gunduz Von Hippel Lindau (VHL) Gene and Protein (pvhl): A Member of the Tumor Suppressor Gene Family 65 Ferah Armutcu, Kadir Demircan and Murat Oznur Application of Cancer Gene Therapy Using Tumor Suppressor Gene p53 81 Hiroshi Tazawa, Shunsuke Kagawa and Toshiyoshi Fujiwara Chapter 6 p63 and p73: Members of the p53 Tumor Suppressor Family 105 Zeynep Ocak, Murat Oznur, Ramazan Yigitoglu, Esra Gunduz and Mehmet Gunduz Chapter 7 Chapter 8 The Emerging Roles of Forkhead Box (FOX) Family Proteins in Tumor Suppression 129 Pang-Kuo Lo Molecular Basis of BRCA1 and BRCA2 and Clinical Approaches to BRCA1/2 Mutation Carriers 183 Kemal Keseroglu, Fatih Aydogan and Mustafa Ozen

6 vi Contents Chapter 9 Chapter 10 Chapter 11 On The Verge of Being a Tumor Suppressor Gene or an Axonal Guidance Molecule: DCC 195 Omer Faruk Karatas, Betul Yuceturk and Mustafa Ozen DKK3, a Mysterious Tumor Suppressor Gene that Possesses Multiple Functions in Tumor Progression 207 Naoki Katase, Tsutomu Nohno and Mehmet Gunduz The Functions and Roles of the Unique Tumor Suppressor Gene PTEN 233 Omer Faruk Karatas, Esra Guzel and Mustafa Ozen Chapter 12 Functions of the Tumor Suppressor Gene APC 253 Tomoki Yamatsuji, Munenori Takaoka, Takuya Fukazawa and Yoshio Naomoto Chapter 13 Structure and Function of the Tumor Suppressor Gene p Zeynep Tarcan, Catherine Moroski Erkul, Bunyamin Isik, Esra Gunduz and Mehmet Gunduz Chapter 14 The Functions and Roles of RB1 in Cancer 287 Erkan Koparir, Asuman Koparir and Mustafa Ozen Chapter 15 Endoplasmic Reticulum Protein 29 (ERp29) and Cancer: Molecular Functions, Mechanisms and Clinical Implication 301 Daohai Zhang Chapter 16 Divergent Roles for Tumor Suppressor Genes in Cancer 333 Marina Trombetta-Lima, Thiago Jacomasso, Sheila Maria Brochado Winnischofer and Mari Cleide Sogayar Index 355

7 In: Tumor Suppressor Genes ISBN: Editors: Mehmet Gunduz and Esra Gunduz 2013 Chapter 1 Physico-Chemical Properties of the Putative Tumor Suppressor Protein, 101F6 Alajos Bérczi 1 and Motonari Tsubaki 2 1 Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary 2 Department of Chemistry, Kobe University Graduate School of Science, Kobe, Japan Abstract Based on structural similarities, the putative human tumor suppressor protein, 101F6, and its mouse ortholog, TSP10, were identified as members of the cytochrome b561 (Cyt-b561) protein family about 10 years ago. Both proteins have been successfully expressed in different yeast expression systems and their basic physico-chemical properties have been established. MALDI-TOF mass analysis indicated that no posttranslational modification of the proteins in yeast cells occurred. These integral membrane proteins have six trans-membrane domains (TMD) and two b-type hemes with different midpoint redox potentials; one heme on each side of the membrane. The two hemes are coordinated by two pairs of His residues that are localized on the four central trans-membrane domains. The recombinant tumor suppressor (TSCytb) proteins could be reduced by ascorbate (ASC) and by dihydrolipoic acid (DHLA) at about the same level but maximal reduction of TSCytb could be reached by dithionite. The 50% reduction of the high-potential and low-potential hemes by ASC could be obtained at about 0.25 mm and 10 mm, respectively. The reaction rates of the electron donation from the ASCreduced human TSCytb to the pulse-generated monodehydroascorbate radical was found to be about 2~5-times faster than in case of other Cyt-b561 proteins, suggesting that human TSCytb is very effective for scavenging monodehydroascorbate radicals in cells. Low-temperature EPR spectroscopy has revealed the presence of a highly anisotropic low-spin (HALS) heme and a rhombic low-spin heme with g z values of 3.61 and 2.96, respectively, for the mouse TSCytb. However, only two overlapping HALS heme signals with g z values around 3.7 appeared for human TSCytb. Resonance Raman spectroscopy could not detect difference between the in-plane and vinyl vibrational modes of the two

8 2 Alajos Bérczi and Motonari Tsubaki hemes as well as any out-of-plane vibrational modes. These results indicate that (1) the local electrostatic environment within the protein at the two hemes are slightly different from that in other Cyt-b561 proteins, (2) the orientation of the imodazole plane of the two heme-coordinating His residues are almost perpendicular at one HALS heme but the other heme might be easily converted from a HALS-type to a rhombic-type heme (where the two imidazole planes are parallel to each other), and (3) both hemes are in relaxed state (no constraint from the protein body) where the central iron lies within the porphyrin plane. Comparison of these results with those obtained for other Cyt-b561 proteins might help in understanding the precise biological function and mechanism of the human 101F6 protein. 1. Introduction Frequent genetic alterations, like allelic loss or homozygous deletions, in the short arm of the human chromosome 3 are among the earliest molecular changes occurring during the tumor developments in lung, breast, kidney, and other organs (Hung et al. 1995, Kok et al. 1997, Wistuba et al. 2000, Zabarovsky et al. 2002). Investigation on this chromosomal region has identified many tumor suppressor gene candidates (TSGs) in the gene-rich 3p21.3 subregion (Sekido et al. 1998; Sundaresan et al. 1998). Identification of nested 3p21.3 homozygous deletions in small cell lung cancers and a breast cancer line directed positional cloning efforts to a 630-kb region, which was subsequently narrowed to a 120-kb subregion by a breast cancer homozygous deletion (Sekido et al. 1998, Lerman and Minna 2000). The 120-kb region contains 8 putative TSGs, one of which is the 101F6 gene. 101F6 mrna is widely expressed in tissues and the mouse mrna was especially abundant in liver, kidney, and lung (Mizutani et al. 2007) while the human protein was most abundant in liver, placenta, and lung (Lerman and Minna 2000). Elevated expression of 101F6 in tumor cells significantly inhibited cell growth; and intratumoral injection of recombinant adenovirus-101f6 gene vectors as well as systemic administration of protamine-complexed adenovirus-101f6 gene vectors significantly suppressed tumor xenograft growth (Ji et al. 2002). Ohtani et al. (2007) recently found that nanoparticle-mediated 101F6 gene transfer and a sub-pharmacological concentration of ASC synergistically and selectively inhibited tumor cell growth by caspase-independent apoptosis or autophagy both in vitro and in vivo. The C-terminal myc-tagged mouse 101F6 protein was expressed in Chinese hamster ovary (CHO) cells, and immunofluorescence microscopy was used to localize the recombinant proteins; they were found in small vesicles, including endosomes and endoplasmic reticulum of the perinuclear region (Mizutani et al. 2007). It was also shown that CHO cells expressing the myc-tagged mouse 101F6 protein showed higher ferric ion and azo-dye reduction level than the control CHO cells. Mizutani et al. (2007) concluded that mouse 101F6 proteins played roles in the ferri-reduction via a yet unresolved mechanism. These results clearly show the tumor suppressor activity of 101F6 protein. At the level of genomic and predicted protein sequences, a human tumor supressor protein (the 101F6 gene product) has been identified as a putative member of the cytochrome b561 (Cyt-b561) protein family (Lerman and Minna 2000, Ponting 2001, Tsubaki et al. 2005). The mouse homologue was also discovered, sequenced, and shown to be 85% and 95% identical with the human sequences on the cdna and protein sequence level, respectively (Lerman and Minna 2000). Both the human and the mouse 101F6 genes were

9 Physico-Chemical Properties of the Putative Tumor Suppressor Protein, 101F6 3 found to encode a protein consisting of 222 amino acids (Figure 1). Both proteins have 6 trans-membrane α-helices, the central four of which make up the cytochrome b561-core, the N- and C-termini in the cytoplasm, and 4 well-conserved histidine residues for binding two heme-b prosthetic groups, one on each side of the membrane. The adrenal cytochrome b561, as a representative and the most studied protein of this family, is a highly hydrophobic protein with a molecular mass of 28 kda and its expression is localized in the secretory vesicle membranes of adrenal chromaffin cells. This protein is involved in a trans-membrane electron transfer reaction from cytosolic ascorbate (ASC) to intravesicular monodehydroascorbate (MDA) radical that replenishes reducing equivalents to maintain physiological levels of ASC inside the vesicles (Kobayashi et al. 1998, Seike et al. 2003). For the efficient electron transfer, the adrenal gland cytochrome b561 contains a putative ASC-binding motif on the cytosolic side and a putative MDA-radical binding motif on the intravesicular side (Okuyama et al. 1998), respectively (Figure 1). Comparative analysis on the amino acid sequences of seven subfamilies of the cytochrome b561 protein family showed that 101F6 protein does not contain the putative MDA-radical binding motif and the ASC binding motif was significantly modified ( modified motif 1 ; Tsubaki et al. 2005). These results suggested that redox active biofactor(s) other than ASC or MDA radical might be responsible for the redox activity of the 101F6 protein. It is very intriguing to consider that the 101F6 protein has a role for trans-membrane redox signaling via unknown redox-linked activity. Clarification of biophysical and biochemical properties of the 101F6 protein is highly essential for understanding the role of this trans-membrane protein as a candidate for the tumor suppressor activity in cancer. Figure 1. Multiple alignment of bovine (Bt) CGCytb, mouse (Mm) TSCytb, and human (Hs) TSCytb by using Clustal W (v ) software ( Predicted transmembrane segments are bold faced, underlined, and obtained by using HMMTOP ( hu/hmmtop/). The highly conserved His residues binding the two hemes are labelled in pairs with stars ( * and +). Gray triangles ( ) show the 10 places where the mouse and human TSCytb are different. The putative ASC-binding site (box with full line) and monodehydroascorbate binding site (box with dashed line) are shown on the bovine protein. While the former one is present, in modified form, in the TSCytb proteins, the latter one is absent in them.

10 4 Alajos Bérczi and Motonari Tsubaki 2. Expression Systems Except for the cytochrome b561 protein localized in the adrenal gland chromaffin granule membrane, all members of the Cyt-b561 protein family are present in a very small amount in cells and tissues. For the biochemical and biophysical characterization of these proteins in detail, high yield expression system to produce the recombinant protein in study might be required. In the case of 101F6 protein, potent expression systems for the mouse and the human protein have recently been established (Bérczi and Asard 2008, Recuenco et al. 2009). While the His 6 -tagged mouse 101F6 protein was expressed in Saccharomyces cerevisiae (Bérczi and Asard 2008), the His 8 -tagged human ortologue was expressed in Pichia pastoris (Recuenco et al. 2009). The His 8 -tagged human 101F6 protein contains a thrombin-specific cleavage sequence (LVPRGS) between the wilde-type 101F6 protein moiety and the His 8 -tag making thus possible the removal of the tag after the His-tag affinity purification step. The name of TSCytb will be used for the recombinant 101F6 proteins. In order to distinguish the native chromaffin granule cytochrome b561 from its recombinant form, the recombinant protein will be called as CGCytb (see also i.e. Bérczi and Asard 2008). 3. Biochemical Properties In the case of a putative electron transporting protein, the value of midpoint potential of redox chromophores, the nature of electron donors and acceptors, and the oxidation-reduction mechanism(s) should be considered as the most characteristic biochemical properties from the point of view of their biological functions Midpoint Redox Potentials Presence of two heme b prosthetic groups with different midpoint redox potential (E 0 ') values is the most characteristic aspect of the Cyt-b561 proteins. Using a broad range of redox mediators and sodium dithionite, the redox titrations under anaerobic conditions resulted in a set of experimental values that could be best approximated by a Nernst-equation containing two independent one-electron redox centers. Although the primary structure of the mouse and human TSCytb proteins differ only by 10 amino acids, the obtained E 0 ' values showed somewhat significant alterations. While the two E 0 ' values were +140 mv and +40 mv for the mouse TSCytb (Bérczi et al. 2010), they were +109 mv and +26 mv for the human protein (Figure 2) (Recuenco et al. 2013). Although the purification protocols for the two His-tagged proteins differred at some points and the TSCytb proteins were obtained in two different detergent micelles (sucrose monolaurate for the mouse protein, octyl-β-d-glucoside for the human protein), redox titrations were performed under similar conditions (50 mm phosphate buffer, ph 7, 10% glycerol, anaerobic condition, redox mediators, dithionite titration). There were 90~100 mv differences between the two E 0 ' values for both proteins, which is, however, in agreement with the results obtained for other members of the Cyt-b561 protein family (Flatmark and Terland 1971, Takeuchi et al. 2001, Nakanishi et al. 2009a, 2009b, Bérczi et al. 2005, 2007, Liu et al. 2007).

11 Physico-Chemical Properties of the Putative Tumor Suppressor Protein, 101F6 5 Figure 2. Determination of the midpoint redox potentials of the purified human TSCytb. Experimental results were analyzed on the basis of the Nernst equation. If two independent one-electron redox components were assumed to exist, the approximation resulted in the midpoint potentials of +109 mv and +26 mv (solid line) for the high-potential and the low-potential hemes, respectively. If only a single one-electron redox center was assumed to exist, the approximation resulted in a midpoint potential of +63 mv and an improper fit (dotted line) Reduction by Ascorbate and Dihydrolipoic Acid Ascorbate-reducibility is the most well-known characteristic of Cyt-b561 proteins. Purified TSCytb proteins are in their fully oxidized state by the end of purification protocols, if no ASC is included in buffers. Gradual increase of the concentration of ASC in the medium up to 100 mm reduced the mouse TSCytb and resulted in an ASC-dependent reduction profile (Figure 3) that could not be explained (and mathematically described) by assuming a single apparent affinity (or binding ) constant but a mathematical equation with two such constants resulted in a satisfactory approximation (Bérczi and Asard 2008). This result is in full agreement with those obtained for other Cyt-b561 proteins (i.e. Bérczi et al. 2005, Bérczi and Asard 2006, Kamensky et al. 2007), although TSCytb does not have the so-called ASCbinding motif (Tsubaki et al. 2005). It should be noted that effective binding of ASC has never been proved with any of the members of the Cyt-b561 protein family. Mentioning apparent affinity constants (or binding constants") in the mathematical descriptions are misleading. These parameters are namely the midpoint ASC concentrations that characterize the redox transition of the hemeb centers in Cyt-b561 proteins while ASC concentration in the medium increases. For the mouse TSCytb, 0.25 mm and 7 mm are the two midpoint ASC concentrations (Bérczi et al. 2013). These values are about 5-10 times higher than those obtained for the mouse CGCytb (Bérczi et al. 2006).

12 6 Alajos Bérczi and Motonari Tsubaki Figure 3. ASC-dependent reduction of purified mouse TSCytb in detergent micelles at ph 7. The experimental points cannot be approximated by assuming only one interaction site between ASC and TSCytb (dashed line) but rather good fit can be obtained by assuming two interaction sites (continuous line). The two interactions are characterized by the c H and c L parameters (ASC concentrations), where H and L refer to the high-potential and the low-potential heme-b centers, respectively. Figure 4. Reduced-minus-oxidized difference spectra of purified mouse TSCytb in detergent micelles at ph 7 by different reducing agents. Dithionite (DTH), ASC, and DHLA concentrations were 1 mm, the concentration of the other reducing agents were 10 mm. DTT refers to dithiothreitol.

13 Physico-Chemical Properties of the Putative Tumor Suppressor Protein, 101F6 7 Detailed spectrum analysis of the α-band (absorption profile between 545 nm and 575 nm) in the spectra of the dithinite-reduced and the ASC-reduced Cyt-b561 proteins revealed that the heme-b that is characterized by the higher E 0 value was reduced first; thus the hemeb center with the higher E 0 value can be characterized by the lower midpoint ASC concentration (Bérczi et al. 2013). This heme is called the high-potential (HP) heme, while the other one is called the low-potential (LP) heme. The mouse TSCytb can be reduced not only by ASC and dithionite but also some dithiol reagents (Bérczi et al. 2013). Reduced pyridine nucleotides (NAD(P)H) or glutathione (GSH) could not reduce this protein more than 2%, as compared to the reduction by dithionite, even above 10 mm concentration of these reducing agents (Figure 4). Dihydrolipoic acid (DHLA) and dithiothreitol however reduced the mouse TSCytb rather efficiently. It was shown earlier that DHLA could reduce the mouse CGCytb more efficiently than ASC did (Lakshminarasimhan et al. 2006) Reduction by Dithionite and Auto-Oxidation There are two indications that TSCytb is an auto-oxidizable protein. First, if no ASC is present in the buffer, or if the ASC-containing buffer is replaced by an ASC-free buffer by a fast desalting chromatography step at the end of the purification protocol, the highly purified TSCytb is always in its fully oxidized form. Secondly, when the protein was reduced by addition of dithionite, the reduced state of TSCytb was lost after a while and the fully oxidized state appeared (Bérczi et al. 2010). The higher the concentration of dithionite was, the longer the protein stayed in its reduced state. This phenomenon was not observed, if reduction of TSCytb by dithionite was carried out under anaerobic conditions (under continuous streaming of humidified nitrogen or argon gas in the cuvette). Since administration of dithionite not only reduces TSCytb but also depletes dioxygen in the medium, the only difference between the experiments under aerobic and anaerobic conditions was the possibility of re-airation of the medium under aerobic conditions. Under conditions used in the experiments, dithionite was always present in great excess to TSCytb; in such cases and in concentrations applied, it is assumed that SO 2 serves as reducing agent (Mayhew 1978). How re-airation resulted in re-oxidation of the reduced TSCytb was left for further studies. This result even does not allow speculating whether hydrogen peroxide generation could occur; a chemical agent that plays a central role in killing cancerous cells (Chen et al. 2005, Du et al. 2010). 4. Biophysical Properties Biophysical properties of TSCytb include a wide range of parameters that can be obtained by using different spectroscopic methods and which characterize either the whole or just a part of the TSCytb molecule. These properties can be either kinetic (dynamic) or steady-state parameters and mostly refer to some structural feature of the protein under study.

14 8 Alajos Bérczi and Motonari Tsubaki 4.1. UV-Vis Spectroscopy The fully oxidized TSCytb has a sharp absorption band, the so-called Soret band, with absorption maximum at 417 nm and a wide, very shallow absorption band between 500 nm and 600 nm with two hardly resolvable absorption maxima, in the visible wavelength region (Figure 5). Addition of ASC causes a characteristic change and the reduced spectrum of TSCytb has three characteristic bands. The Soret band maximum shifts to 427 nm, and the absorbance increases. Parallel with this change, two new bands with absorption maximum at 529 nm (β-band) and 561 nm (α-band) show up. Both bands are asymmetric and have more than one component. Detailed spectrum analyses of the α-band of the reduced spectrum of the ASC-reduced TSCytb revealed the presence of two, distinct, split α-bands (Bérczi et al. 2010, 2013). These results point to the fact that the two b-type hemes are located in a very anisotropic electrostatic field. The anisotropy might origin from the presence of charged amino acid side chains in the vicinity of the hemes. Different side chains result in different local electrostatic fields which can explain the minor differences between the shapes of the split α-bands (Bérczi et al. 2013). Similar results were obtained when mouse TSCytb was reduced by dihydrolipoic acid. Concentration-dependent reduction of TSCytb by reducing agents and the detailed spectrum analysis of the asymmetric α-band of the reduced spectra provide us a rather sensitive experimental tool to detect the changes that affect the electrostatic field in the vicinity of heme pockets. Figure 5. UV-visible absorption spectra of purified human TSCytb in oxidized (solid line), ascorbatereduced (dotted line), and dithionite-reduced (broken line) states. Inset shows a result of pyridine hemochrome assay of the purified sample, indicating the presence of heme B with 1.59 (±0.06) mole of heme B/mole protein.

15 Physico-Chemical Properties of the Putative Tumor Suppressor Protein, 101F Stopped-Flow Analysis on the Reduction by Ascorbate The fast reduction process of oxidized human TSCytb with ASC was analyzed by a stopped-flow method and was found to be independent of ph (Recuenco et al. unpublished). It is in contrast to those observed for chromaffin granule and Zea mays Cyt-b561 proteins in which both cytochromes exhibited very slow rates at ph 5.0 but faster at ph 6.0 and 7.0, although the electron transfer rates were not significantly slower than those previously reported (Takigami et al. 2003, Nakanishi et al. 2009a). This might be reasonable, if we consider the significant difference in the sequence of the putative ASC-binding site. The well-conserved Lys, Arg, and Tyr residues among other members of the Cyt-b561 family (Tsubaki et al. 2005), which are considered to be important for the interaction with ASC (Nakanishi et al. 2009a, Rahman et al. 2012), were replaced with Ala, Ser, and Phe residues, respectively, inferring that ASC might not be a physiological electron donor of human TSCytb or ASC might utilize a different electron transfer mechanism Effects of the Modification with DEPC DEPC is well known as a chemical modification reagent with high selectivity toward a de-protonated nitrogen atom of the imidazole ring of His residues. It was shown previously that DEPC-treatment of bovine adrenal CGCytb in chromaffin granule membranes or in detergent-solubilized and purified state caused a significant inhibition of the electron transfer from ASC (Njus and Kelly 1993, Kipp et al. 2001, Njus et al. 2001, Tsubaki et al. 2000) both in the final reduction level (30~35%) and in the initial reaction rate (~1/400). The inhibition was caused mainly by specific N -carbethoxylation of the heme axial His residue, with the bond between the heme and the axial His residue remaining intact (Takeuchi et al. 2001, Takigami et al. 2003). This observation was central to the proposal of histidine cycle mechanism for the concerted proton/electron transfer from ASC to the heme iron of adrenal CGCytb (Nakanishi et al. 2007, da Silva et al. 2012). Such specific inhibition on the electron transfer from ASC by the DEPC treatment was observed for other Cyt-b561 proteins (Preger et al. 2005, Nakanishi et al. 2009b, Cenacchi et al. 2012). Very interestingly, such inhibition on the electron transfer from ASC was not observed for human TSCytb at all (Recuenco et al. 2013), despite the fact that more than half of total His residues were modified. This observation suggested that human TSCytb might use a different physiological electron donor other than ASC or might use a different electron transfer mechanism, as suggested in the previous section Pulse Radiolysis Analysis on the Reactions with Monodehydroascorbate Radical and Ascorbate Pulse radiolysis experiments were performed with an electronic linear accelerator as described by Kobayashi et al. (1998). The human TSCytb sample solution with 10 mm ASC was radiated with a pulse beam to generate monodehydroascorbate (MDA) radical. Oxidation of the reduced heme of human TSCytb with pulse-generated MDA radical and following rereduction of oxidized heme with ASC were monitored by absorbance changes at 430 nm and 405 nm. The second order rate constant for the electron donation from the ASC-reduced TSCytb to the pulse-generated MDA radical was found to be 5.0 x 10 7 M -1 s -1 (Recuenco et al. unpublished), about two-fold faster than that of bovine CGCytb (Kobayashi et al. 1998) and

16 10 Alajos Bérczi and Motonari Tsubaki about five times faster than that of Zea mays Cyt-b561 (Nakanishi et al. 2009a), suggesting that human TSCytb is very effective for scavenging MDA radicals in cells. In a later time-domain of the pulse-generated reactions, re-reduction of oxidized heme with ASC occurred, as observed earlier for other Cyt-b561 proteins. The second order rate constants for the reaction of the MDA radical-oxidized form of TSCytb with ASC showed significant ph-dependency (Recuenco et al. unpublished), which was very similar to the corresponding re-reduction process of adrenal CGCytb (Kobayashi et al. 1998). Further, the second order rate constant value at ph 6.5 was probably about 3~4 times faster than those of corresponding values for the reaction of oxidized TSCytb with ASC obtained by stoppedflow technique (Recuenco et al. unpublished), which did not show any significant phdependency. These discrepancies are apparently due to the difference in the starting point of the electron transfer reaction. For the re-reduction phase of the MDA radical-oxidized form of TSCytb, an ASC molecule might be already bound at a certain site of TSCytb, being ready for a rapid electron transfer to the oxidized heme on the intravesicular side. On the other hand, in the stopped-flow experiments, the heme reduction processes might be governed by several factors; approaching of ASC to the catalytic site of TSCytb protein, binding of ASC at the catalytic site, and electron transfer from ASC to the oxidized heme by an unknown mechanism EPR Spectroscopy of the Heme-b Centers EPR spectroscopy is a very sensitive technique for studying the properties of molecules having unpaired electrons, particularly for the metal centers including heme iron. It provides information on the coordination structure and electronic state of the metal centers by analyzing the energy needed for the transition of unpaired electrons between the two spin states. The information in an EPR spectrum is obtained as g max or g z value. The larger the g value is, the further the unpaired electron is from the free electron state (g e =2.0023). Both hemes in TSCytb have unpaired electrons when the molecule is in its fully oxidized state. In the fully reduced state, however, there are no unpaired electrons in TSCytb. The transition between the two redox states can be followed not only by UV-Vis spectroscopy (as discussed above) but also by low-temperature EPR spectroscopy. The two b-type hemes in Cyt-b561 proteins have two distinct EPR signals. The two b- type hemes in mouse TSCytb are characterized by g z =2.96 and g max =3.61. While the latter value is in good agreement with results obtained for other Cyt-b561 proteins, the former value is significantly lower than the values between g z = obtained earlier for other Cytb561 proteins (Tsubaki et al. 1997, Takeuchi et al. 2004, Bérczi et al. 2005,2007, Liu et al. 2005,2007, Nakanishi et al. 2009a). EPR spectra of human TSCytb in oxidized state at 5 K showed only a highly anisotropic low-spin (HALS) signal at g z =3.75. However, at 15 and 20 K, another HALS-type signal appeared at g z =3.65 being overlapped with that of g z =3.75. These two HALS-type signals showed distinct power dependency (Recuenco et al. 2013). The rhombic EPR signal at g z =3.16 previously seen in other Cyt-b561 proteins was not observed, neither in the detergent-solubilized purified state nor in the microsomal membrane state. This observation suggested that one of the heme irons would have distinctly different heme environment from those of usual Cyt-b561 members and might be easily converted from a HALS-type to a rhombic-type heme (or vice versa).

17 Physico-Chemical Properties of the Putative Tumor Suppressor Protein, 101F Resonance Raman Spectroscopy of the Heme-b Centers Resonance Raman (RR) spectroscopy is a technique that provide information for the motional freedom of characteristic chemical groups and/or bonds. Heme-b centers in the mouse TSCytb (Bérczi et al. 2010) have already been subject for RR spectroscopy. Resonance excitation occurred in the Soret band at nm, close to the absorption maximum of TSCytb in the Soret band (see above). The most prominent bands characteristic for the different skeletal vibrational modes of the hemes, both for the oxidized and the reduced forms, are located at almost the same frequency (wavenumber) values as those found for the protein-matrix-free heme-b model complex (imidazole) 2 -protoporphyrine IX {[(Im) 2 Fe 3+ PP] + and [(Im) 2 Fe 2+ PP] +, for the oxidized and reduced compounds, respectively; Choi et al. (1982), Choi and Spiro (1983)}. These results are consistent with the presence of fully-oxidized and/or fully-reduced, hexa-coordinated, low-spin heme-b centers. High similarity holds also for the strongest bands characterizing the stretching and in-plane motions of peripheral groups on the porphyrin ring. The presence of these RR bands attributable to motions of the vinyl groups verifies the presence of b-type hemes in TSCytb. There was no indication that would suggest a structural difference between the two heme-b centers in TSCytb. The RR results support the view that both heme-b centers are in a rather relaxed state; e.g. no constraint from the surrounding amino acid side chains can be observed that would result in deformation of the porphyrin ring of heme-b centers. These results are essentially very similar to that reported for the resonance Raman measurements for bovine adrenal chromaffin granule cytochrome b 561 (Takeuchi et al. 2004). 5. Structure-Function Relationship It was generally assumed for decades that the function of a protein was closely linked to its three-dimensional structure (Fetrow and Skolnick 1998). After identifying some intrinsically unstructured proteins (IUPs) with well-defined biological function(s), however, it became necessary to change or at least modify the old paradigm (Wright and Dyson 1999). The Cyt-b561 protein family was identified on the basis of sequence similarity to the amino acid sequence of bovine chromaffin granule cytochrome b 561 (Asard et al. 2001, Verelst and Asard 2003, Tsubaki et al. 2005). The biological function of this protein is transferring electrons across the adrenal gland chromaffin granule membrane from the cytosolic ASC to the intravesicular semidehydroascorbate (SDA) (Kent and Fleming 1987, Seike et al. 2003). According to the structure-function paradigm, TSCytb should have biological function(s) and physico-chemical properties similar to those of the chromaffin granule cytochrome b 561. However, the classical sequence-to-structure-to-function paradigm that was originally worked out for globular and soluble proteins can hardly be applied for TSCytb. As it has been pointed out, very basic structural (6 trans-membrane helices, 2 b-type hemes, highly conserved His residues) and functional (ASC reducibility) properties of TSCytb are common in the Cytb561 protein family, but the biological function of different members of the Cyt-b561 protein family show some variation (McKie et al. 2001, Griesen et al. 2004, Asard et al. 2013). The biological function of TSCytb seems to be coupled to tumor suppression; some experiments suggest that ASC might also be involved. ASC has been used in cancer treatment for many

18 12 Alajos Bérczi and Motonari Tsubaki years in complementary and alternative medicine practices without having a clear action mechanism (Cameron and Campbell 1974, Cameron and Pauling 1978, Chen et al. 2005, Padayatty et al. 2010). Its action mechanism(s) seem(s) to involve hydrogen peroxide generation (Chen et al. 2005, Du et al. 2010), inhibition of cell cycle progression (Belin et al. 2009, Frömberg et al. 2011), gene expression regulation (Belin et al. 2010), ATP depletion (Chen et al. 2012), but somehow all end up at autophagy of cancer cells. How the biophysical and biochemical properties of the tumor suppressor 101F6 protein prevail in this function will be subject for further studies. Progress of X-ray crystallographic studies on TSCytb would be extremely helpful for understanding the biological activity of this protein. Acknowledgments One of the authors (A.B.) expresses his thanks to the Hungary-Romania Cross Border Cooperation Program of the EU (HURO/0901/219) for the financial support. M.T. expresses his thanks to Grant-in-Aid for Scientific Research (C) ( ) from Japan Society for the Promotion of Science for the financial support. References Asard, H., Kapila, J., Verelst, W. and Bérczi, A. (2001). Higher-plant plasma membrane cytochrome b561: a protein in search of a function. Protoplasma, 217, Asard, H., Barbaro, R., Trost, P. and Bérczi, A. (2013) Cytochromes b561: ascorbatemediated trans-membrane electron transport. Antioxidants and Redox Signaling (in press) doi: /ars Belin, S., Kaya, F., Duisit, G., Giacometti, S., Ciccolini, J. and Fontés, M. (2009). Antiproliferative effect of ascorbic acid is associated with the inhibition of genes necessary to cell cycle progression. PLoS One, 4, e4409. Belin, S., Kaya, F., Burtey, S. and Fontés, M. (2010). Ascorbic acid and gene expression: Another example of regulation of gene expression by small molecules? Curr. Genom., 11, Bérczi, A., Su, D., Lakshminarasimhan, M., Vargas, A. and Asard, H. (2005). Heterologous expression and site-directed mutagenesis of an ascorbate-reducible cytochrome b561. Arch. Biochem. Biophys., 443, Bérczi, A. and Asard, H. (2006). Characterization of an ascorbate-reducible cytochrome b561 by site directed mutagenesis. Acta Biol. Szeged., 50, Bérczi, A., Su, D. and Asard, H. (2007). An Arabidopsis cytochrome b561 with transmembrane ferrireductase capability. FEBS Letters, 58, Bérczi, A. and Asard, H. (2008). Expression and purification of the recombinant mouse tumor suppressor cytochrome b561 protein. Acta Biol. Szeged., 5, Bérczi, A., Desmet, F., Van Doorslaer, S. and Asard, H. (2010). Spectral characterization of the recombinant mouse tumor suppressor 101F6 protein. Eur. Biophys. J., 39, Bérczi, A., Zimányi, L. and Asard, H. (2013). Dihydrolipoic acid reduces cytochrome b561 proteins. Eur. Biophys. J., 42,

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20 14 Alajos Bérczi and Motonari Tsubaki deletion region by an adenovirus vector results in tumor suppressor activities in vitro and in vivo. Cancer Res., 62, Kamensky, Y., Liu, W., Tsai, A.-L., Kulmacz, R.J. and Palmer, G. (2007). The axial ligation and stoichiometry of heme centers in adrenal cytochrome b 561. Biochemistry, 46, Kent, U.M. and Fleming, P.J. (1987). Purified cytochrome b 561 catalyzes transmembrane electron transfer for dopamine hydroxylase and peptidyl amidating monooxygenase activities in reconstituted systems. J. Biol. Chem., 262, Kipp, B.H., Kelley, P.M. and Njus, D. (2001). Evidence for an essential histidine residue in the ascorbate-binding site of cytochrome b 561. Biochemistry, 40, Kobayashi, K., Tsubaki, M. and Tagawa, S. (1998). Distinct roles of two heme centers for transmembrane electron transfer in cytochrome b561 from bovine adrenal chromaffin vesicles as revealed by pulse radiolysis. J. Biol. Chem., 273, Kok, K., Naylor, S.L. and Buys, C.H. (1997). Deletions of the short arm of chromosome 3 in solid tumors and the search for suppressor genes. Adv. Cancer Res., 71, Lakshminarasimhan, M., Bérczi, A. and, Asard, H. (2006). Substrate-dependent reduction of a recombinant chromaffin granule Cyt-b561 and its R72A mutant. Acta Biol. Szeged., 50, Lerman, M.I. and Minna, J.D. (2000). The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. Cancer Res., 60, Liu, W., Kamensky, Y., Kakkar, R., Foley, E., Kulmacz, R.J. and Palmer, G. (2005). Purification and characterization of bovine adrenal cytochrome b561 expressed in insect and yeast cell systems. Protein Expr. Purif., 40, Liu, W., Rogge, C.E., Kamensky, Y., Tsai, A.-L. and Kulmacz, R.J. (2007). Development of a bacterial system for high yield expression of fully functional adrenal cytochrome b 561. Protein Expr. Purif., 5, Mayhew, S.G. (1978). The redox potential of dithionite and SO 2 - from equilibrium reactions with flavodoxins, methyl viologen and hydrogen plus hydrogenase. Eur. J. Biochem., 85, McKie, A.T., Barrow, D., Latunde-Dada, G.O., Rolfs, A., Sager, G., Mudaly, E., Mudaly, M., Richardson, C., Barlow, D., Bomford, A., Peters, R.J., Raja, K.B., Shirali, S., Hediger, M.A., Farzaneh, F. and Simpson, R.J. (2001). An iron-regulated ferric reductase associated with the absorption of dietary iron. Science, 291, Mizutani, A., Sanuki, R., Kakimoto, K., Kojo, S. and Taketani, S. (2007). Involvement of 101F6, a homologue of cytochrome b561, in the reduction of ferric ions. J. Biochem., 142, Nakanishi, N., Takeuchi, F. and Tsubaki, M. (2007). Histidine cycle mechanism for the concerted proton/electron transfer from ascorbate to the cytosolic heme b center of cytochrome b 561 : A unique machinery for the biological transmembrane electron transfer, J. Biochem., 142, Nakanishi, N., Rahman, Md.M., Sakamoto, Y., Takigami, T., Kobayashi, K., Hori, H., Hase, T., Park, S.-Y. and Tsubaki, M. (2009a). Importance of conserved Lys83 residue of Zea mays cytochrome b 561 for ascorbate-specific transmembrane electron transfer as revealed by site-directed mutagenesis studies. Biochemistry, 48,

21 Physico-Chemical Properties of the Putative Tumor Suppressor Protein, 101F6 15 Nakanishi, N., Rahman, Md.M., Sakamoto, Y., Miura, M., Takeuchi, F., Park, S.-Y. and Tsubaki, M. (2009b). Inhibition of electron acceptance from ascorbate by the specific N- carbethoxylations of maize cytochrome b 561 : A common mechanism for the transmembrane electron transfer in cytochrome b 561 protein family. J. Biochem., 146, Njus, D. and Kelley, P.M. (1993). The secretory-vesicle ascorbate-regenerating system: a chain of concerted H + /e - -transfer reactions. Biochim. Biophys. Acta, 1144, Njus, D., Wigle, M., Kelley, P.M., Kipp, H. and Schlegel, H.B. (2001). Mechanism of ascorbic acid oxidation by cytochrome b 561. Biochemistry, 40, Ohtani, O., Iwamaru, A., Deng, W., Ueda, K., Wu, G., Jayachandran, G., Kondo, S., Atkinson, E.N., Minna, J.D., Roth, J.A. and Ji, L. (2007). Tumor suppressor 101F6 and ASC synergistically and selectively inhibit non small cell lung cancer growth by caspase-independent apoptosis and autophagy. Cancer Res., 67, Okuyama, E., Yamamoto, R., Ichikawa, Y. and Tsubaki, M. (1998). Structural basis for the electron transfer across the chromaffin vesicle membranes catalyzed by cytochrome b561: Analyses of cdna nucleotide sequences and visible absorption spectra. Biochim. Biophys. Acta, 1383, Padayatty, S.J., Sun, A.Y., Chen, Q., Espey, M.G., Drisko, J. and Levine, M. (2010). Vitamin C: intravenous use by complementary and alternative medicine practitioners and adverse effects. PloS One, 5, e Ponting, C.P. (2001). Domain homologues of dopamine -hydroxylase and ferric reductase: roles for iron metabolism in neurodegenerative disorders. Human Mol. Gen., 10, Preger, V., Scagliarini, S., Pupillo, P. and Trost, P. (2005). Identification of an ascorbatedependent cytochrome b of the tonoplast membrane sharing biochemical features with members of the cytochrome b 561 family. Planta, 220, Rahman, Md.M., Nakanishi, N., Sakamoto, Y., Hori, H., Hase, T., Park, S.-Y. and Tsubaki, M. (2012). Roles of conserved Arg 72 and Tyr 71 in the ascorbate-specific transmembrane electron transfer catalyzed by Zea mays cytochrome b 561. J. Biosci. Bioeng., (in press). Recuenco, M.C., Fujito, M., Rahman, Md.M., Sakamoto, Y., Takeuchi, F. and Tsubaki, M. (2009). Functional expression and characterization of human 101F6 protein, a homologue of cytochrome b 561 and a candidate tumor suppressor gene product. BioFactors, 34, Recuenco, M.C., Rahman, Md.M., Sakamoto, Y., Takeuchi, F., Hori, H. and Tsubaki, M. (2013). Functional characterization of the recombinant human tumor suppressor 101F6 protein, a cytochrome b 561 homologue. J. Biochem., 153, Seike, Y., Takeuchi. F. and Tsubaki, M. (2003). Reversely-oriented cytochrome b561 in reconstituted vesicles catalyzes transmembrane electron transfer and supports the extravesicular dopamine β-hydroxylase activity. J. Biochem., 134, Sekido, Y., Ahmadian, M., Wistuba, I.I., Latif, F., Bader, S., Wei, M.H., Duh, F.M., Gazdar, A.F., Lerman, M.I. and Minna, J.D. (1998). Cloning of a breast cancer homozygous deletion junction narrows the region of search for a 3p21.3 tumor suppressor gene. Oncogene, 16, Sundaresan, V., Chung, G., Heppell-Parton, A., Xiong, J., Grundy, C., Roberts, I., James, L., Cahn, A., Bench, A., Douglas, J., Minna, J., Sekido, Y., Lerman, M., Latif, F., Bergh, J.,

22 16 Alajos Bérczi and Motonari Tsubaki Li, H., Lowe, N., Ogilvie, D. and Rabbitts, P. (1998). Homozygous deletions at 3p12 in breast and lung cancer. Oncogene, 17, Takeuchi, F., Kobayashi, K., Tagawa, S. and Tsubaki, M. (2001). Ascorbate inhibits the carbethoxylation of two histidyl and one tyrosyl residues indispensable for the transmembrane electron transfer reaction of cytochrome b 561. Biochemistry, 40, Takeuchi, F., Hori, H., Obayashi, E., Shiro, Y. and Tsubaki, M. (2004). Properties of two distinct heme centers of cytochrome b561 from bovine chromaffin vesicles studied by EPR, resonance Raman, and ascorbate reduction assay. J. Biochem., 135, Takigami, T., Takeuchi, F., Nakagawa, M., Hase, T. and Tsubaki, M. (2003). Stopped-flow analyses on the reaction of ascorbate with cytochrome b561 purified from bovine chromaffin vesicle membanes. Biochemistry, 42, Tsubaki, M., Nakayama, M., Okuyama, E., Ichikawa, Y. and Hori, H. (1997). Existence of two heme B centers in cytochrome b 561 from bovine adrenal chromaffin vesicles as revealed by a new purification procedure and EPR spectroscopy. J. Biol. Chem., 272, Tsubaki, M., Kobayashi, K., Ichise, T., Takeuchi, F. and Tagawa, S. (2000). Diethylpyrocarbonate-modification abolishes fast electron accepting ability of cytochrome b 561 from ascorbate but does not influence on electron donation to monodehydroascorbate radical: Distinct roles of two heme centers for electron transfer across the chromaffin vesicle membranes. Biochemistry, 39, Tsubaki, M., Takeuchi, F. and Nakanashi, N. (2005). Cytochrome b561 protein family: expanding roles and versatile transmembrane electron transfer abilities as predicted by a new classification system and protein sequence motif analyses. Biochim. Biophys. Acta, 1753, Verelst, W. and Asard, H. (2003). A phylogenetic study of cytochrome b561 proteins. Genome Biol., 4, R38. Wistuba, I.I., Behrens, C., Virmani, A.K., Mele G., Milchgrub, S., Girard, L., Fondon III, J.W., Garner, H.R., McKay, B., Latif, F., Lerman, M.I., Lam, S., Gazdar, A.F. and Minna, J.D. (2000). High resolution chromosome 3p allelotyping of human lung cancer and preneoplastic/preinvasive bronchial epithelium reveals multiple, discontinuous sites of 3p allele loss and three regions of frequent breakpoints. Cancer Res., 60, Wright, P.E. and Dyson, H.J. (1999). Intrinsically unstructured proteins: Re-assessing the protein structure-function paradigm. J. Mol. Biol., 293, Zabarovsky, E.R., Lerman, M.I. and Minna, J.D. (2002). Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers. Oncogene, 21, Reviewed by emeritus professor Takashi Iyanagi (Department of Life Science, University of Hyogo, Japan).

23 In: Tumor Suppressor Genes ISBN: Editors: Mehmet Gunduz and Esra Gunduz 2013 Chapter 2 The Inhibitor of Growth (ING) Gene Family: Potential Use in Cancer Diagnostics and Therapy Mehmet Gunduz 1,, Eyyup Uctepe 1, Catherine Moroski Erkul 1, Senol Dane 2 and Esra Gunduz 1 Department of 1 Medical Genetics, 2 Medical Physiology, Faculty of Medicine, Turgut Ozal University, Turkey Abstract Attempts to explain precisely how ING gene functions have been modified in tumors have produced a number of theories; among these are loss of heterozygosity (LOH), promoter CpG hypermethylation and protein mislocalization. While ING transcript levels are often down-regulated in tumor cells, mutations in these genes are very rare. However, it is known that inactivation of ING family genes at genetic and epigenetic levels plays a major role in the tumorigenesis of many different tumor types. As a family of putative tumor suppressor genes, ING may prove to be of value as a target for diagnosis and molecular therapy in a variety of cancers. Recent studies have demonstrated that ING genes may play a role in regulating the response of cancer cells to chemotherapeutic agents. Despite this compelling evidence, there are few in vitro studies currently in the literature that investigate the potential use of ING family genes in gene therapy. Advancements in knowledge of ING family gene function(s) as well as their interaction with p53 and other as yet unidentified molecules will shed light on their role in the development of human cancers, and aid in determining their potential for use in cancer diagnostics and therapy. Corresponding author: Mehmet Gunduz, MD, PhD, Department of Medical Genetics, Faculty of Medicine, Turgut Ozal University, Turkey. Anadolu Bulvari 16A Gimat Ankara, Turkey. Tel: /7221. Fax: mgunduz@turgutozal.edu.tr.

24 18 Mehmet Gunduz, Eyyup Uctepe, Catherine Moroski Erkul et al. Keywords: Tumor suppressor gene, ING1, ING family, cancer, gene therapy Introduction The first member of the Inhibitor of Growth (ING) family was discovered by Riabowol s group in 1996 via subtractive hybridization [1, 2]. Designated ING1, this gene has been mapped to chromosome 13 at locus 13q33-34 and encodes a 33-kDa protein (p33ing1b). ING genes have been reported in many species including human, mouse, rat, cow, zebra fish, venous, yeast, etc., highlighting their importance in central biological processes [3]. Our group characterized the genomic structure of human ING 1 [4]. Subsequently it is determined that it has four exons and three introns. The ING1 gene can be alternatively spliced to generate p47ing1a, p33ing1b, p24ing1c and p27ing1. The loss of ING 1 gene expression has been demonstrated in numerous cancer types. Furthermore, ING 1 knockout mice were found to be cancer prone [5]. Subsequently, four additional human ING genes (ING2-5) were identified. By homology studies, ING2 (also known as ING1L) was cloned [6]. It was mapped to chromosome 4 at locus 4q35.1 and encodes a single variant. In 2003, ING3was identified through a computational domain search. p47ing3 encodes a 46.8-kDa protein and contains a C-terminal plant homeodomain (PHD) finger motif [7]. It has been mapped to chromosome 7 at the locus 7q31 and as a result of distinct splicing, encodes two variants. The other members of the ING family, ING4 and ING5 were also identified in 2003 through using a computational sequence homology search [8]. The ING4 gene was mapped to chromosome 12 at locus 12q13.3 and encodes 8 variants as a result of alternative splicing. The ING5 gene was mapped to chromosome 2 at locus 2q37.3 and encodes a single variant. Also, an ING-like pseudogene, designated INGX, was identified and mapped to the X chromosome at locus Xq12.9 [9]. As the nomenclature used for the multiple isoforms of the different ING family genes aroused confusion in the literature, a new nomenclature was reported to standardize the naming of the various ING genes and their transcripts [10]. This review summarizes the most recent research on ING family genes in relation to their tumor suppressor function and potential role in cancer therapy. ING Family Structure All ING proteins are characterized by a highly conserved C-terminal domain that is also commonly found in various chromatin remodeling proteins [11]. They have a high degree of homology with one another, but the N-terminal region of each ING protein is unique and determines each ING member s specific role(s) [4, 12] (Figure 1). There is at least one nuclear localization sequence (NLS) and plant homeo domain (PHD) finger found in the C- terminal domain. Additionally, they contain a nuclear conserved region (NCR), which can also direct the ING proteins to the nucleus. This domain might specifically participate in binding to histone acetylase (HAT) and histone deacetylase (HDAC) complexes. There are three nucleolar

25 The Inhibitor of Growth (ING) Gene Family 19 targeting signals (NTS) in ING 1 and ING 2. Two of them target ING 1 to nucleoli in response to different stress.[9, 13] ING members also have other motifs such as a binding motif, polybasic region (PBR), PCNA-interacting protein (PIP) bromodomain (PBD) and leucine zipper like (LZL) motifs. [14] The PIP motif interacts with PCNA in a DNA damage inducible manner. Decreased expression of ING1b causes decline in PCNA monoubiquitination and sensitizes cells in response to UV during S phase [15]. Also it was shown with nuclear magnetic resonance study that ING1b PIP interacts PCNA with a very low affinity suggesting ING1b PIP motif have not a second aromatic residue generally role in the canonical PIP motif [16]. The family, which recognizes binding motifs, and targets proteins to different subcellular localizations. The N-terminus of p33ing1b includes both a PIP and PB motifs, which control proteinprotein interactions during chromatin remodeling. Phylogeny studies have shown that ING1 and ING2 proteins and ING4 and ING5 proteins have an overall high homology with one another and, therefore, could have closely related or redundant functions. Several studies have shown that ING proteins play a significant role in multiple critical cellular processes such as growth regulation, senescence, apoptosis, DNA repair, cell migration, etc [17-19]. p33ing1b was originally described as interacting physically with the tumor suppressor protein p53 and as being necessary for p53 transcriptional activity. Thus, intact p33ing1b is necessary for the efficient negative regulation of cell proliferation by p53 [20]. Further studies have shown that all the ING proteins may have functions in [21] cell cycle arrest, apoptosis and senescence [8, 20, 22-26]. However, a note of caution is necessary as studies on ING1 knockout mice and knockout cells derived from these mice suggest that in physiological conditions, the function of ING1 proteins may be mostly independent of p53 signaling pathways [5, 27]. Figure 1. ING proteins and their basic functions.

26 20 Mehmet Gunduz, Eyyup Uctepe, Catherine Moroski Erkul et al. The ING Family and Their Biological Functions ING family proteins regulate a variety of critical cellular and signaling processes such as growth regulation, apoptosis, DNA repair, DNA demethylation [28], angiogenesis, cell migration, tumorigenesis, cellular senescence, negative regulation of cell proliferation [1, 29, 30], chromatin remodeling [20, 31], hormone responses [32, 33] and regulation of tumor growth via NF-kB (34) and hypoxia inducible factor pathways [35, 36]. They can form complexes with HATs and HDACs [17]. In addition, suppression of ING proteins has been shown to increase cell migration and to inhibit contact inhibition. Some studies have shown that most of the ING proteins are important for proper p53 function [17, 21]. Although more recent mouse model experiments indicate otherwise [27] p33ing1b has been shown to physically interact with p53 and to have an essential role as a cofactor for p53-mediated cell regulation and apoptosis [17, 18, 21]. Studies have also indicated that ING proteins are involved in cell cycle checkpoints and cell cycle progression [17, 21]. ING1 expression is significantly repressed in 44% of human primary breast cancers and 100% of established breast cancer cell lines [18]. Decreased ING1 expression has been found in many other forms of solid and blood tumors [37-41]. Similarly, the expression of ING2, ING3 and ING4 is reduced in human melanomas [42-44]. All ING family proteins have been shown to cooperate with p53 to induce apoptosis and cellular senescence [45-49] and, as a result, the notion that the ING family proteins act as class II tumor suppressors has emerged. It was recently shown that ING1 binds to the DGCR8 promoter, a protein involved in the early steps of microrna biogenesis, and controls its transcription through chromatin regulation [50]. Eapen et al. characterize a mechanism by which ING2 contributes to muscle differentiation. In structure-function analyses, they found that the leucine zipper motif of ING2 drives ING2-dependent muscle differentiation. By contrast, the PHD domain, which recognizes the histone H3K4me3 epigenetic mark, blocks the ability of ING2 to induce muscle differentiation [51]. Helbing et al. demonstrated that ING proteins modulate T helper dependent immune responses and revealed a novel role for ING proteins in hormone signaling which can influence disease states. They suggest that the induction of ING proteins may facilitate TH receptor function during transformation in a tissue-specific manner [52]. Researchers have identified loss of heterozygosity (LOH), reduced mrna expression, loss of nuclear protein expression and mutation of ING genes in different tumors and tumor cell lines (Table 1). Inhibitor of growth-4 has been shown to have a role in innate immunity. It promotes IkappaB promoter activation thereby suppressing NF-kappaB signaling and innate immunity.[53] Zhang et al. showed that in murine embryonic fibroblasts (MEFs) derived from (interaction partners of Inhibitor of cyclin A1) Inca1 (+/+) and Inca1 (-/-) mice, overexpression of ING5 suppressed cell proliferation only in the presence of INCA1, while ING5 had no effect in Inca1 (-/-) MEFs. ING5 overexpression triggered a delay in S-phase progression, which required INCA1. In aggregate, ING5 overexpression accelerated Fasinduced apoptosis in Inca1 (+/+) MEFs, while Inca1 (-/-) MEFs were protected from Fas antibody-induced apoptosis [54]. ING2 was recently established as a novel regulator of spermatogenesis, functioning through both p53- and chromatin-mediated mechanisms, suggesting that anhdac1/ing2/ H3K4me3-regulated, stage-specific coordination of chromatin modifications is very

27 The Inhibitor of Growth (ING) Gene Family 21 important for normal spermatogenesis, and provides an animal model to study idiopathic and iatrogenic male infertility. ING Family Genes and Human Tumors Gene rearrangements in ING1 have been identified in a neuroblastoma cell line and decreased expression is common in primary tumors and cell lines [1, 12]. Our group characterized the genomic structure of the human ING 1 gene and subsequently identified its tumor suppressor role for the first time by demonstrating its chromosomal deletion at 13q34 and tumor-specific mutations in head and neck squamous cell carcinoma (HNSCC) [4]. There are many studies investigating the mrna expression status of ING family genes in different human cancers. Toyama et al. examined mrna expression in breast cancer patients and found 2 to 10 fold decreases in 44% of the tumors tested [18]. In addition, most of the breast cancers exhibiting decreased ING 1 expression had metastasized to regional lymph nodes. In comparison, only a subset of these cancers, which had increased ING1 expression, as compared to adjacent normal tissues, were metastatic [18]. Other studies have also found down-regulation of ING 1 mrna in different cancer types such as lymphoid malignancies, lung cancer, brain tumors and esophagogastric carcinomas, however no comprehensive clinical correlation has been carried out [12, 17, 55] Rare missense mutations in ING 1 have been identified in esophageal carcinomas [39] and colon cancer cell lines [56] while no mutations have been found in studies of leukemias [40, 57], oral cancers [58] and lymphoid malignancies [37]. Although mutations are not common, reduced expression of ING 1 mrna has been detected in breast cancers [13, 45], gastric cancers [56] and lymphoid malignancies [37]. Some theories have been postulated to explain how changes in ING gene functions occur in tumors, such as loss of heterozygosity (LOH), promoter CpG hypermetylation and protein mislocalization [12, 17]. Using methylation-specific PCR, the p33lng1b promoter was determined to be methylated and silenced in approximately a quarter of all cases of primary ovarian tumors [59]. Recent studies failed to find differences in ING expression in myeloid leukemia or melanoma [40, 60]. Reduced ING2 expression has been detected in lung cancer, melanoma, colon cancer and hepatocellular carcinoma (HCC) and was associated with tumor progression and a reduction in overall survival [61]. Our group has also identified frequent deletions of the ING2 locus at 4q35.1, which are associated with advanced tumor stage in HNSCC [62]. ING2 may have a role in melanoma initiation, since decline in nuclear ING2 has been demonstrated in the radial and vertical growth phases in metastatic melanoma as compared with dysplastic nevi. [61]. These epidemiological studies suggest that ING2 loss or reduction may be essential for tumor initiation and progression [1]. The third member of the ING family was recently identified (ING3) by our group [102]. ING3 allelic loss and reduced expression was identified in a limited number of HNSCC cases. In our study a missense mutation at codon 20 of ING3 was identified but was very rare, suggesting that there may be other inactivation mechanisms at play, perhaps via transcriptional mechanisms like promoter methylation. Whatever the precise mechanism, ING3 down-regulation is a significant step in the process of carcinogenesis [102].

28 22 Mehmet Gunduz, Eyyup Uctepe, Catherine Moroski Erkul et al. Table 1. ING Status and Aberrations in Human Malignancies INGs Type of Tumor Mutation/Aberration Status References ING1 ALL Loss of nuclear expression [41, 63] Adenocarcinoma (EGF) Decreased mrna expression [64] Silent and missense mutation Astrocytoma Decreased mrna expression [65] Basal cell carcinoma Missense mutation [66] Bladder cancer Decreased mrna expression [67] Brain tumors Missense mutation, overexpression [1, 67, 68] Breast carcinoma Decreased protein and nuclear expression/decreased mrna expression, mutation [18, 41, 63] Colorectal carcinoma Decreased mrna expression [69, 70] Esophageal carcinoma LOH, mutation [39] Gastric carcinoma Decreased mrna expression [56] Hepatocellular carcinoma Decreased mrna expression/missense mutation,loh [4, 71, 72] HNSCC LOH, mutation [12] Laryngeal squamous cell Mutation [73] carcinoma Melanoma Loss of nuclear expression/missense mutation [19, 74] Meningioma Missense mutation [75] Neuroblastoma Decreased mrna expression [1, 32, 67] NSCLC Nonsense and missense mutation, Decreased mrna [76, 77] expression, LOH Osteosarcoma Nonsense and missense mutation [78] Ovarian carcinoma Decreased mrna expression [59] Pancreatic carcinoma LOH, mutation [79] Papillary thyroid Loss of nuclear expression [63] carcinoma Seminoma Loss of nuclear expression [63] Skin basal cell carcinoma Missense mutation, overexpression [66] ING2 Lung cancer No mutation/decreased mrna expression [80] Melanoma Decreased nuclear expression [66] HNSCC LOH [62] Breast carcinoma LOH [81] ING3 HNSCC Decreased or no mrna expression/missense mutation [3, 82, 83] Melanoma Decreased nuclear expression [83, 84] Ameloblastoma LOH [85] Renal cell carcinoma LOH [86] ING4 Brain Tumors Decreased mrna expression [34, 87] Breast carcinoma Mutation, Chromosomal deletion [88-90] HNSCC LOH, decreased mrna expression [6, 91] Melanoma Decreased mrna expression [92, 93] Ovarian cancer Decreased mrna expression [94] Lung cancer Decreased mrna expression [95] Gastric adenocarcinoma Decreased mrna expression [96] Colorectal Cancer Decreased mrna expression [97] Myeloma Decreased mrna expression [98] Hepatocellular carcinoma Decreased mrna expression [99] ING5 HNSCC, Oral cancer LOH, decreased mrna expression [100, 101]

29 The Inhibitor of Growth (ING) Gene Family 23 In another study, we found that down-regulation of ING3 occurred more frequently in late-stage tumors as compared with early-stage patients and patients with reduced ING3 mrna expression showed worse survival rates as compared to the patients with normal-high ING3 expression [82]. ING4 mrna levels have been found to decreased in glioblastoma and related with advanced tumor progression [34]. Increased expression of IL-8 and osteopontin (OPN) has been associated with reduced ING4 in myeloma [98]. In these reports, higher tumor grade and increased tumor angiogenesis was accompanied by decreased ING4 expression. Also, higherinterleukin-5 and osteopontin expression was identified in myeloma [98]. Expression of ING4 was lower in malignant melanoma as compared to dysplastic nevi and was determined to be an independent factor contributing to poor prognosis in these patients [92]. Another study revealed that ING4 prevented the loss of contact inhibition and unrestrained growth. Finally, some mutations and deletions were found in cell lines originating from human cancers including breast and lung cancer [88]. We previously reported reduced ING4 expression and LOH in HNSCC [103]. Analysis of LOH was performed at the12p12-13 region in 50 HNSCC tumors and allelic loss was estimated at 66% in the informative cases. There were noing4mutationsdetected in these patients. It was recently reported that ING4 may be an important regulator of chromatin acetylation [104]. It was shown that adenovirus-mediated ING4 expression can inhibit tumor invasion and angiogenesis, thereby causing suppression of lung carcinoma cell growth [104]. It was demonstrated that ING4 physically interacts with a HAT, p300, and p53, which causesp53 acetylation. ING4 accelerated the transit of cells through the G1/S phase of the cell cycle, p21 promoter activity and protein expression and apoptotic ratio in cells with wild type p53, however the effect of ING4is lower in cells with mutant p53 [3, 21, 105, 106]. However, no direct relationship with p53 mutation status and HNSCC patients has been determined in our study [103]. Nagahama et al. demonstrated up-regulation of ING4 in a human gastric carcinoma cell line (MKN-l) by triggering mitochondria-mediated apoptosis via the activation of p53 [107]. Our group has also determined that the ING5 chromosome locus is implicated in a human cancer. In our study, using 16microsatellite markers on the long arm of chromosome 2q , we found a high ratio of LOH in oral cancer [100]. Based on these preliminary findings, ING 5 appears to be a strong candidate tumor suppressor; however, it should be noted that several other candidate TSGs such as ILKAP, HDAC4, PPPI R7, DTYMK, STK25, and BOK are also located in this area, where frequent deletions have been found [100]. Still, our ongoing studies have shown mutation and reduced expression of ING5 mrna in oral cancer tumors, supporting its tumor suppressive role in cancer. A summary of alterations of ING genes in human tumors is shown in Table 1. ING Family and mirna In certain cancers, including lymphoid malignancies, lung cancer, gastric cancer, brain tumor, ING1b and ING2a expression loss could be involved in the tumor initiation and/or progression. It is demonstrated that since mir-622 causes to decrease the ING1 mrna expression by targeting the ING1 3 UTR in gastric cancer suggesting decreased ING1b or

30 24 Mehmet Gunduz, Eyyup Uctepe, Catherine Moroski Erkul et al. ING2a mrna could be the result from misregulation of micrornas targeting ING1b or ING2a mrna [108]. Also certain mi-rna s, such as mir-203, are epigenetically regulated by ING1b and may repress cancer cell proliferation by downregulating CDK6, c-abl and Src. [109] Importance of ING Family Genes in Molecular Diagnostics and Therapy Using ING Family Genes in Gene Therapy As noted above, ING transcript levels are often down-regulated in cancer cells but mutations are very rare. Despite the lack of mutations, it is known that the inactivation of ING family genes at genetic and epigenetic levels plays a major role in the tumorigenesis of various cancers. ING family genes may therefore be of value as a target for diagnosis and molecular therapy in a variety of different tumors. There are only a few in vitro studies in the literature in which gene therapy with ING family members is investigated. First, the application of ING1 was reported as a pioneering approach for the treatment of cancer in 1999 [110]. Adenovirus-mediated introduction of ING 1 was able to suppress the growth of glioblastoma cells, and when combined with p53 transduction, there was a synergistic enhancement of apoptosis in these cells [110]. It is therefore suggested that ING1 may itself function as a pro-apoptotic factor as well as enhancing the effect of p53. Another study reported the combined usage of ING1 and p53 in esophageal cancer [111]. Co-expression of ING1 and p53 accelerated cell death as compared to each gene transcript expressed separately in esophageal carcinoma cells. Hence, the synergistic effect between p33ing1 and p53 in the induction of apoptosis has been demonstrated in two different human cancers, i.e., esophageal carcinoma and glioblastoma. Considering these two in vitro studies, combined gene therapy of one or more ING family members with or without p53 has emerged as a potentially useful alternative therapy when the use of p53 alone does not elicit a strong enough response. Another recent study showed that the ING1 splicing isoform p47ing1a is differentially up-regulated after cisplatin treatment in human glioblastoma cells (LN229). This may be part of a DNA damage response triggered by this platinum-containing chemotherapeutic agent. [112]. Increasing ING1 expression may promote tumor progression by enhancing the progression of cells through G1 and thus promote apoptosis more rapidly in response to cisplatin. LN229 cells express ING1 proteins and harbor mutated TP53. It was suggested that ING1 expression levels, independent of p53 status, might predict the relative sensitivity of glioblastoma to treatment with cisplatin and HDAC. It is suggested that combined detection of p33ing1, p53, and Beclin1 genes and proteins will be useful for early diagnosis and prognosis evaluation for NSCLC, and can give experimental evidence for biotherapy of NSCLC [113]. It is found that the efficacy of 5- azacytidine is increased by ING1b suggesting it could be used a therapeutic agent in breast cancer [114]. Expression of p33ing1b is increased by Azidothymidine (AZT), regulating possibly senescence and apoptosis of the TJ905 glioblastoma cells [115].

31 The Inhibitor of Growth (ING) Gene Family 25 Figure 2. Possible effects of ING genes in gene therapy. These results show that gene therapy withing1 could be added to chemotherapeutics in a subset of human cancers (Figure 2). It was recently demonstrated that ING4 may be effective as a novel anti-invasive and anti-metastatic agent in gene therapy for human lung carcinoma [104]. Adenovirus-mediated ING4 expression repressed tumor growth and cell invasiveness in A549 lung cancer cells. These results suggest that ING4, as a potent tumor suppressing agent, may provide significant therapeutic possibilities. Xie et al. reported an interesting finding when they showed that ING4 could down-regulate the expression of MMP-2 and MMP-9, possibly by repressing the NF-kB pathway, in lung cancer cells [104]. Moreover the inhibitory effect of ING4 on MMP- 2 and MMP-9 activities may help to repress melanoma cell invasion [92]. Overexpression of lng4 significantly reduced melanoma cell invasion by 43% and repressed cell migration by 63%. In A549 cells, treatment with Ad ING4 caused down-regulation of the expression of MMP-2 and MMP-9.These findings demonstrate that ING4 could repress the degradation of ECM and basement membrane components. In this regard, the relationship between ING4 and the MMP pathway may present novel opportunities for molecular therapy of cancer. Loss of ING4 may promote microvessel formation and play a role in facilitating the development of ovarian cancer. One study showed that ING4 mrna and protein were significantly down-regulated in ovarian cancer patients as compared to normal controls. ING4 expression correlated negatively with stage and histological grade of ovarian cancers. Although the specific mechanisms are not yet understood, our data suggest that ING4 may be a promising target for the treatment of ovarian cancer [94]. Zhao et al. demonstrated that treatment with either ING4 or 125I radiotherapy could trigger Panc-1 pancreatic cancer cell growth suppression and apoptosis in vitro. Importantly, they also showed that in mouse xenograft models, both treatments could inhibit tumor growth

32 26 Mehmet Gunduz, Eyyup Uctepe, Catherine Moroski Erkul et al. and angiogenesis of Panc-1 pancreatic cancer cells. Furthermore, the combination therapy had a synergistic effect [116]. It has emerged that, both in vitro and in vivo, the synergistic anti-tumor activity exhibited by Ad-ING4-IL24 closely cooperative activation of extrinsic and intrinsic apoptotic pathways and reduced production of the pro-angiogenic factors VEGF and IL-8. Hence, results show that combining two or more tumor suppressors such as ING4 and IL-24incancer gene therapy may constitute a novel and effective therapeutic strategy for cancers [117]. Zhu et al. showed that in vitro adenovirus-mediated ING4 and IL-24 co-expression in A549 lung carcinoma cells had an additive effect on growth suppression and triggered apoptosis. There was also a combined effect on up-regulation of P21, P27, Fas, Bax and cleaved Caspase-8, -9, and -3 and down-regulation of Bcl-2. In an in vivo xenograft experiment, A549 lung carcinoma cells were transplanted subcutaneously into athymic nude mice. Subsequent Ad-ING4-IL-24 treatment had an additive effect on tumor growth inhibition and decreased CD34 and microvessel density [118]. In SMMC-7721 hepatocarcinoma cells, Ad-ING4 plus CDDP (cisplatin) was shown to trigger synergistic growth inhibition and accelerated apoptosis. Together they had an additive effect on up-regulation of Fas, Bax, Bak, and down-regulation of Bcl-2 and Bcl-X (L). An increase in cleaved Bid, cleaved caspase-8, caspase-9, caspase-3 and cleaved PARP was also observed. Importantly, as with the above-mentioned lung xenograft model, when SMMC cells were subcutaneously injected into athymic nude mice, a synergistic suppression of tumor growth, reduced tumor vessel CD34 expression and decreased microvessel density was observed. These results suggest that Ad-ING4 plus CDDP is a potential combination treatment strategy for hepatocarcinoma [119]. Reduced ING4 nuclear and cytoplasm expression were both seen in lung cancer and correlated with tumor grade. Interestingly, compared with normal tissues, tumors have higher ING4 expression in the cytoplasm than in the nucleus. Nuclear ING4 inhibition correlated with tumor stage and lymph node metastasis. Lack of nuclear ING4 correlated with tumor stage and lymph node metastasis. ING4 expression was lower in grade III than in grades I-II tumors and reduced ING4 mrna correlated with lymph node metastasis. Overall reduction of ING4 expression and high cytoplasmic ING4 expression (relative to nuclear expression) may be involved in the initiation and progression of lung cancers, and thus, analysis for ING4 expression may be useful as a clinical diagnostic and prognostic tool for lung cancer [119]. Li et al. demonstrated that Ad-ING4 gene transfer transfectiontomutantp53 MDA-MB- 231 breast carcinoma cells results in a G2/M phase cell cycle arrest and apoptosis. In this model, P21, P27, and Bax were up-regulated andbcl-2, IL-8, and Ang-1 were downregulated. Furthermore, this promoted cytochrome c release from mitochondria into the cytosol and caspase-9, caspase-3, and PARP were activated. Intratumoral injections of Ad- ING4 in nude mice bearing mutant p53 MDA-MB-231 breast tumors markedly inhibited tumor growth and reduced CD34 expression in tumor vessels and also reduced microvessel density. Thus, ING4 is also a potential candidate for breast cancer gene therapy. Borkosky et al. revealed that a significant relationship exists between LOH of D2S 140 (ING5 locus) and solid tumors. LOH of ING3MS (ING3 locus) was also high in solid tumors, showing a near significant association. In addition, a notable tendency toward higher LOH for half of the markers was observed in recurrent cases. LOH of ING family genes is a common genetic alteration in solid ameloblastoma. In work published by Nozell et al. ING4was found to interact physically with NF-kB, causing a decline in p65 phosphorylation, p300 protein

33 The Inhibitor of Growth (ING) Gene Family 27 levels, as well as reduced levels of acetylated histones and H3Me3K4, and an increased amount of HDAC-l [120]. It was also demonstrated that ING4 interacts with p65 and can inhibit NF-kB-induced COX-2 and MMP-9 expression, genes which are known to be regulated by NF-kB and play a role in gliomagenesis [120]. Some research has proposed the notion that the transfer of ING4 into cancer cells by gene therapy could also target molecules that it associates with, such as MMPs and COX-2. Hence, combined treatment with inhibitors of MMP and COX-2 with ING4 gene delivery could be a potent treatment in some cancer types. Targeting ING Protein-Related Complexes in Molecular Therapy HATs and HDACs have a role in regulating the activity of diverse types of non-histone proteins, such as transcription factors and signal transduction mediators. The imbalance between HAT and HDAC levels has been determined in tumors, with particular emphasis on the activity of HDACs human cancers. This has led to the emergence of HDAC inhibitors (HDACI) as a new class of molecular targets for cancer therapy [4, 12, 121, 122]. Acting through their PHD domains, ING proteins can alter chromatin structure by acetylation or de-acetylation of HAT and HDAC complexes, respectively [2, 12, 55]. ING proteins interact with these complexes, which are involved in key cellular processes such as DNA repair, senescence, angiogenesis, apoptosis and tumorigenesis [4, 12, 55, 123]. Because HDACs are part of a transcriptional complex that affects different tumor suppressor genes and it was demonstrated that HDAC inhibitors trigger cell growth arrest and apoptosis in cancer, ING gene products could act as a bridge to bring HAT/HDAC proteins into the molecular targets for the development of enzymatic inhibitors to treat human cancer [21, 121, 122, 124]. HDACIs alter extracellular matrix and ECM related proteins and have a role in cell migration, invasion and metastasis [124]. It has been shown that HDACIs up-regulate some metastasis suppressor genes such as TIMP 1, RECK and tetraspanin and are also able to down-regulate some metastasis activating genes such as MMP-2, MMP- 9 and metastasis associated proteins MTAI, MTA2 and TGFB1.It is known that degradation of the ECM by MMPs is a key step in tumor cell invasion and metastasis. Thus, combined therapy consisting of HDACIs together with ING gene delivery could be promising for cancer therapy. Nagahama et al.reported that [107] forced expression of run-related transcription factor (RUNX3) in a gastric carcinoma cell line up-regulateding1 and ING4, both of which have a significant role in the regulation of apoptosis. RUNX3-inducedup-regulation ofing1 and ING4 accelerated mitochondria-mediated apoptosis via the activation of p53 and suppression of HIF-1. Down-regulation of TXN2 and HSPD1, which suppresses cytochrome c release from the mitochondria, was also observed. This work indicates that ING genes are candidate targets for treatment of cancer either through gene therapy or molecules such as HIF-1. ING4 was proposed as a suppressor of HIF-l and repressor of angiogenesis [35, 98, 125]. HIF-1 is one of the major regulators of hypoxia-responsive genes such as VEGF, IL-8, angiopoetins and OPN [98]. Reduced ING4 expression has been accompanied by upregulated expression of IL-8 and OPN in myeloma cells. Myeloma cells also produce OPN via the activation of the Runx2/Cbfa1 gene [98]. This finding indicates the potential involvement of the NF-kB pathways other than Runx2/Cbfa1 in OPN production. It was also showed by Colla et al that ING4 interacts with the HIF- I -regulating critical enzyme, a HIF

34 28 Mehmet Gunduz, Eyyup Uctepe, Catherine Moroski Erkul et al. prolyl hydroxylase (HPH-2), which is responsible for the hydroxylation of HIF-1, enhancing its proteosomal degradation. Both ING4 and HPH-2 are up-regulated under hypoxic condition which indicates that both molecules are essential for the regulation of HIF-1 activity. The inhibition of ING4 in myeloma cells also increaseshif-1 activity and NIP-3 expression. This finding indicates that ING4 has a role in the antigenic process and exerts an inhibitory effect on the production of HIF-l, IL-8 and OPN. Besides this, one of the ING4/HAT complex subunits, JADE1 (gene for apoptosis and differentiation in epithelia) is stabi1ized by von Hippel Lindau (VHL) tumor suppressor activity [126]. The association of ING4 with HAT as well as HIF-1 may suggest that we focus our attention on identifying the physiological complex in which ING4 is found for investigating new therapeutic candidates. Melanoma antigen A2 (MAGE-A2) was found to interact with and suppress p53 by recruiting HDAC to p53 transcription sites [127]. The association between MAGE-A2 protein expression and resistance to apoptosis was confirmed. The combined treatment of melanoma cells with trichostatin A (TSA) and etoposide (ET) contributed to the p53 response and chemoresistance was reduced. A recent study reported an interaction between the lamin interaction domain (LID) of ING proteins and lamin type V intermediate filament proteins [128]. Lamin type V intermediate filament proteins are thought to play a role in nuclear stability. Lamins also cooperate with oncogenes such as β-catenin. They demonstrated that lamina binds ING1, thereby adjusting its cellular levels and activity, which suggests that ING proteins function as bridges between chromatin and the nuclear lamina. As ING proteins are stoichiometric components of HAT and HDAC complexes [3], this connection between ING and lamin A provides a new clue for exploring the role of ING genes in cellular senescence and tumorigenesis. Known roles for ING proteins in regulating apoptosis and chromatin structure show that loss of lamin A-ING interaction may be an indicator of lamin A loss, thereby contributing to laminopathies and tumor progression [21, ]. Hence further elucidation of this link may be helpful for the development of novel therapies. Using ING Genes as Cancer Biomarkers Sub-Cellular Localization of ING Genes as a Biomarker In the regulation of cell cycle and proliferation, the shuttling of proteins between nucleus and cytoplasm is a critical step. Many TSGs such as p53, BRCA1 and ING1 carry out some of their functions through nucleo-cytoplasmic shuttling. Any impairment in the nuclear cargo system abolishes the subcellular localization of the TSGs and causes cancer development through the mis-localization and altered function of TSG proteins [123]. The mis-targeting of tumor suppressors can lead to direct cellular consequences and potentially trigger initiation and progression of cancer. These kinds of irregularities, which lead to the mis-localization of tumor suppressors have been identified for p53, BRCA1, APC, and ING1.In the tumor suppressor or their partners are generally present during the process of carcinogenesis [123, 133, 134]. Neuman et al. demonstrated that in the development and progression of melanomas, translocation of p33lng 1b from the nucleus to the cytoplasm of melanocytes may have an important role [74]. Cytoplasmic ING 1b immunostained with newly developed monoclonal antibodies (MAb) against GN1 and GN2 was related with malignant melanoma and may be

35 The Inhibitor of Growth (ING) Gene Family 29 an early sign of malignancy, thus indicating that ING1b Mab could be useful in diagnostic approaches. It was shown by Vieyra et al. that subcellular mis-localization of p33 ING1b is a frequent occurrence in glooms and glioblastomas [75]. Overexpression and abnormal localization of ING1b were identified in all 29 of the brain tumors studied. p33ing1b includes a nuclear targeting sequence [12, 20]. It has been shown that altered subcellular localization of p33lng 1b abolishes its proapoptotic functions [135]. Loss of targeting factors which ensure the correct intracellular localization of p33ing 1b or that are required for the physical interaction between ING 1and p53 could be responsible for the aberrant localization of p331ng 1b in tumors. New experimental observations, such as post-translational stabilization of p53 by p33inglb [136] and the discovery of the p53 cytoplasmic anchoring parkin-like ubiquitin ligase (PARC) [137] and its p53-regulatory role shore up the possibility that the interaction of ING1 with p53 could cause the observed aberrant localization. Nuclear localization of ING1 is essential for its proper activity. ING 1b protein phosphorylated at serine residue 199 will bind proteins and subsequently be transported from the nucleus to cytoplasm [138]. Some consequences of nuclear-cytoplasmic transport of ING proteins are presented. Shifting of p33ing1b from the nucleus to the cytoplasm, where the protein is tethered by , take parts in tumorigenesis and progression in HNSCC [139]. It was determined recently by the Riabowol group that ING 1 binds karyopherin proteins and that interruption of this interaction influences localization and activity of ING1 as a transcriptional regulator [138]. The karyopherin/importin family fuctions as adaptors by binding directly to both the NLS of a cargo protein via one domain and to karyopherin β via a second distal region. A novel binding partner of ING4, liprin alpha 1, was recently identified [140]. Liprin α1/ppfia 1 (protein tyrosine phosphatase, receptor type polypeptide) is a cytoplasmic protein necessary for focal adhesion formation and axon guidance interaction with ING4. This causes suppression of cell spreading and migration. Liprin may function in guiding ING4 to its cytoplasmic location. Cytoplasmic ING4 interacts with liprin α1 to control cell migration and with its known anti-angiogenic function, may inhibit invasion and metastasis. All these molecules that are interrelated with the sub-cellular localization of ING proteins could be determined with different molecular and histopathological methods and may be used as a biomarker to characterize the behavior of the tumor. ING Gene Expression Alterations as Prognostic Markers Changes in expression of mrna and/or protein of the ING family genes in various cancers, paves the way to their potential use as biomarkers. A recent study investigated such an association between p331ng1b protein expression and clinical outcome in colorectal cancer. They found that patients whose tumors had normal p33ing1b protein expression have a longer overall and metastasis-free survival rate as compared with patients with decreased p33 ING1b protein expression, but the difference did not show statistical significance [70, 141]. On the other hand, an important correlation between p53 mutation status and overall and metastasis-free survival has been determined. Another study demonstrated allelic loss of ING1 as a novel genomic marker associated with progression to glioblastoma by using comparative genomic hybridization and DNA microarray [141]. Also, it was reported by Takahashi et al. that low levels of ING1 mrna were significantly associated with poor prognosis in neuroblastoma [68]. Takahashi et al.

36 30 Mehmet Gunduz, Eyyup Uctepe, Catherine Moroski Erkul et al. also demonstrated that the expression level of ING1 was closely correlated to survival time. In aggregate, these findings suggest that reduced levels of ING1 mrna and/or protein expression could be an indicator of poor prognosis in advanced stages and/or poor survival for different human cancers. Zhang et al. showed that reduced ING2 mrna as well as protein expressions were accompanied by tumor progression and shortening at survival in HCC. These epidemiological studies proposed that ING2 loss or reduction may be significant for tumor initiation and/or progression [61]. Also, our group demonstrated that high LOH frequency in ING2 locus at 4q35.1 was statistically associated with advanced T stage in HNSCC, suggesting that ING2 LOH might take place in later stages during HNSCC progression [62]. Thus, the importance of ING2 in HNSCC carcinogenesis and its potential prognostic significance are compelling results for future studies. Our group recently measured mrna expression of ING3 in HNSCC and compared this with clinic-pathological characteristics in order to assess its prognostic value as a biomarker [102, 142]. This study revealed that down-regulation of ING3 was more evident in late-stage tumors as compared with early stage cases. Analyses have demonstrated that down-regulation of ING3 may indicate an aggressive form of HNSCC. The newly found connection of ING4 with survival rates and metastasis may prove to be a promising prognostic marker in melanoma [92]. It has been determined that ING4 expression was significantly reduced in malignant melanoma compared with dysplastic nevi, and overexpression of ING4 arrested melanoma cell invasion as compared with controls. Using ING Genes as Chemosensitivity Markers One of the major therapeutic modalities for cancer is chemotherapy. Thus, identification of the genes that predict the response of cancer cells to these agents is very important for improving the efficacy of treatments [17]. Recent studies demonstrated that the ING genes might play a role in regulating the response of cancer cells to chemotherapeutic agents. It was found that there is a correlation between p33lng 1b expression and resistance to vincristine, a mitotic inhibitor, especially in brain tumor cells [143]. Thus, in brain tumors ING 1b mrna levels may be a useful marker to predict chemosensitivity. However, in melanoma cells, there is no association between campthothecin-induced cell death and p33inglb expression and therefore would not be a useful predictor [143]. Subsequent studies have shown that over expression of p33 ING1b in U2OS cells with wt p53 enhanced etoposide-induced apoptosis [144]. This increase in apoptosis maybe p53-dependent since MG63 cells which are mutant for p53 did not undergo clear apoptosis following treatment. Also, it was shown that using taxol (paclitaxel) in the same cell types gave similar results [78, 145, 146], which indicate that p331ng1b might be a significant marker or therapeutic agent for the treatment of metastatic osteosarcoma. Conversely, down-regulation of ING1 in LN229, the p53-deficient glioblastoma cell line, accelerated apoptosis following treatment with cisplatin, illustrating that decreased ING1 expression could predict the sensitivity of some cancer cell types to chemotherapy independent of their p53 status [112]. Although most findings showed that expression of ING genes led to an increase in chemosensitivity, different conditions in various tumors should be tested to predict accurate chemotherapy response. These differences could also be linked to expression variations of the alternative splicing forms of ING genes.

37 The Inhibitor of Growth (ING) Gene Family 31 Finally, curcumin, a chemopreventive agent, triggers ING4 expression during cell cycle arrest by a p53-dependent manner in U251 glioma cells [147]. Thus, ING4 has been proposed as a player in the signaling pathways of the chemotherapeutic agents. Conclusion and Future Prospects Approximately 15 years of studies on the ING1 gene has demonstrated that ING1 modifies chromatin to regulate gene expression and cell growth. Based on its ability to inhibit cell growth and transformation in vitro, ING1 has been suggested to function as a tumor suppressor during development of human cancer. ING family genes have a significant role in human tumorigenesis. Even though it has been demonstrated that this newly emerging family of tumor suppressor genes play a role in multiple cellular processes including cell cycling, cell transformation, angiogenesis, apoptosis, growth regulation, senescence, DNA repair and tumorigenesis, the exact function of the ING genes has not been illuminated. A number of epidemiological studies have implicated changes in ING expression in the development and progression of some cancers and ING proteins likely affect the response of cancer cells to chemotherapy. Although frequent LOH of ING gene loci has been identified in various human cancers, tumor-specific point mutations within ING genes are seldom found. Hence ING mutation is not suggested as the molecular basis of cancer development. However the suppression of ING family genes in some tumors with strong dependence on ING pathways linked to cell cycle, senescence and apoptosis may have relevance for the molecular therapy of some tumors. Participation of ING genes in p53 tumor suppressor pathways and crosstalk between the variants of ING genes and p53 should be explored for their use as diagnostic biomarkers. Advancements in the knowledge of ING family gene functions, as well as investigation of the connection with p53 and other as of yet undefined molecules, will clarify their role in the development of human cancers and help determine their usefulness in cancer diagnostics and therapy. References [1] Garkavtsev I, Kazarov A, Gudkov A, Riabowol K. Suppression of the novel growth inhibitor p33ing1 promotes neoplastic transformation. Nature genetics Dec; 14(4): PubMed. PMID: Epub 1996/12/01. eng. [2] Russell M, Berardi P, Gong W, Riabowol K. Grow-ING, Age-ING and Die-ING: ING proteins link cancer, senescence and apoptosis. Experimental cell research Apr 15; 312(7): PubMed PMID: Epub 2006/03/07. eng. [3] He GH, Helbing CC, Wagner MJ, Sensen CW, Riabowol K. Phylogenetic analysis of the ING family of PHD finger proteins. Molecular biology and evolution Jan;22(1): PubMed PMID: Epub 2004/09/10. eng. [4] Gunduz M, Ouchida M, Fukushima K, Hanafusa H, Etani T, Nishioka S, et al. Genomic structure of the human ING1 gene and tumor-specific mutations detected in head and neck squamous cell carcinomas. Cancer research Jun 15;60(12): PubMed PMID: Epub 2000/06/24. eng.

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51 In: Tumor Suppressor Genes ISBN: Editors: Mehmet Gunduz and Esra Gunduz 2013 Chapter 3 Multifunction New Tumor Suppressor Gene Family from Cancer to Metastasis: A Disintegrin and Metalloproteinases with Thrombospondin Motifs (ADAMTS) Kadir Demircan 1,, Yunus Emre Bilgen 2, Tugrul Celik 2, Yudum Yaral 2, Birsen Dogan 3, Zisan Akcaaga 2, Zahide Nur Unal 3 and Mehmet Gunduz 3 Departments of 1 Medical Biology, 2 Medical Biochemistry, 3 Medical Genetics, Faculty of Medicine, Turgut Ozal University, Ankara, Turkey Abstract ADAMTSs are in the metallopeptidase family and are zinc-dependent matrix enzymes. MMP (matrix metalloproteinase) and ADAM (disintegrin metalloproteinase) are other zinc-dependent proteases are related to ADAMTSs. These proteases have roles in the process of damage and repair of extracellular matrix (ECM). ADAMTSs exert their affect by decomposing structural proteins of ECM like collagen, versican and aggrecan. ADAMTSs are inhibited by TIMP (Tissue inhibitors of metalloproteinase), which are known to be tissue inhibitors of metalloproteases and α2 macroglobulin, which is a serum protein. In this chapter, functions and roles of ADAMTS in various biological events are reviewed. 1. Historical Perspective and Introduction In 1997, Kuno et al. discovered the ADAMTS proteinases (A Disintegrin and Metalloprotinase with Trombospondin motif) that was upregulated in tumors [45]. The Correspondence to: Kadir Demircan, PhD - Department of Medical Biology, Faculty of Medicine, Turgut Ozal University, Ankara, Turkey. kdemircan@turgutozal.edu.tr.

52 46 Kadir Demircan, Yunus Emre Bilgen, Tugrul Celik et al. interest in ADAMTS is increasing day by day due to the role they play in the pathology of many diseases. Another increasing interest in ADAMTS proteinases is that they are also used in drug development studies for the treatment of diseases like osteoarthritis and that they prevent tumorigenesis in cancer [24]. Together with 7 ADAMTS-like (ADAMTSL) members, the 19 mammalian ADAMTS proteases have been discovered [4, 37]. Known functions of ADAMTS proteases include cleaving of procollagens, cartilage proteoglycans and von Willebrand factor [18]. Although ADAMTS are present in many tissues in the body, they are synthesized in the embryonic period as well [38]. ADAMTS proteases play an important role in physiopathological conditions such as coagulation, arthritis, cancer, tumor cell invasion and metastasis diabetes, ovulation, tissue remodeling, angiogenesis and turnover of extracellular matrix proteins [20]. A number of laboratories have focused on this newly discovered proteinases family, providing new insight into the their roles in normal function and disease. Many researchers have made efforts to identify target ADAMTS proteinases for new therapeutic options and ultimately this effort will lead to new drug candidates in the near future. The aim of this chapter is to provide a current knowledge about ADAMTS proteases regarding cancer. 2. ADAMTS Family ADAMTSs, which are from the metallopeptidase family, are zinc-dependent matrix enzymes. MMP (matrix metalloproteinase) and ADAM (disintegrin metalloproteinase) are other zinc-dependent proteases related to ADAMTSs (Figure 1). The roles of these proteases in the process of damage and repair of extracellular matrix (ECM) is known [60]. ADAMTSs exert their affect by decomposing structural proteins of ECM like collagen, versican and aggrecan. ADAMTSs are inhibited by TIMP (Tissue inhibitors of metalloproteinase), which are known to be tissue inhibitors of metalloproteases and α2 macroglobulin, which is a serum protein. TIMP-3 is the only ADAMTS inhibitor currently known [11, 76, 87]. ADAM, ADAMTS and MMP are closely related proteases. ADAMTS and ADAM proteinases are involved in the group of adamalysin. Generally, if we do not consider some of the specialized modules, the adamalysin group of proteases [ADAM, ADAMTS and MMP] consists of different modules [76]. MMP and ADAM proteases contain a transmembrane region localized to the cell membrane, but ADAMTS doesn t have this region [83]. MMPs are zinc- and calcium-dependent endopeptidases families that degrade the components of the extracellular matrix. ADAM proteases play a role in the intracellular communication system and cell-cell adhesion events such as the sperm-egg binding. 23 ADAM genes, and more than 30 MMP genes, have been identified in humans. 19 ADAMTS genes are cloned [4]. ADAMTS are classified according to their tasks (Table 1). For instance, ADAMTS1 and ADAMTS8 are known to be antiangiogenic agents [63, 88]. These two proteases are currently the targets in tumor suppression [41, 63]. Although ADAMTS1 take charge in processes like ovulation, it is a protease that is strongly expressed in heart attacks [18]. In 2009, Hatipoglu et al. demonstrated that hypoxia induces ADAMTS1 with HIF-1 [31]. While ADAMTS1 is expressed in many organs, expression of ADAMTS8 has only been shown in the lung. ADAMTS8, which cannot be expressed in cartilage tissue, is expressed in atherosclerosis in

53 Multifunction New Tumor Suppressor Gene Family from Cancer to Metastasis 47 areas that are rich in macrophage [14, 88, 89]. We also demonstrated, for the first time, that ADAMTS1, -4, -5 and -9, not -8, -15 were expressed in spinal cord tissues in mouse [15]. Figure 1. Domains and motifs of ADAM, ADAMTS and MMP. ADAMTS CLASS Table 1. ADAMTS classification and members of the group CLASS MEMBERS Aggrecanase family ADAMTS1, 4, 5, 8, 9, 15, 16, 18 Anti-angiogenic ADAMTS ADAMTS1, 8, 9 COMP-ADAMTS ADAMTS7, 12 GON-ADAMTS ADAMTS9, 20 Procollagen cleavage ADAMTS2, 3, 14 von Willebrand Factor (vwf) cleavage ADAMTS13 Orphan ADAMTS ADAMTS6, 10, 17, 19

54 48 Kadir Demircan, Yunus Emre Bilgen, Tugrul Celik et al. Figure 2. Fibril organization in normal and abnormal stages. ADAMTS2, ADAMTS3 and ADAMTS14 are known to be procollagen processing enzymes [4]. They cleave the collagen enzymatically and lead to the transformation of procollagen into collagen. ADAMTS2 gene mutations lead to type VIIC Ehlers-Danlos syndrome. In this syndrome, which was first diagnosed in cattle animals, the propeptide domain of the Type I procollagen in the dermis cannot be cleaved by ADAMTSs and a dysfunction in collagen production occurs. As a result, normal collagen fibril formation cannot be completed and this leads to Ehlers-Danlos syndrome, which is characterized by an excessively elastic and fragile dermis (Figure 2). Some ADAMTSs (1, 4, 5, 8, 9, 15, 16 and 18) can enzymatically cuts aggrecan proteoglycan, which is the main component of cartilage [18]. Therefore, this group is also called aggrecanase. Aggrecanases cleave chondroitin sulfate proteoglycans such as aggrecan and collapse ECM integrity. Aggrecanases also cut versican and brevican, which are the ECM proteoglycans. ECM proteoglycans play important and critical roles in the pathogenesis of musculoskeletal diseases e.g. osteoarthritis [24,]. In 1999, ADAMTS4 was called aggrecanase-1 for the first time [84]. In studies on ADAMTS5 with knockout mice, it was demonstrated that these mice acquire resistance against osteoarthritis. After these two studies, ADAMTS5 (aggrecanase-2) came to be classified as a major aggrecanase in mice [26, 77]. ADAMTS13 cuts the von Willebrand factor (vwf), which is an important plasma protein in hemostasis and clotting system. vwf, which enables thrombocytes to sticks to bleeding point, is a great adhesion molecule that is synthesized from endothelium cells and megacaryocytes [2]. The vwf, which is reduced to ideal size by ADAMTS13, interacts with clotting factors like Factor VIII to play a role in clot formation [49]. In Thrombotic Thrombocytopenic Purpura (TTP) disease, vwf becomes excessively large and sticky as a result of ADAMTS13 mutations. In these mutations vwf cannot be cut by ADAMTS13 to

55 Multifunction New Tumor Suppressor Gene Family from Cancer to Metastasis 49 optimal size and leads to hereditary TTP diseases characterized by the intravascular breakdown of erythrocytes, which are called thrombocyte thrombi, embolisms, anemia and thrombositopeny [50]. ADAMTS9 and ADAMTS20 are called as GON-ADAMTS proteases [8, 75]. They are thus named since they have similarities with GON-1 protease of C. Elegans. GON is a metalloprotease, which is expressed in gonad distal type cells in embryonic development. It was shown that ADAMTS20 mutations formed belted white-spotting mutants [69]. In mice with mutations, melanoblast development is defective as a result of defects in neural fissure. GON ADAMTS have the longest TSP repeats in the ADAMTS family. They have 15 TSP repeats [20]. Proteases that decompose cartilage oligomeric matrix protein (COMP) are ADAMTS7 and ADAMTS12 [53]. These two ADAMTS are known as COMP-ADAMTSs (Figure 3). COMP is also known as thrombospondine-5. COMP that can bind calcium of 524 kda is an ECM glycoprotein, which is responsible for structural integrity of cartilage and its interaction with other matrix molecules. Figure 3. COMP degradation by ADAMTSs.

56 50 Kadir Demircan, Yunus Emre Bilgen, Tugrul Celik et al. 3. ADAMTS-Like (ADAMTSL) Proteins ADAMTSs lacking proteolytic activity are called ADAMTS-like proteins, (Figure 4) [46]. They play a role in ADAMTS regulation since they are structurally similar to ADAMTS proteases and bound to extracellular matrix. Papilin and punctin are examples of ADAMTSlike proteins. It was shown that ADAMTSL2 mutations lead to autosomal recessive geleophysic dysplasia characterized with high TGF-beta activity. In geleophysic dysplasia, which is mapped in the 9q34.2 region brachydactyly, shortness, eye anomalies and heart defects are seen. ADAMTS whose functions are not known or which do not have a known substrate are called orphan proteases. To date, they are ADAMTS6, -10, -16, -17, -18 and - 19 [4]. 4. Domain Organizations of ADAMTS The most distinguishing feature of ADAMTS proteins compared to other ADAM and MMPs is that they have thrombospondin repeats (TSP) (Figure 1). ADAMTSs do not have an epidermal growth factor domain and transmembrane module like ADAM proteases. ADAMTSs are composed of a protease domain, which includes an active enzyme area and an ancillary domain composed of thrombospondin repetitions. The protease part is composed of signal peptide, propeptide, catalytic domain and disintegrated-like domains and the ancillary domains are composed of thrombospondin repetitions, a cysteine-rich domain and a link domain, which is called a spacer. Thanks to propeptide, enzymes do not interact with the substrate (Figure 5). Enzymes like furin are responsible for the cutting and removal of the propeptide domain. This enzymatic cutting and activation process is known as the zymogen activation process. Figure 4. ADAMTS like proteins.

57 Multifunction New Tumor Suppressor Gene Family from Cancer to Metastasis 51 Figure 5. Zymogen activation and furin cleavage for ADAMTS activation. Figure 6. Domain organization of some of the ADAMTS proteases.

58 52 Kadir Demircan, Yunus Emre Bilgen, Tugrul Celik et al. The catalytic domain, which includes the zinc-binding domain, is responsible for enzyme activity. All ADAMTS proteases have an active motif. Active motif is composed of a HEXXHXXGXXHD sequence. Here X signifies any amino acid. If there is a mutation in this motif, catalytic activity is completely lost. The disintegrin-like domain plays a role in matrix and cell binding processes. They are called this because the amino acid sequence is similar to the disintegrin motif in ADAM proteases. TSP is an extracellular matrix adhesion glycoprotein released from thrombocytes. The TSP motif of ADAMTSs, bound to extracellular molecules like fibronectine, collagen and laminin, play a role in cell-cell and cell-matrix interaction [44]. The cysteine-rich domain and spacer domain are important for the mechanisms of substrate specification and settlement in the matrix. In addition to these domains, some ADAMTSs have unique domains such as GON, PLAC and CUB. For example, PLAC motif is seen in close to half of ADAMTS family members (ADAMTS2, -3, -10, -12, -14, -16, -17, -18 and -19). Another difference of ADAMTS13, compared to other ADAMTSs, is that it has two CUB (complement C1r/C1s urchin epidermal growth factorbone morphogenetic protein-1) motifs in the C- terminal domain, one being CUB-1 and the other CUB-2 (Figure 6). In brief, ADAMTS proteins consist of two main parts; these are the protease domain and the ancillary domain, which is composed of thrombospondin repetitions. ADAMTS4 did not contain thrombospondin motifs and ADAMTS9 and ADAMTS20 contain 15 TSP motifs. ADAMTS13 has the shortest prodomain sequence. ADAMTS5 and ADAMTS11 is the same enzyme. ADAMTS4 is the first found aggrecanase and called aggrecanase-1. ADAMTS5 is aggrecanase-2. ADAMTS is activated by removal of the propeptide part by the furin enzymes. Zinc is connected with active motif sequence (HEXXH). 5. ADAMTS in Health and Disease ADAMTS proteases play important roles for cells (Figure 7). Its deficiency or defect can lead to serious diseases. For example, complete lack of the ADAMTS13 protease is fatal; mice without the ADAMTS9 gene die in embryonic life, mutations in other ADAMTS genes lead to serious diseases [1, 5, 13, 17, 19, 22, 23, 25, 30, 36, 40, 43, 52, 55, 64, 66, 68, 70, 72, 79, 85, 95]. When we look at the association between some ADAMTS members and diseases, we can see that mrna levels of ADAMTS1 and ADAMTS15 are linked to asthma and that mrna levels in these patients pituitaries are low [32, 65, 74]. However, the level of TIMP-3 mrna, an inhibitor of ADAMTS, is increased. Therefore, it is considered that ADAMTS1 can play role in the renewal of bronchial tissue in asthma. Shute et al reported that fibroblast growth factor-2 (FGF-2), which is found in high amounts in patients with asthma, is an important mediator in fibrosis pathogenesis. It is demonstrated that FGF-2 is controlled by ADAMTS. Why are ADAMTS1 mrna levels in patients with asthma low? Researchers consider the hypothesis that the control of ADAMTS1 on FGF-2 is lost, ECM synthesis in fibroblasts will speed up by FGF-2 stimulation, which will contribute to the renewal of bronchial tissue. It was also reported that ADAMTS4, ADAMTS9 and ADAMTS15 are induced in chronic asthma patients. In a recent study, a new role of ADAMTS5, ADAMTS9 and ADAMTS20 proteases were discovered. It was found that these three proteases play a

59 Multifunction New Tumor Suppressor Gene Family from Cancer to Metastasis 53 role in the removal of cells with apoptosis and cleaning of ECM in embryonic development [47, 57]. Figure 7. ADAMTS associated cellular events and linked molecules. ADAMTS is a large enzyme family, which is involved in many basic physiological processes like extracellular matrix remodeling, angiogenesis, clotting, ovulation and morphogenesis as well as cancer and inflammation. ADAMTSs exert various tasks such as a tumor suppressor gene in cancer, angiogenesis, hypoxia, morphogenesis, apoptosis and inflammation by interacting with nidogen, fibuline, HIF, and NFKB. HRE: Hypoxia response element, vwf: von Willebrand factor, TTP: Thrombotic Thrombocytopenic Purpura, FGF-2: Fibroblast growth factor-2, TSG: Tumor suppressor. Interruption of this process leads to syndactylia. As a result of the large-scale metaanalysis study by Zeggini et al. it was shown that the ADAMTS9 gene is a new locus of gene, which has a tendency to lead to type 2 diabetes [94]. ADAMTS10 gene mutations lead to Weill-Marchesani syndrome, which is a rare collagen disease [13]. WMS is characterized with brachydactyly, shortness, eye anomalies

60 54 Kadir Demircan, Yunus Emre Bilgen, Tugrul Celik et al. and heart defects. It is stated that ADAMTS10 plays an important role in the development of skin, eye and heart. As already mentioned, TTP can lead to damages in organs such as kidneys. As a result of ADAMTS13 mutations there may be clots in the circulation system. Today, more than 65 mutations in the ADAMTS13 gene have been reported. Almost 60% of the mutations are missense mutations [20, 37, 69]. 6. ADAMTS in Cancer Pathogenesis With functional studies it was shown that ADAMTS9 played a role as a tumor suppressor gene in esophageal and nasopharyngeal cancers. However, the ADAMTS9 gene, which is located at 3p14.2, has a significant relation with esophageal squamous-cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC) [54, 92, 95]. It is known that the ADAMTS9 gene is induced with cytokines like IL-1 in human chondrocyte cells [16]. Although ADAMTS9, discovered in 2000, is expressed in all fetal tissues, it is more expressed in heart, kidney, lung, and pancreas tissues in adults. It was reported that aortic anomalies occur in ADAMTS9 deficiency [40]. The association of the ADAMTS9 gene with metastases was investigated and it was demonstrated that ADAMTS9 mrna expression decreased in metastatic tumors [17]. Zhang et al. showed the significant correlation between ADAMTS9 methylation and loss of expression of ADAMTS9 in gastric, colorectal, and pancreatic cancers [94]. It is known that ADAMTS18 protease has a close relation with various cancers. It has been demonstrated that endothelium cells secrete ADAMTS18 and that thrombin induces this secretion. Thrombin cuts down the ADAMTS18 enzymatically and forms a C-terminal fragment of 45 kda and this fragment is regulated in vivo bleeding time and prevents carotid artery thrombus formation [51, 52]. 7. Mechanism of ADAMTS-Related Tumorigenesis: Glioma Lectin-binding proteoglycans or lecticans such as versican and brevican organize the central nervous system (CNS) extracellular matrix [2, 3, 9, 10, 33, 56, 58, 61, 91, 93]. These chondroitin sulfate lecticans are highly expressed in the CNS and limit cell motility in the CNS as a barrier. Brevican is abundant in the brain. Brain ECM, perineuronal net, is a netlike structure between neurons and glial cells [2, 71, 78]. The Fosang group suggested that brevican in the perineuronal net may contribute to neural plasticity. Upregulation of brevican is a hallmark of brain tumors [2, 78]. ADAMTS is responsible for cleavage of brevican, aggrecan and versican in CNS [48]. Brain ECM degradation by ADAMTS has a key role in malignant glioma invasion. In gliomas, it is known that ADAMTS4 and ADAMTS5 are upregulated. Nakada et al. showed that ADAMTS5 is overexpressed by glioblastoma and invading cells [61]. Induced ADAMTS5 promotes invasion of glioma cells through degradation of ECM components. Brevican is a molecular link between hyaluronan and tenascin-r of the brain ECM.

61 Multifunction New Tumor Suppressor Gene Family from Cancer to Metastasis 55 Collapsing of the bridges by aggrecanases may facilitate glioblastoma cell invasion. Fibulin-3 is uniquely upregulated in malignant gliomas and promotes tumor cell motility and invasion [34]. So discovery of inhibitors of ADAMTS in brain has potential as effective therapeutic options. Figure 8. Acting ADAMTS on glioma cells matrix: One possible mechanism of glioma cell invasion by ADAMTS proteinase activation

62 56 Kadir Demircan, Yunus Emre Bilgen, Tugrul Celik et al. 8. ADAMTS Promoter Association with Cancer and Metastasis Recently our group demonstrated that the promoter region of the ADAMTS9 is associated with lymphatic metastasis [17]. We showed that the number of CA repeats in the control group was significantly bigger than that in the non-metastatic group suggesting expansion in CA repeats may not affect tumor formation but may potentially reduce the risk of metastasis in the tumor micro-environment. ADAMTS9 locates to regions known to be frequently deleted in breast cancer and expression of ADAMTS9 in breast cancer tissues was down-regulated [8, 37, 67, 76]. Shimajiri and colleagues investigated CA repeat of the matrix metalloproteinase-9 (MMP-9) gene promoter in esophageal cancer cells (Discussed in the Turkish Journal of Medical Sciences, In Press). In their study, the CA repeat length was thought to regulate MMP-9 gene transcription and enzymatic function. MMP-9 plays an important role in tumor growth, invasion, and metastasis. It is thought that a decrease in the number of CA repeats induces down-regulation of the MMP-9 transcription [12]. 9. Prognostic Marker? Studies in the field of prognostic biomarkers that attempted to predict the course of the disease have gained considerable importance due to their indisputable value in cancer surveillance. Genetic factors are involved in carcinogenesis and are known to influence prognosis. For this purpose, the MMP, ADAM, and ADAMTS gene families are of particular interest for further study. We found that a potential relationship exists between the number of CA repeats in the ADAMTS9 promoter and lymphatic metastasis of breast cancer (Turkish Journal of Medical Sciences, In Press). Therefore, we hypothesized that the ADAMTS9 gene may renew itself by adapting to the host cell and imparting the tumor cell with the ability to metastasize through CA repeat composition. 10. Is ADAMTS a Player in the Signaling Cascade? The transcription factor nuclear factor-kb (NF-kB) plays a central role in regulating inflammatory and anti-apoptotic responses. NF-kB is composed of homodimers and heterodimers of the Rel family proteins including p65/rela, RelB, c-rel, p50/p105 and p52/p100.4,5. The prototypical complex corresponds to a heterodimer of p65 and p50 subunits. The p65 subunit is also phosphorylated by the IKK complex during the process of IkBa s degradation. Stimulus-dependent degradation of IkBa results in the translocation of NF-kB into the nucleus, where it binds to specific binding sites within the promoter of target genes. We recently demonstrated that NF-kB inhibitors, curcumin and BAY117085, effectively inhibited ADAMTS9 gene expression at 10 mm concentration. Curcumin has

63 Multifunction New Tumor Suppressor Gene Family from Cancer to Metastasis 57 been found to inhibit NF-kB-dependent gene transcription in articular chondrocytes (Figure 9) [18]. Conclusion Although ADAMTSs play role in proteolytic destruction of extracellular matrix and have an active role in pathogenesis of many diseases, there is a need for studies for ADAMTS proteinases. Gene mutation analyses like ADAMTS9, ADAMTS10 and ADAMTS13. Promoter analyses of ADAMTS genes and gene polymorphism are fields waiting to be researched. Thanks to the increasing understanding of the relation of ADAMTS genes with transcription factors like NF-kB, NFAT and RunX (Figure 10), our knowledge about ADAMTS proteases will increase day by day. At the same time, many points about ADAMTS proteinases have not been discovered yet. In this mini-review, we have tried to help future studies, so we attempted to provide brief general information about ADAMTS and recent studies on them. Figure 9. Proposed signaling pathways of ADAMTS by cytokines.

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71 In: Tumor Suppressor Genes ISBN: Editors: Mehmet Gunduz and Esra Gunduz 2013 Chapter 4 Von Hippel Lindau (VHL) Gene and Protein (pvhl): A Member of the Tumor Suppressor Gene Family Ferah Armutcu *1, Kadir Demircan 2 and Murat Oznur 3 Departments of 1 Biochemistry, 2 Medical Biology and 3 Medical Genetics, Faculty of Medicine, Turgut Ozal University, Ankara, Turkey Abstract The von Hippel-Lindau (VHL) tumor suppressor gene, responsible for the several malignancies, encodes for a major regulator of the hypoxic response by targeting the transcription factor hypoxia inducible factor (HIF). Inactivation of the VHL gene is responsible for the development of renal carcinomas, pheochromocytomas and tumours in several other organs. The gene product (pvhl) is a central component, and loss of pvhl and subsequent up-regulation of HIF target genes has been attributed to the highly vascular nature of these neoplasms. As such, systemic functions of VHL likely play important roles in the development of VHL disease. The primary aim of this review is to examine and evaluate the current knowledge regarding the pvhl, and the molecular pathogenesis of VHL disease, with subsequent clinical implications. 1. Introduction and Historical Perspective Tumor suppressor genes (TGSs) protect a cell from one step on the path to cancer. Most of the TGSs associated with familial cancer syndromes can be assigned specific cellular functions. The von Hippel-Lindau TGS encodes a multifunctional protein, the mutations of which underlie the genetic defect in the familial von Hippel-Lindau (VHL) syndrome (Kapitsinou and Haase 2008). Germ-line mutations were detected in patients with VHL * Corresponding to: Ferah Armutcu, Department of Biochemistry, Faculty of Medicine, Turgut Ozal University Ankara, Turkey, farmutcu@turgutozal.edu.tr, or drferah@gmail.com

72 66 Ferah Armutcu, Kadir Demircan and Murat Oznur disease, and it is characterized by the development of highly vascularized benign and malignant tumors, including hemangioblastomas of the brain, spinal cord, and retina; renal cell carcinoma; pheochromocytoma. Less frequent VHL gene-related tumors include those pancreatic cysts and neuroendocrine tumors; endolymphatic sac tumors; and epididymal cystadenomas (Barry and Krek 2004; Arjumand and Sultana 2012). In 1904, Eugen von Hippel, a German ophthalmologist, first described retinal lesions in the eye, then Swedish pathologist Arvid Lindau recognized the association between retinal and cerebellar haemangioblastomas and also described the presence of visceral lesions in 1926 (Kim and Kaelin 2004; Arjumand and Sultana 2012). The term von Hippel-Lindau disease was first used by Charles Davison in Melmon and Rosen reviewed the literature on what had come to be known as VHL disease and suggested clinical diagnostic criteria in 1964, and has been in common use since the 1970s (Maher, Neumann et al. 2011). Discovering the VHL gene really started with the development of the tumor suppressor gene theory and the earliest known mechanism for the pathogenesis of VHL disease is explained by Knudson s two-hit model (Knudson 1971; Knudson 1993). The incidence of VHL disease is estimated to be 1 in every 36,000 live births. It is inherited autosomal dominantly trait with a high penetrance, and germline mutations of the VHL gene are associated with VHL disease (Kim and Kaelin 2004). 2. Discovery and Identification of the VHL Gene It is found that VHL gene is located on the short arm of chromosome 3 at position The VHL TSG was characterized in 1993 following positional cloning studies in families with the familial cancer syndrome VHL disease. More precisely, the VHL gene was located chromosome 3 (3p25-26) from base pair 10,183,318 to base pair 10,195,353 (Figure 1). The VHL disease, occurs as a consequence with new mutations accounting for 20% of cases, and the majority of cases have a demonstrable germ-line mutation of the VHL TSG (Seizinger, Rouleau et al. 1988; Latif, Tory et al. 1993). The VHL gene has three exons and encodes a 4.7-kb mrna which is ubiquitously expressed in both fetal and adult tissues. Translation of the VHL mrna gives rise to two different protein products secondary to the presence of two distinct in-frame ATG codons (codon 1 and codon 54), which can both serve as translational initiation sites. The VHL mrna encodes two VHL proteins; a full length 213 amino acid protein (pvhl30) and a smaller protein (pvhl19) that lacks the first 53 amino acids (Iliopoulos, Ohh et al. 1998; Woodward, Buchberger et al. 2000). In most biochemical and functional assays, the two proteins behave similarly and unless otherwise noted are referred to generically as pvhl. pvhl is primarily a cytoplasmic protein but can also be found elsewhere, including the nucleus, the mitochondria and in association with the endoplasmic reticulum (Kaelin 2002). Since its identification, considerable insights have been made regarding the functions of the VHL gene that now appears as a crucial gene influencing many cellular pathways. In the recent past two decades, the scientific data, not only identified molecular basis for VHL disease, but also discovered that pvhl, through its oxygendependent polyubiquitylation of hypoxia-inducible factor (HIF), played a central role in the human oxygen-sensing pathway (Kaelin 2002; Kim and Kaelin 2004). Firstly, in 1988 the

73 Von Hippel Lindau (VHL) Gene and Protein (PVHL) 67 VHL gene was mapped to the short arm of chromosome 3 by linkage analysis and in 1993 the gene was identified as a result of positional cloning strategies performed in VHL kindreds (Seizinger, Rouleau et al. 1988; Latif, Tory et al. 1993). Subsequently in 1999, Maxwell et al., showed that pvhl was pivotal for targeting the α-subunit of HIF for O 2 -dependent proteolysis (Maxwell, Wiesener et al. 1999). HIF is a heterodimeric transcription factor consisting of an unstable α subunit and a stable β subunit. Three HIFα genes (HIF-1α, HIF-2α and HIF-3α) have been identified in the human genome (Semenza 2001). Figure 1. The VHL gene is located on the short arm of chromosome (3p25-p26), and protein VHL product is encoded by three exons. 3. VHL Gene Pathway and HIF-Dependent PVHL Functions The VHL gene pathway is involved in oxygen and energy sensing, and VHL complex targets HIF for ubiquitin mediated degradation. This is an oxygen and iron sensing mechanism; when the cell is low in oxygen or iron, the VHL complex cannot degrade HIF and HIF over-accumulates (Bader and Hsu 2012). Hypoxia-inducible factors are oxygensensitive basic helix-loop-helix transcription factors, which regulate biological processes that facilitate both oxygen delivery and cellular adaptation to oxygen deprivation. There are 3 HIFα genes in the human genome (HIF-1α, HIF-2α, and HIF-3α). HIF1α is ubiquitously expressed whereas the expression of HIF-2α is more restricted. HIF-1α and HIF-2α can bind to specific DNA sequences, hypoxia-responsive elements (HRE), and activate transcription. Both HIF-1α and HIF-2α have two transcriptional activation domains, the N-terminal transactivation domain and the C-terminal transactivation domain, which activate target genes upon DNA binding (Sang, Fang et al. 2002). HIF-1α and HIF-2α share many target genes, but it is also becoming increasingly clear that some genes are preferentially activated by one or the other (Kibel, Iliopoulos et al. 1995; Li and Kaelin 2011). When oxygen levels are high, in the pvhl pathway, HIF-α is hydroxylated on a crucial proline residue by HIF prolyl hydroxylase (PHD); this process requires molecular oxygen, 2-oxoglutarate, ascorbate and Fe +2 as cofactors, facilitating HIF-1α binding to pvhl, and proteasomal degradation by the E3 ubiquitin ligase complex. Namely, presence of oxygen hydroxylates the HIF-α, and hydroxylation of HIF permits recognition by the VHL complex, which therefore targets these transcription factors for proteosomal degradation There are at least three PHDs identified to

74 68 Ferah Armutcu, Kadir Demircan and Murat Oznur date: PHD1 (EGLN2), PHD2 (EGLN1) and PHD3 (EGLN3) (Kaelin 2008; Sudarshan, Karam et al. 2013). Although PHD2 is believed to be the primary hydroxylase for both HIF1α and HIF2α, other studies indicate that PHD3 may be mainly responsible for HIF-2α hydroxylation (Li and Kim 2011). Hydroxylation of one or both proline residues within HIF1α and HIF2α creates a high affinity pvhl binding site. HIF1-α, together with the constitutively expressed HIF1-β subunit, bind to HRE in gene promoters to regulate the expression of genes that are involved in energy metabolism, angiogenesis, erythropoiesis, iron metabolism, cell proliferation, apoptosis and other biological processes (Kaelin 2008). HIF- 1α and HIF-2α mediate transcription of a number of downstream genes thought to be important in cancer including vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), and transforming growth factor alpha (TGF-α). pvhl is part of a multi-subunit ubiquitin ligase complex composed of elongin-b, elongin-c, Cullin-2 and ringbox1 (Rbx1) that targets HIF for ubiquitin-mediated degradation (Kibel, Iliopoulos et al. 1995; Iliopoulos, Levy et al. 1996). Under hypoxic conditions, the PHDs are enzymatically inactive, and similarly, when the VHL gene is mutated, PHD can not hydroxylate HIF-α, therefore inactive PHD or mutant pvhl does not bind to HIF-α resulting in HIF over accumulation (Maxwell, Wiesener et al. 1999; Kaelin 2002), (Figure 2). Figure 2. Summary of the VHL functions, and in briefly HIF targets (Hsu 2012). Early biochemical molecular studies revealed that pvhl forms a multiprotein complex that includes elongin B and elongin C, and additional components of the complex Cul2 and Rbx1. Normal pvhl binds to elongin C, which forms a complex with elongin B and cullin-2 and Rbx1 (Kaelin 2008). HIF is a heterodimer comprising the unique hypoxia-inducible HIF1-α subunit and a second constituvely expressed protein termed HIF-1β (also known as

75 Von Hippel Lindau (VHL) Gene and Protein (PVHL) 69 the ARNT; aryl hydrocarbon nuclear translocator), and activate transcription of numerous target genes involved in cell proliferation, angiogenesis, glucose metabolism, apoptosis and other cellular processes (Woodward, Buchberger et al. 2000). Not binding and stabilized HIF- 1α, causes form active transcription factor complex with HIF-1β, and regulates gene expression via the HRE. RNA polymerase II subunit POLR2G/RPB7 is also reported to be a target of this protein (Maxwell, Wiesener et al. 1999; Arjumand and Sultana 2012). The most investigated of these targets is HIF-1α, a transcription factor that induces the expression of a number of angiogenesis related factors (Barry and Krek 2004). The HIF-α protein then translocates to the nucleus where it dimerizes with HIF-1β and activates the transcription of target genes. Although there have been over 100 genes identified downstream of HIF, HIF-1α and HIF-2α are well characterized proteins regulated by pvhl (Maynard and Ohh 2007). Ultimately, HIF-1α forms heterodimers with HIF-1β and activates transcription of a variety of hypoxia-inducible genes (VEGF, EPO, TGF-α, PDGF-β) (Table 1). In the same way, when pvhl is absent or mutated, HIF1-α subunits accumulate, resulting in cell proliferation and the neovascularization of tumors characteristic of VHL disease (Maynard and Ohh 2007; Roberts and Ohh 2008). Additionally, pvhl has many HIF-independent functions that are also relevant to tumor development, regulation of extracellular matrix (ECM), and apoptosis in certain cell types (Kaelin 2008), (Table 1 and Figure 3). Table 1. List and examples of HIF-responsive gene products (Semenza 2003; Li and Kaelin 2011) ANGPT4 Angiopoietin-4 ANGPT-1, -2 Angiogenic growth factors AURKA Aurora kinase A CA9 Carbonic anhydrase IX CXCR4 C-X-C chemokine receptor type 4 CYCD1 Cyclin D1 EGFR Epidermal growth factor receptor EPO Erythropoietin FLCN Folliculin FH Fumarate hydratase GLUT1,-3 Glucose transporters HGF Hepatocyte growth factor and c-met HMT/HDM Histone methylases and demethylases IGF-1R Insulin-like growth factor 1 receptor IGF-2 Insulin-like growth factor 2 IL-6 Interleukin-6 LDHA Lactate dehydrogenase A LOX Lysyl oxidase PDGF Platelet-derived growth factor ROR2 RTK-like orphan receptor 2 SDF-1 Stromal cell-derived factor-1 SDH Succinate dehydrogenase TGF-α Transforming growth factor α VEGF Vascular endothelial growth factor

76 70 Ferah Armutcu, Kadir Demircan and Murat Oznur Figure 3. VHL protein interactions with HIF-dependent/independent gene products which play important roles in tumorigenesis (Semenza 2000; Li and Kim 2011). 4. Mechanism of VHL Defective Tumorigenesis Given the early onset of life of VHL-associated tumours such as retinal haemangiomas, VHL inactivation is likely to be sufficient for their development. Biallelic VHL inactivation is also common in nonhereditary hemangioblastomas and clear cell renal carcinomas (ccrcc), in keeping with Knudson's two-hit model of carcinogenesis (Knudson 1971; Kaelin 2002). Loss of pvhl function is the most common genetic event associated with sporadic sporadic ccrcc, and HIF has a master regulator of renal cell carcinoma metabolism. As VHL loss is a common event in ccrcc, a significant proportion of the current understanding of this tumor type has been derived from the study of HIF biology. Although there is considerable overlap in the genes that are transcriptionally regulated by HIF-1α and HIF-2α, in vitro and in vivo studies have indicated that HIF-2α is the critical HIF for tumorigenesis in clear cell kidney cancer (Maranchie, Vasselli et al. 2002; Sudarshan, Karam et al. 2013). Clear cell kidney cancers that express both HIF-1α and HIF-2α exhibit enhanced signaling via the mitogenactivated protein kinase (MAPK) and serine/threonine-protein kinase mammalian target of rapamycin (mtor) pathways, whereas clear cell tumors that express only HIF-2α have elevated c-myc activity (Gordan, Lal et al. 2008). In addition to effects on angiogenesis and growth factor expression, HIF has now been shown to have profound effects on cellular metabolism (Sudarshan, Karam et al. 2013). Individuals with this disorder carry a defective

77 Von Hippel Lindau (VHL) Gene and Protein (PVHL) 71 copy of the VHL TSG, typically inherited from either parent but occasionally resulting from a de novo mutation (Barry and Krek 2004). Mutations of the VHL TSG occur in patients with VHL disease and in the majority of sporadic clear cell renal carcinomas (VHL / RCC). Furthermore, overexpression of HIF-1α accompanies p53 upregulation, angiogenesis and metastatic progression in a variety on human tumors, and may occur secondary to loss-offunction muatations affecting the VHL gene in RCCs. Moreover, tumour-associated hypoxia inhibits HIF degradation by inhibiting PHD directly or through TCA cycle generation of ROS. HIF-α and its isoforms, HIF-1α and HIF-2α accumulate then translocate to the nucleus and dimerize with HIF-β. Several clues exist as to why HIF-2α may be more oncogenic than HIF-1α. First, HIF2α is less sensitive than HIF-1α to the inhibition by FIH-1 and is therefore more transcriptionally active under normoxia. Second, HIF-1α more than HIF-2α, remains susceptible to proteasomal degradation in VHL / cell lines (Kim and Kaelin 2006). Third, HIF-2α appears to cooperate with MYC (a protooncogene) to activate MYC transcriptional targets whereas HIF-1α antagonizes MYC transcriptional activation (Gordan, Lal et al. 2008). Interestingly, a recent genome-wide analysis of copy number alterations noted that a region of chromosome 8q encoding MYC is often amplified in both sporadic and VHL disease associated tumours (Li and Kim 2011). The HIF-α/HIF-β complex binds to HRE within the promoters of target genes and thereby regulates transcription of genes involved in cell growth, angiogenesis, anaerobic glucose metabolism, ph regulation, cell survival/apoptosis, cell proliferation, and other genes that modulate various cellular functions (Kaelin 2007; Baldewijns, van Vlodrop et al. 2010) (Figures 1 and 3). Examples of genes up-regulated by HIF in response to hypoxia include VEGF, PDGF, TGF-α, GLUT1, carbonic anhydrase IX, erythropoietin (EPO), and others, all of which are potentially important in the development of ccrcc (Levy, Levy et al. 1997; Iliopoulos, Ohh et al. 1998; Kaelin 2002; Linehan, Pinto et al. 2007). Given the role of pvhl in oxygen sensing, it is also interesting to note that mutations affecting fumarate hydratase (FH) and succinate dehydrogenase (SDH) can, like VHL mutations, give rise to hereditary renal cancer or pheochromocytoma (Baldewijns, van Vlodrop et al. 2010). Downstream HIF- 1α genes such as VEGF and GLUT1 would therefore be critically important to these cancer cells for increasing vasculature and increasing glucose transport. The common endpoint resulting from VHL, FH and SDH mutations is the stabilization of HIF through inactivation of PDH, driving the transcriptional activation of genes that support tumor growth, neovascularization, invasion and metastasis (Linehan, Srinivasan et al. 2010). As a result of mutation or hypermethylation, biallelic VHL inactivation, is common in ccrccs (approximately 50% somatically mutated, 10-20% hypermethylated VHL) and sporadic hemangioblastomas. In the kidney, VHL mrna was differentially expressed within renal tubules suggesting that the VHL gene product may have a specific role in kidney development (Richards, Schofield et al. 1996; Corless, Kibel et al. 1997). On the other hand, micrornas (mirna) regulate gene expression by resulting in direct cleavage of the targeted mrnas or inhibiting translation through complementarity to targeted mrnas (Garzon, Pichiorri et al. 2007). It has been shown that mirnas are aberrantly expressed or mutated in different malignancies, suggesting that they may play a role as a novel class of oncogenes or TGSs (Fulci, Chiaretti et al. 2007; Baldewijns, van Vlodrop et al. 2010). Lately, mirna expression profiling studies identified different expression profiles in RCC. Certain ccrcc oncogenes (mtor, VHL, HIF-1α, PDGF-β) were detected as potential targets of mirnas,

78 72 Ferah Armutcu, Kadir Demircan and Murat Oznur which were up- or down-regulated in ccrcc (Petillo, Kort et al. 2009; Chow, Youssef et al. 2010). Already, the data obtained in a recent a study supporting a connection between mirna-binding site SNPs within the VHL-HIF-1α pathway and RCC risk (Wei, Ke et al. 2012). In brief, more than 100 direct HIF-responsive genes have been described with a number of these genes active in carcinogenesis (Semenza 2001). Recent evidence has accrued to indicate that pvhl has functions other than regulation of HIF-related pathways. The majority of these alternate functions have been discovered through biochemical interactions. However, gene expression studies also support the notion that there are HIF-independent gene expression changes induced by VHL loss (Semenza 2001; Li and Kim 2011). 5. VHL Gene-Related Other Clinical Implications The VHL TSG encodes a multifunctional protein, the mutations of which underlie the genetic defect in the familial VHL disease. In addition to VHL gene, hereditary forms of renal cancers have been related to the following genes, FH, hepatocyte growth factor receptor (c- MET), and Folliculin (FCLN) (Allory, Culine et al. 2011). Several alternative functions of pvhl have been identified that are independent of its role as the substrate-recognition component of an E3 ubiquitin ligase. These diverse functions also suggest that there may not be a simple, unified pathophysiological mechanism that can explain the etiology of VHL diseases. Although cell-autonomous mechanisms in VHL mutant tumors might be explained by up-regulation of cyclin D1, increased Akt-mTOR signaling and, elevated FGF receptor signaling and, disruption of cilia formation, down-regulation of p53, and, regulation of E- cadherin and stabilization Jade-1, among others, it is also well established that VHL mutant cells secret a large repertoire of growth factors and cytokines, including EPO, VEGF, TGF-β, PDGF, TNF-α, among many others (Bader and Hsu 2012), (Table 1). On the other hand, cellular senescence is the phenomenon of irreversible growth arrest in response to DNA damage but is also an important in vivo tumor suppressor mechanism (Kim and Sharpless 2008). Interestingly, it has been recognized that physiological oxygenation can extend the replicative lifespan of cells in culture, which has typically been attributed to a relative decrease in the amount of oxidative stress. Several reports have now confirmed that this phenomenon is at least in part due to stabilization of HIF (Welford, Bedogni et al. 2006; Bell, Klimova et al. 2007). Failure of pvhl to control these functions may also contribute to tumour progression and metastasis (Nyhan, O'Sullivan et al. 2008; Baldewijns, van Vlodrop et al. 2010). pvhl has a critical role in the regulation of the ECM. All VHL disease types have impaired ECM assembly capabilities and sporadic ccrcc cases also show reduced fibronectin staining, which highlights the importance of pvhl for this process (He, Liu et al. 2004). pvhl can bind directly to both fibronectin and hydorxylated collagen IV, and interestingly all pvhl mutants studied to date are defective in this capacity. The inability of VHL deficient cells to bind ECM components results in ineffective ECM organization that is not mediated by HIF (Tang, Mack et al. 2006). Recent studies have reported that pvhl interacts directly with collagen alpha-2(iv) protein (COL4A2) and indirectly with fibronectin (Grosfeld, Stolze et al. 2007; Kurban, Duplan et al. 2008). It is proposed that fibronectin interacts directly with COL4A2, which also binds to pvhl (Petrella and Brinckerhoff 2006). pvhl has also been linked to the regulation of Matrix metalloproteinases (MMPs),

79 Von Hippel Lindau (VHL) Gene and Protein (PVHL) 73 particularly MMP-2 and MMP-9. MT1-MMP expression is mediated by HIF-2α. Therefore loss of pvhl up-regulates the expression of MMPs and may promote ECM degradation and possibly an increase in tumour invasiveness and progression (Struckmann, Mertz et al. 2008). The primary cilium (microtubule-based structure) is important for sensing signals in the extracellular environment and has been proposed to be a negative regulator of cell proliferation. pvhl associates with and is able to stabilize microtubules. This function of pvhl appears to be independent of its ability to either down-regulate HIF and its ubiquitin ligase function. Moreover, pvhl's ability to stabilize microtubules is lost in VHL mutations that predispose to the development of haemangioblastomas and pheochromocytomas, but not those associated with the development of RCC (Hergovich, Lisztwan et al. 2003). However, loss of cilia function in the kidney leads to excessive proliferation of tubular epithelial cells, formation of fluid-filled cysts and kidney failure. pvhl has been localized to the primary cilia in mouse and human proximal tubule epithelial cells and in mouse distal tubules, and pvhl controls the orientation of microtubules and can interact with the Par3-Par6-aPKC complex, which is important for maintenance of the primary cilium (Schermer, Ghenoiu et al. 2006; Thoma, Frew et al. 2007). Furthermore, pvhl's affects on microtubule dynamics is negatively regulated by its phosphorylation by glycogen synthase kinase 3β (GSK-3β) and appears to be HIF-independent, although some studies suggest that HIF dysregulation may play at least a partial role in the loss of microtubule stability imparted by pvhl inactivation. Interestingly, active GSK-3β itself can promote microtubule stability and cilium maintenance in a pvhl-independent manner (Hergovich, Lisztwan et al. 2006; Li and Kim 2011). Analysis of renal cysts from VHL disease patients and ccrcc cell lines demonstrated that these cell types lacked primary cilium, implicating that pvhl may have an important role in the regulation of primary cilium (Esteban, Harten et al. 2006; Lutz and Burk 2006). On the other hand, pvhl has also been identified as a regulator of expression of E-cadherin. Loss of expression of E-cadherin is a hallmark of the epithelial-mesenchymal transition and is associated with loss of the cell-cell adhesion capabilities, which can promote tumour progression. VHL-defective RCC cell lines and patient tissue samples display loss of E- cadherin expression in a HIF-α-dependent manner (Esteban, Tran et al. 2006; Nyhan, O'Sullivan et al. 2008). Current evidence proposes that loss of pvhl results in the stabilization of HIF-α, which leads to the transactivation of E-cadherin repressors that act on the E2 boxes present in the promoter of E-cadherin. Knockdown of E-cadherin in human RCC cells resulted in an increase in the invasive capabilities of these cells. Therefore it can be envisioned that loss of pvhl and subsequent loss of E-cadherin could be critical for ccrcc development and progression (Krishnamachary, Zagzag et al. 2006; Evans, Russell et al. 2007). Interestingly, both HIF and pvhl appear to be able to influence p53 function. HIF can directly bind to and modulate p53 activity, and pvhl is able to regulate p53 function in a HIF-independent manner through suppression of MDM2 (also known as E3 ubiquitin-protein ligase) mediated ubiquitination. Stabilization of p53 resulted in increased p53 transcriptional activity, and loss of pvhl prevents this stabilization. Therefore pvhl loss appears to result in p53 inactivation by both HIF-dependent and HIF-independent effects (Roe, Kim et al. 2006; Sendoel, Kohler et al. 2010) (Figure 3). Jade-1 is a novel protein identified by yeast two-hybrid analysis as a pvhl-interacting protein (Zhou, Wang et al. 2002; Roe, Kim et al. 2006). pvhl interacts with and stabilizes Jade-1 protein, and mutations of the VHL gene disrupt this process and may be associated

80 74 Ferah Armutcu, Kadir Demircan and Murat Oznur with an increase in renal cancer risk (Zhou, Wang et al. 2004; Kapitsinou and Haase 2008). In addition to tumor formation, mutations in the VHL gene can result in the development of polycythemia. At least 10 inherited mutations in the VHL gene have been found to cause familial erythrocytosis, and it is often designated erythrocytosis (ECYT2) (Percy, Chung et al. 2012). Also, in support of a link between VHL and inflammation, it has recently been reported that pulmonary hypertension, a complication of Chuvash polycythemia, is caused by lung fibrosis (Hickey, Richardson et al. 2010). Accumulating evidence suggest that pvhl is a negative regulator of nuclear factor-kappa B (NF- B). In the absence of a functional pvhl, the expression and activity of NF- B are enhanced, which subsequently confer a drug resistant phenotype to RCC by suppressing drug-induced apoptotic pathway. As NF- B is over expressed in RCC, its inhibition as a potential treatment strategy has been a subject of intense research using genetic or chemotherapeutic approaches (Morais, Gobe et al. 2011). Developmental (cardiovascular, mammary gland and skeletal development; adipogenesis, chondrogenesis and embryonic survival) and physiological (hypoxia-induced; -glycolysis, - apoptosis, -erythropoiesis, -pulmonary vascular remodeling, -myocardial preconditioning, - cell cycle arrest and angiogenesis) roles of HIF-1 as established by analysis of HIF-1α-null mice and cell lines (Semenza 2004). 6. Therapeutic Targets and Future Prospects Over the past century, studies focusing on the structure and function of the VHL TSG and its protein product, pvhl, have been highly informative with respect to the pathogenesis of ccrcc as well as the molecular mechanisms of oxygen sensing. Many functions have been attributed to pvhl; however, the one best characterized and most clearly linked to the development of pvhl-defective tumours, is targeting of the HIF transcription factor for proteolytic degradation. In addition to its direct role on tumor growth, HIF-1 has also been implicated in modulating the tumor response to therapies (Arjumand and Sultana 2012). There are a number of compounds reported with anti-hif-1 activity and are mainly classified as direct and indirect inhibitors based on their different modes of action. While direct HIF-1 inhibitors prevent HIF-1 from transactivation, DNA binding, and subsequently initiating transcriptional activity, indirect HIF-1 inhibitors generally block the transcription or translation of HIF-1α or promote the degradation of HIF-1α protein (Wang, Zhou et al. 2011). It is supposed that antiangiogenesis therapy may enhance tumor hypoxia, and when the hypoxia response is abrogated in cancer cells using HIF-1 inhibitors, a robust antitumor activity may be observed. Inactivation of the VHL TSG is a frequent event in ccrcc. The central role of HIF-1α, and particularly HIF-2α, in pvhl-defective ccrcc has spurred interest in the development of HIF antagonists for this disease. Unfortunately, however, there are very few examples of drug-like small organic molecules that are capable of inhibiting the function of DNA-binding transcription factors, with the notable exception of the steroid hormone receptors. Inhibition of mtor kinase decreases the transcription and translation of HIF1α, thereby lowering HIF-1α protein levels. This process might account for the observation that rapamycin-like mtor inhibitors (e.g. temsirolimus and everolimus) have activity in the treatment of ccrcc (Li and Kaelin 2011). Today, currently, it is used many agent in treatment of patients with advanced ccrcc. These agents work either directly on

81 Von Hippel Lindau (VHL) Gene and Protein (PVHL) 75 HIF-regulated targets and their receptors, such as VEGF, VEGFR, PDGFR and other tyrosine kinase receptors, or on mtorc1, which controls cellular growth and increases HIF-1α translation. However, knowledge of the VHL/HIF pathway provides the foundation for the development of novel therapeutic approaches to the treatment of VHL-related tumor and diseases (Linehan, Bratslavsky et al. 2010). Conclusion The pvhl has many functions out of which the best recognized is pvhl ability to target the HIF for ubiquitin-mediated degradation. Loss of pvhl function results in the stabilization of HIF-α and activation of HIF responsive genes. Some of these gene products have been shown to be protumorigenic in the renal cell carcinoma perspective. The product of the VHL gene is a tumor suppressor protein, which targets several proteins for degradation by the proteasomes, and both copies of the gene are inactivated in tumor tissues. The alpha subunits of HIF-1 and HIF-2 are substrates for the product of the VHL gene. In the absence of VHLinduced degradation, HIF-1 and HIF-2 may contribute to increased levels of EPO, VEGF, and other growth factors, providing a stimulus for tumor growth. HIF transcriptionally activates several genes implicated in ccrcc pathogenesis, including VEGF. Multiple VEGF inhibitors have now been approved for this disease based on randomized clinical trial data. Inactivation of the VHL tumor suppressor gene is an early, causal event in the development of ccrcc, and this gene is mutated or silenced in the majority of both hereditary and nonhereditary ccrcc. In addition, VHL mutant tumor cells can secret a number of growth factors and cytokines that can also activate the inflammatory and angiogenic components of the primary tumors. Therefore, it is argue that in designing new treatments for the VHL disease, a systemic approach including targeting the hematopoietic system and the inflammatory response should be considered. Developing knowledge of genetic and epigenetic changes implicated in tumor development and behavior is becoming increasingly important for advancing the efficacy of disease management. Pre-clinical and clinical investigations into HIF-associated pathogenesis have led to the development of new FDA approved targeted therapies, which have produced promising basis for new systemic therapies, including anti-angiogenic drugs and mtor inhibitors. In the future, a more complete understanding of the role of VHL, including its HIF-independent functions, should provide novel therapeutics opportunities. Overall, these all data suggesting a pivotal role of pvhl s in HIF-independent functions of tumor suppression, development and normal tissue physiology. New additional genetic studies into the precise mechanism and biological roles of VHL gene and protein are therefore highly welcome. To discover new therapeutic intervention, more basic and more preclinic study is needed to better understand the pathogenesis of VHL disease. References Allory, Y., S. Culine, et al. (2011). "Kidney cancer pathology in the new context of targeted therapy." Pathobiology 78(2):

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87 In: Tumor Suppressor Genes ISBN: Editors: Mehmet Gunduz and Esra Gunduz 2013 Chapter 5 Application of Cancer Gene Therapy Using Tumor Suppressor Gene p53 Hiroshi Tazawa 1,2, Shunsuke Kagawa 2 and Toshiyoshi Fujiwara 2, 1 Center for Innovative Clinical Medicine, Okayama University Hospital, Okayama, Japan 2 Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan Abstract The tumor suppressor gene p53 encodes a multifunctional transcription factor that regulates diverse cellular processes, including cell cycle arrest, senescence, apoptosis and autophagy; each of these processes suppresses tumor growth and progression. When tumor cells with intact p53 function are exposed to genotoxic stresses, including chemotherapy and therapeutic radiation, many kinds of p53-downstream target genes, such as p21 WAF1, BAX, and DRAM, are directly induced by p53; p21 WAF1 then mediates cell cycle arrest for the repair of the DNA damage, whereas BAX and DRAM activate cell death pathways for the elimination of the damaged cells. Recent evidence demonstrates that p53 induces not only protein-coding genes, but also small non-coding micrornas (mirnas) such as mir-34 and mir-22, and that these mirna act as modulators in the p53-mediated tumor suppression system. The p53 gene is frequently inactivated by genetic alterations in approximately half of all distinct types of human cancers. Tumor cells with impaired p53 function are commonly refractory to genotoxic stresses induced by conventional chemoradiotherapy. Therefore, restoration of p53 function by introducing exogenous p53 expression via a gene delivery system is a promising antitumor strategy because it should result in suppression of tumor growth and progression. This chapter focuses on recent advances in our understanding of the tumor suppressive roles of the p53 gene, which include regulation of p53 target protein-coding genes and of mirnas. Furthermore, the potential application of p53-based cancer gene therapy will be discussed for four types of p53 transfer systems cationic liposome- Address correspondence to this author at the Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho, Okayama , Japan. Phone: ; Fax: ; toshi_f@md.okayama-u.ac.jp.

88 82 Hiroshi Tazawa, Shunsuke Kagawa and Toshiyoshi Fujiwara DNA plasmid complexes, replication-deficient adenovirus vectors, replication-competent adenovirus vectors, and protein transduction systems. A better understanding of the precise molecular mechanism by which p53 mediates tumor suppression would provide us with novel insights that may lead to improvement in p53-based cancer gene therapy. A highly efficient system for inducing p53-mediated cell death may improve the clinical outcomes of patients with any of the many types of p53-inactivated cancers. Keywords: p53, gene therapy, microrna, adenovirus, protein transduction mirna PRIMA htert IRES VEGF DCs CAR HDAC Abbreviations microrna p53 reactivation and induction of massive apoptosis human telomerase reverse transcriptase internal ribosome entry site vascular endothelial growth factor dendritic cells coxsackie and adenovirus receptor histone deacetylase Introduction The tumor suppressor gene p53 encodes a multifunctional transcription factor that regulates diverse cellular processes such as cell cycle arrest, senescence, apoptosis, and autophagy in normal tissues and in tumors [1]. When tumor cells with intact p53 function are exposed to genotoxic stresses (including chemotherapy and therapeutic radiation), many kinds of targets downstream of p53 are transcriptionally induced by activated p53, and these p53 targets cooperatively regulate cellular processes that curb or reverse tumor progression [2, 3]. In response to mild cellular stress, the main effect of p53 activation is the induction of cell cycle arrest, which allows for repair of DNA damage and contributes to cell survival (Figure 1). In contrast, severe cellular stress induces more p53 accumulation, which activates several types of signaling pathways that lead to apoptosis, senescence, and autophagy; therefore, severe stress results in the induction of cell death (Figure 1). Recent evidence demonstrates that p53 regulates transcription of several small non-coding micrornas (mirnas), as well as protein-coding genes, and that these mirnas are important components of the p53-mediated tumor suppression system [4]. A better understanding of the precise molecular mechanisms in the p53-mediated tumor suppression network could lead to novel insights useful for the development of p53-based antitumor therapies. Analyses of the IARC TP53 database ( have shown that epithelial and non-epithelial malignant tumors often harbor somatic mutations in the p53 gene and that the types of p53 mutations and the types of tumors that harbor them vary widely [5, 6]. The p53 gene is frequently inactivated by genetic alterations in approximately half of all distinct types of human cancers. Patients with Li-Fraumeni syndrome, which is a cancer predisposition disorder, each carry a germline mutation in the p53 gene, and they develop

89 p53-based Cancer Gene Therapy 83 early onset tumors [7]. Furthermore, inactivation of p53 via genetic engineering can induce spontaneous tumor growth in mice; several different mouse models of p53 inactivation have been developed, and spontaneous tumors develop in each model [8-10]. These findings support the hypothesis that the p53 gene has a potent, critical role in the tumor suppression network. Furthermore, tumor cells with impaired p53 function are often refractory to the genotoxic stresses induced by conventional chemoradiotherapy [11]. Thus, restoration of wild-type p53 function is a promising antitumor strategy because it could lead to suppression of tumor growth and progression. In principle, there are two ways to restore p53 activity in tumor cells that lack functional p53 [12, 13]. One class of strategies involves the introduction of active, exogenous p53 via any one of several gene delivery systems such as liposome complexes with plasmid DNA, replication-deficient or replication-competent adenovirus vectors, or protein transduction tools (Figure 2). Among these delivery methods, adenovirus-mediated p53 gene therapy has recently emerged in preclinical experiments and clinical studies as a promising antitumor treatment for several types of human cancers [14, 15]. Another class of strategies involves the reactivation of endogenous p53 expression via treatment with chemical compounds such as Nutlin or PRIMA (p53 reactivation and induction of massive apoptosis) (Figure 2). For example, the low-molecular-weight compound PRIMA reportedly induced apoptosis by restoring DNA-binding activity and a functional conformation to mutant p53 protein in human cancer cells that harbor p53 mutations [16]. A different small molecule, Nutlin, induces p53 stabilization in tumor cells that overexpress MDM2 by interacting with MDM2 and thereby inhibit the MDM2-p53 interaction [17]. Fig. 1 Cell Cycle Arrest Mild Stress p53 Cell Survival Cell Death Senescence Apoptosis Figure 1. Conceptual diagram of the different p53-mediated cellular processes that are induce by mild versus severe stresses. Mild stress induces a small amount of p53 activation and therefore cell cycle arrest and repair of DNA damage; these process result in cell survival. In contrast, severe stress induces a large amount of p53 accumulation and activation of three distinct cell death pathways senescence, apoptosis and autophagy that result in elimination of the damaged cells. p53 Severe Stress Autophagy

90 84 Hiroshi Tazawa, Shunsuke Kagawa and Toshiyoshi Fujiwara Fig. 2 mp53 Figure 2. Schematic diagram of the p53-mediated tumor suppression network. Restoration of p53 function induces senescence, apoptosis, or autophagy via activation of three main p53 target genes p21, BAX, or DRAM, respectively. Some chemical compounds, PRIM and Nutlin, reactivate endogenous p53 expression; PRIM induces transcriptional activity of mutant p53 protein (mp53), whereas Nutlin suppresses MDM2 expression. In contrast, some p53 delivery systems DNA plasmid (plasmid), adenovirus (virus), protein transduction tool (protein), or combinations thereof can introduce exogenous p53 activity. However, the therapeutic effects of PRIMA and Nutlin are limited to tumor cells that carry specific p53 gene mutations or to tumor cells that overexpress MDM2, respectively. The level of endogenous p53 expression reactivated by these compounds may be also less effective for inducing tumor cell death when compared to ectopic expression of exogenous p53 gene. Therefore, overexpression of exogenous p53 gene by introduction and overexpression of p53 via any one of several gene transfer methods would be an effective antitumor strategy for the induction of cell death in a variety of p53-inactivated tumor cells. This chapter focuses on the recent advances in our understanding of the tumor suppressive role of p53 through activation of several p53-downstream target genes including protein coding genes and small non-coding microrna genes. Furthermore, we will discuss the potential application of p53-based cancer gene therapies to the treatment of patients with various types of cancers; these therapies involve a liposome-dna plasmid complex, replication-deficient or replication-competent adenovirus vectors, or protein transduction tool. p53 as a Guardian of Human Genome p53-mediated Tumor Suppression System p53 p21 Cell Cycle Arrest PRIMA Senescence Plasmid Virus Protein p53 p53 BAX Apoptosis Cell death The tumor suppressor gene p53 is a main mediator induced by various types of cellular stresses. Activation of wild-type p53 influences the subsequent cell fate trajectories of normal cells and of tumor cells with intact p53 function [2]. There are three cell death pathways Nutlin MDM2 p53 DRAM Autophagy

91 p53-based Cancer Gene Therapy 85 senescence, apoptosis, and autophagy in activated by the p53-mediated tumor suppression system. These cell death pathways are determined by the induction of several p53- downstream target genes, such as p21 WAF1 (p21), BAX, or DRAM (Figure 2). While p21 activation mainly induces cell cycle arrest [18, 19] and subsequent senescence [20], the p53- induced BAX [21] and DRAM [22] lead to apoptosis and autophagy, respectively, resulting in the cell death. In response to mild cellular stress, p21 gene, which is most rapidly and strongly induced during the DNA damage response, mainly induces cell cycle arrest that allows for the repair of DNA damage and consequently contributes to senescence. A recent report suggests that p21-mediated induction of senescence is a promising antitumor strategy [23]. In contrast, in response to severe cellular stress, p53-induced BAX activation induces apoptotic cell death; similarly, p53-induced DRAM activation induces autophagic cell death (Figure 2). Interestingly, p21 activation suppresses apoptotic and autophagic cell death pathways [24, 25]. Therefore, induction of both the apoptotic and autophagic cell death pathways via p53 transactivation would be a more effective antitumor strategy for the suppression of tumor initiation and progression than induction of p21-mediated senescence. Thus, p21 suppression may be an effective strategy for the induction of the apoptotic and autophagic cell death pathways in tumor cells, particularly when the tumor suppressor gene p53 is overexpressed in tumor cells in response to cancer gene therapy. p53-microrna Tumor Suppression Network Recent advances have shown that micrornas (mirnas), small non-coding RNAs comprising 22 nucleotides, are novel potent modulators in the p53-mediated tumor suppression network [4]. In fact, some kinds of mirnas are induced by p53 activation in normal and cancer cells exposed to various cellular stresses, such as chemotherapy and radiation. Among some p53- inducible mirnas, mir-34 was initially identified as a p53-regulated tumor-suppressive mirna; this discovery was made independently in several laboratories in 2007 [26-28]. Ectopic expression of mir-34 induces cell cycle arrest, senescence, or apoptosis in human cancer cells because it can suppress many target genes, including E2F3 (Figure 3). Downregulation of mir-34 expression by promoter methylation occurs in a variety of human malignancies [29-31], suggesting that mir-34 may function as a tumor suppressor mirna. In contrast, mir-22, which is also a p53-inducible tumor suppressor mirna, represses p21 expression and thereby induces apoptotic cell death (Figure 3) [32]. Interestingly, both mir-34 and mir-22 suppress the expression of SIRT1, which negatively regulates p53 by inducing p53 deacetylation; these interactions result in a positive feedback loop that enhances p53 activation (Figure 3) [33, 34]. Interestingly, several p53- inducible mirnas (e.g., mir-143/145 [35], mir-192/194/215 [36], and mir-605) suppress MDM2, another negative regulator of p53 that induces ubiquitin-mediated p53 degradation (Figure 3) [37]. Taken together, these findings suggest that targeting the p53-mir- SIRT1/MDM2 axis as a p53 positive feedback loop may have therapeutic potential. Furthermore, because p53 may be a master modulator of mirna biogenesis [38], many kinds of mirnas may be directly or indirectly regulated by p53 and be involved in p53-mediated tumor suppressor.

92 86 Hiroshi Tazawa, Shunsuke Kagawa and Toshiyoshi Fujiwara Fig. 3 mir-22/34 SIRT1 mir-34 E2F3 Cell Cycle Arrest Senescence p53 Positive Feedback Loop p53 p53 mir-22 p21 Apoptosis Cell death Although the identities of the mirnas that regulate p53-mediated autophagy induction remain unclear, this relationship between p53 and mirna suggests that p53-inducible mirnas positively regulate all three p53-mediated cell death pathways senescence, apoptosis, and autophagy by enhancing p53 expression via a positive feedback loop. Furthermore, mirna-based cancer gene therapy may be a novel antitumor strategy; specifically, introduction of one or more mirnas could induce a p53-mirna positive feedback loop and consequently restore p53 function. Notably, mir-34-based antitumor therapy has been emerging as a novel anticancer strategy [39-41]. We previously reported that ectopic expression of mir-34 induced by mirna mimics suppressed cell viability and induced subsequent senescence-like growth arrest in human colon cancer cells that had either wild-type or mutated p53 alleles [27]. Furthermore, a lentivirus vector-mediated mir-34 gene transfer efficiently induces mir- 34 expression, and this expression results in the suppression of cell proliferation and suppression of tumor growth in human cancer cells [42-45]. Because mirna-based antitumor therapy holds real promise for future clinical application, development of efficient mirna delivery systems is warranted. mir- 143/145/192/ 194/215/605 Autophagy Figure 3. Schematic diagram of a p53-inducible mirna-mediated tumor suppression network. p53- inducible mir-34 mainly induces senescence by suppressing E2F3; in contrast, mir-22 induces apoptosis by suppressing p21. Many kinds of p53-inducible mirnas including mir-22/34, mir- 143/145, mir-192/194/215, mir-605 are involved in the induction of a p53 positive feedback loop because they suppress two negative regulators of p53, SIRT1 and MDM2. MDM2??

93 p53-based Cancer Gene Therapy 87 p53-mediated Suppression of Stemness Property Fig. Recent 4 reports have suggested that p53 plays critical roles not only in tumor suppression in cancer cells, but also in the inhibition of self-renewal and other properties of normal stem cells (Figure 4) [46]. p53 Cancer Cell Suppression of Proliferation Figure 4. Conceptual diagram of p53-mediated suppression of tumor growth and stem cell properties. p53 induces suppression of proliferation and stem-cell-specific properties in cancer cells and normal stem cells, respectively. In cancer stem cells, p53 activation suppresses both tumor growth and stemcell-specific properties. For example, p53 functions as a negative regulator of reprogramming [47] in normal embryonic stem cells [48], induced pluripotent stem cells [49], and a variety of normal stem cells [50-52]. Modulation of mirna networks by p53 is an important factor in the regulation of characteristic properties and differentiation of stem cells [53-55]. The emerging evidence suggests that p53-mediated cancer gene therapy via mirna modulation has significant therapeutic potential with regard to cancer stem cells (Figure 4) [56]. In fact, ectopic expression of the p53-inducible mir-34 inhibits the stemness properties of some cancer stem cells and tumor progression [42, 57-59]. A greater understanding of the underlying molecular mechanisms by which p53-mediated mirna networks affect stem cell biology should facilitate the development of cancer stem cell-targeting therapy. p53-based Cancer Gene Therapy p53 Cancer Stem Cell Suppression of Proliferation Suppression of Stemness Overexpression of exogenous p53 gene by introduction and overexpression of p53 via any one of several gene transfer methods is an effective antitumor strategy for the induction of cell death in a variety of p53-inactivated tumor cells. We will next discuss the potential application of p53-based cancer gene therapies that involve a liposome-dna plasmid complex, replication-deficient or replication-competent adenovirus vectors, or protein transduction tool. p53 Normal Stem Cell Suppression of Stemness

94 88 Hiroshi Tazawa, Shunsuke Kagawa and Toshiyoshi Fujiwara Cationic Liposome Complex with DNA Plasmid In preclinical in vitro experiments, non-viral plasmid DNA expression vectors that encode p53 are usually used to deliver an exogenous p53 gene into tumor cells (Figure 2). Cationic liposomes have been shown to be useful delivery vehicles for transfection of DNA plasmid vectors that encode ectopic p53 into human cancer cells [60-63]. Furthermore, to increase transfection efficiency and tumor-specific delivery of plasmid vectors, an antibodyconjugated immunoliposome has been developed for cancer treatments [64-67]. However, transfection efficacies using either liposome-based method are low and insufficient to induce cell death especially in in vivo tumors. Therefore, a transduction system that is more efficient than are liposome complexes is required to induce exogenous p53 expression in tumor tissues that is sufficient to induce cell death. Replication-Deficient Adenovirus Vectors To induce exogenous expression of p53 in vivo more efficiently than is currently possible with p53-expressing plasmid DNA, a p53-expressing, replication-deficient adenovirus vector (Ad-p53) is frequently used in preclinical in vitro and in vivo experiments (Figure 5) [4, 15, 68, 69]. We previously reported that adenovirus-mediated introduction of wild-type p53 into human lung and colon cancer cells efficiently suppressed cell proliferation and tumor growth [70-73]. Furthermore, infection with the Ad-p53 vector enhanced the in vivo antitumor effect of chemotherapeutic agents because human cancer cells underwent apoptotic cell death [70, 72]. Moreover, Ad-p53-mediated p53 transduction enhanced the chemosensitivity of human sarcoma cells [74-76]. These results suggest that Ad-p53-mediated p53 gene transfer is a potential antitumor therapy that could be used as a monotherapy or in combination with conventional chemotherapy. Overexpression of p21, which is a downstream target of p53, may also have antitumor effects and potential as a pro-senescence, antitumor therapy [23]. We previously compared the antitumor effects of Ad-p53 with those of a p21-expressing replication-deficient adenovirus vector (Ad-p21) in human tumor cells [71]. Ad-p53 infection induced apoptotic cell death, whereas Ad-p21 infection mainly induced cell cycle arrest at G1. When Ad-p53 and Ad-p21 were co-transduced, Ad-p53-mediated p53 induction overcame the Ad-p21- mediated cell cycle arrest and induced apoptotic cell death. Thus, Ad-p53-mediated gene therapy is a promising antitumor therapy for induction of profound apoptotic cell death in tumor cells. In many clinical studies, Ad-p53 (Advexin; INGN-201; Introgen Therapeutics Inc.) reportedly induces antitumor effects in patients with various types of cancers, including nonsmall-cell lung cancer (Figure 5) [77, 78], head and neck squamous cell carcinoma [79], glioma [80], ovarian cancer [81], esophageal squamous cell carcinoma [82]. In these studies, Ad-p53 infection exhibited a safe profile and clinical benefit as a monotherapy or in combination with chemotherapy and radiotherapy [83]. Moreover, intratumoral Ad-p53 injection alone or in combination with cisplatin was feasible and well tolerated in patients with advanced non-small-cell lung cancers [78].

95 p53-based Cancer Gene Therapy 89 Figure 5. Schematic diagrams of DNA structures of several adenovirus vectors. Ad-p53 (Advexin, Gendicine) is a p53-expressing replication-deficient adenovirus; the p53 gene expression is under the regulation of the CMV promoter and this cassette is inserted into the E1 region; the E3 region is deleted. In Telomelysin (OBP-301), the htert gene promoter element drives the expression of E1A and E1B genes, which are linked with an IRES. OBP-702 is a p53-expressing telomerase-specific replication-competent oncolytic adenovirus; p53 gene expression is under the regulation of the Egr-1 gene promoter, and this cassette is inserted into the E3 region. In the Ad-p53-treated tumor tissues, p53 transgene expression was confirmed by quantitative PCR analysis. Of the 15 patients who received intratumoral injection of Ad-p53, 13 could be assessed to determine the efficacy of the treatment; one patient had a partial response, 10 patients had stable disease during at least 9 months, and two patients had progressive disease. Furthermore, Ad-p53 (Gendicine, Shenzhen SiBiono GeneTech Co.) was approved by the State Food and Drug Administration of China for clinical use in 2003 (Figure 5) [84-86]. Recent reports have demonstrated that Ad-p53 in combination with chemotherapy and radiotherapy is clinically effective in patients with advanced hepatocellular carcinomas [87-89]. Taken together, the accumulating evidence indicates that Ad-p53-mediated cancer gene therapy is a promising anticancer therapy. However, Ad-p53 is a replication-deficient adenovirus; therefore, it is impossible to induce exogenous p53 expression in every tumor cell via this vector. The low transduction rate of p53 gene transfer via the replication-deficient Ad-p53 vector is major problem that needs to be overcome in order to improve clinical outcomes of patients with advanced cancers. Based on findings from preclinical experiments, there are several potential

96 90 Hiroshi Tazawa, Shunsuke Kagawa and Toshiyoshi Fujiwara approaches by which Ad-p53-mediated p53 expression and therefore Ad-p53-mediated cell death has been enhanced; here, we describe three such approaches (Figure 6). Fig. 6 OBP-301 p21 sirna E1A Replication p53 p21 Cell Cycle Arrest Senescence Ad-p53 p53 p53 BAX Apoptosis Cell death Figure 6. Schematic diagram of Ad-p53-mediated induction of two programmed cell death pathways and strategies for enhancing the associated Ad-p53-mediated antitumor effects. Replication-deficient Ad-p53 infection induces apoptosis and autophagy and therefore results in cell death, rather than cell cycle arrest, when combined with replication-competent OBP-301, replication-deficient adenovirus vectors (Ad-E2F1, Ad-ARF, Ad-FHIT), chemical compound (Nutlin), or p21 sirna. Ad-p53 induces apoptosis or autophagy through activation of BAX or DRAM, respectively. OBP-301 enhances Adp53-mediated p53 expression through E1A -induced replication of an E1-deleted Ad-p53. Infection with Ad-E2F1, Ad-ARF or Ad-FHIT or treatment with Nutlin enhances Ad-p53-mediated p53 expression through MDM2 suppression. Transfection with p21 sirna induces both Ad-p53-mediated cell death pathways because p21 is downregulated. Ad-p53-mediated p53 expression is enhanced if combined with E1A-expressing oncolytic adenovirus. We previously developed a telomerase-specific replication-competent oncolytic adenovirus, OBP-301 (Telomelysin), that induces tumor-selective oncolytic cell death in a telomerase-dependent manner; in this construct, the promoter from htert (human telomerase reverse transcriptase) drives the expression of two adenoviral genes, E1A and E1B, that are linked to an internal ribosome entry site (IRES) (Figure 5) [90-92]. A combination therapy that involves co-transduction of Ad-p53 and oncolytic adenovirus OBP- 301 enhanced p53 expression; this combination therapy resulted in a more profound antitumor effect and enhanced apoptotic cell death when compared to monotherapy with either OBP-301 or Ad-p53 [93]. Adenoviral E1A expression induced by oncolytic adenovirus supports the replication of Ad-p53 vector and subsequently enhance the Ad-p53-mediated p53 expression (Figure 6). Nutlin p53 E2F1 DRAM ARF MDM2 Autophagy Ad-E2F1 Ad-ARF Ad-FHIT

97 p53-based Cancer Gene Therapy 91 Another method is to inhibit the expression of MDM2, which functions as a negative regulator of p53 via ubiquitin-mediated p53 degradation, and stabilize exogenous p53 expression induced by Ad-p53 (Figure 6). Suppression of MDM2 expression via treatment with Nutlin [94] or via adenovirus-mediated overexpression of the tumor suppressor FHIT gene [95] enhances Ad-p53-mediated p53 expression and apoptotic cell death in human cancer cells. Furthermore, overexpression of the ARF gene introduced via a recombinant adenovirus vector, Ad-ARF [96] or Ad-E2F1 [97], induces enhancement of Ad-p53-mediated p53 expression and antitumor effects because the exogenous ARF downregulated MDM2 in human cancer cells. MDM2 is downregulated by oncogenic stress-mediated ARF activation in a mouse model of oncogene K-Ras-induced lung tumorigenesis, and this MDM2 downregulation is necessary for p53 overexpression to have a substantive antitumor effect in this model of tumorigenesis [98, 99]; therefore, combining a MDM2-downregulating therapy with Ad-p53 should be a more effective antitumor strategy than Ad-p53 monotherapy. Ad-p53-mediated cell death in tumors could be enhanced via p21 suppression. Suppression of p21 expression by genetic deletion [25] or an exogenous p21-targeted sirna [100] enhances Ad-p53-induced apoptosis (Figure 6). Since p53-downstream target p21 functions as a suppressor of apoptosis and autophagy [24, 25], p21 suppression may be a critical factor for inducing both apoptosis and autophagy in response to p53 overexpression. Each of these three strategies for enhancing Ad-p53-mediated cell death should improve the clinical outcomes of Ad-p53-mediated cancer gene therapy. Fig. 7 VEGF VEGF VEGF VEGF Ad-p53 Suppression of Angiogenesis p53 Infiltration of Neutrophils X X X X p53 X X X X VEGF Figure 7. Conceptual diagram of Ad-p53-mediated bystander effects on neighboring, uninfected tumor cells. When tumor cells are infected with Ad-p53, p53 overexpression induced cell death in the infected tumor cells. In contrast, uninfected tumor cells are also eliminated via bystander effects, which can include suppression of angiogenesis by VEGF downregulation and infiltration of neutrophils by CD95L expression. CD95L N N CD95L N CD95L N CD95L

98 92 Hiroshi Tazawa, Shunsuke Kagawa and Toshiyoshi Fujiwara Apparently, Ad-p53 transduction causes antitumor effects in not only Ad-p53-infected cells, but also in uninfected tumor cells, because the exogenous p53 induces bystander effects [101]. In fact, Ad-p53-mediated p53 gene transfer induces bystander effects to neighboring tumor cells via multiple mechanisms in preclinical in vivo settings (Figure 7). For example, Ad-p53 infection markedly inhibited the expression of an angiogenic factor and vascular endothelial growth factor (VEGF) and increased the expression of a antiangiogenic factor; together, the effects of p53 expression result in the suppression of neovascularization in tumor tissues [73, 102]. Additionally, Ad-p53-mediated p53 transfer induced overexpression of CD95 ligand (CD95L) in tumor cells and consequently massive infiltration of neutrophils into tumor tissues [103]. Overexpression of CD95L was also partially responsible for the Ad-p53-induced apoptosis that was mediated by the Fas receptor/ligand system [104]. Furthermore, when bone marrow-derived dendritic cells (DCs) infected with Ad-p53 were intratumorally injected into subcutaneous xenograft tumors, we observed antitumor effects in both DC-injected and non-injected tumor tissues [105], suggesting that the administration of Ad-p53-infected DCs caused a systemic immune response. Natural killer cells may be the immunological mediators of some bystander effects caused by Ad-p53-mediated cancer gene therapy [106]. These findings suggest that adenovirus-mediated p53 overexpression is a promising antitumor therapy that has antitumor effects because angiogenesis is suppressed and immune responses are induced within infected and uninfected tumor tissues. Similarly, a combination therapy that involves Ad-p53 and bevacizumab [107], a monoclonal antibody specific for VEGF-A, or FasL transduction [108] may be effective in completely eradicating tumor cells. Replication-Competent Oncolytic Adenovirus Tumor-specific, replication-competent oncolytic viruses are being developed as novel vectors for anticancer gene therapies; in these vectors, the promoters of cancer-related genes are used to regulate virus replication in a tumor-dependent manner [ ]. OBP-301 (Figure 5) is a replication-competent oncolytic adenovirus that induces tumor-selective oncolytic cell death in a telomerase-dependent manner [90-92]. A phase I clinical trial of OBP-301 in patients with advanced solid tumors has been recently completed, and OBP-301 was well tolerated by the patients [113]. Sixteen patients with a variety of solid tumors were enrolled. However, the antitumor effect of OBP-301 was limited in some of the OBP-301- injected tumors; one patient had a partial response of the injected malignant lesion and seven patients had stable disease at day 56 after treatment. Recently, armed oncolytic viruses that express therapeutics transgenes have been genetically engineered to enhance the antitumor effect of an oncolytic virus [114, 115]. Among the many possible therapeutic transgenes, p53 is an excellent candidate for such a novel approach because it is a potent therapeutic transgene and can induce cell cycle arrest, senescence, apoptosis, autophagy, or some combination thereof (Figure 1) [2]. Oncolytic adenoviruses that express p53 and are armed, tumor-specific, and replication-competent induce higher p53 expression and stronger antitumor effects than does Ad-p53 or a non-armed oncolytic adenovirus [ ]. Moreover, we also generated an armed OBP-301 variant that expresses wild-type p53 (Figure 5); this construct, which is designated OBP-702, suppresses the viability of various types of epithelial malignant cells more efficiently than does OBP-301 [122]. However, the molecular

99 p53-based Cancer Gene Therapy 93 mechanism by which p53 enhances the antitumor effect of oncolytic adenovirus remains unclear. OBP-702 mediates profound antitumor effects because OBP-702-mediated p53 overexpression induces two types of programmed cell death, apoptosis and autophagy, in malignant human cells, epithelial or non-epithelial, and thereby improves on the antitumor effects of the OBP-301 precursor [122, 123]. The p53-mediated enhancement of the antitumor effects of the oncolytic adenovirus could result from either or both of two possible mechanisms (Figure 8). One possibility is that p53 activation is enhanced because of virus replication and E1A-dependent MDM2 suppression in tumor cells. When human cancer cells were infected with a similar dose of OBP-702 or Ad-p53, the level of p53 expression induced by replication-competent OBP-702 was much higher than that induced by replicationdeficient Ad-p53 [122]. However, although OBP-702 induced high p53 expression, one of the main p53-target genes, MDM2, was induced at a lower level in the OBP-702-infected tumor cells than in the Ad-p53-infected tumor cells. This difference in MDM2 expression was due to adenoviral E1A-mediated MDM2 suppression in cells infected with OBP-702. Furthermore, this MDM2 suppression enhanced the adenovirus-mediated p53 expression and the consequent apoptotic cell death. Another possibility is that E1A mediated p21 suppression. Activation of p21 often suppresses p53-induced apoptotic and autophagic cell death pathways [24, 25]. Therefore, p21 suppression enhances the effects of the p53-mediated induction of apoptotic and autophagic cell death pathways in tumor cells. Fig. 8 E2F1 E2F1 Rb mir-93 mir-106b p53 p21 Cell Cycle Arrest E1A Senescence OBP-702 Figure 8. Schematic diagram of OBP-702-mediated induction of programmed cell death pathways. Replication-competent OBP-702 infection induces apoptosis and autophagy, resulting in the cell death; these effects are dependent on p53-mediated BAX/DRAM upregulation and adenoviral E1A-dependent p21 downregulation via E2F1-inducible mir-93/106b activation. p53 p53 BAX Apoptosis E1A Cell death p53 DRAM MDM2 Autophagy

100 94 Hiroshi Tazawa, Shunsuke Kagawa and Toshiyoshi Fujiwara In fact, inactivation of p21 by adenoviral E1A can enhance apoptosis in human colon cancer cells that overexpress p53 and have been treated with chemotherapeutic drugs [124]. Additionally, genetic deletion of p21 can induce autophagy in mouse embryonic fibroblasts that have been treated with C(2)-ceramide or gamma-irradiation [24]. In contrast, p21 overexpression can inhibit the induction of apoptosis that is mediated by Ad-p53 [25]. Furthermore, E1A-dependent activation of the transcription factor E2F1 induces the upregulation of two mirnas, mir-93 and mir-106b, that efficiently suppress p21 expression in OBP-702-infected tumor cells; this suppression of p21 leads to the enhancement of p53- induced apoptosis and autophagy in these cells (Figure 8) [123]. Interestingly, E2F1 also suppresses MDM2 expression by inducing upregulation of mir-25/32, which targets MDM2 [125]; therefore, cooperation between the MDM2-p53-p21 pathway and the E2F1-miRNA pathway may be involved in the induction of the apoptosis and autophagy that is cause by OBP-702. Thus, E1A-mediated downregulation of p21 and MDM2 would enhance p53- induced apoptosis and autophagy in OBP-702-infected cells (Figure 8). Protein Transduction Method Adenovirus-mediated p53 gene transfer systems are expected to induce exogenous p53 expression in various types of human cancer cells more efficiently than plasmid-based delivery systems; nevertheless, adenovirus infection is mainly mediated by interactions between virus particles and CARs (coxsackie and adenovirus receptors) that are expressed on host cells. Therefore, CAR-expressing tumor cells are the main targets for any adenovirusmediated exogenous gene transfer system, and tumor cells that lack CAR can escape from being killed by adenovirus-mediated p53 transduction. In fact, CAR expression can often be downregulated as tumors progress [126, 127]. Furthermore, CAR-expressing tumor cells often become refractory to Ad-p53-induced cell death because of decreases in CAR expression [128]. In tumor cells with low CAR expression, a histone deacetylase (HDAC) inhibitor can elevate CAR expression [129, 130]. However, CAR-negative tumor cells may be less sensitive or insensitive to the CAR upregulation that is mediated by the HDAC inhibitor. These findings indicate that development of a novel p53-based cancer gene therapy targeted specifically against CAR-negative tumor cells is imperative. Membrane-permeable peptides may be useful as tools for introducing exogenous, therapeutic p53 protein into tumor cells. Recently, 11 polyarginine peptides have been used as a delivery system to introduce the p53 protein into cells, and this exogenous p53 protein induced the p21 gene promoter, a downstream target of wild-type p53, as efficiently as Adp53-mediated p53 transduction [131]. Modified isoforms of p53 that are resistant to MDM2- mediated ubiquitination are more able to activate transcription of downstream targets and induce profound antitumor effects than is the wild-type p53 protein [132]. Alternatively, when pyrenebutyrate was used in combination with a fusion protein comprising three polyarginine peptides and wild-type p53, the p53 fusion protein was efficiently taken up by cancer cells and transported into the nucleus where it activated transcription of downstream target genes [133]. Recently, this fusion protein transduction system has been used to show that a fusion protein including only the carboxy-terminal region of p53 was able to efficiently induce apoptosis and autophagy in human cancer cells [134, 135]. These results suggest that a

101 p53-based Cancer Gene Therapy 95 p53 protein transduction method that involves polyarginine peptides and pyrenebutyrate is a promising p53-based cancer therapy that is independent of CAR expression on target cells. Conclusion Cancer gene therapy is defined as the treatment of malignant tumors via the introduction of a therapeutic tumor suppressor gene or the abrogation of an oncogene. One of the most potent therapeutic tumor suppressor genes is the multifunctional transcription factor p53, which regulates diverse cell fates, including cell cycle arrest, senescence, apoptosis, and autophagy. Recent advances in tumor biology indicate that p53-based cancer gene therapy has substantial therapeutic potential because a p53-mediated mirna network suppresses both tumor growth and stem cell properties in cancer cells. Gene replacement therapy that involves any one of several delivery systems to introduce the tumor suppressor p53 is a promising antitumor strategy because active p53 can induce tumor suppression in response to genotoxic therapeutic agents. For example, the replicationdeficient Ad-p53 adenovirus (Advexin, Gendicine) has antitumor effects against many types of cancers in preclinical experiments and clinical experience trials. However, Ad-p53- mediated p53 activation is insufficient for inducing cell death pathways in tumor tissues; therefore, many kinds of strategies for enhancing Ad-p53-mediated p53 activation are warranted. The enhancement of adenovirus replication, suppression of negative regulators of p53 or of p53-related cell death pathways, activation of a mirna-mediated p53 positive feedback loop, or some combination of these strategies may be effective for improving the clinical outcomes of p53-based cancer gene therapies. Given the underlying molecular mechanisms of the p53-mediated tumor suppression system, we should endeavor to develop safe and effective cancer gene therapies that are based on the potent tumor suppressor p53. Acknowledgments This study was supported by grants from the Ministry of Health, Labour, and Welfare of Japan and from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] Lane D, Levine A. p53 Research: the past thirty years and the next thirty years. Cold Spring Harb. Perspect. Biol., 2010;2:a [2] Vousden KH, Prives C. Blinded by the Light: The Growing Complexity of p53. Cell, 2009;137: [3] Aylon Y, Oren M. Living with p53, dying of p53. Cell, 2007;130: [4] Hermeking H. MicroRNAs in the p53 network: micromanagement of tumour suppression. Nat. Rev. Cancer, 2012;12: [5] Olivier M, Hollstein M, Hainaut P. TP53 mutations in human cancers: origins, consequences, and clinical use. Cold Spring Harb. Perspect. Biol., 2010;2:a

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111 In: Tumor Suppressor Genes ISBN: Editors: Mehmet Gunduz and Esra Gunduz 2013 Chapter 6 p63 and p73: Members of the p53 Tumor Suppressor Family Zeynep Ocak 1, Murat Oznur 2, Ramazan Yigitoglu 3, Esra Gunduz 2, and Mehmet Gunduz 2 1 Department of Medical Genetics, Faculty of Medicine, Abant Izzet Baysal University, Bolu, Turkey Departments of 2 Medical Genetics and 3 Biochemistry, Faculty of Medicine, Turgut Ozal University, Istanbul, Turkey Abstract The p53 transcription factor plays a significant role in cellular homeostasis and controls the transition between the G1 and S phases during cell division. Thus, replication of damaged DNA and initiation of apoptosis are prevented. Damaged DNA cannot be repaired in the absence of homozygous p53; thus, mutations occur. However, some studies have tentatively reported that mice lacking the p53 tumor suppressor protein can survive normally. It is thought that some proteins with functions identical to those of p53 might be present; thus, the p53-related proteins p63 and p73 have been investigated. The p63 and p73 proteins have high structural similarities as well as some common functional properties. Nevertheless, p63 and p73 mutat distinct from p53 in human carcinomas. P63 and p73 genes/proteins are members of the p53 gene family and show some structural and functional similarities with p53. Keywords: p53, p63, p73, tumor suppressor, cell cycle Corresponding author: Esra Gunduz, DMD, PhD, Department of Medical Genetics, Faculty of Medicine, Turgut Ozal University, Turkey, Anadolu Bulvari 16A Gimat Ankara, Turkey, egunduz@turgutozal.edu.tr.

112 106 Zeynep Ocak, Murat Oznur, Ramazan Yigitoglu et al. 1. Introduction p53 is one of the most-studied genes in human biology. The p53 gene is located on the short arm of chromosome 17 at the 17p13-1 position and is a tumor suppressor gene. The p53 gene consists of 11 exons and is approximately 20 kb in length [1]. When the p53 gene was initially investigated it was thought to be simply an oncogene; however, later it was understood that only the mutated form plays an important role in abnormal cell growth and that the normal p53 gene acts as a tumor suppressor [2]. Two genes that have prominent structural and sequence homology with p53 have been identified. One of these, p73, is localized at 1p36, while the other is p63, which is localized at 3q27-29 [3, 4]. The p63 and p73 proteins encoded by these two genes have a high similarity in the N- terminal transactivation region, central DNA-binding region, and C-terminal oligomerization region [5,6]. Furthermore, these two genes stimulate p53-sensitive promoter regions and trigger programmed cell death when expressed excessively. Remarkably, these three genes have similar intron and exon sequences [7] p63 General Information: Symbols, Location, Size, Cofactors, Subunits HGNC approved gene symbol: TP63 Alternative names and symbols: tumor protein p73-like, TP73L, p53-related protein p63, p63, KET Cytogenetic location: 3q27-q29 Subcellular location: nucleus Size: 680 amino acids; Da Cofactor: Binds 1 zinc ion per subunit Subunit: Binds DNA as a homotetramer. Isoform composition of the tetramer may determine transactivation activity. Belongs to the family of homeodomain-interacting protein kinase (HIPK) genes discovered 13 years ago. HIPK2, the most-studied member of the family, acts as co-regulator of an increasing number of transcription factors and modulates many basic cellular processes, such as apoptosis, proliferation, DNA damage response, differentiation, and development. Most of these effects are mediated by phosphorylation and activation of the p53 oncosuppressor protein [8]. Alpha and gamma isoforms of the p63 gene interact with HIPK2. They also interact with structure-specific recognition protein 1, which functions as a co-activator of the transcriptional activator p63. Isoforms 1 and 2 interact with the WW domain containing E3 ubiquitin protein ligase 1 (WWP1). Isoform 5 (via activation domain) interacts with nucleolar complex associated 2 homolog (

113 p63 and p73: Members of the p53 Tumor Suppressor Family p63 Gene Structure The p63 gene is found on chromosome 3q27-29 and is composed of 15 exons and six isoforms [4]. Figure 1. P63 gene structure. Structurally, it contains an N-terminal transactivation region, a central DNA-binding region, and a carboxy-terminal oligomerization region (Figure 1). The p63 protein containing the transactivation region is called Tap63 and the noncontaining p63 protein is called ΔNp63 [9, 10]. The p63 and p53 DNA binding regions have 60% sequence identity. The N-terminal transactivation and C-terminal tetramerization regions are similar to the orders of 22 and 37% (Figure 1). The TA and ΔN isoforms of p63 have opposite functions. The TA isoforms have tumor suppressor activity, whereas the ΔN isoform has oncogenic activity. mrnas of p63 yield three proteins known as α, β, and γ. These products have six forms that differ according to transactivation region, as follows: Tap63α, Tap63β, Tap63γ, ΔNp63α, ΔNp63β, and ΔNp63γ. These various p63 proteins have distinct features and functions [10]. P63 has a sterile alpha motive region (SAM) that is not in p53. Indeed, this region is found only in α forms of p63. The SAM region is thought to play a significant role in cellular lifespan, similar to apoptosis, transcriptional transactivation, focal adhesion, chromatin formation, receptor tyrosine kinase stimulation, and SUMOylation [9]. Unlike p53, p63 is transcribed under the control of two promoter regions. One is located in the internal region and encodes the N-terminal transactivation noncontaining region protein. The other is encoded as a normal N-terminal transactivation containing region protein [10].

114 108 Zeynep Ocak, Murat Oznur, Ramazan Yigitoglu et al p63 Gene Function Much research has focused on dissecting the specific contributions of each class of isoforms to the functions of p63. Although there is still no definitive evidence regarding the mechanistic role of each isoform, several lines of evidence indicate that TAp63 isoforms induce apoptosis [11]. P63 induces p53 target genes by binding p53, which stops the cell cycle at G1 phase and triggers apoptosis. Table 1. Mouse models of Trp63 Mouse Phenotype Reference Trp63 / p63 -/- mice have major defects in their limb, craniofacial, and epithelial development. Yang et al Trp63 / Brdm2 p63 -/- mice have severe limb and skin defects Mills et al Conditional Cre/lox TrTAp63 / TAp63 essential for Ras-induced senescence; TAp63 / increases proliferation/oncogenesis Keyes et al Loricrin ΔNp63α Normal epidermal development; UV-B challenge exhibit 45% less apoptosis Liefer et al Trp63 / Brdm3 Embryonically lethal; no epidermis Mills et al RU486-lunginducible ΔNp63α No major phenotypic changes have been seen yet Koster et al RU486-lunginducible Lung SCC, K5/K14 expression, with hyperproliferation; Koster et al. TAp63α epidermal expression, accelerated chemical-induced tumor 2004 development and progression, resulting in EMT to spindle cell carcinomas and lung metastases Trp63 +/ Higher tumor burden and metastasis compared with p53 +/ mice Flores et al Conditional Cre/lox TrTAp63 / No defects found so far Suh et al K5 TAp63α No major phenotypic changes have been seen yet Candi et al Trp63 / K5 Embryonically lethal; no epidermis Candi et al. TAp63α 2006 TrΔNp63 / knockdown Skin fragility and erosion with suprabasal defects Koster et al Trp63R279H Limb and cranofacial defects, ectodermal dysplasia Lo Iacono et al ΔNp63α and ΔNp63β Lung epithelium exhibit squamous metaplasia Romano et al Conditional Cre/lox TrTAp63 / No limb defect, deterioration of hair morphology, ulcerated wounds, reduced wound healing, accelerated aging, develop metastatic tumors Su et al However, this does not induce ΔNp63 transcription. The ΔNp63 isoform binds directly to the p53 and TA isoforms or acts as a negative dominant by competing with the wild type isoform. Thus, the p53 and TA isoforms are inactivated. Therefore, ΔNp63 has anti-apoptotic specificity. Tap63 proteins ensure cell differentiation, while ΔNp63 induces cell proliferation. ΔNp63 is a dominant isoform of p63

115 p63 and p73: Members of the p53 Tumor Suppressor Family 109 that is expressed on the epithelial basal layers of the skin, breast, prostate, and uterine cervix. Expression of ΔNp63 might thereby contribute to the proliferation of basal or progenitor cells by blocking the induction of apoptosis by p53. TAp63 and ΔNp63 have distinct and overlapping functions in normal and cancer tissues. The development of epithelial tissues in humans is a highly complex process. p63 plays an important role in embryogenesis, ectodermal differentiation, and packaging of epithelial progenitor cells [12]. Studies of p63-null (p63-/-) mice, indicated that p63 plays a key role in regulating epithelial proliferation and differentiation programs. Yang and Mills demonstrated that p63 is required for ectodermal differentiation during embryogenesis [4, 5] (Table1). The absence of p63 elicits skin, breast, and lacrimal glandular and prostate agenesis. The mice were born with developmental abnormalities and died immediately after birth [13]. Hair follicles, teeth, breast glands, and other epidermal mesenchymal originating structures do not develop in mice lacking p63 [5] (Table1). Thus p63 is necessary for ectodermal differentiation during embryogenesis. The embryonic epidermis of p63-/- mice undergoes an unusual process of nonregenerative differentiation, culminating in the striking absence of all squamous epithelia and their derivatives, including mammary, lacrimal, and salivary glands [13]. p63 protein localization and expression on the epidermis, hair follicles, sweat glands, cervix, tongue, esophagus, breast glands, prostate, and urogenital canal have been identified by immunohistochemical evaluation. p63 expression could be used as a stem cell marker of the endometrial, cervical, breast, and prostate containing epithelial cell types. P63 is expressed continuously on epidermal basal cell nuclei, germinative hair matrix cells, and the outer stem sheath of the hair follicle Regulation of p63 Expression of the p63 protein, including TAp63 and ΔNp63, occurs only during some physiological processes in the body. ΔNp63 is expressed plentifully in basal epithelial cells, such as in the basal layer of the skin and prostate, the myoepithelial cells of the breast, and thymic epithelial cells [14, 15]. In addition, TAp63 is expressed at significant levels in the female germline cells, but not in those of males [16]. However, TAp63 cannot be identified in basal epithelial cells by either Western blotting or immunostaining techniques. TAp63 transcript expression in epithelial cells can be detected by reverse transcription polymerase chain reaction, albeit at lower levels than ΔNp63 expression [17, 18]. ΔNp63 and TAp63 expression are regulated differently at the transcriptional, post-transcriptional, and post-translational levels. ΔNp63α expression can be induced in isolated dental epithelia and in the mouse lamboidal junction at the transcriptional regulation level, which is a maturation feature during morphogenesis, and is under the control of a binding element on the mouse Trp63 gene locus that is highly conserved in mammals. Similarly, TAp63 expression can be induced in certain carcinoma cell lines [19, 20]. Micro RNAs (mirna) play a significant role in post-transcriptional regulation. Each type of mirna targets different expression patterns, and the effect of ΔNp63-TAp63 transcription on skin development, epidermal stem cell proliferation and clonogenic capacity, myeloid cell proliferation, G2 cell cycle progression and glioblastoma cells occurs at the post-translational

116 110 Zeynep Ocak, Murat Oznur, Ramazan Yigitoglu et al. level [21,22]. mirnas act as sequence-specific DNA binding transcriptional activators or repressors. Previous studies have indicated that the co-activator and histone acetyltransferase (HAT) p300 binds to TAp63 and stimulates TAp63-dependent transcription of the p21cip1 gene. The novel INHAT repressor (NIR) is an inhibitor of HAT. Figure 2. Development Notch Signaling Pathway. The central portion of the NIR binds to the transactivation and C-terminal oligomerization domains of TAp63. NIR is highly expressed during the G2/M phase of the cell cycle and only weakly expressed during G1/S. Isoforms of the p63 gene contain a varying set of transactivation and auto-regulating transactivation inhibiting domains, thus showing isoform-specific activity [23, 24]. Isoform 2 activates RIPK4 transcription and may be required in conjunction with TP73/p73 to initiate p53/tp53-dependent apoptosis in response to genotoxic insults and in the presence of activated oncogenes. Isoform 2 is involved in Notch signaling, likely by inducing JAG1 and JAG2. It also plays a role in the regulation of epithelial morphogenesis (figure 2). The ratio of DeltaN- and TA*-type isoforms may govern the maintenance of epithelial stem cell compartments and regulate the initiation of epithelial stratification from the undifferentiated embryonic ectoderm. Isoform 2 is required for limb formation from the apical ectodermal ridge and activates transcription of the p21 promoter [23, 24] Role of p63 in the Cell Cycle The role of p63 in cell cycle regulation depends on the cell type and context. Exogenous TAp63γ responds to genotoxic stress and upregulates p21 expression, stops cell cycle progression in erythroleukemia cells, whereas exogenous ΔNp63α binds directly to the p21 promoter to inhibit reporter expression [25]. Endogenous p63 expression in human primary keratinocytes increases p21 expression and leads to reduced proliferation and G1 arrest. Cyclin D1, CDK4, and CDK2 expression in mouse primary keratinocytes diminishes when endogenous ΔNp63α is reduced.

117 p63 and p73: Members of the p53 Tumor Suppressor Family 111 Thus, this pattern prevents cyclin D1 and CDK4 downregulation and rescues the proliferation defects caused by the p63 deficiency [26, 27] Senescence and Aging Cellular senescence and aging is related to p63. For example, germline disruption of p63 expression results in a dramatic increase in senescence-associated β-galactosidase (SA β-gal) staining in mouse embryos, which is phenocopied by somatic disruption of p63 expression in basal epithelial cells using a K5 promoter. Importantly, blocking p63 expression with the ΔNp63 isoform is affected by the K5 promoter [28]. As mentioned previously, disrupting TAp63 expression in the basal epithelial surfaces does not renew the skin defects that arise after disruption in adults. Furthermore, genomic instability and increased DNA damage emerge as a result of the absence of TAp63 in dermal precursor cells. Nevertheless, exogenous expression of TAp63 isoforms in cell culture leads to increased cellular senescence independently of p53, as evidenced by increased SA β-gal staining and decreased proliferation. These observations highlight the importance of both ΔNp63 and TAp63 in cell senescence and demonstrate that positive or negative modulation of either p63 isoform may result in senescence by different molecular mechanisms [28, 29]. 2. Significance of the p63 Gene in Diseases Human p63 heterozygous germline mutations are strongly associated with human autosomal dominant developmental diseases [30]. p63 gene mutations in humans are characterized by lip anomalies and/or ectodermal dysplasia. Figure 3. p63 are associated with diseases.

118 112 Zeynep Ocak, Murat Oznur, Ramazan Yigitoglu et al. Mutations in the p63 gene are known to cause at least five different syndromes and two non-syndrome human disorders; these include isolated split hand/foot and non-syndromic cleft lip [31] (figure 3). AEC: Ankyloblepharon-Ectodermal Defects-Clefting Syndrome (Hay-Wells sendromu) ADULT: Acro-dermato-ungual-lacrimal-tooth LMS: Limb-Mammary Syndrome UMS: Rapp-Hodgkin Syndrome 2.1. Ectrodactyly, Ectodermal Dysplasia, and Cleft Lip/Palate Syndrome 3 Ectodermal dysplasia (ED), as well as cleft lip and/or cleft palate, sparse hair, skin, malformed ears, fingers and toes, and partial or complete syndactylyis, is an autosomal dominant syndrome of ED [32]. Previous studies have identified frequent mutations of five amino acids, including R204, R227, R279, R280, and R304 in the EEC population, all located in CpG islands. These five mutations occur in almost 90% of patients with EEC syndrome [33]. P63 mutation analyses have identified eight mutations that prevent DNA binding in 9 unrelated EEC3 families. These eight mutations are amino acid changes, while the ninth is a frame-shift mutation. To date, 34 mutations involving 20 amino acids have been reported in EEC. Only the following two mutations are outside the DNA-binding domain: one insertion (1572 InsA), and one point mutation (L563P) in the SAM domain [31] (figure 3). The five p63 arginine hotspot mutations, and probably the other DNA binding domain mutations, that are found in EEC syndrome appear to impair p63 protein binding to DNA. In this syndrome, slight mental retardation and liver malfunction have been reported in some cases. Ectrodactylia is defined as a defect described as the fusion or the absence of the fingers in the central part of hands and feet [34]. Thus, the description of syndactylia is included in the description of ectrodactylia. 84% of the patients with EEC syndrome present ectrodactylia, while 77% present ectodermic dysplasia, 68% present cleft palate and/or lip, 59% present lachrymal duct anomalies, 23% present genitourinary tract anomalies, 14% present deafness and 7% present mental retardation [35]. The major diagnosis criteria of the syndrome are: ectodermic dysplasia, ectrodactylia, cleft palate and/ or lip, lachrymal duct anomaiesl. If the patient does not present with any other findings suggesting some other syndrome, the presence of at least two of the criteria is characteristic. Considering the diagnosis criteria of the syndrome, even without cleft palate and/or lip and lachrymal duct anomaly, patients with ectodermic dysplasia and ectrodactylia meet the criteria requirements of diagnosis [37] Split-Hand/Foot Malformation (SHFM) 4 SHFM is a congenital malformation characterized by deep median clefts of the hands and feet. SHFM may occur in isolation or as part of a multisystemic syndrome. Transmission is autosomal dominant. The non-syndromic from of SHFM4 is caused by several mutations dispersed throughout the p63 gene, including a point mutation in the TA (R58C), a splice-site

119 p63 and p73: Members of the p53 Tumor Suppressor Family 113 mutation in front of exon 4 (3 ss intron 4), four missense mutations in the DBD (K193E, K194E, R280C, R280H), and two nonsense mutations in the TI-domain (Q634X, E639X) [31, 37]. Several SHFM4 mutations cause alterations in p63 protein activation and stability. Q634X and E639X are known to disrupt the sumoylation site and increase the stability and transcriptional activity of the p63a isoform. Furthermore, amino acids K193 and K194 are required for ubiquitin conjugation by E3 ubiquitin ligase (Itch), and naturally occurring mutations in these amino acids result in a more stable p63 protein [38] (figure 3) Ankyloblepharon-Ectodermal Defects-Clefting (AEC) Syndrome Hay Wells syndrome, also known as AEC syndrome, is a rare autosomal dominant disorder characterized by congenital ectodermal dysplasia, including alopecia, scalp infections, dystrophic nails, hypodontia, ankyloblepharon, and cleft lip and/or cleft palate. Analysis of the p63 gene in eight patients with AEC syndrome identified a missense mutation in the TP63 gene in one patient. A duplication of 11 bp in the p63 gene has been identified in a patient with Rapp Hodgkin signs [32] (figure 3) Acro-Dermato-Ungual-Lacrimal-Tooth (ADULT) Syndrome ADULT syndrome is characterized by ectrodactyly, excessive freckling, onychodysplasia, obstruction of lacrimal ducts, and hypodontia and/or early loss of permanent teeth. Clinical presentation is variable, and transmission is autosomal dominant. Fourteen cases have been described to date. All families and one of the sporadic cases had a point mutation in exon 8, changing R298 in the DNA-binding domain into either a glutamine or a glycine [39] (figure 3) Limb-Mammary (LMS) Syndrome LMS is a rare disease, and <50 cases have been described in the literature. This syndrome is characterized by severe hand and/or foot anomalies and hypoplasia/aplasia of the mammary gland and nipple. The clinical presentation is extremely variable. LMS is an autosomal dominant disease caused by loss-of-function mutations in exons 13 and 14 of the TP63 gene, localized to the subtelomeric region of chromosome 3 (3q27) [40]. One large LMS family (29 affected members) has a point mutation in exon 4, causing a G76W substitution in the DNspecific putative TA2. One other point mutation (S90W) is also located between the TA domain and DBD [41]. Other LMS mutations have been detected in the C-terminus, including a TT deletion in exon 13 and an AA deletion in exon 14. These affect only the p63a protein isoforms, in which they are predicted to cause a frame-shift mutation and a premature stop codon [42]. Additionally, a stop mutation in the transcription factor inhibitory domain (TI) (K632X) has been identified in a patient with sporadic LMS. The latter mutation is predicted to impair

120 114 Zeynep Ocak, Murat Oznur, Ramazan Yigitoglu et al. the suppressive effect of the TI domain on the TA domain, thus increasing transactivation activity [31] Rapp Hodgkin Syndrome (RHS) RHS is characterized by the association of anhidrotic ectodermal dysplasia with cleft lip/palate. The syndrome is usually evident at birth but its prevalence is unknown, as < 100 cases have been reported in the literature [43]. RHS mutations are located in the C-terminus of the p63 protein, and RHS is transmitted as an autosomal dominant trait. RHS mutations are located in the C-terminus of the p63 protein. They are either point mutations in the SAM domain or deletions in the SAM or TI domains. A mother and daughter with RHS syndrome and corneal dystrophy revealed a deletion in the gene 1BP of the tumor protein p73-like (TP73L) gene. A heterozygous missense mutation in the TP73L gene was identified in a 14- year-old boy with RHS syndrome [31]. 3. Role of the p63 Gene in Cancer Researchers have identified that ΔNp63α, but not the ΔNp63γ or TAp63 isoforms, modulate Erk2 signaling to inhibit mammary cell migration, invasion, and metastasis (figure 4). Furthermore, another study showed that Sharp-1 and cyclin G2 are two clinically relevant p63 targets that inhibit cell migration and invasion. p63 inhibits RCP-mediated integrin α5β1 recycling to the plasma membrane via an unknown mechanism, thus downregulating downstream signaling from these complexes to Akt and thereby inhibiting cell migration and invasion. H1299 cells express mainly TAp63 isoforms, and TAp63α over-expression reverts cell invasion induced by mtp53 back to control levels in this system [11]. There are many studies on the role of the p63 gene in neoplastic transformation. The p63 gene is widely investigated because it is thought to play a role in the regulation of neoplasm proliferation and differentiation due to its determination in various solid tumors and epithelial cells [44]. According to the analyses of the p63 gene in cancer studies, this gene is defined as an oncogenous agent and not as a tumor repressing gene. The mutation of p63 is rare in human cancers. Gene amplification and protein over expression are more frequent in skin, cervix, oral cavity, bladder, nasopharynx, head and neck carcinoma [45,46]. As p63 gene expression is limited to the epithelial cells, it can be involved in proliferation and differentiation of premalignant and malignant lesions of epithelial origin. Di Como et al. studied the presence of p63 expression in various human tumors using microarray technology. While a high level of p63 expression is observed in basal and squamous cell carcinoma and transitional cell carcinoma, no expression was observed in adenocarcinoma including in organs such as breast and prostate [47,48]. High level of p63 gene expression has been observed in thymoma. While this gene is expressed in some Non Hodgkin lymphoma, no expression has been detected in tumors such as soft tissue sarcoma, mesothelioma or hepatocellular carcinoma.

121 p63 and p73: Members of the p53 Tumor Suppressor Family 115 Table 2. Major p63 expression and gene changes in cancer Cancer Notes Reference Cervical p63 marker of squamous differentiation; HPV-p63 Wang et al carcinoma association Cervical p63 preferentially expressed in immature squamous cells; Quade et al carcinoma useful for differential diagnosis in early stages Distinct p63 expressed in thymomas, non-hodgkin's lymphoma, Di Como et al cancers basal cell/scc, not in adenocarcinomas of breast/prostate Distinct ΔNp63 is the most expressed isoform Nylander et al cancers Bladder cancer Loss of ΔNp63 associate with progression/invasion Urist et al Lung SCC Increased copy number in 88% SCC, 42% LCC, 11% AC; Massion et al ΔNp63α predominant protein, associated with better prognosis Head and neck Head and neck SCC p63+ ΔNp63α predominant protein Sniezek et al cancer Head and neck ΔNp63α predominant protein and reflects platinum Zangen et al cancer response and favorable outcome Cancer Notes Reference Bladder cancer Low p63 associates with higher TNM and low β-catenin; Barbieri et al p63 prognostic effect is independent of TNM Bladder cancer ΔNp63 associate with invasiveness and upregulation of N- cadherin Fukushima et al Lung SCC Correlation of podoplanin/cd44/ p63 in a hierarchical Shimada et al manner Prostate cancer p63 marker of early neoplastic lesion (intraepithelial Hull et al2009. neoplasia versus adenocarcinoma) Prostate cancer ΔNp63α more abundant than TAp63; 1 mutation exon 8; Kellogg Parsons et al Pre-cancerous p63+ in dysplastic oral mucosa, with loss of E-cadherin Das et al conditions Breast cancer p63+ prognostic factor in ER+ Pt; no correlation with Hanker et al standard parameters Lung SCC p63 distinguish AC, ( ve) from SCC, (+ve) Terry et al Lung SCC ΔNp63 is the isoform that distinguish AC from SCC Uramoto et al Bladder carcinoma ΔNp63 correlates with high MW cytokeratins in basal cancer, more aggressive with poor prognosis Karni-Schmidt et al Breast cancer p63 improves identification of myoepithelial cells Aikawa et al Breast cancer MFG-E8 (ligand of integrin αvβ3-5) is a p63 target in vivo Yang et al Lung SCC NSCLC differential diagnosis: p63+ in 100% SCC, 10% AC); p63 & cytokeratin5/6 allow accurate classification Mukhopadhyay and Katzenstein % cases Lung SCC Cytokeratin5/7, TTF1, p63 allow accurate classification of Righi et al all NSCLC cases Lung SCC Higher ΔNp63/TAp63 ratio in NSCLC indicating poor Iacono et al outcome Lymphoma TAp63 is the most expressed isoform Iacono et al Thyroid carcinoma p63+ in 67% follicular adenoma, 41% papillary, 29% follicular carcinoma Tan et al.2011.

122 116 Zeynep Ocak, Murat Oznur, Ramazan Yigitoglu et al. The p63 gene is known to be expressed in 93% of SCC of the lung, 10% of breast ductal carcinomas, and 25% of ovary endometrial carcinomas. A low level of p63 gene expression has been reported to be rare in breast, lung or prostate adenocarcinoma. The ΔNp63 gene has been detected to be over expressed in neuroblastoma, colorectal carcinoma, bladder cancer, nasopharynx cancer, head and neck SCC and hepatocellular carcinoma. Figure 4. The p63 protein inhibits metastasis via multiple mechanisms Role of p63 in Epidermis, Cutaneous Adnexal and Skin Cancer Ivan et al. have studied the relation between the p63 gene and primary cutaneous tumors and Skin adenocarcinoma mestastasis. as According to their results, the p63 gene was found to be a sensitive and specific determinant for benign or malignant adnexal tumors. There has been observed no expression of p63 in metastatic carcinoma,thus, they proposed that the p63 gene may be helpful in the differentiation of primary skin cancer and metastatic skin adenocarcinoma [49]. Parsa et al. showed that the p63gene is expressed in vitro in normal cells as well as neoplastic keratinocytes and that this gene constitutes an indicator for the proliferation capacity of keratinocytes. It has also been postulated that the p63 gene can be a diagnosis indicator of anaplasia in keratinosid tumors [50]. Laurikalla et al. studied the expression of 2 isoforms of the p63 gene in embryonic molar teeth of mice; they observed that p63 gene expression is high at a specific step of the tooth enamel epithelium. The absence of Np63 expression in internal epithelial cells that differentiate into ameloblasts has been reported. It has been demonstrated that Np63 gene expression continues in the epidermis as well as in tooth and hair follicle epithelial cells and that it is down regulated during the differentiation of keratinocytes and ameloblasts.. No transactivated TA p63 isoform expression has been observed. Kumonoto et al. studied p63 and p73 gene expression in tooth germ and ameloblasts (48 healthy and 5 malign). They observed that the

123 p63 and p73: Members of the p53 Tumor Suppressor Family 117 p63 gene expression is high in desmoplastic ameloblastoma, acanthomatous and granular cell ameloblastoma. They also found the TA p63 gene in 5 of 8 tooth germs andin 16 of 34 ameloblastomas (5 malignant ameloblastomas). Finally, Np63 has been observed in all developing and neoplastic odontogenic tissues Role of p63 in Bladder Cancer Urist et al. studied the p63 expression rate in 102 bladder variant epithelial cell carcinoma (VECC). The expression rates of of p63 in the highly differentiated papillary surface tumors was 93% and in the less differentiated surface cells it was 68%. The expression of p63 decreased in invasive tumors (16%). In relation with this, the phase and grade-based urothelial differentiation in bladder VECC is proposed to be accompanied by p63 gene expression [46]. In VECC, the decrease of p63 gene expression can induce the loss of p63 isoforms that inhibit growth and differentiation. It has been proposed that the p63 gene is not necessary for the formation of bladder epithelium but is required for its differentiation [46] Role of p63 in Breast Cancer In a study about the relation between p63 and p53 gene expression in normal and neoplastic breast tissue, no p63 gene expression was observed in intraductal carcinoma, tubular carcinoma, lobular carcinoma, medullar carcinoma and grade I and II invasive ductal carcinoma tumor cells. However, P63 gene expression has been detected in grade III invasive ductal carcinoma. These results lead to the hypothesis that p63 gene is an indicator of cells with pathological differentiation in breast cancer. P63 displayed expression in the myoepithelial layer surrounding the normal ductal and alveolar epithelium [51]. In 21.17% of breast carcinoma presented p53 expression, 11.76% of these carcinoma presented p63 expression. The expression of p63 is associated with poor prognostic parameters such as the phase, size, histological differentiation, lymph node metastasis and estrogen receptor negative status of the tumor. According to these results, the p63 gene is thought to be an indicator of the aggressiveness of breast carcinomas [52] Role of p63 in Cervix Cancer Based on some studies, it has been proposed that the p63 gene is expressed in basal and immature cervical squamous epithelium. In a study including 96 cases of cervix SCC, Wang et al. detected p63 gene expression in97% of cases SCC, while no expression was observed in adenocarcinomas. They observed a significant relation between p63 gene expression and squamous differentiation in the cervical transformation area. They did not detect any p63 in normal mature endocervical epithelium [53]. It has been proposed that p63 may be used as a marker for the basal cells of the ectocervix epithelium, for the identification of cervical stem cells [53, 54].

124 118 Zeynep Ocak, Murat Oznur, Ramazan Yigitoglu et al Role of p63 in Lung Cancer In a study of Pelosi G about the lung SCC, a high level of p63 immunoreactivity was observed in precursor lesions such as squamous metaplasia and dysplasia, and in gradient neuroendocrine tumors. In another study of p63 in lung tumors, expression was found to be 96.9%, 30%, and 37% in SCC, adenocarcinoma and in large cell carcinoma, respectively. In neuroendocrine tumors, p63 expression increased with tumor grade, and an immunoreactivity level of 1.9%, 30.8%, 50%, 76.9% was detected for classical carcinoids, atypical carcinoids, large cell neuroendocrine carcinoma, and small cell carcinoma, respectively [71]. While p63 expression was observed in basal and suprabasal cells of the bronchial epithelium in lung SCC, a loss of expression was seen in more superficial, differentiated cell layers. p63 expression increased with increasing intesity of bronchial epithelial dysplasia. The maximum level of p63 expression was reported in SCC. In the same study [74], no significant difference was observed between p53 and p63 expressions Role of p63 in Oral Cancer One of the most frequently encountered malignant pathologies in the oral cavity is squamous cell carcinoma. The relation between p63 expression and squamous cell carcinoma and prognosis has been studiedextensively. The prognostic indicator of oral squamous cell carcinoma is lymphatic tissue metastasis and 3q21-29 chromosome (p63 gene location) amplification. Chen et al. observed that p63 expression is correlated with the level of oral epithelial dysplasia. According to these observations, the increase of p63 expression has been proposed as an important actor in early oral carcinogenesis [48]. In a similar study, Lo Muzio et al. described the correlation between p63 expression in SCC and tumor differentiation. Cases with diffuse p63 expression have been observed to be more aggressive and poorly differentiated. The overexpression of p63 is found to be related to poor prognosis in SCC Role of p63 in Head Neck Cancer The loss of expression of TAp63 has been related to tumor progression in laryngeal carcinoma. In basal and suprabasal layers of the epithelium in normal laryngeal mucosa, nuclear immunoreactivity with p63 has been detected in the myoepithelial cells of the seromucinous glands. In laryngeal intraepithelial neoplasia, p63 immunoreactivity is not limited to the basal layer. p63 expression has been observed in all layers of the epithelium. p63 expression has also been observed in tumor cells of all larynx SCC at a rate varying between 10% and 98%. Furthermore, tumor localization, lymphatic node metastasis, clinical phase and smoking are found to be associated with tumor recurrence. As p63 expression has been detected in laryngeal intraepithelial neoplasia, p63 is suspected to play a role in early stages of the development of larynx cancer. Sniezek et al. studied the expression of p53 and p63 in head and neck SCC. Out of a total of 36 cases, p53 expression was observed in 72% and p63 expression was observed in 100% of patient samples.. In the 10 patients whose cancers were not positive for p53 expression, a high level of p63 expression was detected. The difference between p53 and p63 expression was statistically significant. In relation to

125 p63 and p73: Members of the p53 Tumor Suppressor Family 119 this, it has been proposed that p63 may play an oncogenic role by antagonizing the p53 pathway in head and neck SCC [18,55]. In the same study, Western Blot analysis revealed that the ΔNp63α is the most dominant isoform in head and neck SCC and that it is more highly expressed in tumors when compared to normal contiguous tissue. It has been proposed that the ΔNp63α isoform plays an anti-differentiation and anti-apoptotic role in mucosal epithelium in head and neck SCC and that it may lead to the development of tumors. Besides, higher p63 gene expression is associated with poor prognosis and disease free survival [56]. 4. p73 General Information: Symbols, Location, Size, Cofactor, Subunit HGNC Approved Gene Symbol: TP73 Cytogenetic location: 1p36.3 Subcellular location: nucleus and cytoplasm. Size: 636 amino acids; Da Cofactor: binds one zinc ion per subunit Subunit: found in a complex with p53/tp53 and CABLES1. The C-terminal oligomerization domain binds to the ABL1 tyrosine kinase SH3 domain and interacts with HECW2. The beta isoform interacts homotypically with p53/tp53, whereas the alpha isoform does not. The gamma isoform interacts homotypically with all p73 isoforms. The delta isoform interacts with the gamma and alpha isoforms homotypically. The alpha and beta isoforms interact with HIPK2. The alpha isoform interacts with RANBP9, and the beta isoform with WWOX. Interacts (via SAM domain) with FBXO45 (via B30.2/SPRY domain). Interacts with YAP1 (phosphorylated form). Interacts with HCK (via SH3 domain); this inhibits TP73 activity and degradation ( pl?gene=tp73) p73 Molecular Structure The TP73 gene is composed of 14 principal exons. Primary transcripts generated from two alternative promoters (P1 and P2) undergo differential splicing to generate multiple isoforms. p73 (TA-p73) contains a N-terminal transactivation domain (TAD), followed by a proline-rich sequence (PR), a central DNA-binding domain (DBD), and a C-terminal oligomerization domain (OD), involved in the formation of tetramers (figure 5). The sequences of the DBD, TAD, and OD regions of p73 and p53 exhibit 63%, 29%, and 38% similarities [57] p73 Gene Function Evidence from in vitro studies indicates that p73 has a tumor suppressive role. Isoforms containing the transactivation domain are pro-apoptotic, whereas those lacking the domain are

126 120 Zeynep Ocak, Murat Oznur, Ramazan Yigitoglu et al. anti-apoptotic and block the function of p53 and transactivating p73 isoforms. P73 stimulates the transcription of genes like p21waf1/cip1, RGC (Ribosomal Gene Cluster), mdm2, bax, cyclin G, GADD45, IGF-BP3 (insulin-like growth factor-binding protein 3) whose transcription are regulated by p53 and can pause the cell cycle in G1 phase. Figure 5. Structure of the p73 gene and encoded proteins. Studies have demonstrated that p73 is required for the construction of specific neural structures. One of these neural structures is a bipolar neuron named Cajal-Retzius. These neurons are responsible for the cortex organization of the hippocampus. In p73 knock-out mice, these neurons have been selectively lost thus leading to hippocampus dysgenesis. These mice also present many limbic telencephalon malformations. In p73 knock-out mice (p73-/-), there are neurological, pheromone and inflammatory defects but there is no spontaneous tumor development [57] Regulation of p73 Protein Levels TA-p73 and ΔN-p73 isoform protein levels might be regulated by other factors. The intracellular p73 level does not change after applying DNA damaging agents such as actinomycin D or UV. Degradation of p73 occurs through binding to p53 and by mouse double minute 2 (MDM2), which directs the p53 protein to the ubiquitin-proteosome pathway. MDM2 does not trigger p73 ubiquitination. However, it can catalyze p73 neddylation (the conjugation of the small ubiquitin-like protein NEDD8), which inhibits p73 transcriptional activity. These observations show that although p73 can perform the functions of p53, it is not stimulated by DNA damage and is regulated in a manner different than is p53. The NEDD4-like ubiquitin ligase Itch is an important regulator of the p73 protein level. It recognizes a PY motif (the PPxY amino acid sequence) in the C-terminal region of p73 that is not present in p53. c-jun prevents degradation of TA-p73. Degradation of ΔN-p73 is triggered at the same time by the nonclassical polyamine-induced antizyme (Az) pathway.

127 p63 and p73: Members of the p53 Tumor Suppressor Family 121 The Az antizyme (a small protein initially identified as an inhibitor of ornithine decarboxylase) is the first key enzyme in the polyamine biosynthesis pathway. Az expression is promoted by c-jun upon genotoxic stress, leading to proteasomemediated ubiquitin-independent degradation of ΔN-p73. The p73-induced RING 2 protein (PIR2), a ring-finger domain ubiquitin ligase, regulates the TA- to ΔN-p73 isoform ratio. Notably, PIR2 is a transcriptional target of TA-p73 that preferentially degrades ΔN-p73, thus releasing TA-p73 and triggering apoptosis following DNA damage. Figure 6. The p73 pathway and its regulators p73 as a Novel Target of Anticancer Therapies The p53 homolog p73 is frequently overexpressed in cancers. In particular, the transactivation domain truncated isoform DNp73 has oncogenic properties, and its upregulation is associated with poor patient survival. The p73 pathway is an attractive target for cancer drug development. Experimental and clinical evidence demonstrates that TA-p73 isoforms have the potential to alter p53 functions in cancer cells by inducing apoptosis after DNA damage. Conventional chemotherapeutic drugs can increase TA-p73 levels by activating the E2F1-TA-p73 axis. TA-p73 activities are frequently dampened in tumors by mutation or deregulated expression of p73 modulators or co-factors. The anticancer drugs used in selection of the p73 target are presented in Table 3.

128 122 Zeynep Ocak, Murat Oznur, Ramazan Yigitoglu et al. Table 3. List of Drugs Targeting the p73 Pathway Drug Target Mechanism of Action Effects Reference Prima-1, Prima- 1MET Mutant-p53 Reactivation of mutant p53 through covalent binding to Apoptosis in tumor cells expressing Bykov VJ, et al (APR-246) their core domain mutant-p53, alone or in combination with cisplatin Arsenic trioxide (ATO) PML-RAR Reduction of N-p73 levels and increase of p300- p73-mediated apoptosis, alone or Lunghi P, et al mediated acetylation of p73 in APL cell lines. Increased expression of both TA- and N- p73 expression in primary APL cells. by co-administration with MEK1 inhibitors (PD98059, PD184352) Nutlin-3 MDM2 Displacement of MDM2- E2F1-p73 complex Apoptosis in p53- null or mut-p53 expressing cells in Vassilev LT, et al Pierce SK, et al combination with chemotherapeutic drugs (doxorubicin) PD9805, PD MEK1 Alteration of TA/ N-p73 ratio: reduction of N- p73 levels and accumulation p73-mediated apoptosis alone or in co-administration Lunghi P, et al Lunghi P, et al and tyrosine phosphorylation of TA-p73 with ATO 37aa peptides iaspp Disaasembly of iaspp-p73 complex p73-dependent apoptosis in vitro Bell HS, et al and in tumor xenograft in vivo RETRA Mutant p53 (?) Disassambly of mutp53/p73 complex p73-dependent inhibition of cell Kravchenko JE, et al growth in vitro and in vivo in tumor xenografts Aptamers Mutant p53 Unknown Apoptosis in mutp53 expressing cells Guida E, et al MLN8054 Aurora kinase A Induction of TA-p73 expression p73-dependent apoptosis in p53- Dar AA, et al null cells SIMP peptides Mutant p53 Disassembly of mutp53/p73 complex p73-dependent apoptosis in mutp53 Di Agostino, et al expressing cells in combination with chemotherapeutic drugs (doxorubicin) Curcumin mtor, NF- kb NF-kB and mtor inhibition and TA-p73 accumulation and activation p73-dependent apoptosis Chakraborty J, et al Beevers CS, et al Enzastaurin (LY HCL PKCkinases Accumulation of -catenin, that promotes c-junp73-dependent apoptosis in multiple Raab MS, et al dependent induction of p73 myeloma cells Forodesine Unknown Increased TA-p73 transcription Apoptosis in CLL cells, alone or in combination with bendamustine and rituximab Alonso R, et al

129 p63 and p73: Members of the p53 Tumor Suppressor Family 123 Drug Target Mechanism of Action Effects Reference Rapamycin FKB12 Direct mtor inhibition and increased TA-p73 levels p73-dependent cell death, increased by cisplatin coadministration in Rosenbluth JM, et al Wong SW, et al basal-like triple negative breast cancer cells Lenalidomide (CC-5013, or Unknown Induction of CD154 expression, that trigger the CD95-mediated or fludarabine-induced Lapalombella R, et al evlimid) c-abl-mediated activation of p73 c-abl/p73 dependent apoptosis in p53- deficient CLL cells Panobinostat (LBH589) HDACs E2F1- and myc-mediated transcription of mir-106b, TA-p73 induced apoptosis in CLL Sampath D, et al that targets the p73 ubiquitin ligase Itch cells Thymoquinone Unknown Increased TA-p73 protein level p73-dependent cell cycle arrest and apoptosis in acute lymphoblastic leukemia (ALL) Jurkat cell line Alhosin M, et al References [1] Isobe M, Emanuel BS, Givol D, Oren M, Croce C M.Localization of gene for human p53 tumour antigen to band 17p13. Nature, 320: 84-85, [2] Lane DP P53, A dieth in the life of p53. Nature, 358: [3] Sheikh MS, Fornace AJ Role of p53 family members in apoptosis. Journal of cellular physiology, 1982: [4] Yang A, Kaghad M, Wang Y et al P63, a p53 homolog at 3q27-29, encodes multiple products with transativating, death inducing and dominant-negative activities. Mol. Cell, 2: [5] Mills AA, Zheng BH, Wang XJ, Vogel H, Roop DR, Bradley A p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature, 398(4): [6] Moll UM, Slade N p63 and p73: Roles in development and tumor formation. Mol. Cancer Res., 2(7): [7] Kaelin WG The p53 gene family. Oncogene, 18(11): [8] Lavra L, Rinaldo C, Ulivieri A, Luciani E, Fidanza P, Giacomelli L et al The Loss of the p53 Activator HIPK2 Is Responsible for Galectin-3 Overexpression in Well Differentiated Thyroid Carcinomas. PLoS One, 6(6): e [9] Deutsch, G. B, Zielonka, E. M, Coutandin, D, Weber, T. A, Schafer, B, Hannewald et al DNA damage in oocytes induces a switch of the quality control factor TAp63- alpha from dimer to tetramer. Cell, 144: [10] Westfall MD, Pietenpol JA p63: molecular complexity in development and cancer. Carcinogenesis, 25 (6): [11] Bergholz J, Xiao ZX Role of p63 in Development, Tumorigenesis and Cancer Progression. Cancer Microenvironment, 5:

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135 In: Tumor Suppressor Genes ISBN: Editors: Mehmet Gunduz and Esra Gunduz 2013 Chapter 7 The Emerging Roles of Forkhead Box (FOX) Family Proteins in Tumor Suppression Pang-Kuo Lo The Department of Biological Sciences, University of South Carolina, Columbia, SC, US Abstract The FOX gene family comprises gene members that encode forkhead box transcription factors, which contain an evolutionarily conserved DNA-binding domain termed the forkhead box or winged helix domain. FOX protein family members have been recognized as pivotal transcriptional factors to regulate a wide spectrum of biological processes, including metabolism, development, differentiation, proliferation, apoptosis, cellular mobility, vascularization and longevity. Given that FOX proteins control these imperative developmental and homeostatic processes, dysregulation of expression and functions of FOX gene family members can lead to alterations in cell fate, tumorigenesis and cancer progression. In this chapter, the emerging roles of FOX family transcription factors in tumor suppression are discussed based on the growing evidence. This comprehensive chapter covers topics regarding functional roles of these FOX tumor suppressors in cancer, pathological mechanisms giving rise to down-regulation of their expression as well as inhibition of their functions in cancer, and the potential of FOX proteins as targets for therapeutic intervention in cancer. In addition to the main focus on the tumor-suppressor roles of FOX proteins, the complexity of their dual roles as tumor suppressors and oncogenes in tumorigenesis and cancer progression is also discussed. Keywords: Forkhead box transcription factors (FOXs), forkhead box-o (FOXO), forkhead box-f (FOXF), forhead box-l (FOXL), forkhead box-p (FOXP), tumor suppressor Correspondence to: Pang-Kuo Lo, Ph.D. Research Assistant Professor, Department of Biological Sciences, University of South Carolina, 715 Sumter Street, CLS 601, Columbia, SC 29208, TEL: , FAX: LOP@mailbox.sc.edu; pklo9867@yahoo.com.

136 130 Pang-Kuo Lo Abbreviations AMPK AMP-activated protein kinase ATG12 autophagy-related 12 homolog ATM ataxia telangiectasia mutated ATR ataxia telangiectasia and Rad3 related BCL6 B-cell lymphoma 6BNIP3 BCL2/adenovirus E1B 19kDa interacting protein 3 CAF cancer-associated fibroblasts CBP CREB-binding protein CDK2 cyclin-dependent kinase 2 CDKI cyclin-dependent kinase inhibitor CK1 casein kinase 1 CML chronic myeloid leukemia CREB camp response element-binding protein CSCs cancer stem cells DYRK1A dual-specificity tyrosine-phosphorylated-regulated kinase 1A 4E-BP eukaryotic translation initiation factor 4E binding protein EMT epithelial-to-mesenchymal transition ERK1/2 extracellular signal-regulated kinases 1 and 2 FHL2 four and a half LIM 2 G6Pase glucose 6 phosphatase GABARAPL1 GABA-receptor-associated protein-like 1 HAUSP herpesvirus-associated ubiquitin-specific protease HIF1 hypoxia-inducible factor 1 IHC Immunohistochemistry IKK IkappaB kinase InsR, insulin-like growth factor receptor IRS1 insulin receptor substrate-1 IRS2 insulin receptor substrate-2 JNK c-jun N-terminal kinase LC3 microtubule-associated protein 1A/1B-light chain 3 LICs leukemia initiating cells MAPK mitogen-activated protein kinases MDM2 RING-finger E3 ligase murine double minute 2 MLL myeloid/lymphoid or mixed lineage leukemia MnSOD Mn superoxide dismutase MST1 mammalian orthologue of the ste20-like protein kinase mtorc1 mammalian target of rapamycin complex 1 mtorc2 mammalian target of rapamycin complex 2 NES nuclear export sequence NLS nuclear localization signal OGCTs ovarian granulosa cell tumors PEPCK phosphoenolpyruvate carboxykinase PGC-1 peroxisome proliferative-activated receptor- coactivator 1 PI3K phosphoinositide 3-kinase

137 The Emerging Roles of Forkhead Box (FOX) Family Proteins 131 PKB protein kinase B PTEN phosphatase and tensin homolog Raptor regulatory associated protein of mtor Rictor rapamycin-insensitive companion of mtor ROS reactive oxygen species S6K S6 kinase SGK serum and glucocorticoid inducible kinase SKP2 S-phase kinase-associated protein 2, E3 ubiquitin protein ligase Treg regulatory T cells TSC1/2 tuberous sclerosis complex 1 and 2 VHL von Hippel-Lindau tumor suppressor ubiquitin ligase Introduction The forkhead box (FOX) gene family is composed of genes encoding evolutionarily conserved transcriptional regulators with a common DNA-binding domain named the forkhead box or winged helix domain [1, 2]. The forkhead box DNA-binding domain of approximately 100 amino acids in length was originally discovered and defined as the conserved protein region among Drosophila Fork head and mammalian FOXA family proteins [3, 4]. Since the first forkhead box protein was identified in Drosophila melanogaster, a transcription factor that promotes terminal rather than segmental development, there are at least 41 genes currently identified in humans, which are classified into 17 FOX gene subfamilies [1]. FOX gene family members are evolutionarily expanded from worms to mammals to fulfill the needs of increased developmental and tissue complexity. In spite of a conserved forkhead DNA-binding domain, different subfamilies of FOX proteins have differential regulation and functional diversification, which are attributable to protein sequence variations outside of the forkhead domain [1]. These non- DNA-binding protein regions are engaged in interaction with components of transcriptional activators, transcriptional repressors, or DNA repair complexes to regulate gene transcription and DNA repair [5]. FOX protein family members are imperative for a wide array of biological processes, including developmental embryogenesis as well as organogenesis, metabolism, immune responses, differentiation, proliferation, apoptosis, migration, invasion and longevity [2, 5-9]. Due to the essential roles of FOX proteins in regulating these developmental and homeostatic processes, it is expected that dysregulating the expression and functions of FOX protein family members can alter cell fate and give rise to tumorigenesis. Indeed, it is known that many FOX subfamilies such as FOXO, FOXM, FOXP, FOXC, FOXA, FOXE, FOXQ, FOXR and FOXF have been linked to tumorigenesis and the progression of certain cancers [5, 9]. Genetic and epigenetic deregulation of the functions of these FOX proteins acting as oncogenes, tumor suppressors or bi-functional factors are involved in initiation, progression and metastasis of cancer [5]. Therefore, an understanding of dysregulating FOX protein expression and function is crucial to addressing the potential that FOX proteins are employed as direct targets and/or indirect effectors of therapeutic intervention.

138 132 Pang-Kuo Lo Forkhead subfamily FOXOs FOXP3 FOXFs FOXL2 Table 1. Dysregulation of FOX tumor suppressors in cancer Tumor-suppressor roles Transactivates target genes implicated in cell cycle arrest, apoptosis, autophagy, DNA repair, ROS detoxification. An X-lined tumor suppressor in epithelial cells acts as a transcriptional repressor for HER2, SKP2 and c- MYC genes, but an activator for the p21 gene. Negatively regulates E2F target genes to inhibit the DNA replication process and activates DNA repair genes. Transcriptionally activates target genes with roles in cell cycle arrest, apoptotic regulation, ROS detoxification and inhibits metastasisrelated genes Mechanisms of dysregulation in cancer Chromosomal translocations, deletions, cytoplasmic mislocalization, proteasomal degradation, microrna silencing Chromosomal deletions, somatic inactivating mutations, alternative splicing variants, cytoplasmic mislocalization Chromosomal deletions, epigenetic silencing, microrna silencing, cytoplasmic mislocalization Somatic inactivating mutations, loss of or reduced expression Types of cancer with dysregulation Prostate cancer (deletion), alveolar rhabdomyosarcoma (translocations), leukemia (translocations), various types of epithelial carcinomas with deregulated PI3K-AKT, MEK-ERK, and IKK signaling (e.g. breast, lung, gastric cancers) Breast, prostate and ovarian cancers Breast, colorectal and prostate cancers Ovarian Granulosa Cell Tumors (OGCTs) Here, this chapter focuses on the recent advances in the emerging roles of tumorsuppressor FOX proteins in cancer, their cancer-specific dysregulation and therapeutic implications in cancer therapy. Therefore, this chapter encompasses FOXO, FOXP, FOXF and FOXL subfamilies due to their roles in tumor suppression (Table 1). Since oncogenic FOX factors are not in the scope of this chapter, the readers can refer to several recent review articles covering the functional roles and dysregulation of oncogenic FOX proteins (e.g., FOXA, FOXM, FOXC and FOXQ) in cancer if readers are interested in these topics [5, 9]. In addition, we discuss that tumor-

139 The Emerging Roles of Forkhead Box (FOX) Family Proteins 133 suppressor FOX proteins can exhibit bi-functional characteristics as oncogenes or tumor suppressor genes in a context-dependent manner. A. FOXO Subfamily Genes 1. The Biological Functions of FOXO Genes The forkhead box-o (FOXO) subfamily genes comprise FOXO1 (FKHR), FOXO3 (FKHRL1), FOXO4 (AFX) and FOXO6. The RXRSCTWPL motif in the N-terminal protein region and the RRRAXSMD motif in the forkhead box domain are conserved in all members of the FOXO subfamily, whereas the longer motif with a sequence RXRXXSNASXXSXRLSP in the middle protein region is only conserved among FOXO1, FOXO3, FOXO4 proteins [5]. In line with this, according to the homology analysis of FOXO subfamily protein members, FOXO1, FOXO3 and FOXO4 proteins are significantly homologous to each other, but they are less related to FOXO6 in mammals [10]. It is known that FOXO transcription factors can bind to the consensus DNA sequence, TTGTTTAC or BBTRTTTTD [11-13], or interact with other transcription factors (e.g. SMAD proteins) to regulate the transcription of target genes [14, 15]. Besides, transcriptional co-activators (e.g. histone acetyltransferase and CREB binding protein CBP/p300) [9, 16] or repressors (e.g. histone deacetylase SIRT1) have been found to interact with FOXO proteins [17-19], which ultimately determine the outcome of gene-transcriptional regulation by FOXO proteins. Currently there are numerous identified FOXO target genes functionally involved in regulating cell cycle progression, DNA repair, apoptosis and homeostasis of reactive oxygen species (ROS) [20] (Figure 1). For example, activation of FOXO transcription activity leads to up-regulating the expression of cyclin-dependent kinase inhibitor (CDKI) genes p21 Cip1, p27 Kip1, p15 INK4b, p19 INK4d and retinoblastoma-like p130rb2, resulting in cell cycle arrest at the G0/G1 phase [21-24]. For apoptotic regulation, FOXO transcription factors enhance the transcription of genes encoding the apoptotic inducers or mediators to elicit the onset of apoptosis, such as Fas ligand (FasL), TRAIL, the Bcl-2-like protein BIM and B-cell lymphoma 6 (BCL6), which mediates transcriptional down-regulation of the pro-survival factor BCL-X L [12, 25-31]. Moreover, gene products implicated in DNA damage responses (e.g. GADD45 with the functional abilities to arrest cell cycle progression and repair damaged DNA) and in protecting cells from ROS-induced DNA damages (e.g. Mn superoxide dismutase MnSOD and catalase) are transactivated by FOXO proteins [14, 32-34], which are cell survival mechanisms mediated by FOXO proteins. Therefore, FOXO proteins control cell survival and death in a context-dependent manner. In addition to the well-know roles of FOXO factors in regulating cell cycle progression, DNA repair and apoptosis, they also play crucial roles in the regulation of cellular autophagy/atrophy and glucose metabolism (Figure 1). It has been initially highlighted that FOXOs are important mediators of the muscle-related atrophy process. Forced expression of a constitutively active form of FOXO3 in fully differentiated skeletal and cardiac muscle cells results in atrophy [35-37]. Particularly, FOXO-triggered muscular atrophy results from a reduction in cell size, not from apoptosis [35].

140 134 Pang-Kuo Lo FOXO AKT Ubiquitination-mediated proteasomal degradation Oxidative Stress & DNA Repair Mn-SOD Catalase GADD45 ATM DDB1 PDK FOXO AKT PIP3 TSC1/2 mtorc2 Rictor Cell Cycle Arrest Cyclin G2 p15 p19 p21 p27 p130rb2 GADD45 PTEN AMPK FOXO Growth Factor Figure 1. The regulation of FOXO activity by the growth factor-triggered RTK-IRS-PI3K-AKTmTORC and stress-induced JNK/MST1 signaling pathways. Upon activation of receptor tyrosine kinases (RTK) by growth factors, activated PI3K increase the production of PIP3, which in turn triggers activation of PDK1-AKT. PTEN inhibits the PI3K-mediated generation of PIP3. SGK is concurrently activated with AKT by PI3K signaling in some circumstances to phosphorylate FOXO proteins (not shown in this figure). Activated AKT phosphorylates FOXOs to create the docking site for The binding of to FOXOs leads to nuclear export of FOXOs to the cytoplasm for ubiquitinationmediated proteasomal degradation. Activated AKT also inhibits TSCs to indirectly activate mtorc1, which in turn phosphorylates the downstream substrates including S6K and 4EBP1. The mtorc1- mediated phosphorylation stimulates S6K activity and activated S6K inhibits IRS1/2 to form the negative feedback loop regulation. Upon activation of JNK and MST1 by cell stress, FOXOs are activated by JNK- and MST1-mediated phosphorylation, which in turn transcriptionally activates the expression of FOXO target genes involved in cell cycle arrest, apoptosis, autophagy/atrophy, oxidative stress responses, DNA repair, metabolism, etc. FOXOs can transactivate the expression of Sestrin 3 to inhibit mtorc1 activity. Transcriptional induction of Rictor by FOXOs leads to AKT activation. The FOXO-induced reduction in muscular cell size is due to a decrease in overall cellular protein levels, which is mediated by the FOXO-induced increased expression of atrogin-1, a muscle-specific ubiquitin ligase that promotes protein degradation and muscle atrophy [35, PIP2 PI3K mtorc1 IRS1/2 Sestrin 3 FOXO target genes Apoptosis BIM FasL TRAIL BCL6 RTK S6K 4EBP1 Autophagy & Atrophy ATG12 Atrogin-1 LC3 BNIP3 BNIP3L GABARAPL1 Stress JNK FOXO MST1 Metabolism G6Pase PEPCK

141 The Emerging Roles of Forkhead Box (FOX) Family Proteins ]. Consistent with these findings, a transgenic mouse model with the genetically engineered overexpression of FOXO1 in muscle tissue manifests the atrophy symptom with the features of a reduction in size of type I and type II muscle fibers [38]. Therefore, given the role of FOXO factors in promoting cell atrophy, growth factor-induced PI3K-AKT/SGK signaling (discussed in the next section) can increase cell size by negatively regulating FOXO factors to enhance protein synthesis and decrease protein degradation. Furthermore, an increasing number of reports indicate that FOXO factors can transactivate the expression of several protein components involved in the lysosomal protein degradation pathway, such as LC3 (microtubule-associated protein 1A/1B-light chain 3), BNIP3 (BCL2/adenovirus E1B 19 kda-interacting protein 3), ATG12 (autophagy-related 12 homolog), and GABARAPL1 (GABA-receptor-associated protein-like 1), to regulate the autophagy response [39]. Autophagy has long been regarded as a critical process to promote cellular survival under nutrient deprivation by eliciting lysosome-mediated eradication of damaged or surplus organelles [10]. Hence, the role of FOXOs in autophagy regulation is imperative for cells to adapt to nutrient deprivation stress. In glucose metabolism, FOXO transcription factors are implicated in facilitating gluconeogenesis by up-regulating the expression of glucosemetobolism-regulatory genes, such as glucose 6 phosphatase (G6Pase) which catalyzes the conversion of glucose 6 phosphate to glucose and phosphoenolpyruvate carboxykinase (PEPCK) which catalyzes the conversion of oxaloacetate to phosphoenolpyruvate [40-43]. Thus, the effects of insulin on glucose metabolism are mediated in part by the activation of PI3K-AKT signaling to negatively regulate FOXO function (discussed in the next section). 2. Regulation of FOXO Transcription Factors In normal mammalian cells, two modes of cellular signaling predominantly regulate FOXO transcriptional activity; regulatory signaling pathways elicited by growth factors and stress stimuli (Figure 1). Growth-factor-mediated signaling (e.g. insulin) is one of main signal transduction pathways to regulate FOXO activity through the phosphoinositide-3-kinase (PI3K)-AKT (protein kinase B, PKB)/SGK (serum and glucocorticoid inducible kinase) signaling axis [20]. FOXO proteins are directly phosphorylated by AKT or SGK on their three conserved Ser/Thr residues, giving rise to enhancing binding of the adaptor protein to FOXO proteins [12, 20]. Binding of elicits nuclear export of FOXO proteins and consequently abrogates FOXO-mediated transcription [12, 20, 44] (Figure 1). Intriguingly, AKT and SGK have their preferential phosphorylation sites on FOXO proteins [45], indicating that external stimuli can regulate FOXO phosphorylation in a sophisticated manner to modulate subcellular localization, degradation and transcriptional activity of FOXO proteins through their differential effects on AKT and SGK activation. The major outcome of FOXOs phosphorylation by AKT and SGK is to facilitate the relocalization of FOXO proteins from the nucleus to the cytoplasm through the enhancement of their binding to chaperone proteins [12, 44, 46] (Figure 1). There are several mechanisms proposed for the inhibitory effect of the protein on FOXO transcription factors. It has been shown that binding promotes the releasing of FOXO proteins from a nuclear DNA anchor [47], indicating that binding of to FOXO proteins alters the FOXO protein conformation and in turn suppresses FOXO DNA binding ability. Regarding the effect of on promoting the nuclear exclusion of phosphorylated FOXO proteins,

142 136 Pang-Kuo Lo two lines of evidence suggest that binding either leads to a conformational change in FOXO proteins, which exposes their nuclear export sequence (NES) for allowing the interaction with Exportin/Crm [44] or masks the nuclear localization signal (NLS) of FOXO proteins to prevent their nuclear import [48, 49]. Another study also indicates that growth factors (e.g. IGF-1) can elicit PI3K-PDK1-AKT-induced, CK1 (casein kinase 1)-mediated phosphorylation at Ser322 and Ser325 of FOXO1, which accelerates FOXO1 relocalization to the cytoplasm by facilitating the interaction between FOXO1 and the Ran-containing export machinery [50]. Furthermore, through mutational studies of FOXO proteins, it has been unraveled that each AKT/SGK-targeted phosphorylation site contributes to the nuclear export of FOXO proteins [45]. Therefore, the regulation of subcellular FOXO protein localization by phosphorylation is known to be an effective mechanism for modulating the transcriptional activity of FOXO proteins in different cell types or in response to different signal stimuli (Figure 1 and Table 2). Besides regulating the nucleocytoplasmic shuttling of FOXO proteins, modulating the degradation of FOXO transcription factors is found to be another layer of the regulatory mechanism to alter their protein stability (Figure 1 and Table 2). Given that alterations in the mechanisms to regulate FOXO protein degradation are highly associated with tumorigenesis [51, 52], the elucidation of these regulatory mechanisms is crucial to the understanding of their roles in initiating cancer development. To reveal these mechanisms, many research groups worked on this issue and found that the ubiquitin-proteasome mechanism is responsible for the degradation of FOXO proteins [51-56]. The followed studies further demonstrate that both AKT-mediated phosphorylation and cytoplasmic localization are required to be successfully ubiquitinated by E3 ubiquitin ligases and subsequently degraded [52, 55, 56]. The ubiquitin ligase accounting for the ubiquitination of the FOXO1 protein has been identified as the F-box protein SKP2 (S-phase kinase-associated protein 2), a component of the Skp1/Cull/F-box (SCF) E3 ubiquitin ligase protein complex [52, 56] (Table 2). Intriguingly, SKP2 only specifically interacts with FOXO1, not with FOXO3 and FOXO4, raising the possibility that different FOXO family proteins are degraded by their specific E3 ubiquitin ligase machinery complexes. Besides the AKT-mediated degradation of FOXO proteins, I B kinase (IKK ) has been found to mediate an AKT-independent mechanism responsible for the proteasome-dependent degradation of the FOXO3 transcription factor [51] (Table 2). IKK catalyzes the phosphorylation at Ser644 of the FOXO3 protein, which in turn gives rise to the cytoplasmic retention and ubiquitination-mediated proteasomal degradation of FOXO3 [51]. The role of the IKK -dependent degradation mechanism in tumorigenesis was revealed by ectopic overexpression of either IKK or FOXO3 in cells. Constitutive expression of IKK enhances cell proliferation and tumorigenesis, whereas the forced expression of FOXO3 counteracts IKK s effect to suppress carcinogenesis [51]. Therefore, both AKTmediated and IKK -mediated degradation mechanisms are involved in the regulation of FOXO protein stability. However, the exact physiological role of IKK in the regulation of FOXO3 in normal cells is still unclear and needs further investigations. In addition to growth factor signaling, cellular stress (e.g. oxidative stress, heat shock, DNA damage) can elicit multiple signaling pathways to modulate FOXO transcriptional activity (Figure 1).

143 The Emerging Roles of Forkhead Box (FOX) Family Proteins 137 Table 2. The post-translational modifications of FOXO transcription factors and their biological effects The modification type Enzyme The biological effect on FOXO proteins Phosphorylation AKT/SGK Inhibition of FOXOs. FOXO proteins are subjected to nuclear export and SKP2-mediated polyubiquitination as well as proteasomal degradation. ERK1/2 Inactivation of FOXOs. FOXO proteins are subjected to nuclear export and MDM2-mediated polyubiquitination as well as proteasomal degradation. IKK Suppression of FOXO3. The FOXO3 protein is subjected to nuclear export and polyubiquitinationmediated proteasomal degradation. CK1 Inhibition of FOXO1. The nuclear export of FOXO1 is increased. DYRK1A Inhibition of FOXO1. The nuclear export of FOXO1 is increased. CDK2 Inhibition of FOXO1. The nuclear export of FOXO1 is increased. AMPK Nuclear import of FOXOs and their transcription activities are enhanced. JNK Upon stress, activated JNK phosphorylates FOXO4 and promotes its nuclear import as well as transcriptional activity. MST1 Upon stress, activated MST1 phosphorylates FOXOs and promote their nuclear import as well as transcription activities. Acetylation CBP/p300 CBP-binding promotes FOXO activity, but CBPmediated acetylation of FOXOs inhibits FOXO activity. PCAF Similar to CBP, PCAF-catalyzed acetylation of FOXOs inhibits FOXO activity. Deacetylation SIRT1/2/3 SIRT-mediated deacetylation of FOXOs activates or inhibits FOXO activity in a context-dependent manner. Ubiquitination SKP2 SKP2-mediated polyubiquitination of FOXO1 promotes proteasomal degradation of the FOXO1 protein. MDM2 MDM2-mediated polyubiquitination of FOXOs promotes proteasomal degradation of FOXO proteins, but MDM2 also catalyzes monoubiquitination of FOXO4 and enhances its activity. Deubiquitination HAUSP HAUSP-mediated deubiquitination of monoubiquitinated FOXO4 inhibits FOXO4 transcriptional activity. The c-jun N-terminal kinase (JNK), a MAPK (mitogen-activated protein kinases) family kinase that can be activated by stress stimuli, is implicated in the regulation of FOXO proteins

144 138 Pang-Kuo Lo in several organisms [57-59]. It has been reported that JNK can phosphorylate FOXO1, FOXO3 and FOXO4 in vitro. However, only FOXO4 has been reported to be phosphorylated by JNK at Thr447 and Thr451 [57], and the JNK-targeted phosphorylation sites on FOXO1 and FOXO3 protein remain elusive. In comparison to AKT-mediated phosphorylation, JNK-mediated phosphorylation has an opposite effect to trigger the relocalization of FOXO4 from the cytoplasm to the nucleus, which activates the FOXO4 transcriptional activity and induces FOXO target genes such as MnSOD and catalase for detoxification of ROS in response to oxidative stress [57] (Figure 1 and Table 2). This regulatory mechanism is also conserved in worms and flies and implicated in regulating longevity [58, 59]. Although the mechanism whereby stress stimuli and JNK cause the relocalization of FOXO proteins to the nucleus is still under investigation, a line of evidence indicates that JNK can phosphorylate the protein and in turn release binding proteins such as FOXO factors [60]. Besides JNK, the mammalian orthologue of the ste20-like protein kinase (MST1), which is also activated by oxidative stress, has been reported to phosphorylate and activate FOXOs [61] (Figure 1 and Table 2). It has been reported that the activation of FOXOs by JNK/MST1-mediated phosphorylation overrides the inhibitory phosphorylation by AKT [17, 62]. Intriguingly, other MAPK members, extracellular signal-regulated kinases 1 and 2 (ERK1/2), are also implicated in the regulation of FOXO proteins but play an opposite role compared with JNK s role (Table 2). Activation of ERK by Ras-Raf-MEK signaling promotes the interaction between ERK and FOXO3 and ERK-mediated phosphorylation of FOXO3, leading to cytoplasmic sequestration and proteasomal degradation of FOXO3 (63). ERK-induced proteasomal degradation of FOXO3 is mediated by RING-finger E3 ligase murine double minute 2 (MDM2) [63] (Table 2). Therefore, the ERK-MDM2 mechanism is analogous to the AKT-SKP2 mechanism for the negative regulation of FOXO factors. Regarding the MDM2 role in the regulation of FOXO proteins, it has been known that MDM2 acts as a general E3 ligase for promoting ubiquitination of various FOXO factors including FOXO1, FOXO3 and FOXO4 [63-65]. In addition to promoting polyubiquitination of FOXO proteins, another study has shown that MDM2 also catalyzes multiple monoubiquitination of FOXO4 rather than polyubiquitination [64]. Intriguingly, monoubiquitination of FOXO4 promotes nuclear localization of FOXO4, which is observed in cultured cells in response to oxidative stress [66] (Table 2). In the nucleus, the deubiquitinating enzyme herpesvirus-associated ubiquitin-specific protease (HAUSP/USP7) has been found to be involved in the deubiquitination of monoubiquitinated FOXO4 [66], which acts as a mechanism to balance the effect of MDM2 on FOXO4 (Table 2). Moreover, it has been posited that monoubiquitinated FOXO proteins can be further converted into polyubiquitinated forms by branching E3 ligases such as SKP2 [64]. As mentioned above, several kinases have been found to phosphorylate FOXOs for repressing (e.g. AKT, IKK, ERK) or activating (e.g. JNK) the transcriptional activity of FOXOs (Table 2). In addition to these kinases, more kinase pathways have been identified to phosphorylate FOXOs and regulate their transcriptional activity; the AMP-activated protein kinase (AMPK) [67, 68] is involved in the activation of FOXOs activity and the dual-specificity tyrosinephosphorylated-regulated kinase 1A (DYRK1A) is to inhibit FOXOs (69) (Table 2). Therefore, the combined outcome from the relative ratio between active FOXO-targeting

145 The Emerging Roles of Forkhead Box (FOX) Family Proteins 139 kinase levels (e.g. AKT, IKK, ERK, JNK, etc.), MDM2 levels, HAUSP levels and FOXOtargeting E3 ligase levels (e.g. SKP2) determines the subcellular localization and protein stability of FOXO transcription factors. Besides phosphorylation of FOXO proteins as the post-translational modification mechanisms to regulate the subcellular localization and degradation of FOXO proteins, the acetylation/deacetylation modifications also play critical roles in regulating FOXO transcriptional activity (Table 2). It has been known that FOXO factors bind to coactivator or corepressor complexes and the acetylation status of FOXO proteins are modulated by these factors [19, 70, 71]. Transcriptional coactivators (e.g. PGC-1, peroxisome proliferativeactivated receptor- coactivator 1) have been reported to bind to FOXO proteins and potentiate their transcriptional activity [43]. However, some of these coactivators (e.g. CBP/p300 and PCAF) are also acetyltransferases and thus catalyze the acetylation of FOXO proteins at several conserved lysine residues, which actually have the opposite effect to inhibit their transcriptional activity [71]. Therefore, coactivator binding and coactivatormediated acetylation of FOXO proteins dynamically regulate the transcriptional activity of FOXO factors. It is well-known that protein acetylation can be reversibly regulated by deacetylasecatalyzed deacetylation. This acetylation/deacetylation bidirectional regulatory mechanism is involved in modulating the transcriptional activity of FOXO proteins. Multiple lines of evidence have shown that the Sir2 family deacetylases are responsible for the deacetylation of FOXO proteins [17-19, 72-74] (Table 2). The Sir2 family is classified as class III deacetylases that use NAD + as a cofactor for the enzymatic activity [75]. The studies have shown that Sir2 deacetylases extend the longevity of organisms such as yeast, worms and flies (76-79). For the connection between FOXO and Sir2, several research groups have demonstrated that SIRT1 is able to directly catalyze the deacetylation of FOXO proteins in vitro and also to participate in the in vivo deacetylation of these factors within cells [17-19, 72-74]. According to multiple studies, the consensus for the effect of SIRT1-mediated deacetylation on FOXO function is to preferentially activate FOXO s transcriptional ability for transactivating a subset of FOXO target genes, in particular stress resistance, cell cycle arrest and DNA repair genes, but simultaneously inhibit FOXO factors to induce proapoptotic gene expression [17-19, 80]. Therefore, through binding to FOXO factors and catalyzing their deacetylation, SIRT1 can regulate FOXO protein function towards stress resistance but away from cell death, which is in line with the role of Sir2 family proteins in extending longevity. However, the underlying mechanisms for SIRT1-mediated regulation of FOXO factors remain unclear. Therefore, the investigation of whether the binding of SIRT1 to FOXO factors and SIRT1- catalyzed deacetylation of these proteins have differential regulatory roles in FOXO activity will be critical to decipher the mechanisms. Although this chapter covers most of the key aspects of the roles for post-translational modifications of FOXO family proteins in regulating FOXO function, readers can find the more information in the recent comprehensive review specific to this topic by Zhao et al. [81].

146 140 Pang-Kuo Lo 3. The Roles of FOXOs in Coordinating the Activities of PI3K-AKT and Targets of Rapamycin Complexes (TORCs) As indicated above, growth factors (e.g. insulin, IGF-1) trigger the activation of PI3K- AKT signaling, which in turn inactivates the transcriptional functions of FOXOs. However, multiple reports indicate that FOXOs are actually implicated in coordinating the negative feedback circuit mediated by the mtorc1 (mammalian target of rapamycin complex 1)-S6 kinase (S6K) axis, which is the downstream target signaling axis of PI3K-AKT and activated by active AKT (10, 62, 82) (Figure 1). There are two types of mammalian targets of rapamycin complexes (mtorcs); mtor complex 1 (mtorc1) is a conserved downstream mediator of AKT and mtor complex 2 (mtorc2) is a conserved upstream activator of AKT. Each mtor complex contains its defining subunit; the regulatory associated protein of mtor (Raptor) in mtorc1 and the rapamycin-insensitive companion of mtor (Rictor) in mtorc2 [83]. When growth factors are deficient, the mtorc1 activity is indirectly inhibited by tuberous sclerosis complexes (TSCs). TSCs formed by the heterodimer of the tuberous sclerosis complex 1 and 2 (TSC1/2) possess GAP activity and suppress Rheb activity, a small GTPase indispensable for the activation of mtorc1 [84, 85]. The TSC2 activity is positively controlled by intracellular ATP levels and AMPK activity [86]. In addition, it has been reported that AMPK phosphorylates Raptor to inhibit mtorc1 [87]. In contrast, AKT can maintain intracellular ATP levels and attenuate AMPK activity by activating mtorc1 [88]. The major physiological functions of mtorc1 are to increase mrna translation and fatty acid biosynthesis. To enhance mrna translation, mtorc1 activates the downstream mediator S6K and inhibits the eukaryotic translation initiation factor 4E binding protein (4E-BP), a repressor of mrna translation [85] (Figure 1). Besides the effect on protein synthesis, mtorc1 possesses anabolic activity to promote fatty acid biosynthesis through the activation of the sterol-regulatory-element-binding protein (SREBP1), a transcription factor engaged in transcriptionally activating the expression of enzymes responsible for fatty acid synthesis (89). Furthermore, mtorc1 has an intrinsic activity to inhibit the autophagy process via phosphorylating proteins essential for the initiation of autophagy [90]. By using TSC1/2-null cell model systems, it has been demonstrated that there is a negative feedback mechanism involving the inhibitory effect of the mtorc1-s6k signaling axis on insulin receptor substrates (IRS1/2), which mediate PI3K-AKT activation by insulin and IGF-1 [91] (Figure 1). The recent reports demonstrate that FOXOs play a central role in coordinating this negative feedback mechanism through their downstream target genes, Sestrin 3 and Rictor [82, 92-94] (Figure 1). Sestrin 3 belongs to the Sestrin family members that were originally identified in mammals as antioxidants (95, 96). Sestrin 3 plays a dual functional role downstream of FOXO signaling; it acts as a scavenger of ROS to mediate FOXO-induced ROS detoxification, and as an activator of AMPK to inhibit the mtorc1- S6K signaling axis. Thus, FOXO-induced Sestrin 3 expression relieves the mtorc1- mediated negative feedback effect on IRS1/2 and in turn activates the PI3K-AKT signaling axis [82, 92] (Figure 1). Intriguingly, the FOXO-Sestrin 3-AMPK-mTORC1-S6K signaling axis is highly analogous to the reported p53-sestrin 1/2-AMPK-mTORC1-S6K signaling axis [97] and both regulatory mechanisms are implicated in negatively regulating mtorc1- mediated cell proliferation and anabolic activity. Therefore, AKT activity is activated by the FOXO-elicited inhibition of the mtorc1-s6k-mediated negative feedback loop circuit.

147 The Emerging Roles of Forkhead Box (FOX) Family Proteins 141 However, besides this mechanism, FOXOs also can enact another mechanism that transcriptionally induces the expression of Rictor, which increases mtorc2 activity and in turn activates AKT activity [82] (Figure 1). Additionally, it has been shown that FOXOs can elevate insulin-like growth factor receptor (InsR) and IRS2 mrna levels to amplify growth factor signaling [62, 98]. FOXO was also reported to induce the expression of HER2/HER3 tyrosine kinase receptors in several cancer cell lines (91). Therefore, the suppression of the mtorc1-s6k signaling axis by FOXOs could further augment AKT activity by the FOXOmediated elevation of InsR and IRS2 expression. The mechanisms mentioned above are not the sole ones to regulate AKT and mtorc1. It has been shown that FOXO can inhibit the expression of the pseudokinase tribbles 3 (TRB3), an AKT inhibitor, to activate AKT [99, 100]. Moreover, FOXO has been reported to elevate the expression of BNIP3 with activity to inhibit mtorc1 function [101, 102]. A recent study also reported that FOXO3 transcriptionally elevates the expression of TSC1 to suppress mtorc1 activity [103]. Therefore, FOXOs play a master role in coordinating both InsR-IRS-PI3K-AKT-mTORC1-S6K and InsR-IRS-PI3K-mTORC2-AKT arms to regulate AKT and mtorc1 activities for modulating the intracellular balance of energy metabolism, ROS homeostasis, cell proliferation, cell survival and autophagy. When cells are under physiological stress conditions, FOXOs are activated to keep AKT activation and concurrently to inhibit mtorc1 activity. The inhibition of mtorc1 leads to a reduction in its anabolic activity to consume cellular energy and the activation of AKT causes an increase in cellular energy metabolism for maintaining cellular energy homeostasis. 4. The Roles of FOXO Proteins in Cellular Responses to DNA Damage, Oxidative Stress and Hypoxia In addition to the critical role of p53 tumor suppressor protein in mediating the downstream effects of ATM/ATR-dependent DNA damage responses, FOXO protein members have been also found to be involved in ATM-triggered DNA damage responses [104, 105]. FOXO3 has been reported to induce ATM gene transcription [105] and interact with ATM to promote its kinase activity and downstream DNA-damage responsive signaling, such as intra-s-phase, G2-M cell-cycle checkpoints and the repair of damaged DNA [104]. Furthermore, FOXO proteins are required for the expression of DNA repair genes such as GADD45 and DDB1, which are essential for maintaining the integrity of DNA repair machinery [20, 34]. Besides cell cycle arrest and DNA repair, induction of cellular apoptosis by FOXO factors is also essential to maintain genomic stability especially when cellular DNA is severely damaged. Therefore, this functional aspect of FOXO proteins in response to DNA damage is well-accepted as one of their imperative tumor-suppressive properties. A study has shown the important role of cyclin-dependent kinase 2 (CDK2) in the regulation of FOXO1 in response to DNA damage. It has been known that the function of CDK2 is frequently abrogated as cells respond to DNA damage [106]. In the normal circumstance, CDK2-mediated phosphorylation of FOXO1 at Ser249 leads to cytoplasmic localization and inhibition of FOXO1 [106]. This CDK2-mediated effect can be abolished by the Chk1/2-dependent DNA damage response, which in turn activates FOXO1 to induce cellular apoptosis for the elimination of severely damaged cells [106]. Therefore, this

148 142 Pang-Kuo Lo molecular interaction between CDK2 and FOXO1 provides a mechanism to regulate apoptotic cell death during DNA damage responses. Besides their roles in DNA repair and DNA damage responses, FOXO protein members are known to play important roles in regulating cellular antioxidant capacity through upregulating the expression of free radical scavenging enzymes, including Mn superoxide dismutase (MnSOD) and catalase, which function primarily to scavenge mitochondrial respiration-derived reactive oxygen species (ROS) [14, 20, 32]. Thus, FOXO transcription factors are responsible for two aspects of cellular resistance to stress: detoxification of ROS and repair of DNA damages elicited by ROS. These FOXO-mediated protective mechanisms for cell survival are responsible for the essential roles of FOXO proteins in cell longevity [20]. Under the condition of low oxygen levels (e.g. insufficient blood supply due to rapid growth of embryonic or tumorigenic tissue), the hypoxia-inducible factor (HIF) complex are stabilized and become active to transactivate hypoxia-responsive genes, which are required for tissue cells to adapt to hypoxia and to modulate angiogenesis. The well-known mechanisms to regulate the stabilization and transcriptional activity of HIF1 proteins are posttranslational protein hydroxylation and ubiquitination-dependent protein degradation. Under normal oxygenation conditions (normoxia), the HIF1 protein is consistently hydroxylated by oxygen-dependent hydroxylases and subsequently degraded by the action of the von Hippel- Lindau tumor suppressor ubiquitin ligase (VHL) [107, 108]. The hydroxylation-dependent modification of HIF1 also prevents the association of HIF1 with the transcriptional coactivator p300, which in turn inhibits HIF1 transcriptional activity [109]. However, a VHLindependent mechanism to negatively regulate HIF1 activity has been identified, which is mediated by the action of FOXO proteins. The first evidence is from the finding that FOXO4 negatively regulates the levels of HIF1 expression in a VHL-independent manner, thereby compromising the responsiveness to hypoxia [110]. Another FOXO-dependent negatively regulatory mechanism is achieved by competing with HIF1 for binding to p300, a transcriptional co-activator required for HIF1 transcriptional activity. This competition is mediated by binding of FOXO proteins [10, 111] or the FOXO-transcriptional target CITED2 (CBP/p300-interacting transactivator 2) to p300 proteins [109, 111], leading to the suppression of HIF1 transcriptional activity. In addition to these mechanisms, the effect of FOXO proteins on reducing the levels of ROS also negatively regulates HIF1 transcriptional activity because ROS has been shown to be required for activating HIF1 [112]. Therefore, the relative ratio between FOXOs and HIF1 determines the cellular response to hypoxia. 5. Are FOXO Transcription Factors Bona Fide Tumor Suppressors? Given that FOXO proteins possess tumor-suppressor characteristics that include the blockade of cell cycle progression, the negative regulation of ROS production and the repair of damaged DNA, their roles as tumor suppressor genes had been investigated. From in vitro studies, it has been demonstrated that ectopic expression of FOXO proteins with mutations at residues required for inactivation by AKT/PKB-mediated phosphorylation can inhibit colony formation of cells with aberrantly activated PI3K/AKT signaling (e.g. RAS-transformed cells or cells deficient for the phosphatase and tensin homolog PTEN) [22]. Furthermore, another line of evidence has shown that a dominant-negative form of FOXO (dnfoxo) can replace

149 The Emerging Roles of Forkhead Box (FOX) Family Proteins 143 RAS to collaborate with MYC for oncogenic transformation of cells [113], indicating that PI3K/AKT-mediated phosphorylation to inactivate FOXO transcriptional activity is crucial to cell transformation. Besides, several studies have shown that ectopic expression of FOXO in human cancer cell lines attenuates their in vivo xenograft tumor formation in nude mice [51, 114]. Therefore, these lines of evidence, taken together, indicate that FOXO proteins potentially function as tumor suppressors. Recent in vivo animal studies have provided the solid evidence to support FOXO proteins as genuine tumor suppressors. For example, Paik et al. performed broad somatic deletion of all three FOXO genes in mice and found that inactivation of FOXOs resulted in the narrow tumor spectrum, characterized as thymic lymphomas and hemangiomas [11]. In contrast, the genetic deletion of only one or two FOXO genes leads to no or a moderate tumor-prone phenotype [11, 115, 116], indicating that FOXO genes are a functionally redundant tumor suppressor family. The evidence from their studies demonstrates that the mammalian FOXO transcription factors are indeed bona fide tumor suppressors. However, it is unclear that abrogation of FOXOs only induces tumorigenesis of hematopoietic lineages, which is contradictory to the ubiquitous expression patterns of FOXO proteins in tissue. The possible explanations for these findings are that hematopoietic cells more rely on FOXOs to maintain genomic integrity and their high turnover rates might promote the tumorigenic progression under the inactivation of FOXOs. Another line of evidence has shown that the sable introduction of a dominant-negative FOXO (dnfoxo) gene moiety, which functionally abrogates all of FOXOs, into Emu-myc transgenic hematopoietic stem cells enhances lymphomagenesis in recipient mice [117]. This enhanced tumorigenesis is attributable to the effect of dnfoxo to attenuate Myc-induced apoptosis [117]. Furthermore, forced expression of dnfoxo in Emu-myc:p53 (+/-) progenitor cells overcomes the pressure to inactivate the remaining p53 allele during lymphomagenesis by inhibiting the p19arf signaling axis [117]. Given that p19arf is known to mediate oncogenic stress-induced activation of p53 and in turn to engender cell cycle arrest and/or apoptosis [118], this finding points the mechanistic interaction between FOXO and p53. However, results from the studies using dnfoxoexpressing murine lymphoma models should be interpreted with caution because ectopic expression of dnfoxo not only abrogates FOXO functions but also disturbs the normal physiological functions of FOXO-interacting partners by protein-protein interaction, which might be also involved in enhanced lymphomagenesis in the Emu-myc model. Nevertheless, based on gathering evidence from the studies of genetic mouse models, FOXO proteins indeed are bona fide tumor suppressors with in vivo activities to prevent both spontaneous tumor formation and Myc-induced development of lymphoma. In addition, two molecular mechanisms underlying FOXO-mediated effects to interfere with oncogenic functions of Myc have been deciphered. According to the studies by Chandramohan et al., ectopic Myc expression has a suppressive effect on the expression of cyclin-dependent kinase inhibitor p27 Kip1, whereas FOXO proteins counteract this inhibitory effect from Myc and promote the transcriptional activation of p27 Kip1 [119]. Another line of evidence indicates that the PI3K- AKT-FOXO signal cascade can modulate Myc transcriptional function and in turn affect Myc-regulated gene expression. Delpuech et al. found that FOXO3 can suppress the expression of Myc target genes via transcriptional induction of MxiI, a member of Mad/Mxd family transcriptional repressors [120]. MxiI is known to compete with Myc to bind to Max for the formation of the MxiI-Max heterodimerized transcriptional repressor complex that has a transcriptionally repressive effect on the expression of Myc target genes [120]. Although

150 144 Pang-Kuo Lo these two lines of evidence shed new light on how FOXO proteins counteract oncogenic functions of Myc, it is not clear to what aspects of FOXO functions contribute to FOXOmediated in vivo tumor suppression. Hence further advanced studies are still mandatory to address these issues. 6. Genetic Alterations in FOXO Subfamily Genes It was initially identified that FOXO subfamily genes participate in chromosomal translocations found in alveolar rhabdomyosarcomas (PAX3-FOXO1, PAX7-FOXO1) and some forms of leukemia such as secondary leukemia and acute lymphoblastic leukemia (MLL, myeloid/lymphoid or mixed lineage leukemia, MLL-FOXO3, MLL-FOXO4) [ ]. All of these translocations occur at a breakpoint in the intron 2 of FOXO subfamily genes. These discovered chromosomal translocations predominantly lead to chimeric proteins in which the C-terminal domains of FOXOs are fused to the N-terminal domains of other transcription factors (e.g. PAX3, PAX7, MLL). Although the absence of a functional FOXO allele may contribute to some extent of the tumorigenic effect derived from these translocations due to the tumor-suppressive roles of FOXOs, it has been recognized that the chimeric fusion proteins PAX3/PAX7-FOXO1 or MLL-FOXO3/FOXO4 mostly contribute to tumorigenesis [128, 129]. However, a study has shown that the MLL-FOXO4 fusion has an effect to transdominantly interfere with the expression of the remaining intact FOXO4 allele, suggesting that this transdominant effect can potentiate the oncogenic effect derived from the MLL-FOXO4 chimeric protein [130]. Notably, the subsequent knock-in animal studies examining the oncogenic potential of the chimeric allele did not recapitulate tumor phenotypes, raising the possibility that the disruption of the functional FOXO allele independently causes or collaborates with the chimeric allele to elicit tumorigenesis [93, 131]. In addition to chromosomal translocations, the genetic deletion of FOXO genes has been discovered. For example, the frequently deleted chromosomal region in prostate cancer encompasses the FOXO1 gene, which is highly associated with the observations that FOXO1 mrna expression is frequently downregulated in prostate cancer [132]. To date the identified genetic aberrations in FOXO genes are not common in cancers and restricted to some types of cancer mentioned above. The growing evidence has shown that the inactivation of FOXOs tumor suppressive function in cancer frequently occurs at the protein level, which is discussed in the next section. 7. Dysregulation of FOXO Gene Expression and Function in Cancer Since FOXO factors possess the ability to induce cell cycle arrest, DNA repair and apoptosis, these tumor-suppressive features make FOXOs potential candidates as tumor suppressors. A series of xenograft and genetic knockout studies also strongly indicates that FOXOs are genuine tumor suppressors. Therefore, it is a well-accepted consensus that dysregulation of FOXOs expression and function is the crucial mechanism leading to the development of a variety of cancers. In addition to genetic alterations in FOXO genes such as chromosomal translocations and deletions as mentioned above, currently there are other four different levels of dysregulation that can lead to the inactivation of FOXO transcription

151 The Emerging Roles of Forkhead Box (FOX) Family Proteins 145 factors, including the dysregulation at the levels of mrna synthesis, subcellular localization, protein degradation and FOXO protein partners. As mentioned in the prior section, the FOXO1 gene loci are within the commonly deleted genomic region in prostate cancer, which is thought as a potential mechanism to cause downregulation of FOXO1 expression [132]. Chromosomal translocations found in alveolar rhabdomyosarcoma and leukemia are also a mechanism leading to loss of half expression of FOXO mrnas [ ]. Moreover, it has been reported that the EWS-Fli1 oncogenic fusion protein functions as a transcriptional repressor to suppress the expression of FOXO1 in Ewing s sarcoma cells [133], which inactivates the tumor-suppressive function of FOXO1 at the mrna synthesis level. Intriguingly, the recent evidence has revealed that mir-499-5p targets the FOXO4 mrna and negatively regulates its levels in colorectal cancer cells [134], which is a novel tumorigenic mechanism to inactivate FOXO function via micrornamediated silencing. Multiple aberrant oncogenic mechanisms occurring in cancer have been found to cause the activation of PI3K-AKT signaling, which in turn leads to the inhibition of FOXO function by promoting cytoplasmic retention and protein degradation of FOXOs. The most known mechanism is the frequent mutation or deletion of the phosphatase and tensin homolog (PTEN) gene in a large spectrum of human cancer types such as brain, breast, prostate and kidney cancers [135, 136]. PTEN is a lipid phosphatase which antagonizes the effect of PI3K [137, 138] (Figure 1). Therefore, loss of PTEN gives rise to an increase in the levels of phosphatidylinositol [3,4,5] trisphosphate (PIP3) in the plasma membrane, which in turn results in activation of AKT [135, 136]. Activation of AKT by loss of PTEN leads to the generation of phosphorylated FOXO proteins whose transcriptional functions are inactivated by sequestrating phospho-foxos in the cytoplasm via binding and by promoting the depletion of phosphorylated FOXOs via SKP2-mediated ubiquitination and proteasomal degradation (Figure 1). Besides loss of PTEN, the frequent mutation of the PIK3CA gene, coding for the catalytic subunit p110 of class IA phosphatidylinositol 3-kinases (PI3Ks), in human cancer is another pathological mechanism resulting in activation of AKT [139]. The prevalent mutants of p110 (e.g. E542K, E545K, and H1047R) show a gain of enzymatic function in vitro and are oncogenic in vitro and in vivo by activating AKT kinase activity [139]. The in vivo tumorigenicity of PIK3CA mutants in an avian species strongly suggests a crucial role for these mutated proteins in human malignancies [139]. Thus, activation of AKT by the constitutively activated PIK3CA mutants in cancer cells leads to the inhibition of FOXOs tumor-suppressor function, which contributes to tumorigenesis induced by mutated PIK3CA. Cytoplasmic retention of the FOXO3 transcription factor has been found in breast cancer tissue sections and correlates with poor survival of breast cancer patients. Intriguingly, cytoplasmic FOXO3 detected in a subset of breast cancer cases is not correlated with the active form of phospho-akt and the further studies demonstrate that IKK is responsible for the AKT-independent inactivation of FOXO3 [51]. IKK physically interacts and phosphorylates FOXO3 at Ser644, and then subjects phosphorylated FOXO3 to the cytoplasm for ubiquitination-dependent degradation by the proteasome complex [51]. In addition to IKK signaling, Ras-Raf-MEK-ERK signaling that is frequently activated in many types of cancer has been identified as another AKT-independent mechanism to negatively regulate FOXO3 [63]. Activated ERK catalyzes the phosphorylation of FOXO3 at

152 146 Pang-Kuo Lo multiple residues, which in turn is subjected to MDM2-mediated ubiquitination and proteasomal degradation [63]. Therefore, ERK-MDM2-mediated proteasomal degradation of FOXO3 is also one of vital mechanisms for promoting cell proliferation and tumorigenesis. Besides FOXO3, MDM2 also targets FOXO1 and FOXO4 and promotes their ubiquitination and proteasome-mediated degradation [63-65]. In light of that gene amplification and overexpression of MDM2 have been found in breast cancer, glioblastoma, osteosarcoma and liposarcoma [140], the tumor-suppressor function of FOXO factors is inactivated in these cancer types via the high expressed levels of MDM2 to enhance FOXO degradation. To maintain cell survival and proliferation, it has been reported that cancer cells specifically rely on some FOXO-interacting proteins to conquer the tumor-suppressive effects of FOXO factors. For example, sirna-mediated knockdown of SIRT1 in several types of epithelial cancer cells is able to trigger FOXO-dependent apoptosis and/or cell cycle arrest, whereas silencing of SIRT1 has no significant effect on cell survival and growth of normal human epithelial cells and normal human diploid fibroblasts [80]. These findings indicate that SIRT1 imposes restraint on FOXOs to maintain the survival and proliferation of epithelial cancer cells. Moreover, another line of evidence has revealed that four and a half LIM 2 (FHL2) interacts with FOXO1 to promote the binding of FOXO1 to SIRT1, resulting in SIRT1-mediated FOXO1 s deacetylation and suppression of FOXO1-dependent apoptosis and target gene expression in prostate cancer cells [74]. Particularly, the latent protein LANA2 from the Kaposi-sarcoma-associated herpesvirus has the direct interaction with FOXO3 and proteins to enhance nuclear export of FOXO3 [141], which is the first evidence that viral proteins can inhibit FOXO transcriptional activity as a mechanism to trigger cell transformation and tumorigenesis. Therefore, the tumor-suppressor functions of FOXO transcription factors are abrogated in cancer cells through the multiple mechanisms that include chromosomal translocations or deletions of FOXO genes, mirna-mediated silencing of FOXO mrnas, kinase-mediated cytoplasmic sequestration of FOXO proteins, ubiquitination-mediated proteasomal degradation of FOXO proteins and protein-proteininteraction-mediated inhibition of FOXO transcriptional function. B. FOXP Subfamily Genes 1. The Biological Functions of FOXP Genes The human FOXP subfamily is composed of four genes, FOXP1-4. All of FOXP genes encode proteins that contain the highly conserved C-terminal tetramerization domain comprising zinc-finger and leucine-zipper domains and a DNA-binding forkhead box domain [142]. FOXP proteins function as sequence-specific transcription factors which are found to participate in the development of speech and language regions of the brain during embryogenesis, the development as well as function of regulatory T cells, tumor suppression and oncogenesis. Among these four FOXP genes, FOXP1 and FOXP3 have been convincingly connected to tumorigenesis. The FOXP1 transcription factor has been known to potentially play a dual role as either an oncoprotein in a number of types of lymphomas and hepatocellular carcinoma or a tumor suppressor in breast cancer [reviewed in [5, 143]]. However, molecular mechanisms underlying the roles of FOXP1 in tumorigenesis remain

153 The Emerging Roles of Forkhead Box (FOX) Family Proteins 147 largely unknown. Therefore, this chapter section does not cover the details of the recent advances in FOXP1 studies. The readers who are interested in FOXP1 can refer to these recent review articles [5, 143]. In comparison to FOXP1, the FOXP3 gene at Xp11.23 has been well-studied and defined as an X-linked tumor suppressor gene in epithelial carcinomas (e.g. breast cancer, prostate cancer, ovarian cancer, etc.) [reviewed in [ ]]. Besides its critical role in tumor suppression, actually the Foxp3 gene was originally identified from the scurfy mice bearing a lethal X-linked recessive immunodysregulation [ ]. Since its discovery, FOXP3 has been well-studied in immune cells and known as a master regulator in the development and function of CD4 + CD25 + regulatory T cells (Treg) which are involved in immunosuppression [reviewed in [144, 147, 152]]. Although immunological studies have shown that the role of FOXP3 in Treg is involved in the immunosuppression-related tumorigenesis, this chapter section only focuses on the functional roles of FOXP3 in epithelial cells and what pathological mechanisms lead to inactivation of FOXP3 s tumorsuppressive function in epithelial carcinomas. The readers interested in the immunological aspect of FOXP3 can refer to the recent review articles [144, 147, 152]. The expression of mouse Foxp3 in epithelial cells was first documented in Rag2 -/- mice which are a genetic mouse model with the Scurfy mutation and a notable lack of T lymphocytes. The study has shown that the expression of the Foxp3 mrna and its encoded nuclear protein has been found in epithelial cells of breast, lung and prostate, but not liver, kidney and intestine [153]. Regarding the role of FOXP3 in epithelial cancer, there are multiple important lines of recent evidence demonstrating that FOXP3 is a bona fide X-linked tumor suppressor gene in breast, prostate and ovarian cancers [ ]. By studying a female mouse model with a heterozygous Scurfin mutation at the Foxp3 gene [Foxp3(sf/+)], Zuo et al. found that the mutant mice developed mammary carcinomas at a high rate [154]. The further studies have also shown that FOXP3 functions as a transcriptional repressor of HER2 and SKP2 oncogenes and a transcriptional activator of p21 gene in breast epithelial cells (154, 155, 158) (Figure 2). HER2 is a well-known oncoprotein that triggers oncogenic signaling pathways such as PI3K-AKT-mTORC1 and MEK-ERK [159]. SKP2 is aberrantly overexpressed in a wide spectrum of cancers [160] and involved in ubiquitination and degradation of the cyclindependent kinase inhibitor (CDKI) p27 and tumor-suppressor FOXOs. Since overexpression of HER2 and SKP2 is commonly present in breast cancer and accounts for or contributes to breast tumorigenesis [154, 155], FOXP3-mediated suppression of the expression of these two genes is critical for its tumor-suppressive effect. Consistently, there is a reverse expression correlation between FOXP3 and these two oncogenes in breast cancer tumor specimens [154, 155]. Moreover, FOXP3 can transcriptionally activate the CDKI gene p21 promoter and induce its mrna expression [158]. The functionality of p21 has been demonstrated to be crucial to FOXP3-mediated tumor suppression because shrna-mediated silencing of the p21 gene rescues cell growth inhibition induced by FOXP3 transduction [158]. Immunohistochemistry (IHC) analysis of human breast cancer tissue microarrays has also deciphered the positive correlation between the protein expression of FOXP3 and p21 [158]. The findings that genomic deletions, functionally significant somatic mutations, and down-regulation of the FOXP3 gene are commonly found in human breast cancer samples further strengthen its role as a genuine tumor suppressor gene in female breast cancer [154].

154 148 Pang-Kuo Lo Epithelial Cancers Genomic Deletions Somatic Mutations Alternative Splicing FOXO Apoptosis FOXP3 SKP2 p21 HER2 c-myc p27 Cell Cycle Arrest Cytoplasmic Mislocalization Figure 2. A schematic view of the cancer-specific deregulation of FOXP3 function that has a physiological tumor-suppressor role in epithelial cells. In epithelial cancers (e.g. breast, prostate, ovarian cancer), FOXP3 is inactivated by multiple pathological mechanisms including genomic deletions, somatic inactivating mutations, alternative splicing, cytoplasmic mislocalization, etc. In epithelial cells, FOXP3 acts as a tumor suppressor to transcriptionally repress the expression of HER2, SKP2 and c-myc oncoproteins and to transactivate p21 (a CDKI) expression. SKP2 mediates the polyubiquitination of p27 (a CDKI) and FOXO proteins for promoting their proteasomal degradation in the cytoplasm. Therefore, FOXP3-mediated inactivation of SKP2 can restore the levels and functions of p27 and FOXOs, leading to cell cycle arrest and apoptosis. Induction of p21 expression by FOXP3 also results in cell cycle arrest. FOXP3-mediated cell cycle arrest and apoptosis in epithelial cells play a protective role to prevent the occurrence of tumorigenesis and cancer progression. HER2 signaling can activate both PI3K-AKT and Ras-Raf-MEK-ERK signaling cascades to enhance cell survival and proliferation. The c-myc protein functions as a promoter of cell proliferation. Suppression of HER2 and c-myc expression by FOXP3 reduces cell survival and inhibits proliferation, which have the effects to block cancer development and progression. PI3K AKT Ras/Raf MEK ERK1/2 Cell Survival and Proliferation Tumorigenesis and Cancer Progression

155 The Emerging Roles of Forkhead Box (FOX) Family Proteins 149 In addition to its role in breast epithelial cells, the FOXP3 gene also plays an imperative tumor-suppressor role in prostate epithelial cells. It has been reported that the X-linked FOXP3 gene at Xp11.23 is frequently inactivated in human prostate cancer by genomic deletions, somatic inactivating mutations and expression down-regulation [156]. By analysis of lineage-specific ablation of FoxP3 in prostate epithelial cells in mice, Wang et al. found that inactivation of Foxp3 resulted in prostate hyperplasia and prostate intraepithelial neoplasia, suggesting the importance of Foxp3 in suppressing early prostate tumorigenesis in male mice [156]. Importantly, in both human normal and neoplastic prostate tissues, FOXP3 is essential to transcriptionally repress the expression of the c-myc gene, a most frequently overexpressed oncogene in prostate cancer [156] (Figure 2). These findings demonstrate that FOXP3 is an X-linked prostate tumor suppressor gene in males. Given that males have only one X chromosome, these studies provide a paradigm of "single genetic hit" to inactivate X- linked tumor suppressor genes [145, 156]. In the study of ovarian cancer, FOXP3 expression has been shown to be down-regulated or lost in ovarian cancer cells compared to normal ovarian epithelia [157]. Ectopic expression of FOXP3 in ovarian cancer cells displays the tumor-suppressive effects to inhibit cell proliferation, migration and invasion [157]. Taken together, FOXP3 principally acts as a tumor suppressor at least in breast, prostate and ovarian epithelia to prevent tumorigenesis. 2. Alterations in FOXP3 Expression and Function in Cancer The aberrant mechanisms causing the inactivation of FOXP3 in epithelial carcinomas involve genomic deletions, functionally significant somatic mutations, abnormal alternative splicing, epigenetic gene silencing and unknown expression down-regulation [ ] (Figure 2). The prevalence of somatic mutations ( > 20 mutations found) at the FOXP3 gene has been found in breast (36%) and prostate (25%) cancer cases (145, 146, 154, 156). The frequency of the FOXP3 deletion has also been reported in breast (13%) and prostate (14%) cancers [145, 154, 156]. IHC analysis of tumor samples was also performed to reveal the frequency of FOXP3 protein expression in different types of cancer. However, the interpretations of IHC results from different research groups are controversial, which might have been complicated by the use of different anti-foxp3 antibodies and the presence of cytoplasmic FOXP3 in examined tumor tissues [145]. Nevertheless, the consensus from these IHC studies is that normal epithelia commonly exhibit nuclear FOXP3 staining and cytoplasmic localization of FOXP3 is predominantly present in FOXP3-positive epithelial cancer cells, which is thought as an inactive form of the FOXP3 protein [ ]. Although it is still not completely clear, cytoplasmic localization of FOXP3 in tumor epithelial cells is suggested to be attributable to somatic mutations and/or alternative splicing variants, which inactivate or remove nuclear localization signals of the FOXP3 protein [ , 154, 156, 157, 161]. It is noteworthy that the alternative splicing forms of the FOXP3 protein have been found to be predominantly expressed in some types of cancers such as breast and ovarian cancers, malignant melanomas and malignant T cells of Sezary syndrome. These alterations in alternative splicing of the FOXP3 pre-mrna can lead to the truncation of the FOXP3 protein and inactivation of its function [146, 154, 157, 161, 162]. In spite of reported deletions, somatic mutations and alternative splicing, these mechanisms can not completely explain high frequencies of FOXP3 loss or down-regulation in examined tumor specimens

156 150 Pang-Kuo Lo [145]. Exploring other potential mechanisms (e.g. DNA methylation, histone deacetylation and the regulation by transcription modulators) is mandatory for future investigation. C. FOXF Subfamily Genes 1. The Biological Functions of FOXF Genes The FOXF subfamily has two member genes FOXF1 and FOXF2. The human FOXF genes were first cloned and characterized by Hellqvist et al. and named as Forkhead RElated ACtivator (FREAC)-1 and -2 [163]. In addition to the forkhead DNA-binding domain, the C- terminal portion of FOXF proteins possesses the characteristics of the transcriptional activation domain [164, 165]. Intriguingly, FOXF2 has been reported to contain another transactivation domain mapped in the central protein part [164]. The multiple studies have shown that FOXF factors transcripitionally modulate the expression of tissue-specific genes (e.g. lung, intestine, adipose, etc.) [163, ]. According to published literature, the expression of FOXF transcription factors is found in lung, placenta, breast, prostate, colon, brain and head tissue [163, ]. The in vitro hybridization studies have shown that Foxf mrna expression is predominately distributed in the mesoderm-derived tissues [176, 177], suggesting their expression is mesenchyme-specific and their functions are important for generation of descendants of splanchnic mesoderm. The studies of Foxf genetic knockout mice have shown that the functions of Foxf transcription factors are essential for embryonic development, organogenesis (e.g. lung, gut, liver, gall bladder) and vasculogenesis [167, 176, ]. In line with these findings in mice, the germline deletions and inactivating mutations of human FOXF1 gene have been discovered and associated with genetic disorders in lung and gut organs such as alveolar capillary dysplasia (ACD), esophageal atresia and VACTERL association (Vertebral anomalies, Anal atresia, Cardiac malformations, Tracheo- Esophageal fistula, Renal and Limb malformations) [ ]. Although the roles of Foxf factors in mouse development have been extensively documented, the biochemical roles of their functions in mammalian cells are largely unknown. However, several studies have shown that the mammalian forkhead box-f1 gene expression is regulated by the hedgehog (Hh) signaling pathway and Hh-downstream mediators Gli transcription factors [167, 168, 179], suggesting that Foxf1 is a key downstream mediator of hedgehog signaling that is critically implicated in modulation of organ morphogenesis. Moreover, a further study has shown that inactivation of one allele each of Foxf1 and Foxf2 results in defects in gut development, a decrease in stromal Bmp4 expression as well as an increase in stromal Wnt5a expression, and causing epithelial hyperproliferation [167]. This study indicates that stromal Foxf factors are essential for regulating the polarity and proliferation of epithelial cells during gut development. The crucial role of the Foxf1 function in regulating the migration of normal fibroblasts has also been reported [187]. Owing to the limited studies of FOXF2, its biochemical function remains largely unknown. However, a recent study has deciphered a novel role of FOXF2 in the regulation of growth factor signaling and the homeostasis of glucose metabolism in addition to its well-known function in modulating the expression of lung-specific genes [169]. Ectopic overexpression of Foxf2 down-regulates IRS1 mrna and protein expression levels in

157 The Emerging Roles of Forkhead Box (FOX) Family Proteins 151 adipocytes and concurrently lowers insulin-mediated glucose uptake compared with wild-type adipocytes [169]. Therefore, these findings suggest that Foxf2 may be also implicated in cellular and systemic whole body glucose tolerance via, at least in part, the down-regulation of IRS1 expression [169]. Besides this role in glucose tolerance, the Foxf2-mediated suppression of growth factor signaling (e.g. insulin, IGF-1) may have a tumor-suppressive effect in cancer cells, which is worthy of further investigation. 2. Aberrant Regulation of FOXF Gene Expression and Function in Cancer There are several lines of recent evidence indicating that FOXF transcription factors are potential tumor suppressors. In prostate cancer, the FOXF1 gene is located in the common deleted regions of 16q23-qter [172]. In line with this, FOXF1 expression is markedly reduced in prostate tumor specimens compared to normal prostate tissue [172] (Figure 3). We also recently discovered that human FOXF1 plays a novel tumor-suppressor role in cell-cycle regulation in breast epithelial cells, which is frequently silenced in breast cancer through epigenetic mechanisms [174] (Figure 3). Breast Cancer Epigenetic Silencing Mitotic genes ANAPC2 ANAPC4 CUL3 G1-regulatory genes CDK6? upregulate DNA repair genes BRCA1 MRE11A Gene transcriptionregulatory genes CCNT1 (Cyclin T1) Colorectal Cancer Cytoplasmic Mislocalization FOXF1 E2F-induced genes MCM3 RB1 Cyclin B1 Cyclin B2 Importin 2 Survivin Prostate Cancer Genomic Deletions downregulate S-phase activating genes CDC34 CDK5R1 CDK5RAP1 Figure 3. Dysregulation of FOXF1 expression and function in cancer. FOXF1 expression is lost or down-regulated in cancer (e.g. breast and prostate cancer) through pathological mechanisms such as chromosomal deletions and/or epigenetic gene silencing. FOXF1 expression is also underexpressed in colorectal cancer; however, the underlying mechanism is unclear. Although it is still uncertain, epigenetic gene silencing is a potential mechanism leading to the down-regulation of FOXF1 in colorectal cancer. FOXF1 is also inactivated in colorectal cancer by cytoplasmic mislocalization. FOXF1 is able to up-regulate and down-regulate the expression of genes functionally implicated in multiple biological pathways for maintaining the stringency of DNA replication and genomic stability.

158 152 Pang-Kuo Lo Our functional studies have shown that ectopic overexpression of FOXF1 in FOXF1- negative breast cancer cells inhibits the CDK2-Rb-E2F signaling cascade to block the G1-S transition and inactivation of endogenous FOXF1 in FOXF1-expressing breast cancer cells by sirna knockdown leads to genomic DNA rereplication [174]. In addition, the expression profiling study indicates that inactivation of FOXF1 function up-regulates the expression of E2F target genes implicated in DNA replication initiation and S-phase cell cycle progression, suggesting that FOXF1 is involved in negatively controlling DNA replication [174] (Figure 3). FOXF1 also up-regulates the expression of genes functionally involved in DNA repair (BRCA1, MRE11A), mitosis (ANAPC2, ANAPC4, and CUL3) and G1 progression (CDK6) (Figure 3), suggesting that FOXF1 may regulate multiple biological processes to induce the tumor-suppressive effect [174]. Up-regulation of DNA repair genes by FOXF1 further supported the tumor-suppressor role of FOXF1 in maintaining genomic stability. Besides breast cancer, our very recent report has revealed that FOXF1 expression is predominantly silenced in colorectal cancer cell lines with the inactive p53 gene [188]. The biological function of FOXF1 in colorectal cancer cells has been deciphered by the sirna knockdown study. Abrogation of FOXF1 function results in DNA rereplication (also called over-replication, a mechanism known to cause genomic instability) in colorectal cancer cells with a defect in the p53-p21 checkpoint [188]. Therefore, these findings, taken together, imply that FOXF1 might play a tumor-suppressor role in colorectal carcinogenesis. Indeed, our most recent clinical studies have shown that human colorectal adenocarcinomas exhibit either a reduction in FOXF1 expression or cytoplasmic mislocalization of the FOXF1 protein [175] (Figure 3). This is the first report revealing that the FOXF1 protein is overexpressed as well as mislocalized in colorectal tumor epithelial cells and underexpressed/lost in tumorassociated stromal fibroblasts. Thus, in addition to the aberrant epigenetic mechanisms, cytoplasmic mislocalization is another pathological mechanism to inactivate FOXF1 function in cancer due to the fact that the functional FOXF1 protein is located in the nucleus. However, the underlying mechanism causing FOXF1 mislocalization is unclear and thus the future investigation is warranted to address this question. This study also indicates that cytoplasmic FOXF1 is a promising prognostic marker owing to its statistically significant association with the malignancy and metastasis of colorectal cancer. Regarding the role of FOXF2 in cancer, a recent interesting report has shown that FOXF2 is the target of mir-301, whose overexpression in lymph node negative (LNN) invasive ductal breast cancer has been implicated as a poor prognostic indicator [189]. In this study, the authors posited that FOXF2 is an anti-proliferation factor by inhibiting WNT5A signaling, which is negatively regulated by mir-301 in breast cancer [189]. D. FOXL Subfamily Genes 1. The Biological Functions of FOXL Genes The FOXL subfamily is comprised of FOXL1 and FOXL2, which both are single-exon genes located at 16q24 and 3q23, respectively. FOXL1 functions as a mesenchymal transcription factor that has been linked to the regulation of the Wnt/ -Catenin signal axis, a vital pathway for gastrointestinal development and tumorigenesis [190, 191]. The in vivo

159 The Emerging Roles of Forkhead Box (FOX) Family Proteins 153 animal studies of Apc (Min/+) ;Foxl1 -/- mice indicate that loss of Foxl1 results in a significant increase in tumor multiplicity in the colon of mice with the Apc (Min/+) genetic background compared to Foxl1 +/+ control mice [191]. In addition to promoting colorectal tumorigenesis, Apc (Min/+) ;Foxl1 -/- mice also develop gastric neoplasia that are not present in Apc(Min) control mice. These outcomes are attributable to accelerated loss of heterozygosity (LOH) at the Apc locus in gastrointestinal tissue [191]. These studies have revealed that Foxl1 is a potential tumor suppressor playing a key role in gastrointestinal tumorigenesis. However, Foxl1 -/- mice only manifest abnormal intestinal epithelia, postnatal growth retardation and defective intestinal glucose uptake, but do not develop gastrointestinal neoplasia [192, 193], suggesting that single loss of Foxl1 is not sufficient for the neoplastic development in the gastrointestinal tract. Furthermore, Foxl1 has been shown to collaborate with Foxf1 in mediating hedgehog signaling and controlling epithelial proliferation in the developing stomach and intestine [168]. It has also been reported that Foxl1 is involved in liver repair and the regulation of bipotential hepatic progenitor cells [194, 195]. Despite these critical roles in mice, the biological functions of human FOXL1 in cancer still remain unknown and need to be deciphered by future investigation. The forkhead transcription factor FOXL2 is emerging as a central transcription factor in regulating the development of ovary and the growth as well as maturation of ovarian follicles [reviewed in [196, 197]]. FOXL2 expression is mainly found in the periocular region, the pituitary and ovarian granulosa cells [198, 199]. The FOXL2 gene was first identified and cloned by Crisponi et al. from the Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome (BPES) region on human chromosome 3q23 [200], a genetic disorder with germline mutations at the FOXL2 gene locus. Patients with BPES manifest a characteristic eyelid dysplasia with associated symptoms such as premature ovarian failure (POF) and infertility in affected females [201]. In line with these clinical findings in humans, the studies from two independently created Foxl2 -/- knockout mouse models have revealed that loss of Foxl2 impairs granulosa cell differentiation, follicle development and ovary maintenance [202, 203]. Therefore, the FOXL2 forkhead transcription factor is an essential ovarian transcription factor crucial to ovarian development, female sex determination and the postnatal ovary as well as follicle maintenance. The diverse transcriptional activities of FOXL2 are likely modulated by posttranslational modifications (e.g. Sumoylation, phosphorylation, acetylation, etc.) and binding to other key protein partners implicated in the regulation of granulosa cells [196, 197, ]. Besides germline mutations in BPES patients, somatic mutations and down-regulation of the FOXL2 gene were identified in human ovarian granulosa cell tumors (OGCTs) [reviewed in [204]]. These studies of FOXL2 in OGCTs suggest that it may act as a tumor suppressor in ovarian tumorigenesis. In line with clinical findings, the recent advances in the studies of FOXL2 biological function also support a paradigm that FOXL2 is a potential tumor suppressor (Figure 4), at least in ovary. FOXL2 are known to interact with several partner proteins that function as tumor suppressors (Figure 4). For example, FOXL2 has been reported to be able to heterodimerize with the transcription factor SMAD3, a crucial mediator responsible for the TGF cytostatic effect. Their interaction is attributable to the binding of the C-terminal portion of the FOXL2 forkhead domain to the SMAD3 N-terminal domain [207, 208]. Intriguingly, this heterodimerized transcriptional complex is involved in up-regulating the expression of the Follistatin gene, encoding a cytostatic protein to inhibit BMP15-stimulated granulosa cell proliferation [208].

160 154 Pang-Kuo Lo Rb FOXL2 DDX20 LATS1 SMAD3 Cell Cycle Arrest FST p16ink4a Apoptosis Regulation ATF3 BCL2A1 CH25H IER3 TNFAIP3 Oxidative Stress Mn-SOD SIRT1 Cell Adhesion MMP23 Cell Cycle Arrest Apoptosis Figure 4. An illustrated view of the regulation of FOXL2 and FOXL2-mediated biological effects. FOXL2 expression is positively regulated by the Rb protein. FOXL2 may interact with multiple protein partners to execute its effects on cell cycle arrest and apoptosis. FOXL2 functions are implicated in cell cycle arrest, apoptotic regulation, oxidative stress responses and cell adhesion via its downstream target genes. Up-pointing arrows, up-regulation by FOXL2; down-pointing arrows, down-regulation by FOXL2. Furthermore, it has also been shown that FOXL2 is able to interact with DDX20 (also named as DP103 or Gemin3), a DEAD-box helicase that potentiates the ability of FOXL2 to induce apoptosis in CHO cells [209]. Through the oncogenomics-based in vivo RNAi screening, Ddx20 has been recently unraveled as a potential tumor suppressor in hepatic carcinomas [210], implying that the interaction between FOXL2 and DDX20 may assist FOXL2-mediated tumor suppression in ovary. Besides these two protein partners, FOXL2 can also interact with the serine/threonine kinase LATS1 (large tumor suppressor 1, also named as WARTS) [204]. Lats1 has been demonstrated as a bona fide tumor suppressor gane in Lats1-deficient mice, evidenced by suppressing the development of ovarian stromal cell tumors and soft-tissue sarcomas [211]. Additionally, the cell model studies have shown that LATS1 is involved in the G2/M checkpoint and can induce G2 arrest by inhibiting CDC2 kinase activity, a critical effector mediating the G2/M transition [212]. Thus, it will be an interesting issue of whether the interaction with LATS1 plays a key role in FOXL2-driven tumor suppression, which warrants future investigation. Another line of evidence also links FOXL2 to a bona fide tumor suppressor, the Retinoblastoma (Rb) protein. Expression of Foxl2 has been found to be significantly decreased in Rb -/- granulosa cells, suggesting that Rb functions as an upstream modulator to positively regulate Foxl2 expression [213]. Since inactivation of Rb is also found in ovarian cancer [214], it will be important to investigate whether both molecules are synergistically implicated in preventing ovarian tumorigenesis. Some of identified FOXL2 downstream target genes have been shown to have tumorsuppressive characteristics, which may mediate FOXL2-driven tumor suppression (Figure 4). One of the examples is Foxl2-Smad2-upregulated Follistatin gene expression as mentioned above [208]. Moreover, the p16-ink4a protein encoded from the CDKN2A locus is expressed in a FOXL2-dependent manner [204, 215, 216]. The p16-ink4a protein is a well-known CDKI with the ability to arrest cells in the G1 phase [217]. In line with the fact that FOXL2 expression is generally down-regulated or lost in the most aggressive OGCTs, FOXL2 has

161 The Emerging Roles of Forkhead Box (FOX) Family Proteins 155 been reported to negatively regulate matrix metalloproteinases (e.g. MMP23), suggesting that loss of or low FOXL2 expression will favor the metastatic progression. Through the transcriptome perturbation study of a granulosa cell model, numerous apoptotic regulators were identified as potential FOXL2 targets [215] (Figure 4). These identified FOXL2 targets include pro-apoptotic CH25H, TNF-R1 (tumor necrosis factor-receptor 1) and Fas (CD95/APO-1) [218, 219], anti-apoptotic TNFAIP3 [220], and modulators with dual roles in the pro- or anti-apoptotic effect dependent on the cellular context such as BCL2A1, ATF3 and IER3/IEX-1 [215, 221, 222]. These discoveries suggest that FOXL2 might function as either a pro- or anti-apoptotic effector in a context-dependent manner (e.g. the differentiation status of granulosa cells, severity of cellular stresses, etc.). Finally, a recent study has shown that oxidative stress activates the transcriptional ability of FOXL2 to induce the expression of stress-responsive genes (e.g. MnSOD, SIRT1) [223] (Figure 4). Interestingly, its target SIRT1 deacetyase actually inhibits FOXL2-mediated transactivation, which forms a negativefeedback loop to counterbalance stress-induced FOXL2 transcriptional activity for restoration of cellular status back to the steady state [223]. Taken together, by regulating the expression of stress-responsive and apoptosis-regulatory genes, it has been proposed that FOXL2 is a key actor to mediate cell stress responses in ovarian granulosa cells for the maintenance of cell survival as well as genomic stability and the elimination of severely damaged cells [204]. 2. Aberrations in FOXL Genes in Cancer FOXL1 expression has been shown to be elevated in low-grade fibromyxoid sarcomas (LGFMS) [224]. However, its role in this type of tumors is completely unknown. As discussed above, FOXL2 expression is lost or markedly reduced in more than half of juvenile OGCT ovarian tumor cases [216]. A complete extinction of FOXL2 expression is further observed in recurring juvenile OGCT cases [216]. The pathological mechanisms leading to these alterations in FOXL2 expression in OGCTs remain elusive and are currently under investigation. Through genome-wide next-generation sequencing analysis of the transcriptome in OGCTs, Shah et al. identified the somatic mutation p.cys134trp in the protein sequence of FOXL2 [225]. The prevalence of this mutation has been confirmed in 97% of examined adult OGCTs according to the published report [225]. The impact of this somatic mutation on FOXL2 function has been studied, which has shown that the FOXL2 mutant protein is unable to trigger the full apoptotic response compared to wild-type FOXL2 [219]. In line with this result, this mutation attenuates the ability of FOXL2 to up-regulate pro-apoptotic effectors TNF-R1 and Fas [219]. It is also worth noting that FOXL2 mutant has been shown to be able to dimerize with the wild-type protein and has a dominant-negative effect to interfere with wild-type FOXL2 to regulate its downstream target genes [226]. This finding suggests that the FOXL2 mutant protein can impair the normal transcriptional activity and target-gene specificity of the wild-type FOXL2 protein, a mechanism potentially leading to ovarian tumorigenesis. Indeed, through ectopic overexpression of wild-type and mutant FOXL2 in the granulosa cell tumor (GCT) line (COV434) with a lack of FOXL2 or the silencing of mutant FOXL2 expression in the GCT line (KGN), mutant FOXL2 protein has been shown to be able to differentially regulate the cellular transcriptome compared to the wild-type protein [227]. Taken together, these findings indicate that in addition to loss of or

162 156 Pang-Kuo Lo reduced FOXL2 expression, this somatic mutation is another crucial mechanism accounting for the development and aggressiveness of OGCTs. E. The Bi-Functional Roles of FOX Transcription Factors in Cancer 1. The Bi-Functional Roles of FOXO Transcription Factors in Cancer FOXO transcription factors are known to render cells resistant to oxidative stress, premature aging and cellular senescence. Hence, FOXOs are critical to cell survival and longevity. These surviving functions of FOXOs are mediated by their effects on ROS detoxification, the enhancement of DNA repair, the induction of cell cycle arrest, the inhibition of the mtorc1-s6k signaling axis, and the promotion of cellular atophy and autophagy. Although these FOXOs activities are commonly thought as their tumor suppressor functions, the recent studies also indicate that cancer cells, especially cancer stem cells (CSCs), can hijack these FOXO-mediated cell surviving mechanisms for the tumor development and resistance to radiotherapy and chemotherapy. A recent study reported that the expression of FOXO and anti-oxidant FOXO target genes is elevated in the radiotherapy-resistant subset of the breast CSC population compared with untreated control cells [228]. These findings indicate that FOXO-mediated ROS detoxification renders CSCs resistant to oxidizing damage from therapeutics and potentially contributes to relapse and the poor patient survival rate [228)]. In addition, FOXO1 expression has been found to be upregulated in paclitaxel-resistant ovarian cancer cells, which protects cells by lowering ROS production and alleviating drug-induced cytotoxicity (229). In doxorubicin-treated leukemia cells, FOXO3 has been shown to be responsible for induced expression of the multi-drug resistant ABCG1 gene and PIK3CA gene, which is considered as a potential mechanism leading to therapeutic resistance [230, 231]. Similarly, it has also been reported that the expression of multiple drug resistant 1 (MDR1) in doxorubicin-resistant breast cancer cells relies on FOXO1 function [232]. In light of multiple studies demonstrating the role of FOXO proteins in the DNA repair pathway [34, 104], it is plausible that therapy-triggered FOXO activation may be beneficial to cancer cell (or cancer stem cell) survival by facilitating DNA repair in addition to ROS detoxification. These lines of contradictory evidence raise issues of whether they are a general phenomenon in all kinds of cancers as they become resistant to anti-cancer therapeutics, what cell subset in heterogeneous cancer cell populations displays this phenomenon and whether this therapeutics-resistant cell population must have CSC-like properties. These raised questions warrants future investigation due to their importance to the development of therapeutics targeting FOXOs. As indicated above, through induction of multiple downstream target genes (e.g. InsR, IRS2, TSC1, Sestrin 3, Rictor, HER2/3, BNIP3, etc.), FOXO factors can potentiate AKT activity to maintain cell survival by inhibiting the mtorc1-s6k-mediated negative feedback loop, activating mtorc2 and augmenting the upstream RTK and IRS input signaling. Apparently, these FOXOs effects contradict their roles as tumor suppressors. These contradictory roles of FOXOs may give an explanation for the observations that overall AKT

163 The Emerging Roles of Forkhead Box (FOX) Family Proteins 157 activity is decreased in FOXO-deficient cells and there are no epithelial origins of tumors developed in FOXO-knockout mice [11]. Therefore, these FOXO-mediated effects may potentially promote cancer cell survival in response to therapeutic treatments especially when FOXO-mediated pro-apoptotic effects are abrogated in cancer cells. Indeed, a recent report demonstrates that FOXOs are responsible for the development of the acquired resistance to AKT inhibitors [91]. The AKT inhibitor (MK-2206) has been shown to inhibit mtorc1 signaling but to activate FOXO-dependent elevated expression of RTKs (e.g. HER2/3, IGF1R) and RTK downstream signaling modules (91). Furthermore, through the downstream autophagy-related target genes (e.g. LC3, BNIP3, ATG12, GABARAPL1) and the inhibition of mtorc1 activity, FOXOs are known to promote the autophagy process, which is a cellular mechanism to eliminate defective mitochondria by macroautophagy for increasing cell survival. Thus, transformed cancer cells can employ this aspect of FOXO function to escape from a cellular energy crisis, increased ROS and DNA damage. Indeed, it has been reported that autophagy is required for Ras-mediated tumorigenesis and the development of pancreatic cancers [233, 234]. In light of all lines of evidence as mentioned above, it is warranted to further investigate the functional significance of FOXO-mediated activation of ROS detoxification, DNA repair, RTK signaling and autophagy in tumor maintenance and survival upon metabolic, oxidative and DNA-damaged stress. In mammals, cellular senescence is considered as one of the defensive mechanisms against the in vivo tumor development. It has been reported that FOXOs are able to elicit cellular senescence in melanoma cells harboring the oncogenic mutated BRAF gene, where high ROS levels and activated JNK stimulate FOXO-induced p21 expression and p21- dependent senescence [235]. On the other hand, other studies have shown the opposite role of FOXOs in the regulation of cellular senescence. In cultured human endothelial cells, constitutively activated AKT induces p53-p21-dependent cellular senescence, which is mediated by the inhibition of FOXO activity and its effect on lowering ROS levels that are required for activation of p53 [236]. It has been a consensus that hyper-activation of oncogenic signaling can trigger p53-dependent cellular senescence. The hyper-activation of mtorc1 has been found as one of such mechanisms that induce senescence by evoking cellular checkpoint responses [237]. The hyper-activation of mtorc1 also leads to loss of a response to growth factor stimuli, which promotes the occurrence of senescence [237]. A recent related study has also shown that loss of PTEN induces senescence in prostate cancer cells. In particular, the underlying mechanism for this type of senescence does not involve hyper-proliferation and DNA damage response. In fact, it is mediated by the p53 action that is triggered by PTEN loss-induced hyper-activation of mtorc1 [238]. In light of the fact that FOXOs inhibit mtorc1 activity and induce cell cycle arrest upon cellular stresses, it is plausible that FOXOs can block mtorc1-triggered senescence. Therefore, the investigations of whether FOXOs possess intrinsic ability to antagonize cellular senescence in many cell types and what role of this function in tumorigenesis are mandatory. The reported role of FOXOs in maintaining leukemia initiating cells (LICs) in chronic myeloid leukemia (CML) also indicates another side of FOXOs. The animal studies have shown that FOXO3-deficient LICs fail to be successively transplanted in the syngenic mouse model of Bcr-Abl-induced CML [239].

164 158 Pang-Kuo Lo The further study elucidates that abrogation of FOXO attenuates the TGF -FOXO pathway, which is indispensable for the survival and maintenance of the LIC fraction [239]. Noticeably, the combined treatment with a tyrosine kinase inhibitor (Imanitib) and TGF inhibitor (LY364947) more effectively depletes LICs and improves overall survival by suppressing both AKT and TGF -FOXO [239]. Furthermore, it has been shown that FOXO factors are essential for maintaining the long-term homeostasis of hematopoietic as well as neural stem cells and regulating their responses to physiological stresses [ ]. Whether FOXOs have a parallel function in maintaining relatively quiescent cancer initiating cell population in particular types of tumors is a critical issue that needs to be addressed by future investigation. In prostate cancer, FOXOs also play a dual role to act as either tumor suppressors to repress the expression of androgen receptor in androgen-dependent prostate cancer cells [243] or oncoproteins to transcriptionally up-regulate VEGF expression following androgen-ablation therapy, giving rise to the development of metastatic and androgen-refractory tumors [244]. In conclusion, the exact roles of FOXO transcription factors in cancer cells depend on the cellular context, disease stages, tissue types and environmental cues. 2. The Bi-Functional Roles of FOXP Transcription Factors in Cancer As discussed above, FOXP1 potentially functions as either an oncoprotein in various types of lymphomas (e.g. Diffuse large B-cell lymphoma and mucosa-associated lymphoid tissue lymphoma) and liver cancer or a tumor suppressor in numerous tissues and organs [reviewed in [5, 143]]. These differentially functional aspects of FOXP1 are attributable to tissue-type-specific and cancer-type-specific expression levels and/or alternative splicing variants of the FOXP1 protein [5, 143]. In contrast, FOXP3 is a bona fide X-linked tumor suppressor in breast, ovarian and prostate cancer. However, the immunosuppressive role of FOXP3 in regulatory T lymphocytes has been known to be positively associated with tumor development because FOXP3 + Treg cells are abundantly present in infiltrated tumor tissues and experimental Treg reduction could dampen tumor growth and enhance the efficacy of tumor immunotherapy [reviewed in [144, 147]]. Therefore, by being expressed in either epithelial cells or regulatory T lymphocytes, FOXP3 has a bi-functional role in human cancers. 3. The Bi-Functional Roles of FOXF Transcription Factors in Cancer As indicated above, our evidence supports the tumor-suppressor role of FOXF1 in breast and colorectal tumorigenesis [174, 175, 188]. However, on the other hand, there are two lines of opposite evidence supporting that FOXF1 acts as an oncoprotein. Saito et al. reported that FOXF1 is required for the contractility of cancer-associated fibroblasts (CAF), CAF-mediated production of hepatocyte growth factor and fibroblast growth factor-2, and CAF-mediated stimulation of lung cancer cell migration and xenografted tumor growth [245]. In addition to its role in lung cancer, another study has revealed that ectopic overexpression of FoxF1 in a mouse mammary

165 The Emerging Roles of Forkhead Box (FOX) Family Proteins 159 epithelial cell line leads to the induction of epithelial-to-mesenchymal transition (EMT), increased invasiveness of epithelial cells in vitro and enhanced growth of breast xenograft tumors in vivo [246]. These findings are strengthened by the correlation between FOXF1 expression and a mesenchymal phenotype in human breast cancer cell lines [246]. Therefore, these two lines of evidence have deciphered the oncogenic aspect of FOXF1 that can enhance tumor-promoting properties of cancer-associated fibroblasts in lung cancer and induce an EMT phenotype in mammary epithelial cells [245, 246]. Their studies suggest that FOXF1 plays oncogenic roles in lung tumorigenesis and breast cancer metastasis. In contrast, we identified another aspect of FOXF1 function that acts like a tumor suppressor to negatively regulate DNA rereplication for maintaining the stringency of DNA replication and genomic stability. According to expression analysis of FOXF1 gene in 20 breast cancer cell lines [174, 188], FOXF1 expression is preferentially detected in basal-like (mesenchymallike) breast cancer cell lines (e.g. BT20, BT549, MDA-MB-157, HCC1937 and HCC1187), but mostly silenced in luminal (epithelial-like) breast cancer cell lines. These results are in line with observations from Nilsson et al. [246]. This raises a possibility that FOXF1 exhibits the tumor-suppressive function in luminal breast cancer cells but displays tumor-promoting features in basal-like (or metastatic) breast cancer cells. However, how basal-like breast cancer cells can escape the FOXF1-mediated tumor-suppression control needs to be further elucidated. Thus, our and other studies conclusively support a paradigm that FOXF1 plays a dual role in tumorigenesis as an oncoprotein or a tumor suppressor in a context-dependent manner. Owing to the high homology between FOXF1 and FOXF2 protein sequences and their functional redundancy, it will be interesting to decipher whether FOXF2 also has a dual role in tumorigenesis and metastasis. 4. The Bi-Functional Roles of FOXL Transcription Factors in Cancer In Apc(Min) mice, Foxl1 acts like a tumor suppressor to inhibit gastrointestinal tumorigenesis [191]. However, Foxl1 collaborates with Foxf1 to regulate gastrointestinal morphogenesis by mediating hedgehog signaling [168] and also positively modulates Wnt/ -Catenin signaling [190]. Both hedgehog and Wnt signaling pathways have been known to be implicated in gastrointestinal stem cell regulation and tumorigenesis [ ]. Therefore, it is possible that FOXL1 may play a dual role in promoting either tumor suppression or tumorigenesis in the gastrointestinal system depending on tissue cell types, tissue-environmental stimuli, tumor stages, etc. Similarly, in different tissue types, FOXL2 performs different roles that either activate or inactivate tumorigenesis. In ovary, FOXL2 functions as a tumor suppressor to inhibit OGCT tumor development [196, 197, 204]. Thus OGCTs exhibit the frequent inactivation of FOXL2 function via markedly reduced expression of FOXL2 or somatic mutations on its gene [204]. However, in breast cancer, FOXL2 is a transcriptional inducer of the aromatase CYP19A1 gene which encodes an enzyme required for the estrogen biosynthesis. FOXL2-induced production of estrogen is essential for the growth of ER-positive breast tumors, but concurrently renders breast cancer cells susceptible to tamoxifen therapy [250].

166 160 Pang-Kuo Lo F. The Potential of FOX Proteins as Therapeutic Targets 1. Therapeutic Targeting of FOXO Proteins in Cancer Among these FOX subfamilies discussed above, FOXO subfamily draws most attention in recent targeting therapeutics. Inactivation of FOXOs tumor-suppressor functions in multiple cancer types is mediated by various mechanisms such as chromosomal translocation, genomic deletion, mirna regulation and post-translational dysregulation by a variety of oncogenic signaling pathways (e.g. AKT, IKK and ERK). Reintroduction of FOXOs into cancer cells defective of FOXO activity gives rise to in vitro growth inhibition of cancer cells and in vivo tumor suppression. Therefore, due to the strong anti-tumor activities of FOXO factors, anti-cancer agents that can reactivate FOXOs functions in cancer cells become compelling therapeutics for cancer treatments. For example, there are several preclinical/clinical chemotherapeutic agents reported to be able to reactivate FOXO3 and its downstream pro-apoptotic (e.g. BIM) or CDKI (e.g. p27) target gene in various cancer types (Table 3); (1) Paclitaxel activates FOXO3 in breast carcinoma cells by inhibiting AKT and activating JNK [251, 252]. (2) KP372-1, a multiple kinase inhibitor, activates FOXO3 in acute myeloid leukemia cells by reducing AKT activity [253]. (3) OSU-03012, a novel anticancer agent, activates FOXO3 and its target p27 expression in ER-negative breast cancer cells by inhibiting AKT, which sensitizes ER-negative breast cancer cells to Tamoxifen therapy [254]. (4) AZD6244 activates FOXO3 in lung and gastric cancer cells by suppressing ERK [255, 256]. (5) Imatinib activates FOXO3 and its pro-apoptotic target gene (BIM) expression in chronic myeloid leukemia cells via the inhibition of BCR-ABL fusion oncoprotein [257, 258]. (6) Trastuzumab and cetuximab, antibodies to negatively target epidermal growth factor receptors (HER family receptors), activate FOXO3 and transcriptional induction of FOXO3 s pro-apoptotic target BNIP3L gene in breast, prostate, kidney, ovarian and lung cancer cells by interfering with activation of PI3K-AKT signaling [9, 259]. In addition to these chemotherapeutic agents that target upstream kinases responsible for negatively regulating FOXOs, there are several novel small molecules that have been developed to inhibit the nuclear exclusion of FOXO proteins (Table 3). In light of the antitumor activity of FOXO1 in PTEN-null cancer cells, Kau et al. employed a high-throughput chemical genetic screening to successfully identify novel general inhibitors to block FOXO nuclear export [260]. These identified inhibitors either react with CRM1 (nuclear exporter) or inhibit PI3K/AKT signaling [260]. Moreover, Schroeder et al. screened a collection of marine natural product extracts for the identification of compounds that can compensate for loss of tumor-suppressive functionality in PTEN-null cells [261]. They discovered the most effective extract (psammaplysene A) that compensates for loss of PTEN by inhibiting the nuclear exclusion of FOXO1 [261]. By using a cell-based imaging model system that tracks the cellular translocation of FOXO proteins, Link et al. screened 33,992 small chemical molecules and identified pyrazolopyrimidine derivatives as potent FOXO relocators [262]. These compounds actually are the biochemical inhibitors of PI3K. Based on virtual

167 The Emerging Roles of Forkhead Box (FOX) Family Proteins 161 screening and molecular modeling, they synthesized a most potent PI3K inhibitor ETP with a strong effect to suppress tumorigenicity in vitro and in vivo [262]. Table 3. FOX tumor suppressors as anticancer therapeutic targets Cancer therapeutics FOX factor targeted Tested cancer type The underlying therapeutic mechanism Paclitaxel FOXO3 Breast cancer Inhibition of AKT and activation of JNK (251, 252) KP372-1 FOXO3 Acute myeloid leukemia A reduction of AKT activity (253) OSU FOXO3 Breast cancer Inhibition of AKT and sensitizing ER-negative breast cancer cells to Tamoxifen (254) AZD6244 FOXO3 Lung and gastric Inhibition of ERK (255, 256) cancer Imatinib (Glivec) FOXO3 Chronic myeloid leukemia Inhibition of BCR-ABL fusion oncoprotein, leading to FOXO3 dephosphorylation and BIMdependent apoptosis (257, 258) Trastuzumab, cetuximab, and lapatinib FOXO3 Breast, prostate, kidney, ovarian and lung cancer Inhibition of EGFR and HER2 signaling, suppression of PI3K- AKT, activation of FOXO3 and induction of pro-apoptotic FOXO3 target BNIP3L (9, 259) Psammaplysene A FOXO1 Kidney cancer Inhibiting the nuclear exclusion of FOXO1 (261) ETP FOXO3 Osteosarcoma, Inhibition of PI3K activity (262) breast carcinoma Adenovirusmediated transfer FOXO3 Melanoma Activation of FOXO3 and induction of apoptosis (263) of constitutively active FOXO3 Anisomycin FOXP3 Breast cancer Induction of FOXP3 expression and apoptosis (269) Doxorubicin FOXP3 Breast and colorectal cancer p53-dependent induction of FOXP3 expression (270) These studies provide examples to screen and identify active therapeutic agents to target oncogenic PI3K/AKT signaling based on their effects on relocalizing FOXO to the nucleus. Besides reactivation of FOXO by chemical agents, reintroduction of constitutively active FOXO3 into melanoma cells via the adenovirus-mediated transfer was also reported and this strategy induces apoptosis in melanoma cells [263]. This therapeutic strategy may be beneficial to the killing of FOXO-deficient cancer cells with or without the combination with other therapeutics. Owing to the frequent occurrence of FOXO inactivation in cancers, the combination with therapeutic agents that reactivate FOXO activity may potentially enhance the treatment efficacies of traditional anti-cancer agents. These combined therapeutic

168 162 Pang-Kuo Lo regimens are also potentially beneficial to the treatment of drug-resistant cancers derived from the inactivation of the FOXO signaling pathway. It is noteworthy that ionizing radiation has been reported to activate FOXO3-induced BIM up-regulation and concurrent apoptosis in p53-null osteosarcoma cells [264]. This suggests that the combination of radiotherapies with chemotherapies targeting FOXOs may be a potential therapeutic regimen to sensitize radioresistant tumor cells to radiation therapy. Additionally, previous reports have shown that the resistant tumor cells developed in treatment with MEK inhibitors manifest hyper-activated AKT activity [256, ]. It has been known that inhibition of MEK-ERK signaling can reactivate FOXO activity for inducing tumor-suppressive effects (e.g. BIM, p27). However, AKT is hyper-activated during the development of drug resistance, which in turn inhibits FOXO activity. Therefore, reactivation of FOXO by therapeutic agents is a promising strategy to conquer cancer resistance to the MEK inhibitors [255]. Conclusively, these studies implicate that FOXO reactivation may be a prospective therapeutic target and pharmacological indicator to potentiate and predict the efficacies of MEK inhibitors in clinics. Although most of studies emphasize the beneficial effects of FOXO reactivation on cancer therapy, the roles of FOXOs in negative feedback regulation loop complicate the applications of targeting FOXO to cancer treatment. The inhibitory effect of FOXO activation on mtorc1 signaling could potentially benefit cancer therapy. Despite these benefits from FOXO-mediated tumor-suupressive effects, it could not be ignored that the effects of FOXOs on the coupling to the activation of AKT and upstream RTK signaling may raise the question of whether the abrogation of FOXOs is preferred over their activation for cancer therapy. Therefore, more future investigations are warranted for the understanding of what signaling context in cancer cells is suitable for which therapeutics based on either activation or inhibition of FOXO activity. 2. Therapeutic Reactivation of the FOXP3 Protein in Cancer X-chromosome inactivation occurring during embryogenesis is a vital mechanism to silence gene expression from one of the two X chromosomes in females. Given that the majority of the somatic mutations and deletions of the FOXP3 gene are heterozygous in human female cancers, it is possible to reactivate the remaining wild-type FOXP3 allele for cancer therapy [145, 146, 268]. Indeed, it has been reported that anisomycin treatment triggers an unknown cellular response to induce the expression of FOXP3 (Table 3). Induced FOXP3 elicits apoptosis in various breast cancer cell lines and inhibits xenograft mammary tumor formation in vivo [269]. Intriguingly, the FOXP3 gene is the downstream target of the p53 transcription factor and the chemotherapeutic agent doxorubicin, a DNA damaging drug to activate p53, has been reported to induce the p53-dependent transcription of the FOXP3 gene in vitro [270] (Table 3). In light of these two lines of evidence, a paradigm that anticancer therapeutics can be implemented by the restoration of FOXP3 function in cancer cells might have great potential to be translationally applied to effective therapy of human malignancies.

169 The Emerging Roles of Forkhead Box (FOX) Family Proteins 163 Conclusion It is convincing that FOX transcription factors are crucial regulators in a variety of biological processes, embryonic development, organogenesis and tissue regeneration. Given that the expression of a wide spectrum of direct and indirect target genes is under FOX regulation, dysregulation of FOX expression and function can significantly contribute to or is sufficient for tumorigenesis and metastasis. As mentioned in this chapter, the functions of FOX genes with tumor-suppressor characteristics are inactivated in cancer through multiple mechanisms such as chromosomal translocations, genomic deletions, somatic inactivating mutations, aberrant alternative splicing, cytoplasmic mislocalization, protein degradation, epigenetic gene silencing, microrna deregulation, etc. For those pathological mechanisms involving cytoplasmic mislocalization and protein degradation, there are therapeutic opportunities to target these aberrant mechanisms to correct these alterations and in turn restore the functions of FOX tumor suppressors (e.g. FOXO3, FOXP3) in cancer cells. For those FOX genes (e.g. FOXF1) silenced by the epigenetic mechanism, their expression can be potentially restored by current clinically used epigenetic drugs (e.g. demethylating agents and histone deacetylase inhibitors) [271]. Based on these viewpoints, FOX-targeted therapies look promising in the future. However, the complicated natures of these FOX proteins, being able to act as either oncoproteins or tumor suppressors in a context-dependent manner, render the clinical implications of FOX-targeted therapeutics questionable. Therefore, before the translational applications of FOX-targeted therapies to clinics, the context-dependency of FOX functions should be comprehensively investigated and defined in various types of human cancers and also in different stages of cancers (e.g. non-metastatic and metastatic). Such information will be crucial to further rationally optimize the combination of FOX-targeted therapies with current therapeutic regimens. In light of recent innovations in both next-generation sequencing and bioinformatics technologies [272], the genetic and epigenetic landscapes of FOX family genes as well as FOX-binding sites within the regulatory regions of FOX-target genes in human cancers can be characterized and defined in a genome-wide, high-throughput manner. These future efforts could promote understanding of the mechanisms underlying FOXregulated tumorigenesis and the development of novel cancer diagnostics, prognostics and anti-cancer therapeutics. Acknowledgments I thank Dr. Saraswati Sukumar at the Johns Hopkins University for previously supporting the FOXF studies and Drs. Vicki Vance and Richard Showman at the University of South Carolina (USC) for supporting and assisting my research at USC. This review chapter was supported in part by the Advanced Support Program for Innovative Research Excellence-I (ASPIRE-I) Grant A026 to P. K. Lo, awarded by the Office of the Vice President for Research at USC.

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189 In: Tumor Suppressor Genes ISBN: Editors: Mehmet Gunduz and Esra Gunduz 2013 Chapter 8 Molecular Basis of BRCA1 and BRCA2 and Clinical Approaches to BRCA1/2 Mutation Carriers Kemal Keseroglu 1,, Fatih Aydogan 2, and Mustafa Ozen 1, 3, 1 Department of Medical Genetics, Istanbul University, Cerrahpasa Medical School, Istanbul, Turkey 2 Department of General Surgery, Breast Division, Istanbul University, Cerrahpasa Medical School, Istanbul, Turkey 3 Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, US Abstract The human BRCA1 and BRCA2 genes are tumor suppressor genes. Whereas BRCA1 is mainly expressed in the cells of thymus and testes, BRCA2 has the highest expression in mammary glands and thymus. Both BRCA1 and BRCA2 proteins are nuclear proteins and whilst BRCA1 protein has functions in DNA repair of double-stranded breaks, ubiquitination, transcription and transcriptional regulation; BRCA2 has functions in homologous recombination and DNA repair. Due to their essential functions in the cell, BRCA1/2 mutations lead to tumor formation, especially breast and over cancer. Lifetime risk of BRCA1/2 mutation carriers is 35-87% for breast cancer and 16-60% for ovarian cancer and the lifetime risk for other cancers are also elevated. Genetic counseling must be given according to recommendations for risk assessment to hereditary breast and over cancer. There are three possible results of the BRCA1/2 testing: (i) Negative, which shows that no mutation was detected (ii) Positive, which means that the risk of developing breast and ovarian cancer for the client is increased (iii) and inconclusive result. Correspondence: Mustafa Ozen M.D. Ph.D. Department of Medical Genetics, Istanbul University, Cerrahpasa Medical School, Istanbul, Turkey, mozen@bcm.edu, mozen@istanbul.edu.tr. These authors contributed equally.

190 184 Kemal Keseroglu, Fatih Aydogan and Mustafa Ozen The recommendations for increased cancer risk in carriers of BRCA1/2 mutations are: (i) surveillance, which includes clinical exams, mammography and such blood tests, (ii) prophylactic mastectomy, where the incidences of breast cancer and ovarian cancer decrease up to 90% and 80%, respectively, in women that carry BRCA1/2 mutation, and (iii) chemoprevention. Two agents, Tamoxifen and Raloxifen, which are the selective estrogen modulators, are approved up to now to decrease breast cancer risk. Keyaords: BRCA1, BRCA2, breast cancer, ovarian cancer, BRCA mutation carriers, risk assessment BRCA1 and BRCA2 Structures, Expressions and Functions The human BRCA1 and BRCA2 genes are tumor suppressor genes [1, 2], which produce the breast cancer type 1 susceptibility protein (BRCA1) and breast cancer type 2 susceptibility protein (BRCA2), respectively. The human BRCA1 gene spans base pairs (bps) on chromosome 17q21. It contains 23 exons and it is transcribed into 7094 bps product; BRCA1 has Da weight and 1863 residues in translation length. The human BRCA2 gene, on the other hand, spans bps on chromosome 13q12.3; the transcript of the gene has 27 exons, bps; the protein has Da weight and 3418 residues in translation length (Figure 1). BRCA1 is widely expressed in a variety of cells and tissues such as thymus, testes, breast, ovary, uterus, spleen, liver and lymph nodes [3], however, as seen in Table 1, the majority of the mutations of the gene causes breast and ovarian cancers where damaged DNA is failed to be repaired. The subcellular localization of the encoded protein is reported to be primarily in nucleus [4], but alternatively it is also in cytoplasm [5], centrosome [6], perinuclear region [7] and mitochondrion [8]. The BRCA1 protein contains four major protein domains; (i) the Zinc finger Cys3HisCys4-RING domain where it interacts with a homologous region of BARD1 [9] and is an essential region for ubiquitination [10], (ii) the BRCA1 serine domain where the phosphorylation sites are concentrated in [11] and (iii-iv) two BRCA1 C Terminus (BRCT) domains those interact with each other to control cellular responses to DNA damage [12] (Figure 1b). Thus, the human BRCA1 protein has multiple functions in cells such as DNA repair of double-stranded breaks [13], ubiquitination, transcription and transcriptional regulation [14]. The highest expression levels of the human BRCA2 gene are detected in mammary glands and thymus [15] and its subcellular localization is primarily seen in nucleus [16], whilst alternatively its cytoplasmic [16] and extracellular [17] presence is also documented. It has six phosphorylation sites and can directly bind the single strand DNA [18] (Figure 1d). BRCA2 is involved in different molecular pathways including homologous recombination [19], Fanconi anemia pathway [20], pathways in pancreatic cancer [21] and other cancers [22]. BRCA2 has several motifs for interaction with the recombinase RAD51 [23], which functions in homologous recombination and DNA repair [24]. Therefore, the mutation of BRCA2 causes mismatch repair, which leads to loss of stability of the human genome, dangerous gene rearrangements and so tumor formation [22]. After DNA damage is occurred, both BRCA1 and RAD51 proteins accumulate at the damaged region and BRCA1 has shown to be necessary for subnuclear Rad51 cluster [25]. During this process, BRCA1 is phosphorylated by active ATM, ATR and/or Chk2 and its

191 Molecular Basis of BRCA1 and BRCA2 185 phosphorylation has demonstrated to either directly or indirectly regulates homologous recombination, non-homologous recombination and S/G 2 phase checkpoint mechanisms [26-29]. Figure 1. Structures of BRCA1 and BRCA2 genes (a, c), transcript (b, d) and tertiary conformations of BRCA1 and BRCA2 proteins (e, f), respectively.

192 186 Kemal Keseroglu, Fatih Aydogan and Mustafa Ozen Figure 2. Molecular pathways of BRCA1 and BRCA2 proteins. Gene Table 1. Number of mutations of BRCA1 and BRCA2 according to disease Breast Cancer Breast and/or Ovarian Cancer Ovarian Cancer Prostate Cancer BRCA BRCA Others Besides, BRCA2 directly interacts with RAD51, which keeps it in inactive form and facilitates Rad51 filament formation [25, 30] Contrary to BRCA1, BRCA2 only acts in homologous recombination (Figure 2). BRCA1 is also involved in ubiquitination of proteins via interaction and making heterodimer with BARD1. Both BRCA1 and BARD1 have RING-finger motif close to their amino termini, which are documented to be associated with their ubiquitination activities [31]. Ubiquitination function of BRCA1 has shown to be related to its DNA damage repair activity, due to occurring as a consequence of replication stress [32]. Meanwhile, ubiquitination activity of BRAC1 has shown to be related to Fanconi anemia pathway through BRCA1-associated helicase, BACH1 (also known as FANCJ) and BRCA2 (also known as FANCD1) [33, 34]. Clinical Management of BRCA Mutation Carriers a. Risk Assessment 5 10% of familial breast cancers and 7-10% of familial ovarian cancers stem from inherited gene mutations [35]. Among these genes, in many cases, BRCA1 and BRCA2 are responsible for the hereditary breast and ovarian cancer. Although the BRCA mutations are not common (1/800 1/1000) in the most populations, its prevalence varies in different ethnic

193 Molecular Basis of BRCA1 and BRCA2 187 groups and geographical regions. Founder effects have been reported in multiple populations, most notably in Ashkenazi Jewish, Icelandic, Dutch, and French Canadian [36]. It has been published that if a BRCA1/2 mutation carrier woman was once affected by breast cancer, propensity for another independent breast or ovarian cancer development is notably increased [37-39]. Thus, for the BRCA1/2 mutation carriers, identification of the mutation is highly important not only for herself, but also for her family members. Lifetime risk of BRCA1 and BRCA2 mutation carriers is 35% to 87% for breast cancer and 16-60% for ovarian cancer (40-42). BRCA mutation carriers also have high risks for developing other cancers, such as prostate cancer, pancreatic cancer and colon cancer (Table 1, 2) [42, 43]. Genetic testing is recommended by The American Society of Clinical Oncology when these criteria are met: (i) an individual who has a personal or family history that suggests an inheritable cancer situation, (ii) insufficient interpretation of the test and (iii) results that will help to diagnose or find the effect of the medical or surgical approaches for the affected individual or family members [44]. Many countries and organizations have proposed their own criteria for that a patient should be referred for genetic counseling and testing (Table 3, 4) [45, 46]. Table 2. BRCA1 or BRCA2 Mutations and Associated Cancer Risks (by age 70) BRCA1 Mutation Breast cancer (initial)...: 65% Breast cancer (second)...: 3% per year Male breast cancer...: 1% Ovarian...: 39% Prostate cancer...: None to 2- to 3- fold increase Pancreatic...: 1-4% Colon cancer...: slight increase BRCA2 Mutation Breast cancer (initial)...: 45% Breast cancer (second)...: 3% per year Male breast cancer... : 6% Ovarian...: 11% Prostate...: % Pancreatic...: 2-7% Table 3. Recommendations for genetic testing of hereditary breast and ovarian cancer according to American Society of Breast Surgeons Early onset breast cancer (diagnosed before age 50) Two primary breast cancers, either bilateral or ipsilateral Family history of early onset breast cancer Male breast cancer Personal or family history of ovarian cancer (particularly nonmucinous types) Ashkenazi (Eastern European) Jewish heritage in the setting of a newly diagnosed breast cancer or family history of breast cancer A previously identified BRCA1 or BRCA2 mutation in the family Triple negative (ER-, PR-, Her2 normal) breast cancer diagnosed prior to age 60. Pancreatic cancer associated with a family history of hereditary breast and ovarian related

194 188 Kemal Keseroglu, Fatih Aydogan and Mustafa Ozen Table 4. Recommendations for genetic testing of hereditary breast and ovarian cancer according to European Society of Medical oncology Three or more breast and/or ovarian cancer cases, at least one <50 years Two breast cancer cases <40 years Male breast cancer and ovarian cancer or early onset female breast cancer Ashkenazi Jew with breast cancer of <60 years Young onset bilateral breast cancer Breast and ovarian cancer in the same patient b. Genetic Counseling Genetic counseling is a communication process, which considers the genetic-related health problems of the patient and helps both the individual and family according to occurrence or incidence of a genetic disease, carried out by one or more health professions [47]. National Society of Genetic Counselors has reported a guideline about recommendations for risk assessment and genetic counseling in hereditary breast and over cancer [48]. Genetic counseling must be guided by these recommendations and must be carried on by educated people. The first step in genetic testing for the cancer patient or his/her relative is giving adequate information and preparing a family tree. Information given to the person must include all the aspects like the meaning of genetic counseling, the way it is done, the limitations, the benefits, the cost, probable test results and the alternative approaches. The information given must be suitable with the patient's educational and cultural degree and if necessary must be supported by visual materials. For the persons who are not thought to be psychologically ready to be informed about the test results, especially for the young women, psychiatric consultation can be requested. Genetic testing must be applied if the clinical approach for the person and the family is going to change. Patients without a known mutation may be candidates for analysis of large rearrangements in BRCA1/2. The mutation must be searched for in the cancer patient first; if it is found, then the other members of family must be screened too. c. Interpreting Genetic Test Results There are three possible results of the BRCA1 and BRCA2 testing: (i) Negative (ii) Positive (iii) Inconclusive result. A negative test shows that no mutation was detected in the BRCA1 or BRCA2 gene. Additionally, having no mutation as has been previously reported in the family for an index is called a true negative test result, although it does not mean that the person will not be affected by cancer; contrary, it is thought that the risk of developing breast and ovarian cancer

195 Molecular Basis of BRCA1 and BRCA2 189 would be same as that of the general population. It is essential to share the possibilities of a false-negative test with clients receiving a negative test result, A positive result gives an explanation for the inheritance of the cancer that is observed in a family and means that the risk of developing breast and ovarian cancer for the client is increased. Current risk estimates for BRCA1 and BRCA2 mutations are 35-87% lifetime risk for breast cancer and 16-60% lifetime risk for ovarian cancer [40-42]. Further tests are recommended for the relatives who have high risks, however, the health and social consequences for family members, even for future generations, should be cared. Thirdly, finding a variation in BRCA1 or BRCA2 that has not been defined previously as a reason for cancer, the test result is accepted as inconclusive. Having a variant of uncertain or unknown clinical significance is reported in 10-15% individuals who have done genetic testing for BRCA1/2 mutations [49]. Recommendations for BRCA1 and BRCA2 mutation carriers who have increased cancer risk are: surveillance, prophylactic mastectomy and chemoprevention. The aim of surveillance is following the mutation carriers closely and making early diagnosis. The surveillance methods for breast cancer include clinical breast examination, mammography and magnetic resonance imaging; for ovarian cancer, the methods consist of clinical exams, blood tests for CA-125 antigens and transvaginal ultrasound. d. Prophylactic Surgery Prophylactic bilateral mastectomy: Prophylactic bilateral mastectomy was shown to decrease the occurrence of breast cancer up to 90% in women carrying BRCA1/2 mutation [50]. After the surgery in the same session some reconstructive attempts are made for these patients to promote the life quality. But it is impossible to determine the psychological and physical deprivation of the patients, which is caused by the operation. Because of that reason, a preventive surgical intervention like this must be offered to the patient only after a full psychological assessment [51]. Prophylactic bilateral salpingho-ooferektomy: It decreases the occurrence of breast cancer 50% and the ovarian cancer 80% in women who are having BRCA1/2 mutations. The preventive effect is shown to be the same in both BRCA1 and BRCA2 mutations [52]. Because this surgery is recommended for premenopausal women, the menopausal symptoms and osteoporosis, which may occur after surgery, must be taken in consideration. This kind of preventive surgery is only recommended for women who have BRCA1/2 mutation [53]. e. Chemoprevention Two agents received approval up to now to decrease breast cancer risk. Tamoxifen and Raloxifen are the selective estrogen modulators, which bind the estrogen receptors competitively showing both agonistic and antagonistic effect. The most of the cancers occurring with BRCA1/2 mutation are known to be estrogen negative. There isn't any prove of chemoprevention to decrease the incidence of breast cancer in women with BRCA1 and BRCA2 mutation. In NSABP (National Adjuvant Surgical Breast and Bowel Project)-P1 study, while use of tamoxifen was shown to decrease the frequency of occurrence of breast

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201 In: Tumor Suppressor Genes ISBN: Editors: Mehmet Gunduz and Esra Gunduz 2013 On The Verge of Being a Tumor Suppressor Gene or an Axonal Guidance Molecule: DCC Chapter 9 Omer Faruk Karatas, 1,2 Betul Yuceturk 1 and Mustafa Ozen 1,3, 1 Department of Medical Genetics, Istanbul University Cerrahpasa Medical School, Istanbul, Turkey 2 Molecular Biology and Genetics Department, Erzurum Technical University, Erzurum, Turkey 3 Department of Pathology & Immunology Baylor College of Medicine, Houston, TX, US Abstract Towards the end of the 1980s, the efforts to reveal putative tumor suppressor genes in colorectal cancer were mostly concentrated on loss of heterozygosity (LOH) studies, which have been shown to be a crucial mechanism in inactivation of tumor suppressor genes. As a consequence of these studies, the DCC (deleted in colorectal cancer), localized on 18q21, has been found to be a tumor suppressor gene in colorectal cancer. Further studies confirmed the reduced level of DCC in several cancers. However, rare detection of point mutations within the coding region of DCC raised the doubts about the candidacy of DCC as a tumor-suppressor gene and the tumor suppressor status of DCC has been challenged since its discovery. In the mean time, additional roles and functions are attributed to DCC such as axon guidance in the following years. DCC is involved in axon attraction through mediating neuronal growth cones towards Netrin-1 expressing regions. Besides, DCC behaves as a dependence receptor, which is defined as having the functional property of inducing cell death when its ligand is detached. In this chapter we have reviewed and summarized the most current evidences that implicate the importance of DCC in cellular functioning in association with its tumor Correspondence: Mustafa Ozen M.D. Ph.D. Department of Medical Genetics, Istanbul University, Cerrahpasa Medical School, Istanbul, Turkey, mozen@bcm.edu

202 196 Omer Faruk Karatas, Betul Yuceturk and Mustafa Ozen suppressive role through unraveling the recent insights into its expression, function and activity in normal and pathological states. We believe that this chapter will help investigators not only working in cancer research field, but also studying DCC in neuroscience, to delineate the functions of DCC. Keywords: Tumor suppressor, cancer, DCC, Netrin-1, axonal guidance Introduction The DCC (deleted in colorectal cancer) is a tumor suppressor gene that is so called due to its identification firstly in colorectal cancer. Towards the end of 1980s, the efforts, to reveal putative tumor suppressor genes in colorectal cancer, was mostly concentrated on loss of heterozygosity (LOH) studies, which has been shown to be a significant mechanism in inactivation of tumor suppressor genes [1]. These studies identified several chromosomal regions such as 5q, 17p and 18q that are affected through LOH [2, 3]. Among them, loss of 18q region, as a promising marker detected in almost 70% of primary colorectal cancers especially in advanced ones with hepatic metastasis, was preferentially attracted the attentions knowing the fact that no known tumor suppressor genes has been localized in this region [4]. Further studies revealed the DCC gene localized in the 18q21 region, as a candidate tumor suppressor gene involved in colorectal carcinogenesis [5]. Thereafter, LOH of chromosome 18q have been evaluated as causing haploinsufficiency at DCC [6] and being responsible for reduced level of DCC, which is detected in a large percentage of colorectal tumors [7, 8]. Moreover, sequencing analysis revealed point mutations of the DCC gene in about 6% of sporadic colorectal cancers [9]. On the contrary, the tumor suppressor function of this gene has been called into question in the following years [4, 10] because of several reasons that will be discussed in the chapter, however, additional roles and functions are attributed to DCC such as axon guidance [11]. This chapter summarizes the importance of DCC in cellular functioning in association with its tumor suppressive role through unraveling the recent insights into its expression, function and activity in normal and pathological states. Structure of DCC Having a homolog in mammals, called neogenin [12], DCC has conserved orthologs in C. elegans (UNC40), Drosophila (Frazzled) and Xenopus [13-15]. In human, DCC gene spans about 1.2 Mb on chromosome 18q21 and includes 29 exons. It encodes for a 1447-amino-acid transmembrane protein with several protein isoforms produced as a consequence of alternative splicing [16, 17]. All characterized isoforms are reported to be type I transmembrane glycoproteins of approximately 175 to 190 kda with a single membranespanning domain. DCC is a member of the immunoglobulin superfamily of cell adhesion molecules, and its extracellular domain, which is made up of approximately 1,100 amino acids, contains four immunoglobulin-like domains and six fibronectin type III-like motifs (Figure 1). This domain shares a high level of structural similarity with the extracellular domains of certain types of cell adhesion molecules, such as NCAM [9]. The cytoplasmic

203 On The Verge of Being a Tumor Suppressor Gene 197 domain, however, that is made up of approximately 325 amino acids, shares very little similarity with other proteins [4]. Nonetheless, P1, P2 and P3 domains within the cytoplasmic domain have shown to be more specifically conserved throughout the DCC orthologs and are proposed to be involved in DCC activity [18]. DCC Expression and Function DCC encodes for a receptor protein that is targeted by the axon guidance molecule Netrin-1 (Figure 1). Its expression is detected in many of the developing and adult tissues [16, 19]. Most of the tissues, especially the basal lamina of several epithelia including the gastrointestinal tract, skin, lung and bladder exhibit low levels of expression in both mrna and protein levels [19-22]. The highest level of DCC expression is observed in the nervous system during development [23]; and in the brain in adults [16]. Development of the visual and olfactory system has been shown to involve DCC expression [24-26]. Figure 1. A schematic representation of DCC receptor and its ligand Netrin-1.

204 198 Omer Faruk Karatas, Betul Yuceturk and Mustafa Ozen Particular axon populations, especially those forming large tracts of fasciculated axons, like the lateral olfactory tract, the internal capsule, corpus callosum, anterior commissure, the fimbria/fornix complex, the fasciculus retroflexus and the estria medularis has been reported to express DCC [27]. Moreover, its expression is detected in the components of peripheral nervous system; and developing spinal, segmental and sciatic nerves, sensory ganglia and their axonal projections, and the early developmental phase of the enteric nervous system has been reported to exhibit high levels of DCC expression [28]. DCC is involved in axon attraction and it mediates axon guidance of neuronal growth cones towards Netrin-1 expressing regions [29, 30]. Netrin-1 is a laminin-related diffusible protein, which has both chemo-attractive and chemo-repulsive potential for axons and neurons via interaction with its receptors [23, 31-33]. DCC, being the main receptor for Netrin-1, is a chemo-attractant for commissural axons in the vertebral spinal cord [34] and it accomplishes axon attraction upon ligand binding and through interaction of its cytoplasmic tail with the tyrosine kinases Src and focal adhesion kinase (FAK, also known as PTK2) [35-37]. It is also proposed to play role in axon repulsion through interaction with UNC5 receptors via forming heterodimers [38, 39]. Besides, in the absence of its ligand, DCC behaves as a dependence receptor, which is defined as having the functional property of inducing cell death when its ligand is detached. DCC has shown to accumulate in the lipid rafts and cause apoptosis, when Netrin-1 is disengaged from DCC [40, 41]. DCC and Cancer LOH of chromosome 18q, which contains DCC, as well as other genes, has been found frequently in colorectal cancer [5]. Allelic losses of 18q have been detected mostly in primary colorectal carcinomas and in almost all of their hepatic metastases, however, very rarely seen in early stage tumors such as small adenomas. This suggests that allelic loss of 18q primarily contributes to the progression of colorectal cancer rather than its initiation [4]. In more than 90% of the 18q LOH occasions in the primary colorectal cancer, DCC is involved in the allelic loss region [42]. Further studies revealed the reduced level of DCC in several cancers, including neuroblastoma [43], hematologic malignancies [44], gastric [45], prostate [46], endometrial [47], ovarian [48], esophageal [49], breast [50], testicular [51], and glial cancers [52]. DCC mutations detected in different cancer types are summarized in Table 1. Besides, restoration of DCC expression through introduction of an intact copy of chromosome 18 into a colorectal cancer cell line that has reduced endogenous DCC expression resulted in inhibition of tumor growth [53]. As a consequence of these observations, DCC was accepted as a tumor suppressor gene. Nevertheless, sequencing analysis resulted in rare detection of point mutations within the coding region of DCC [7], raising the doubts about the candidacy of DCC as a tumor-suppressor gene. Additionally, no tumor predisposition phenotype has been observed in heterozygous mice with an inactivating mutation of the DCC gene [54] and other novel candidate tumor suppressor genes such as deleted in pancreatic cancer 4 (DPC4, also known as SMAD4), and mothers against decapentaplegic homolog 2 (MADH2, also known as SMAD2) have been identified in the allelic loss region of chromosome 18q [18, 55]. Their nonsense, frame-shift and missense mutations have been found in a variety of cancers [18, 42].

205 On The Verge of Being a Tumor Suppressor Gene 199 Table 1. DCC mutations detected in different cancer types MutationTypes CancerType References Colorectal [5] Lung [71] Loss of Heterozygosity Prostate [72, 73] Endometrial [74] Bladder [46] Testicular [51] Homozygous Mutations Pancreatic [61] Biliary [61] Somatic Mutations Breast [50] Epigenetic Alterations Gastric [58] Head and Neck [75-77] Among these genes SMAD4 has shown to be also associated with cancer progression [56]. Taking above-mentioned reasons into account, the tumor suppressor status of DCC has been challenged since its discovery. However, it should be kept in mind that low levels of DCC might stem from the mutations, out of the coding region, that would result in the reduction of DCC expression. For instance, as a common somatic mutation in colorectal cancer, in roughly % of all cases, a 120 to 300 bp expansion is found in a dinucleotide repeat region in the intron residing between exon 7 and exon 8. DCC level was reported to be low in all of the tumors where dinucleotide expansion is observed. Although there is no clear evidence between the expressional reduction of DCC in these tumors and nucleotide repeat expansion in the intron, with further confirmation experiments, it can be proposed that the mutations are not necessarily confined to the coding regions of DCC to cause loss of expression [57]. Besides, aberrations in epigenetic regulation might be involved in the DCC inactivation, which might also explain the low frequency of somatic mutations of DCC. As an example, DCC expression was inhibited via abnormal methylation of DCC promoter in gastric cancer [58]. Moreover, in nude mice, increase of DCC expression to the wild type level suppressed the tumor growth [59] and suppressed expression of DCC in normal rat cells through antisense oligonucleotide targeting caused anchorage-independent cell growth in vitro and tumor formation in nude mice [60]. Besides, in a study, it has been reported that in a subset of pancreatic and bilary cancers with homozygous deletions on 18q, SMAD4 has been shown to be intact, where DCC is inactivated [61]. As a result, although there are some controversies and still too much questions to be answered about the role of DCC in carcinogenesis, in support of the above-mentioned arguments, it still remains as a putative tumor suppressor at 18q21. DCC and Neuronal Guidance Netrin proteins carry out their axonal guidance function through interacting with its receptors including DCC [62] and promoting the outgrowth of commissural axons toward the

206 200 Omer Faruk Karatas, Betul Yuceturk and Mustafa Ozen midline in a variety of organisms [11, 34]. Unc-6 in C-elegans has been previously shown to mediate the circumferential migration of axons to the midline in nematodes [14, 22]. Further discovery of Unc-6 homologs, net-1 and net-2 in the chick [33], NetA and B genes in Drosophila, where they act as guidance cues and direct proper neuronal migration [63-65], proposed the involvement of Netrin family proteins in axonal guidance. Similarly, Netrin-1 in mice has also demonstrated to be one of the important factors mediating migration of commissural axons [33]. Furthermore, DCC represents homology with the unc-40 gene of C. elegans, which encodes for a receptor protein that contributes to the axonal guidance and neuronal migration through its interaction with the ligand UNC-6 [15, 66]. Besides, loss of function mutants of DCC and Netrin family proteins in C. elegans [11, 66], D. melanogaster [14, 64, 65], and mice displayed similarities in terms of axon guidance phenotypes such as axonal projection defects [54]. These similarities and further detection of DCC expression in the axonal growth cones of retinal ganglion cells [24, 67, 68] suggested a putative role for DCC as an axonal guidance molecule. Dcc mutations in mice caused lack of main commissures of the central nervous system, since many commissural axons failed to reach midline [54]. This aberrancy lead to the failure in the development of corpus callosum, hippocampal commissure and pontine nuclei and formation of a new ectopic commissure in the interface between the midbrain and the hindbrain in Dcc mutant mice. In these mice anterior commissure has also been reported to be underdeveloped [54]. In addition, DCC has been implied in the control of spatial distribution of Net protein in D. melanogaster, which has been postulated to provide positional information for other Net receptors in neighboring axons [69]. Interestingly, neither any abnormalities concerning intestinal growth, differentiation or morphogenesis nor elevated risk for tumorigenesis was observed as a result of DCC loss of function, which called the tumor suppressor function of this gene into question [2, 70]. Since there is no in vivo evidence for DCC to promote carcinogenesis when it is lost in animal models for cancer, the tumor suppressor potential of DCC still remains to be controversial. However, having the capacity to induce apoptosis through acting as dependence receptor, it is still believed that DCC can play crucial roles in tumor progression [70]. Conclusion Almost 70% of primary colorectal cancers especially in advanced ones with hepatic metastasis have been demonstrated to lack 18q region. Further studies showed that DCC is involved in 90% of the 18q LOH occasions in the primary colorectal cancer, which suggests DCC gene as a novel candidate tumor suppressor contributing colorectal carcinogenesis. However, rare detection of point mutations within the coding region of DCC raised the suspects about the candidacy of DCC as a tumor-suppressor gene. Besides, heterozygous mice for Dcc displayed no tumor predisposition phenotype and other novel candidate tumor suppressor genes have been found in the allelic loss region of chromosome 18q. Although these findings called the tumor suppressor function of this gene into question, a number of studies supported the notion that DCC is a tumor suppressor. For example, in nude mice, the tumor growth is inhibited as a result of increase of DCC expression to the wild type level.

207 On The Verge of Being a Tumor Suppressor Gene 201 Figure 2. DCC as a chemo-attractant, chemo-repellent and dependence receptor. Although the tumor suppressor function of this gene has been called into question additional roles and functions are attributed to DCC such as axon guidance. Recent studies demonstrated that DCC is involved in axonal guidance through interacting with its ligand Netrin-1 and promoting the outgrowth of commissural axons toward the midline in a variety of organisms. DCC, being the main receptor for Netrin-1, may serve as a chemo-attractant and chemo-repellent for commissural axons in the vertebral spinal cord. DCC also behaves as a dependence receptor in the absence of its ligand, accumulates in the lipid rafts and causes apoptosis. In conclusion, although great progress has been made in understanding of the mechanisms underlying the roles and functions of DCC in axon guidance and tumorigenesis, there is still much work to be done. Further investigations are needed to provide novel insights into the mechanisms and pathways that regulate this controversial tumor suppressor protein and its involvement in neuronal guidance. References [1] Knudson AG, Jr. Hereditary cancer, oncogenes, and antioncogenes. Cancer research Apr;45(4): [2] Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, et al. Genetic alterations during colorectal-tumor development. The New England journal of medicine Sep 1;319(9): [3] Vogelstein B, Fearon ER, Kern SE, Hamilton SR, Preisinger AC, Nakamura Y, et al. Allelotype of colorectal carcinomas. Science Apr 14;244(4901):

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213 In: Tumor Suppressor Genes ISBN: Editors: Mehmet Gunduz and Esra Gunduz 2013 Chapter 10 DKK3, a Mysterious Tumor Suppressor Gene that Possesses Multiple Functions in Tumor Progression Naoki Katase 1*, Tsutomu Nohno 1 and Mehmet Gunduz 2 1 Department of Molecular and Developmental Biology, Kawasaki Medical School 577 Matsushima, Kurashiki, Okayama, Japan 2 Departments of Medical Genetics and Otolaryngology, Head & Neck Surgery, Faculty of Medicine, Turgut Ozal University, Ankara, Turkey Abstract The Wnt/β-catenin pathway is one of the most important pathways in morphogenesis and cell differentiation, and is frequently deregulated in a wide range of cancers. Wnt pathways are regulated by secreted Wnt inhibitory molecules, which are also down regulated by promoter methylation. The DKK family, consisting of DKK1, 2, 3 and 4, is one of these Wnt inhibitors. The Wnt pathway inhibitory ability differs between the DKK members. DKK3 is a very mysterious gene in DKK family members. Although the Wnt inhibitory ability of DKK3 is still unclear, its down-regulation is reported in almost all kinds of malignancies. Hence, DKK3 is thought to be a powerful target for cancer therapy. In this chapter, aberrant expressions of DKK3 and its function in carcinogenesis, tumor angiogenesis and apoptosis is reviewed. Introduction Cancer is an aberrant cell mass that consists of abnormal cells with autonomic proliferation ability and uncontrolled cell growth. In a recent survey, it is reported that * Corresponding Author: Naoki Katase, TEL: , katase@med.kawasaki-m.ac.jp. Corresponding Author: Mehmet Gunduz, TEL: , mgunduz@turgutozal.edu.tr.

214 208 Naoki Katase, Tsutomu Nohno and Mehmet Gunduz approximately 12.7 million new cancers cases were diagnosed and 7.6 million people died of cancer worldwide. According to the estimation of GLOBOCAN 2008, death by cancer represents approximately 13% of all deaths each year. Now cancer is the major cause of death both in the developed and developing world [1]. Generally, the carcinogenesis step includes multiple processes in which cellular regulation is lost, and abnormal cells potentially possess invasive and metastatic properties. Cancer is essentially a disease caused by an aberration in key cancer-critical genes or oncogenic signals. However, the deregulation in the cancer-critical genes are not genetically inherited ones, but are mostly caused by environmental factors, including exposure to chemical mutagens such as tobacco smoking, viral or bacterial infection, obesity caused by dietary problems or physical inactivity, physical agents and radiation [2-11]. Therefore, by changing lifestyle habits, cancer may be preventable, unless important critical genes or pathways are involved. Meanwhile, the true nature of the cancer, i.e. abnormalities in cancer critical genes, includes the loss of tumor suppressor genes (TSGs) or activation of oncogenes. Functional aberrations in TSGs are caused by loss-of-function mutation, genetic deletion or DNA methylation that results in reduced expression of tumor suppressive protein. On the other hand, activation of oncogenes is the result of gain-of-function mutation, elevated protein activity or amplification of oncogenes by increased gene copy number or chromosomal abnormality. In normal condition, TSGs (for typical example, Rb, p53, CDKN2A etc.) are also important genes that regulate cell cycle, DNA repair and apoptosis [12-14]. Oncogenes exist as a proto-oncogene in the normal condition, which code functional proteins triggering signal induction including transcription factor (c-myc), receptor tyrosine kinase (epidermal growth factor receptor: EGFR), regulatory GTPases (Ras protein) and cytoplasmic serine/threonine kinases (Raf kinase) [15-18]. These pivotal genes are functioning in the orchestra of interaction among genes, thus aberrant regulation in one gene will influence the others, resulting in abnormal regulation of the important cellular signaling that controls cell proliferation, cellular growth, differentiation, cell survival or apoptosis. It is well known that cancer is a heterogeneous disease, and its clinical features differ case by case. Some show a high aggressive phenotype including high invasiveness, and is likely to become metastatic, and some exhibit resistance to the chemotherapeutic agent or radiation therapy. Considering this cancer heterogeneity, it is implied that cancer phenotypes are governed not just by a single pathway, but also by complicated interactions between an oncogenic tumor and suppressive signaling circuits [19, 20]. Intriguingly, recent research established specific mechanics to seek actual TSG/oncogene aberrations and their involved signaling in cancers. Investigating the gene signature or pathway signature makes it possible to know the group of genes or pathways in the cancer cells. This signature-centric or pathway-centric way is quite strategic for developments in targeted molecular therapy [21, 22]. Gene signature or pathway signature analyses revealed that deregulation in major pathways (for example: Ras, Wnt/β-catenin, Src, Myc, p53, NFκB, E2F3 and p21) are commonly observed regardless of the difference in cancer type, organ involved, or pathological subtypes [20-22]. The Wnt signaling pathway is one of the most important pathways in which abnormality is commonly reported in a wide range of malignancies. Wnt signaling participates in in the physical process in adult tissue that is involved in homeostasis, and plays a critical role in the developmental process, such as stem cell homeostasis, cell fate determination, differentiation

215 DKK3, a Mysterious Tumor Suppressor Gene 209 and proliferation [23]. Wnt ligands, which consist of 19 highly conserved members, act as a multifunctional growth factor that drives cellular signaling. There are three pathways in Wnt signaling, Wnt/β-catenin pathway [24], planar cell polarity (PCP) pathway [25] and Wnt/Ca 2+ pathway [26]. The Wnt/β-catenin pathway is called the canonical pathway and the latter two are called non-canonical pathways. In the Wnt/β-catenin pathway, cytoplasmic β-catenin is ubiquitinated and degraded without Wnt ligand binding. When Wnt ligands bind to the receptor complex, Frizzled and Lrp5/6, cytoplasmic β-catenin is stabilized and translocated into the nucleus, inducing TCF/LEF mediated transcription. Distinct secreted Wnt inhibitors, including the secreted Frizzled-related protein (sfrp) family, Wnt inhibitory factor (WIF) and Dickkopf (DKK) family, tightly regulate Wnt signaling. The sfrp and WIF directly sequester Wnt ligands, but the DKK family will interfere with the binding between Wnt ligands and the receptor complex [27]. DKKs bind to its cell surface receptor complex, LRP5/6 and Kremen, mediating internalization of Lrp (Figure 1). Figure 1. In Wnt/β-catenin pathway, cytoplasmic beta-catenin is ubiquitinated and degraded without Wnt ligand binding. When Wnt ligands bind to the receptor complex, Frizzled and Lrp5/6, cytoplasmic β-catenin is stabilized and translocated into the nucleus, inducing TCF/LEF mediated transcription. DKK family members antagonize this pathway by binding Lrp5/6 and Kremen. Binding of DKKs with LRP5/6 and Kremen complex resulted in endocytosis of Kremen. Among these extracellular Wnt antagonists, DKK3 of the Dkk Family shows unique characteristics. So far, aberrant regulation in the DKK3 is also repeatedly reported in malignancies. DKK3 was firstly found as an REIC (Reduce expression in cancer) or RIG (regulated in glioma), and later identified as a member of the DKK family. DKK3 may function not only as a tumor suppressor but also as an apoptosis inducer or tumor vessel inducer, suggesting its multifunction in cancer condition. Moreover, even possible oncogenic function is reported.

216 210 Naoki Katase, Tsutomu Nohno and Mehmet Gunduz In this chapter, the details of DKK3, a mysterious and absorbing molecule is discussed, including the history, gene and protein structure and their expression profile, the function. DKK3 Gene/Protein Structure and its function (1) DKK3 Gene Structure DKK3 gene is 46,367 bp long, containing nine exons that span over 50 kbp of genomic DNA [28]. There are two exons in exon1, which are alternatively used in two different transcripts. Three transcript variants are known in total. The DKK3 gene is transcribed into three different isoforms (NM_015881, 2650bp, NM_013253, 2635bp, and NM_ , 2587bp). Two of them result from alternative use of the first exon (i.e. exon 1a and exon1b, although they are both non-coding). One more variant lacks exon1. All the variants share exons 2 to 8, and code 350 amino acid (aa) functioning protein (Figure 2). Figure 2. DKK3 gene location at chromosome 11p15.2, and its annotated transcripts. Exons are indicated as boxes, White boxes present untranscripted region (UTR), and black boxes represent translated region. The start codon (ATG) in exon2, and stop codon (TAG) in exon 8 are indicated as arrows. Human DKK3 DNA/RNA expression is widely observed in normal human tissues. Northern blotting analyses reveal that DKK3 mrna is expressed in the brain, heart, lung, liver, pancreas, spleen, kidney, small intestine, colon, skeletal muscle and placenta. Amongst them, DKK3 expression is particularly high in the heart and brain. The pseudogene for DKK3 is not reported, and neither the germinal nor somatic mutation is reported. 5 single nucleotide polymorphisms (SNP) are known (rs , rs ,

217 DKK3, a Mysterious Tumor Suppressor Gene 211 rs , rs , and rs ). The DKK3 homolog is conserved over species, in vertebrates including zebrafish, murine, rat, chicken, dog, cow, Rhesus monkey and chimpanzee and invertebrate, such as Dictyostelium, cnidarian, tunicate and ascidian. In vertebrates, DKK proteins consist from 4 members (i.e. DKK1, 2, 3 and 4). Although all these proteins possess two cysteine-rich domains, the homology among DKK1, 2 and 4 is 41-50%, whereas that between DKK3 and other members is 37-40% [23, 27]. (2) DKK3 Protein The DKK3 protein possesses several defined regions, which may confer multiple functions to the protein. Amino acid 1-21 is a signal peptide (SP) that characterizes this protein as a secreted protein. Four putative N-glycosylated sites and O-glycosylated at one site region (aa 26-46) suggest that the protein may undergo posttranslational modification before its secretion. Two cysteine-rich domains are conserved over species. N-terminal one is DKK_N (formerly called Cys-1) and C-terminal one is called the Colipase fold (formerly called Cys-2). The two domains contain 10 cysteine residues and are separated by 12aa linker region (Figure 3). Figure 3. All the DKK3 gene transcripts encode 350aa, 38.3kDa glycoprotein. DKK3 protein contains N-terminal signal peptide, two cysteine rich domains (i.e. DKK-type Cys-1 and DKK-type Cys-2). DKK-type Cys-1 is located within the DKK_N (Dickkopf N-terminal cysteine rich region) region. DKK-type Cys-2 include prokineticin region. Two coiled-coil regions are present in the N-terminal side and the C-terminal side. Putative N-glycosylation sites are indicated. The Colipase fold features lipid hydrolysis and may contribute to lipid binding (interacting with cell surface LRP5/6, for instance.) The colipase fold is solved to form an interactive surface with finger-like structure. The presence of a coiled-coil domain suggests possible protein-protein interaction. All these structural features facilitate Wnt/DKK

218 212 Naoki Katase, Tsutomu Nohno and Mehmet Gunduz interactions. Moreover, DKK3 possesses potential proteolytic cleavage sites by furin-type proteases, suggesting that the protein is subject to posttranslational processing. DKK3 protein is Extracellular secreted protein. Its intracellular localization is observed in cytoplasm, organelle and endoplasmic reticulum. (3) Biological function of Dkk3 DKK family was firstly identified in Xenopus embryogenesis [29], and named after its role as head inducer, Dickkopf (dick=thick, kopf=head in German). Dkk binds to the Wnt coreceptor, lipoprotein receptor-related protein 5/6 class (LRP5/6), and exerts antagonistic function for Wnt induced beta-catenin stabilization [30, 31]. Dkk plays an important role in vertebrate antero-posterior axial patterning, limb formation, eye formation and bone formation [23]. The Wnt signaling inhibitory ability differs between the DKK members; DKK1 and 4 can inhibit the Wnt/β-catenin pathway, and DKK2 can both inhibit and activate β-catenin signaling [32], and the co-receptor class of Kremen protein facilitates DKK1, 2, and 4 binding to block Wnt signaling [33]. However, DKK3 neither binds to LRP5/6 nor Kremen [34, 35]. The receptor for DKK3 is yet to be investigated and its Wnt/β-catenin inhibitory function is still elusive [27]. However, the Wnt modulating function of DKK3 is reported in several kinds of malignancies including glioma [36], breast cancer [37], prostate cancer [38,39], and lung cancer [40]. And because of its obvious tumor suppressor function, DKK3 is regarded as a tumor suppressor. Recently, intracellular function of DKK3 is noted. Cytoplasmic DKK3 may bind to beta TrCP, and facilitate beta-catenin degradation [41]. In cancers, DKK3 and mrna expression is down-regulated by promoter methylation, but there is a discrepancy between mrna expression and protein expression in tissue samples, which may reflect tumor heterogeneity. DKK3 function in tumors Reflecting the alias of this gene, RIG (Regulated in glioma) or REIC (Reduced expression in cancer), DKK3 mrna and protein expression is deregulated in a wide range of tumors, including glioma, gastric carcinoma, colorectal carcinoma, hepatocellular carcinoma, pancreatic cancer, leukemia, renal cell carcinoma, bladder carcinoma, prostate cancer, testicular carcinoma, ovarian carcinoma, cervical cancer, breast cancers, non-small cell lung cancer, mesothelioma and skin cancers. The abnormal conditions of the Wnt/β-catenin pathway in malignancies are summarized in Table 1 and 2. This downregulation in mrna expression is caused by the promoter hypermethylation. Thus, Dkk3 is thought to be a potential tumor suppressor, and is focused as a therapeutic target. However, in DKK3 protein expression level, some reports show that the DKK3 protein expression is up-regulated, suggesting a cancer specific expression pattern and a potential alternative role in cancer invasion.

219 Table 1.

220 Table 1. (Continued)

221 SCC: squamous cell carcinoma, CIS: carcinoma in-situ, CLL: chronic lympahtic leukemia, AML:Acute myeloid keukemia, ALL: Acute lymphoblastic leukaemia.

222 Table 1. (Continued)

223 CIN: cervical intraepithelial neoplasia, RCC: renal cell carcinoma, RCCC: renal clear cell carcinoma, NSCLC: non-small cell lung cancer, AAH: atypical adenomatous hyperplasia, BCC: basal cell carcinoma

224 218 Naoki Katase, Tsutomu Nohno, Mehmet Gunduz The following is a detailed exposition of DKK3 aberration status in each type of malignancy. (1) Brain tumors As indicated in the name, RIG (Regulated in glioma), alias name for DKK3, DKK3 protein expression is down regulated in glioma and deregulation of DKK3 is also present in several kinds of brain tumors. Glioma is most common brain tumor, which accounts 25-30% of the brain tumor. Glioma is a collective term for the neuroglia-derived tumors that includes distinct tumors: astrocytic tumors (diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, etc.), oligodendrogliomas, oligoastrocytoma, ependymal tumors and choroid plexus tumors. According to the WHO classification, gliomas are divided into 4 different malignancy grades [42]. Low expression of DKK3 protein is widely reported from grade II/III tumor (diffuse astrocytoma) to grade IV tumor (glioblastoma), and its expression loss is reported to correlate to tumor grade [43, 44]. The reduced DKK3 protein expression was attributed to DNA hypermethylation, and its forced expression in the glioblastoma derived cell line resulted in induction of JUN phosphorylation-mediated apoptosis [36] In neuroblastoma, DKK3 mrna expression is down regulated. DKK3 functions as a tumor suppressor, and its expression is negatively regulated via mir92, which is up regulated by MYCN [44, 45]. Low DKK3 expression in neuroblastoma correlates with poor prognosis. On the other hand, DKK3 expression is high in ganglioneuroma [46]. DKK3 gene expression is observed in normal organs, and its expression is highest in the brain and heart, thus DKK3 protein expression level or DNA methylation status may indicate neuroblastic tumor maturation. (2) Head and neck cancer More than 90% of carcinomas in the oral, head and neck region are squamous cell carcinoma (SCC), and SCC represents 5% of all cancers in men and 2% in women [47]. SCC is thought to arise as a cumulative genetic or epigenetic alteration in cancer associated genes, yet the specific genes that play a pivotal role in cancer invasion, metastasis or clinical diverseness remain unidentified. DNA methylation and subsequent mrna expression reduction is a commonly observed feature in adenocarcinomas in the digestive tract. In a similar fashion, it is also reported in oral SCC tissue sample and cell lines [48]. Aside from this, frequent LOH in Dkk3 locus (11p15.2) is reported, suggesting that DKK3 may play a role in carcinogenesis of the squamous epithelium [49]. However, DKK3 protein expression status and its biological role of DKK3 in SCC are unaccountable. DKK3 may play an oncogenic role in oral, head and neck SCC. Indeed, LOH in DKK3 locus is frequently seen, but LOH in DKK3 locus inversely correlates with lymph nodal metastasis and overall survival [49]. Despite frequent LOH in DKK3 locus, protein is dominantly expressed in oral SCC tissue samples and cell lines. And DKK3 protein expression correlates with shorter disease-free survival, and metastasis-free survival [50].

225 DKK3, a Mysterious Tumor Suppressor Gene 219 Moreover, most recent reports demonstrated DKK3 mrna expression is conserved in oral SCC derived cell lines, and its knockdown decreased cellular invasion and migration [51]. In association with these series of reports, DKK3 expression increases from epithelial dysplasia, carcinoma in situ to invasive cancer, and is thought to be independent with Wnt/β-catenin pathway [52]. It is suggested that DKK3 may be involved in SCC carcinogenesis in the oral, head and neck region. However, its detailed function has yet to be investigated. (3) Gastrointestinal cancers Aberration of the Wnt/β-catenin pathway is a well-known key event in carcinogenesis steps in gastrointestinal adenocarcinomas. Deregulation of the signaling is traditionally attributed to mutations in Axin, adenomatous polyposis coli (APC), and β-catenin, which cause pathway hyper-activation. Moreover, Wnt/β-catenin signaling is also modulated through various other mechanisms, down-regulation of the Wnt inhibitor, cross talk with other altered signaling pathways are examples of them [53]. Therefore, down-regulation of the Wnt inhibitor, including DKK3, is a hot topic in this field. In the esophagus, most of the cancers are squamous cell carcinoma. Although there are only a few reports on DKK3 expression in esophageal SCC, some reports demonstrated that DKK3 DNA is hyper-methylated in esophageal cancer patient samples and cell lines and concluded that methylation of DKK3 predicts risk of recurrence [54, 55]. However, another report indicates that the DKK3 protein is overexpressed and that the DKK3 protein expression correlates with invasive depth, lymph nodal metastasis and an advanced TNM stage [56]. In gastric adenocarcinoma cell lines, DKK3 mrna expression is down regulated and methylation of DKK3 is a prognostic predictor for shorter survival [55, 57, 58]. However, in tissue samples, DKK3 protein expression is also observed in the tumor endothelium adjacent to the cancer tissue, and DKK3 protein expression in cancer cells is associated with pt-stage and UICC stage and correlates with a favorable prognosis [59]. In conclusion, reduced DKK3 mrna expression by CpG methylation is thought to be involved in gastric cancer development, and might be a potential clinical target. In colorectal adenocarcinoma cell lines, DKK3 expression is down-regulated both in the mrna and protein level. Forced overexpression of DKK3 mrna results in G0/G1 cell cycle arrest, induction of apoptosis and reduced cell proliferation. Increased cytoplasmic β-catenin is also noted [60]. In clinical tissue samples, DKK3 protein expression is decreased compared to corresponding normal tissues, and DKK3 expression correlates with invasion depth, TNM stage and dedifferentiation [61]. From these reports, DKK3 might be involved in carcinogenesis of colorectal cancer via the Wnt/β-catenin pathway. (4) Liver cancer The most representative liver cancer is hepatocellular carcinoma (HCC). In HCC and cirrhosis-related HCC tissue samples, DKK3 mrna expression is low because the promoter hypermethylation and hypermethylation of DKK3 may correlate to shorter progression-free

226 220 Naoki Katase, Tsutomu Nohno and Mehmet Gunduz survival in cirrhosis-related HCC. Hypermethylation is more frequent in high-grade tumors [62, 63]. However, as for protein expression, one report describes that DKK3 protein expression is up-regulated in HCC and hepatoblastoma tissue samples [64]. DKK3 may be involved in tumorigenesis of HCC and associated with dedifferentiated nature. (5) Pancreatic cancers Pancreatic cancer is one of the most aggressive human cancers, with an exceedingly poor prognosis because of its late disclosure of symptoms, rapid progression, frequent metastasis and insensitivity to chemotherapy and radiotherapy [65]. Therefore, identification of genes and pathways that contribute to pancreatic cancer progression is eagerly anticipated. DKK3 is a possible target for pancreatic cancer therapy. DKK3 expression is low in pancreatic cancer cell lines (MIA PaCa-2 and AsPC-1), due to DNA methylation. DKK3 expression in transfection of expressing plasmids decreases cell proliferation and β-catenin expression [66]. However, another report indicates that DKK3 expression is overexpressed in PANC-1 cell line (derived from human pancreatic ductal carcinoma), and that its down-regulation results in a reduction in cellular proliferation [67]. DKK3 may be involved in carcinogenesis in pancreatic carcinoma via Wnt/β-catenin signaling. (6) Hematopoietic neoplasm and leukemia The possible function of DKK3 as an immune modulator and involvement in hematopoietic neoplasms are reported. As for chronic lymphatic leukemia (CLL), CLLderived cell lines demonstrated DKK3 methylation ranging from 23-37%. DKK3 methylation is also observed in CLL patients, ranging from % [68]. A small population of acute myeloid leukemia (AML) patients shows DKK3 methylation [69, 70]. DKK3 methylation is also reported in acute lymphatic leukemia (ALL) derived cell lines and patients, and DKK3 methylation is a prognostic predictor of disease free survival [71]. As for the function of the immune modulator function, it is reported that recombinant DKK3 may alter CD14+ monocyte into novel phenotypes, which demonstrate dendritic cell like appearances and IL-4, GM-CSF. Administration of recombinant DKK3 results in tumor regression with CD11c+, CD8+ T-cell infiltration [72]. From these reports, immunological aspects of DKK3 are noted. (7) Gynecological cancers Gynecologic malignancies include cancer of the uterus, ovaries, cervix, and endometrial cancer. The estimated incidence of cases are more than 80,000 cases per year, which accounts for more than 11% of all malignancies in women [73]. Particularly, ovarian cancer exhibits the most aggressive nature of all gynecologic cancers. Overall cure rates for ovarian cancer are limited to 30% [74]. Therefore, a new therapeutic strategy is urgently needed. Although the number is a few, possible involvement of Wnt/β-catenin signaling and its inhibitors is also discussed.

227 DKK3, a Mysterious Tumor Suppressor Gene 221 In cervical squamous cell carcinoma (SCC) tissue samples and cell lines, DNA methylation of DKK3 is reported [75]. Overexpression in the cervical SCC cell line results in reduction of the cellular β-catenin level [41]. DKK3 methylation is also reported in cervical adenocarcinoma, and DKK3 DNA methylation status may correlate with a larger tumor size and shorter disease-free survival [76]. Taken together, DKK3 methylation and aberrant Wnt/β-catenin signaling may be involved in cervical SCC. As for ovarian cancer, DKK3 mrna expression is decreased in ovarian cancer tissue [77], and low DKK3 mrna levels correlate with high-stage and a high incidence of lymph nodal metastasis. Another report suggested that serum DKK3 protein level is low in ovarian cancer patients compared to non-cancerous subjects, and low serum DKK3 levels correlate with a high frequency of lymph nodal metastasis [78]. In endometrial cancer tissue samples, DKK3 mrna expression is down regulated and overexpression in endometrial cancer cell lines results in reduced cell proliferation and betacatenin mediated TCF activity [79]. As shown above, DKK3 may be involved in a wide range of gynecological malignancies. (8) Breast cancer Breast cancer remains the most commonly diagnosed malignancy among females [80]. Positive associations between environmental and individual factors and increased risk of breast cancer were pointed out. Thus, prevention is a highly feasible approach to breast cancer control [81]. Indeed, breast cancer sometimes becomes difficult to control. The involvement of DKK3 in breast cancer is discussed in the context of aberration in Wnt/β-catenin signaling. DNA hypermethylation of DKK3 is reported both in breast cancer tissue samples and cell lines, and DKK3 DNA methylation status may be a prognostic factor for disease free survival and overall survival [82, 83, 84]. Forced expression in breast cancer cell lines result in induction of JNK-mediated apoptosis and reduction of anticancer drug resistance [85]. The knockdown of DKK3 by shrna transfection revealed the possible function of DKK3 as a modulator of the Wnt/beta-catenin signaling modulator in breast cancer [37]. In summary, DKK3 may be involved in carcinogenesis of breast cancer, and may modulate Wnt/β-catenin signaling. (9) Urologic cancers The involvement of DKK3 and its possibility for use in cancer treatment is most actively reported in the field of urology. As for bladder cancer, DKK3 is thought to be a candidate therapeutic target. The details will be discussed below, in the section, DKK3 as a candidate cancer treatment target. In renal cell carcinoma (RCC), DKK3 mrna expression is down regulated because of promoter CpG island methylation. Stable transfection of DKK3 in RCC cell lines does not affect the Wnt/β-catenin pathway, but induces apoptosis via the JNK pathway [86]. Methylation of DKK3 is also observed in renal clear cell carcinoma (RCCC) [87].

228 222 Naoki Katase, Tsutomu Nohno and Mehmet Gunduz So far, mutation in the DKK3 gene is not reported in any kind of malignancy, but SNP in the DKK3 gene is reported in RCC, and rs SNP correlates with distant metastasis [88]. DKK3 methylation is observed in bladder cancer, and forced expression in cancer cell lines induces JNK mediated apoptosis [89, 90, 91]. In conclusion, DKK3 methylation may be involved in carcinogenesis in RCC and bladder carcinoma. (10) Prostate cancer and testicular cancer Similar to urologic cancers, DKK3 is focused as a therapeutic target for prostate and testicular cancer. In prostate cancer, mrna and protein expression are down regulated and DKK3 protein expression in prostate cancer decreases gradually in prostate carcinogenesis. DKK3 protein expression loss may correlate to tumor grade. Overexpression of DKK3 in the prostate cancer model may ameliorate tumor progression [39, 92]. And high DKK3 protein levels are reported in the seminal plasma of prostate cancer patients [93]. Overexpression in prostate cancer cell lines induces JNK-mediated apoptosis [38] and decreases lymph nodal metastasis in the prostate cancer mice model [94, 95]. In testicular cancer, DKK3 expression is down regulated and forced expression in cancer cell lines induce JNK-mediated apoptosis [96]. As shown above, DKK3 methylation may be involved in carcinogenesis in prostate and testicular cancers. (11) Lung cancers Reduced DKK3 mrna level is first reported in human non-small cell lung cancer (NSCLC) tissue samples [97]. Decreased expression ofdkk3 mrna is due to DNA methylation, and DKK3 may regulate cancer cell growth via the Wnt/β-catenin pathway [40]. DKK3 methylation is also observed in precarcinomatous lesions, atypical adenomatous hyperplasia [98]. In addition to lung NSCLC cases, DKK3 expression is also down regulated in the mesothelioma cell line, and overexpression of DKK3 induces JNK-mediated apoptosis [99]. These reports suggest that DKK3 may be involved in NSCLC via Wnt/β-catenin signaling regulation. (12) Skin and bone cancer DKK3 protein expression is down regulated in skin cancers [100]. In malignant melanoma tissue sample and cell lines, DKK3 mrna expression is strongly reduced, and the stable expression of DKK3 in malignant melanoma cells reduces cellular migration [101]. As for bone tumors, the osteosarcoma-derived cell line Saos2, shows decreased expression of DKK3, which may modulate Wnt/β-catenin signaling [102].

229 DKK3, a Mysterious Tumor Suppressor Gene 223 As mentioned above, aberrant expression of DKK3 is reported in a wide range of cancers in almost all organs. However, the function of DKK3 is not fully clarified, and is sometimes conflicting report by report. Indeed DKK3 is a potential therapeutic target, however, further investigation is necessary. In the following articles, the multifunctional aspect of DKK3, particularly in tumor vessel formation, and possible clinical use are discussed. DKK3 and tumor vessels Recent reports revealed the existence of a new function of DKK3 in tumor angiogenesis. It is well known that tumor nests require abundant blood vessels in order to supply nutrition and oxygen. Thus, tumor cells stimulated tumor stroma, facilitating production of neoangiogenesis [103]. Nowadays, the differences between tumor vessels and normal vessels are discussed, and it is revealed that tumor endothelial cells possess a distinct nature at the molecular level, compared with normal endothelial cells [104]. This concept indicates tumor vessels to be a new therapeutic target. DKK3 protein expression in tumor vessels is noted in glioma, melanoma [105], oral squamous cell carcinoma and its precursor lesion [50,52], gastric cancer [59], prostate cancer [92], pancreatic cancer [106] and colorectal cancers [107]. In colorectal cancers, immunohistochemical analysis revealed that vessels in/adjacent to the cancer tissue shows DKK3 protein expression, whereas normal vessels do not. This implies pro-angiogenic function of the DKK3 protein [105,107]. In pancreatic cancer, DKK3 protein expression in tissue samples revealed that DKK3 protein expression is observed both in cancer cells and tumor endothelium. Moreover, DKK3 expressing endothelium is sensitive to anticancer drugs. Low DKK3 protein expression in tumor endothelium correlates with a worse clinical outcome [106]. Supporting this research with clinical samples, stable overexpression of DKK3 increased microvessel density in melanoma mice model [105]. The detail about DKK3 endothelial expression is still unclear. However, one possible explanation for this is regulation among receptor tyrosine kinase Axl, angiopoietin-2 (ANG- 2) and DKK3 in endothelial cells [27]. Generally, Axl expresison is up-regulated in cancer cells and tumor endothelium. Knockdown of Axl expression in human umbilical vein endothelial cells (HUVEC) results in up-regulation of Axl-2 and down-regulation of DKK3. And DKK3 knockdown results in reduced tube formation [108]. Although further investigations are required, DKK3 may be a potent anti-cancer therapeutic target. DKK3 as a candidate cancer treatment target As the reduced expression ofdkk3 mrna or protein is observed in all kinds of malignancies, it is no wonder that we hypothesize that overexpression of DKK3 might ameliorate hopeless cancers. Indeed, pre-clinical evidence includes the adenovirus-mediated DKK3 transfection model, which is known as Ad-REIC/DKK3 [28, 85, 90, 91, 94, 96, 109-

230 224 Naoki Katase, Tsutomu Nohno and Mehmet Gunduz 114]. Investigations using Ad-REIC are especially focused on the urology field (prostate cancer, testicular cancer) As for prostate carcinoma, Ad-REIC treated PC3 cells showed significant tumor reduction and growth inhibition in the BALB/c mice tumor injection model [109, 110]. Similarly, when RM-9 cells after Ad-REIC treatment was injected into prostate tissue of C57/BL6 mice, tumor growth and lymph nodal metastasis was significantly decreased [94] In testicular cancer, NCCIT cells treated with Ad-REIC was injected into BALB/c mice, then 40% of the tumor disappeared and the rest of the tumors showed reduced tumor growth [96] In the mice gastric scirrhous carcinoma model, intraperitoneal administration of adenovirus vector carrying DKK3 significantly decreases tumor dissemination and increases recruitment of killer T cells [113]. Ad-REIC treatment was also reported in breast cancer. Intratumoral injection of Ad-REIC in mice resulted in delayed tumor growth and upregulation of apoptosis [85]. The mechanism that Ad-REIC decreases tumor growth and ameliorates disease in animal models is mainly due to JNK-mediated apoptosis induction, which is independent of Wnt/βcatenin canonical pathway. However, it bears on a wide variety of aspects, such as apoptosis induction, and inhibition in lymph nodal metastasis. All the effects contribute to better survival in animal models. Ad-REIC might be a powerful tool for cancer treatment. But as indicated previously, DKK3 possesses multiple functions including a possible oncogenic role. Further investigation will substantialize clinical use of Ad-REIC. Conclusion Wnt/β-catenin signaling and its regulators are one of the important pathways often deregulated in various kinds of cancers. The DKK family is one of the Wnt regulators that antagonizes Wnt ligands, and is commonly down regulated in cancers by CpG methylation. DKK3, a member of DKK family, of which Wnt inhibitory activity is still questionable, but regarded as a putative Wnt signaling inhibitor and tumor suppressor. DKK3 behaves as a multifunctional gene, including tumor suppression via inhibition of Wnt/β-catenin signaling, participation in tumor vessel formation and apoptosis induction. Currently, DKK3 research is an engrossing theme that has potential for therapeutic applications. References [1] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011; 61(2): [2] Anand P, Kunnumakkara AB, Sundaram C, Harikumar KB, Tharakan ST, Lai OS, Sung B, Aggarwal BB. Cancer is a preventable disease that requires major lifestyle changes. Pharm Res. 2008; 25(9): [3] Sasco AJ, Secretan MB, Straif K. Tobacco smoking and cancer: a brief review of recent

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239 In: Tumor Suppressor Genes ISBN: Editors: Mehmet Gunduz and Esra Gunduz 2013 Chapter 11 The Functions and Roles of the Unique Tumor Suppressor Gene PTEN Omer Faruk Karatas, 1,2 Esra Guzel 1 and Mustafa Ozen 1,3, 1 Department of Medical Genetics, Istanbul University Cerrahpasa Medical School, Istanbul, Turkey 2 Molecular Biology and Genetics Department, Erzurum Technical University, Erzurum, Turkey 3 Department of Pathology & Immunology Baylor College of Medicine, Houston, TX, US Abstract Phosphatase and tensin homolog (PTEN), is a well-documented tumor suppressor gene, which was discovered in 1997 as a result of identification of a frequently lost region on chromosome 10q23. PTEN primarily acts at the plasma membrane and negatively regulates PI3K/AKT signaling pathway through converting PIP3 to PIP2. PTEN signaling pathway conveys the signals from cell surface receptors to the effector proteins in association with other tumor suppressor and oncogenic signaling pathways. It is one of the most frequently mutated or deleted tumor suppressors in human sporadic cancers worldwide. Since it is known as not belonging to a protein family, it is a unique protein, which has been shown to participate in carcinogenesis and whose aberrant expression has been detected in several primary tissues or established tumor cell lines. In this chapter we have reviewed and summarized the most current evidences that implicate the importance of PTEN in cellular functioning in association with its tumor suppressive role through unraveling the recent insights into its expression, function and activity in normal and pathological states. We believe that this chapter will help investigators not only working in the cancer research field, but also studying PTEN in other diseases, to understand the functions of PTEN. Keywords: Tumor suppressor, cancer, PTEN, PI3K/AKT mozen@bcm.edu.

240 234 Omer Faruk Karatas, EsraGuzel and Mustafa Ozen Introduction Phosphatase and tensin homolog (PTEN), also known as MMAC1 (mutated in multiple advanced cancers-1) or TEP1 (tensin-like Phosphatase-1), was originally discovered in 1997, as a result of identification of a frequently lost region on chromosome 10q23 [1, 2] and named as so due to encoding a phosphatase and exhibiting high homology to tensin and auxilin. Being the second most frequently mutated or deleted tumor suppressor [3] in human sporadic cancers including brain, bladder, breast, melanoma, lung, prostate, renal and endometrial cancers [4], the relevance of PTEN in development and cancer was further revealed with the utilization of germline knockout Pten mice mutants. Homozygous loss of Pten caused embryonic lethality, while heterozygous mice for Pten, developed dysplasia of several tissues, cancers of multiple origins, and a lethal haploinsufficient lymphoproliferative disease [5-7]. Altered PTEN expression through promoter methylation has also been reported in several tumors [8, 9]. In addition to cancer, germline loss and mutations of PTEN are associated with several hereditary autosomal dominant disorders such as Cowden disease, Bannayan-Riley-Ruvalcaba syndrome, Lhermitte-Ducros disease (LDD) and Proteus syndrome, which share common symptoms such as neurological disorders, multiple hamartomas and cancer susceptibility [10, 11]. This chapter summarizes the importance of PTEN in cellular functioning in association with its tumor suppressive role through unraveling the recent insights into its expression, function and activity in normal and pathological states. Structure of PTEN PTEN gene spans 105 kb on chromosome 10q23 and is made up of 9 exons (Figure 1A). It is a relatively small, multi-domain polypeptide (55 kda) that is made up of 403 amino acids. As it is demonstrated in Figure 1C, PTEN contains five domains consisting of 1) an amino terminal phosphatase domain homologous to chicken tensin, 2) a PIP2 binding domain, 3) two consecutive PEST (rich in proline, glutamate, serine, and threonine residues) homology domains, which regulates protein stability via the ubiquitin-proteasome pathway, and 4) a PDZ-binding domain, and 5) the C2 regulatory domain, which is implicated in recruitment of PTEN to plasma membrane [12] and may be related to protein stability [13]. As to the phosphatase domain of PTEN, it has shown to contain a central five-stranded - sheet and 6 α-helices wrapping the core [14]. The targets of dephosphorylation activity of PTEN are demonstrated to be Tyr, Ser and Thr phosphorylated residues of highly acidic substrates in vitro [15]. Although the tumor suppressor potential is mostly associated with the N-terminal lipid phosphatase activity, at least 40% of the mutations are localized in the C- terminal domain [16] (Figure 1B). Therefore, it has been suggested that PTEN may have additional functions through its C terminal domain, which is involved in tumor suppressor capability of PTEN [17]. Moreover, crystal structure analysis of PTEN carried out by Lee et al. indicated that C2 regulatory domain has a -sandwich structure, which is interpreted as a basis for interaction of PTEN with DNA and proteins [18].

241 The Functions and Roles of the Unique Tumor Suppressor Gene PTEN 235 Figure 1. Phosphatase and tensin homolog (PTEN), is a unique tumor suppressor that is localized on chromosome 10q23. A simplistic schematic representation of PTEN gene (A), mutation hot-spots regions throughout its exons (B) and the functional domains of PTEN (C). Furthermore, PTEN contains a catalytic signature motif, which is a common active site signature of protein tyrosine phosphatases [19]. This catalytic signature motif constitutes the phosphate-binding domain and includes the hot-spot bases for cancer associated PTEN mutations [20, 21]. Residing in the amino terminal region, the catalytic motif shows homology to the actin binding protein tensin 1 (TNS1) and auxilin, the cofactor of ATPase heat shock cognate 70 (HSC70), which are not actually related to catalytic activity of PTEN [1, 2]. Regulation of PTEN Expression Expressional regulation of PTEN involves a series of mechanisms involving transcriptional activation and repression, epigenetic silencing, microrna regulation and posttranslational regulation. Further interactions with PTEN-interacting proteins also play roles in the activation and repression of PTEN function. PTEN expression is strictly controlled, and although it is assumed to be constitutively expressed in normal tissues, it is, later showed to be both transcriptionally activated and repressed by several regulators [22]. Transcription factors such as early growth regulated transcription factor 1 (EGR1) [23], peroxisome proliferator-activated receptor (PPAR ) [24], p53 [25] and human sprouty homolog 2 (SPRY2) [26] activates transcription of PTEN through binding its promoter. On the other hand, regulators such as Mitogen-activated protein kinase kinase-4 (MKK4) [27], Transforming growth factor (TGF ) [28], JUN [29], JNK

242 236 Omer Faruk Karatas, EsraGuzel and Mustafa Ozen nuclear factor-κb (NF-κB, [27] has shown to suppress PTEN transcription through direct or indirect mechanisms. In addition, epigenetic mechanisms are also involved in transcriptional repression of PTEN by, for instance, a zinc finger transcription factor called sal like protein 4 (SALL4), which recruits an epigenetic repressor complex containing a chromatin remodelling ATPase and a histone deacetylase to the PTEN promoter [30]. In several types of cancer, dramatic reduction of PTEN gene expression is detected because of promoter silencing by DNA methylation [31-33]. In the post-transcriptional level, micrornas (mirnas), which are small, approximately nucleotide-long, non-coding and endogenously synthesized ribonucleic acids (RNAs), are involved in the regulation of gene expression [34]. They bind to 3 -untranslated regions (3 UTR) of target mrnas and cause either mrna degradation or translational blockade [35]. It is thought that mirnas modulate the expression of at least 60% of all protein coding human genes through working in combination with each other and fine-tuning of the target mrna levels [36, 37]. They are shown to be involved in several crucial biological processes such as development, proliferation, and apoptosis through their spatial and temporal expression [38, 39]. Expressional deregulation of mirnas is associated with pathogenesis of several diseases, including human cancers and they can act as potent tumor oncogenes or tumor suppressor genes [40, 41]. MiRNAs have recently been demonstrated to be targeting PTEN and resulting in tumorigenesis and formation of PTEN associated diseases through inhibiting its expression. For example, deregulation of mir-17~92 cluster is associated with lymphoproliferative disease and autoimmunity [42], whereas altered expression of mir 19 is related to leukemia and Cowden disease [43]. In addition, deregulation of mir 21 has been demonstrated to alter the expression of PTEN in multiple cancers [44, 45]. PTEN Expression and Function Since PTEN is known as not belonging to a protein family, it is a unique protein, which means in times of PTEN loss, there are no other family members to substitute for it. Therefore, the loss of PTEN is catastrophic for cells [46]. Its expression has been shown to be detected in embryonic cells starting from the time when blastocysts aged 3.5 days [47] and ubiquitous expression of PTEN is maintained until late stages of the embryonic development (E15-19) [6]. In addition to its expression in extra-embryonic tissues, which has been demonstrated by both in situ hybridization and immunohistochemistry analyses, PTEN is predominantly expressed in embryonic tissues/organs including central nervous system components, bone, liver, heart, skin and gastrointestinal tract. Similar expression pattern is maintained throughout the lifetime and its roles in the development and functioning of these organs have also been reported [6, 48, 49]. PTEN expression is associated with several biological functions, differing from embryogenesis to sexual organ development in adults. Controlling the organ size through modulating the checkpoints for cell proliferation and soma growth is another key role of PTEN through development [50]. Furthermore, induction of apoptosis and cell cycle arrest along with maintaining chromosomal integrity are among

243 The Functions and Roles of the Unique Tumor Suppressor Gene PTEN 237 the well-defined functions of PTEN in the cellular level [18, 51, 52]. Cell migration is another cellular mechanism that is postulated to be negatively regulated by PTEN through directly dephosphorylation of p125fak and alteration of MAP kinase activity [52, 53]. Besides, its expression is also important for the regulation of stem cell self-renewal and proliferation [54, 55]. PTEN, a multifunctional phosphatase, has both potential to dephosphorylate lipids and proteins. It carries out its lipid dephosphorylation activity, which is associated with tumor suppression capability of PTEN [1, 56] mostly at the plasma membrane. Recruitment of PTEN to the membrane, via interaction with other proteins such as MAGI1b, MAGI2, MAGI3, MAST3 and SAST, through its PDZ-binding domain (Figure 2), is required because of the localization of its target, PIP3, at the plasma membrane [22]. PTEN expression, however, is also observed in both cytosol and nucleus within the cell [57, 58]. Detection of PIP3 and PIP2 in the nucleus ascribed putative roles for PTEN in the nucleus, although it is not as well-described as the role of PTEN at the plasma membrane [59]. Further studies revealed that loss of nuclear PTEN resulted in formation of neoplasias and tumorigenesis, attributing a tumor suppressor role for nuclear PTEN [60]. In addition, nuclear PTEN has shown to reduce the levels of cyclin D1 and phospho- MAPK, playing important roles in the cell cycle arrest. Meanwhile, cytoplasmic PTEN intervenes apoptosis through downregulating phospho-pkb levels and upregulating p27kip1 levels [61, 62]. Figure 2. PIP3 represents the major substrate of the lipid phosphatase activity of PTEN. It is responsible for the activation of the serine-threonine kinase, AKT. Phosphorylation helps the stabilization of PTEN and the ubiquitin ligase NEDD4-1 has shown to be responsible for both mono- and poly-ubiquitylation of PTEN. Recruitment of PTEN to the membraneis performed via interaction with other proteins such as MAGI1b, MAGI2, MAGI3, MAST3 and SAST.

244 238 Omer Faruk Karatas, EsraGuzel and Mustafa Ozen As a lipid phosphatase, PTEN demonstrates phosphatase activity against the 3 position of phosphatidylinositol 3,4,5-triphosphate (PIP3), the second messenger that is produced by a potent proto-oncogenic lipid kinase, phosphoinositide 3-Kinase (PI3K), and therefore, negatively regulates the PI3K/AKT signaling pathway and suppresses tumorigenesis [63]. PIP3, representing the major substrate of the lipid phosphatase activity of PTEN [9, 21], is responsible for the activation of the serine-threonine kinase, Akt, and known to be playing crucial roles in antiapoptosis, proliferation and oncogenesis [9] (Figure 2). Therefore, loss of PTEN lipid phosphatase function causes elevated levels of PIP3 and subsequent derepression of PI3K/AKT pathway, which stimulates proliferation and tumorigenesis [20, 47]. PTEN is also known to demonstrate phosphatase activity against protein substrates. The phosphatase activity results in subsequent inactivation of related proteins. Focal adhesion kinase FAK (PTK2), a non-receptor protein tyrosine kinase [64], p130cas, a FAK downstream effector [52], Shc, PDGFR and PTEN itself [52, 65, 66] are among the identified direct protein targets of PTEN. Additionally, receptor tyrosine kinases are also found to be targeted by PTEN. For example, the receptor of platelet-derived growth factor physically interacts with PTEN and it is dephosphorylated as a result of protein phosphatase activity of PTEN [17]. PTEN also targets and inactivates upstream molecules of MAP kinase such as RAS and IRS-1 [67, 68], and downstream effectors of the same pathway including ETS-2, a transcription factor whose DNA-binding ability is controlled by phosphorylation [69] and another transcription factor Sp1, through its protein phosphatase activity [70]. On the other hand, it has been reported that dephosphorylation activity of PTEN is restricted to the intracellular signaling molecules and extracellular signal-regulated kinases cannot serve as direct targets of PTEN for its phosphatase activity [71]. PTEN has also ascribed functions independent of its phosphatase activity [22]. JNK has demonstrated to be a functional target of PTEN, in which no direct involvement of phosphatase activity detected [71]. Post-translational Regulation of PTEN PTEN post-translational regulations involve phosphorylation, acetylation, oxidation and ubiquitination. Phosphatase activity of PTEN is regulated through phosphorylation of its C terminal tail at Ser380, Thr382, Thr383 or Ser385 positions, which results in inhibition of its lipid phosphatase activity. In the meanwhile phosphorylation helps the stabilization of PTEN through promoting a closed PTEN conformation, which blocks its membrane localization [72, 73] (Figure 2). Moreover, PTEN is susceptible to acetylation at Lys125 Lys128 residues by p300/creb binding protein (CBP) associated factor (PCAF) and at Lys402 residue by CBP, which has shown to be linked to inhibition of its catalytic activity. Acetylation also improves protein-protein interactions with PDZ domain containing proteins [74]. As to the oxidation of PTEN, a catalytic Cys nucleophile is oxidized, which is important in the modulation of PTEN s catalytic activity. Furthermore, having two canonical PEST domains, which is a signature of involvement of the ubiquitin pathway for ubiquitin-mediated proteasomal degradation [75, 76], regulation of PTEN is also controlled by ubiquitylation. The ubiquitin ligase NEDD4-1 (neural precursor cell expressed, developmentally downregulated gene 4-1) has shown to be responsible for both mono- and poly-ubiquitylation of

245 The Functions and Roles of the Unique Tumor Suppressor Gene PTEN 239 PTEN [77], whilst only poly-ubiquitylation is recognized as a target for proteasomal degradation through the C-terminal PEST domain of PTEN (Figure 2). Mono-ubiquitylation at the Lys13 and Lys289 residues, which are reported to be mutated in somatic cancers and Cowden disease, respectively, is necessary for the PTEN nuclear cytoplasmic shuttling [78]. Interestingly, in certain types of cancer samples, wild-type PTEN gene has been observed with low and even in undetectable levels, which is most probably associated with abnormal post-translational regulation of PTEN [79, 80], pointing the importance of post-translational regulation. PTEN-Controlled Signaling Pathways PTEN signaling pathway conveys the signals from cell surface receptors to the transcription factors in association with other tumor suppressor and oncogenic signaling pathways. PTEN primarily acts at the plasma membrane and negatively regulates PI3K/AKT signaling pathway through converting PIP3 to PIP2. As ligand-bound growth factor receptor tyrosine kinases or Ras activates PI3K, which is a heterodimeric kinase, PIP3 is produced via phosphorylation of PIP2 by PI3K [81]. PIP3 binds and recruits AKT, the major downstream target of PI3Ks [82], to the plasma membrane. Binding of PIP3 to AKT results in colocalization of AKT and phosphoinositide-dependent kinase 1 (PDK-1) through interaction of their pleckstrin homology (PH) domains. Thereafter, PDK-1 phosphorylates AKT at Thr 308 residue in its kinase domain [83]. Additional phosphorylation of AKT at Ser 473 residue within the carboxyl-terminal regulatory domain by phosphoinositide-dependent kinase 2 (PDK2) is needed for the complete activation of AKT signaling [84]. Upon activation of AKT, it is transported to the nucleus, where it exhibits its functions. Activated AKT modulates the downstream targets to be involved in a variety of cellular functions such as cell survival, cell cycling, metabolism and angiogenesis [85]. Moreover, PTEN also regulates mtor/s6k signaling pathway, and by this way it controls translation mechanisms and thus influence the cell size [86, 87]. Mammalian target of rapamycin (mtor, also known as mechanistic target of rapamycin or FK506 binding protein 12-rapamycin associated protein 1) is serine/threonine protein kinase that is involved in the regulation of several cellular processes such as, cell growth, proliferation, cell motility, cell survival, protein synthesis, and transcription [88, 89]. Germline mutations of PTEN, as a negative regulator of mtor/s6k signaling have been shown to dysregulate the mtor pathway and cause development of familial cancer syndromes [88, 90]. In addition to PI3K/AKT and mtor signaling pathways, PTEN has also been reported to interact with TGF- /SMAD pathway andwnt/ -catenin pathway, and contribute to expressional regulation of homeobox genes, such as NKX3.1 [91] and hepatic nuclear factors [92], pointing the role of PTEN in multiple levels through the development. PTEN and Cancer Analysis of either primary tissues or established tumor cell lines showed that PTEN mutations and deletions have been observed in a variety of human [1, 93-97], especially with

246 240 Omer Faruk Karatas, EsraGuzel and Mustafa Ozen a high frequency in glioblastoma, endometrial and prostate cancers [17]. These mutations include homozygous deletion, frame-shift, inframe deletion, truncation and point mutations [1, 98]. Missense mutations, as well as nonsense and frame-shift mutations of PTEN, has been documented to reside frequently in the phosphatase domain [97, 99]. Most of the missense mutations have shown to result in significant reduction of phosphatase activity through induction of early termination of translation [21, ]. Common missense point mutations of PTEN include H123Y (endometrial cancer), L57W (glioblastoma), G165R (glioblastoma), T167P (breast cancer) and C124S [102]. On the other hand, even though identified mutations seems to disperse throughout the PTEN gene and the tumor suppressor potential is mostly associated with the N-terminal lipid phosphatase activity, almost 40% of all germline mutations occur in the 5 th exon, which only encodes for 20% of the whole protein. This represents the biological significance of the phospholipid domain of PTEN that is encoded from the 5 th exon [103]. Studies has also revealed that truncated PTEN products lacking the C2 domain and the PDZ-biding motif, because of nonsense and frame-shift mutations occurred within the C2 domain C-tail junction, lost their stability and functionality [102]. Moreover, a loss of PTEN has been frequently detected especially in late-stage brain, prostate and endometrium tumors. This implies that PTEN s involvement in carcinogenesis occurs mostly during tumor progression instead of initiation [1, 2]. PTEN and Other Diseases In addition to cancer, PTEN is also implicated in a variety of familial cancer predisposition syndromes such as Cowden disease (CD), Bannayan-Riley-Ruvalcaba syndrome (BRRS), Lhermitte-Ducros disease (LDD) and Proteus syndrome (PS), which have been collectively called as PTEN hamartoma tumor syndromes (PHTSs) [17, 104]. PHTSs have common symptoms like neurological disorders, multiple hamartomas and predisposition to cancer [17, 105]. 85% of Cowden disease patients have shown to be carriers for germline mutations of PTEN [106]. Besides, 65% of Bannayan-Riley-Ruvalcaba syndrome cases [107] and 20% of Proteus syndrome and Proteus-like syndrome cases have also shown to be positive for germline mutations of PTEN [108]. CD, being discovered in 1963 by Lloyd and Dennis, is a rare autosomal dominant hereditary genodermatosis disorder [109], which is characterized by mucocutaneous skin findings in more than 95% of the cases and multiple hamartomatous polyps of ectodermal, mesodermal, and endodermal origins [110]. There is also increased susceptibility to cancer like breast, thyroid, endometrial, colon and renal cell carcinomas [111, 112]. For example, predisposition to breast cancer in women with CD, has shown to be elevated, in which the lifetime risks has been estimated to increase from 11%, which was documented as the risk for normal population, to 25-50% [113, 114]. Besides, Lhermitte Duclos disease, a dysplastic gangliocytoma of the cerebellum, is another hamartoma syndrome, which is often associated with CD and accepted as pathognomonic for it. LDD is diagnosed with diffuse hypertrophy of the stratum granulosum of the cerebellum (106). Furthermore, Bannayan-Riley-Ruvalcaba Syndrome is also a rare autosomal dominant PTEN hamartoma tumor syndrome, which is characterized by macrocephaly, multiple lipomas, intestinal hamartomatous polyps, haemangiomas and pigmented macules of the

247 The Functions and Roles of the Unique Tumor Suppressor Gene PTEN 241 glans penis [ ]. Additionally, having clinical features like hamartoses, lipomas, and overgrowth, a Proteus-like syndrome patient has been reported to be positive for a germline PTEN R335X mutation and shown to be a carrier for a second hit mosaic R130X mutation in his affected tissues [118]. The most frequently detected PTEN mutations in PHTSs and their germline mutation frequency in total are summarized in Table 1. Deregulation of PTEN function is also implicated in diseases other than PHTS and cancer, such as diabetes and autism [22]. Experiments carried out in conditional PTEN-knockout mice demonstrated that, specific deletion of PTEN from adipocyte or pancreatic cells, didn t result in development of carcinomas, instead, in addition to being healthy and fertile, and they were protected from streptozotocin-induced diabetes [119, 120], although the mice lacking PTEN specifically in pancreatic -cells were shown to be significantly smaller than control animals [121]. Table 1. The most frequently detected PTEN mutations in PHTSs and their germline mutation frequency in total for each disease PHTSs Mutations Ref. Frequency of Germline Mutation of PTEN Ref. G129E [10] R233TER [10] 85% [125] H123R [126] C124R [126] R130TER [126] IVS6, T-G, +2 [127] 1-BP DEL, 696A [127] Cowden M35R [127] Disease L70P [128] R130Q [129] R335TER [130] 1-BP INS, A [131] C124S [128] 1-BP DEL, 802G [132] 5-BP DEL, NT347 [132] -764A-G [133] -861G-T [133] R233TER [117, 128] 65% [107] S170R [128] R130TER [126] Bannayan- 1-BP DEL, 1390C [116] Riley- Y178TER [116] Ruvalcaba Q214TER [116] Syndrome E256TER [116] R335TER [130] DEL [133] Lhermitte- E157TER [104] DucrosDisease L112P [135] 50% [134] Proteus-like R335TER [133] Syndrome 1-BP DEL, 507C [108] 20% [136]

248 242 Omer Faruk Karatas, EsraGuzel and Mustafa Ozen Besides, conditional PTEN-knockout mice, in which PTEN is deleted in the cerebral cortex neurons and hippocampus demonstrated several aberrant social behaviors, augmented response to sensory stimuli and learning disabilities [122], which resembles the symptoms of autism spectrum disorders. In another study, which includes 18 patients with autism spectrum disorders, germline missense PTEN mutations in 3 patients have been found [123]. All the above-mentioned diseases, which are associated with several PTEN mutations, indicate the wide-range of clinical reflections of PTEN alterations, and attributes novel functions independent of its tumor suppressor role. Conclusion More than ten years of investigation has showed the importance of PTEN in cellular functioning in association with its tumor suppressive role. Although it has shown to be primarily responsible for conveying the signals from cell surface receptors to the transcription factors through acting at the plasma membrane and negatively regulating the PI3K/AKT signaling pathway, functions independent of its phosphatase activity, which is mostly carried out within the nucleus, has also been ascribed. In addition, since its discovery, it has demonstrated to be involved in regulation of several cellular processes such as cell cycle transition, chromosomal integrity, cell migration, stem cell self-renewal and proliferation. Most importantly, PTEN, being the first designated lipid phosphatase, is one of the most frequently affected genes in cancers [124]. In addition to carcinogenesis, its aberrant expression and mutations are implicated in many familial cancer predisposition syndromes, such as Cowden disease, Bannayan-Riley-Ruvalcaba syndrome, Lhermitte-Ducros disease and Proteus syndrome, which have been collectively called as PTEN hamartoma tumor syndromes. Knowing the fact that its expression is controlled through a series of mechanisms involving transcriptional activation and repression, epigenetic silencing, microrna regulation and post-translational regulation, novel therapeutic and diagnostic approaches can be developed via targeting these mechanisms. For example, it is interesting to note that mirnas have been recently demonstrated to be targeting PTEN and resulting in tumorigenesis and formation of PTEN associated diseases through inhibiting its expression. Thus, profiling the changes in mirna expression in cancer tissues originated from PTEN aberrancy is very important for enlightening possible mechanisms of pathogenesis. This might also help to develop mirna biomarkers for the early detection, diagnosis of these cancers, since mirnas are promising candidates for novel therapeutic applications against cancer. As a conclusion, although great progress has been made in the understanding of the mechanisms underlying the PTEN regulation and along with its roles and functions in cell growth and tumorigenesis, there is still much work to be done. Further investigations are needed to provide novel insights into the mechanisms and pathways that regulate this unique tumor suppressor protein and develop novel therapeutic approaches and tools for cancer prevention and therapy.

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255 The Functions and Roles of the Unique Tumor Suppressor Gene PTEN 249 [94] Dreher T, Zentgraf H, Abel U, Kappeler A, Michel MS, Bleyl U, et al. Reduction of PTEN and p27kip1 expression correlates with tumor grade in prostate cancer. Analysis in radical prostatectomy specimens and needle biopsies. Virchows Archiv : an international journal of pathology Jun;444(6): [95] Dahia PL. PTEN, a unique tumor suppressor gene. Endocrine-related cancer Jun;7(2): [96] Aveyard JS, Skilleter A, Habuchi T, Knowles MA. Somatic mutation of PTEN in bladder carcinoma. British journal of cancer May;80(5-6): [97] Ali IU, Schriml LM, Dean M. Mutational spectra of PTEN/MMAC1 gene: a tumor suppressor with lipid phosphatase activity. Journal of the National Cancer Institute Nov 17;91(22): [98] Shin KH, Kim JM, Rho KS, Park KH, Oh JE, Min BM. Inactivation of the PTEN gene by mutation, exonic deletion, and loss of transcript in human oral squamous cell carcinomas. International journal of oncology Nov;21(5): [99] Maehama T, Taylor GS, Dixon JE. PTEN and myotubularin: novel phosphoinositide phosphatases. Annual review of biochemistry. 2001;70: [100] Han SY, Kato H, Kato S, Suzuki T, Shibata H, Ishii S, et al. Functional evaluation of PTEN missense mutations using in vitro phosphoinositide phosphatase assay. Cancer research Jun 15;60(12): [101] Myers MP, Pass I, Batty IH, Van der Kaay J, Stolarov JP, Hemmings BA, et al. The lipid phosphatase activity of PTEN is critical for its tumor supressor function. Proceedings of the National Academy of Sciences of the United States of America Nov 10;95(23): [102] Maehama T. PTEN: its deregulation and tumorigenesis. Biological & pharmaceutical bulletin Sep;30(9): [103] Hollander MC, Blumenthal GM, Dennis PA. PTEN loss in the continuum of common cancers, rare syndromes and mouse models. Nature reviews Cancer Apr;11(4): [104] Mester J, Eng C. Estimate of de novo mutation frequency in probands with PTEN hamartoma tumor syndrome. Genetics in medicine : official journal of the American College of Medical Genetics Sep;14(9): [105] Liu W, Zhou Y, Reske SN, Shen C. PTEN mutation: many birds with one stone in tumorigenesis. Anticancer research Nov-Dec;28(6A): [106] Eng C. Will the real Cowden syndrome please stand up: revised diagnostic criteria. Journal of medical genetics Nov;37(11): [107] Marsh DJ, Kum JB, Lunetta KL, Bennett MJ, Gorlin RJ, Ahmed SF, et al. PTEN mutation spectrum and genotype-phenotype correlations in Bannayan-Riley-Ruvalcaba syndrome suggest a single entity with Cowden syndrome. Hum. Mol. Genet Aug;8(8): [108] Smith JM, Kirk EP, Theodosopoulos G, Marshall GM, Walker J, Rogers M, et al. Germline mutation of the tumour suppressor PTEN in Proteus syndrome. J. Med. Genet Dec;39(12): [109] Lloyd KM, 2nd, Dennis M. Cowden's disease. A possible new symptom complex with multiple system involvement. Annals of internal medicine Jan;58:

256 250 Omer Faruk Karatas, EsraGuzel and Mustafa Ozen [110] Bae BG, Kim HJ, Lee SG, Choi JR, Hwang C, Lee JH, et al. A novel PTEN mutation in a Korean patient with Cowden syndrome and vascular anomalies. Acta dermatovenereologica Jan;91(1): [111] Trufant JW, Greene L, Cook DL, McKinnon W, Greenblatt M, Bosenberg MW. Colonic ganglioneuromatous polyposis and metastatic adenocarcinoma in the setting of Cowden syndrome: a case report and literature review. Human pathology Apr;43(4): [112] Pilarski R. Cowden syndrome: a critical review of the clinical literature. Journal of genetic counseling Feb;18(1): [113] Starink TM, van der Veen JP, Arwert F, de Waal LP, de Lange GG, Gille JJ, et al. The Cowden syndrome: a clinical and genetic study in 21 patients. Clinical genetics Mar;29(3): [114] Longy M, Lacombe D. Cowden disease. Report of a family and review. Annales de genetique. 1996;39(1): [115] Fargnoli MC, Orlow SJ, Semel-Concepcion J, Bolognia JL. Clinicopathologic findings in the Bannayan-Riley-Ruvalcaba syndrome. Archives of dermatology Oct;132(10): [116] Longy M, Coulon V, Duboue B, David A, Larregue M, Eng C, et al. Mutations of PTEN in patients with Bannayan-Riley-Ruvalcaba phenotype. J. Med. Genet Nov;35(11): [117] Gorlin RJ, Cohen MM, Jr., Condon LM, Burke BA. Bannayan-Riley-Ruvalcaba syndrome. American journal of medical genetics Oct 1;44(3): [118] Zhou XP, Marsh DJ, Hampel H, Mulliken JB, Gimm O, Eng C. Germline and germline mosaic PTEN mutations associated with a Proteus-like syndrome of hemihypertrophy, lower limb asymmetry, arteriovenous malformations and lipomatosis. Human molecular genetics Mar 22;9(5): [119] Kurlawalla-Martinez C, Stiles B, Wang Y, Devaskar SU, Kahn BB, Wu H. Insulin hypersensitivity and resistance to streptozotocin-induced diabetes in mice lacking PTEN in adipose tissue. Molecular and cellular biology Mar;25(6): [120] Komazawa N, Matsuda M, Kondoh G, Mizunoya W, Iwaki M, Takagi T, et al. Enhanced insulin sensitivity, energy expenditure and thermogenesis in adipose-specific Pten suppression in mice. Nature medicine Nov;10(11): [121] Stiles BL, Kuralwalla-Martinez C, Guo W, Gregorian C, Wang Y, Tian J, et al. Selective deletion of Pten in pancreatic beta cells leads to increased islet mass and resistance to STZ-induced diabetes. Molecular and cellular biology Apr;26(7): [122] Kwon CH, Luikart BW, Powell CM, Zhou J, Matheny SA, Zhang W, et al. Pten regulates neuronal arborization and social interaction in mice. Neuron May 4;50(3): [123] Butler MG, Dasouki MJ, Zhou XP, Talebizadeh Z, Brown M, Takahashi TN, et al. Subset of individuals with autism spectrum disorders and extreme macrocephaly associated with germline PTEN tumour suppressor gene mutations. Journal of medical genetics Apr;42(4): [124] Song MS, Salmena L, Pandolfi PP. The functions and regulation of the PTEN tumour suppressor. Nature reviews Molecular cell biology May;13(5):

257 The Functions and Roles of the Unique Tumor Suppressor Gene PTEN 251 [125] Image interpretation session. Mycetoma of the right foot. Radiographics Jan;12(1): [126] Nelen MR, van Staveren WC, Peeters EA, Hassel MB, Gorlin RJ, Hamm H, et al. Germline mutations in the PTEN/MMAC1 gene in patients with Cowden disease. Hum. Mol. Genet Aug;6(8): [127] Olschwang S, Serova-Sinilnikova OM, Lenoir GM, Thomas G. PTEN germ-line mutations in juvenile polyposis coli. Nat. Genet Jan;18(1):12-4. [128] Marsh DJ, Dahia PL, Caron S, Kum JB, Frayling IM, Tomlinson IP, et al. Germline PTEN mutations in Cowden syndrome-like families. Journal of medical genetics Nov;35(11): [129] Kurose K, Araki T, Matsunaka T, Takada Y, Emi M. Variant manifestation of Cowden disease in Japan: hamartomatous polyposis of the digestive tract with mutation of the PTEN gene. Am. J. Hum. Genet Jan;64(1): [130] Celebi JT, Tsou HC, Chen FF, Zhang H, Ping XL, Lebwohl MG, et al. Phenotypic findings of Cowden syndrome and Bannayan-Zonana syndrome in a family associated with a single germline mutation in PTEN. J. Med. Genet May;36(5): [131] Raizis AM, Ferguson MM, Nicholls DT, Goodisson DW. A novel 5' (4041insA) mutation in a patient with numerous manifestations of Cowden disease. J. Invest. Dermatol Mar;114(3): [132] Fackenthal JD, Marsh DJ, Richardson AL, Cummings SA, Eng C, Robinson BG, et al. Male breast cancer in Cowden syndrome patients with germline PTEN mutations. J. Med. Genet Mar;38(3): [133] Zhou XP, Waite KA, Pilarski R, Hampel H, Fernandez MJ, Bos C, et al. Germline PTEN promoter mutations and deletions in Cowden/Bannayan-Riley-Ruvalcaba syndrome result in aberrant PTEN protein and dysregulation of the phosphoinositol-3- kinase/akt pathway. Am. J. Hum. Genet Aug;73(2): [134] Eng C. PTEN: one gene, many syndromes. Hum. Mutat Sep;22(3): [135] Sutphen R, Diamond TM, Minton SE, Peacocke M, Tsou HC, Root AW. Severe Lhermitte-Duclos disease with unique germline mutation of PTEN. Am. J. Med. Genet Feb 12;82(4): [136] Breathnach CS. The stability of the fetal oxygen environment. Ir. J. Med. Sci Jul;160(7):

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259 In: Tumor Suppressor Genes ISBN: Editors: Mehmet Gunduz and Esra Gunduz 2013 Chapter 12 Functions of the Tumor Suppressor Gene APC Tomoki Yamatsuji, Munenori Takaoka, Takuya Fukazawa and Yoshio Naomoto Department of General Surgery, Kawasaki Medical School Keywords: APC: adenomatous polyposis coli; FAP: familial adenomatous polyposis; colorectal cancer Introduction Adenomatous polyposis coli (APC) is a multifunctional protein commonly mutated in colon cancer. Mutations in adenomatous polyposis coli are associated with most colon cancers however the precise mechanisms how these mutations induce neoplasms are not cleared [1]. Over twenty years ago, mutations in the APC gene were identified as the causative lesion in autosomal dominant colorectal cancer or familial adenomatous polyposis coli (FAP)[2]. In addition, over the eighty percent of sporadic colon cancers have been reported to possess truncating mutations of APC gene [3]. Following these great discoveries, APC is recognized as one of the most important tumor suppressor genes, and the mutations in the APC gene may directly result in colorectal cancer [4]. In this chapter, we will start describing the history of the APC, cloning, location and structure of the gene. Later we will discuss the protein function of APC, oncogenic mechanisms of APC mutations, interaction for many cellular molecules, the specific relationship to the Wnt signaling pathway, and a newly discovered candidate for gene targeted therapy. Phone fax , ynaomoto@med.kawasaki-m.ac.jp.

260 254 Tomoki Yamatsuji, Munenori Takaoka, Takuya Fukazawa et al. Cloning of APC Gene In terms of a discovery of APC gene, in 1987 its location was firstly identifiedon Chromosome 5, and in 1991 cloned simultaneously and independently by two groups [5-12]. According to their findings, the APC gene has an 8,538 bp open reading frame, and consists of 15 transcribed exons [8 10], on the long (q) arm of chromosome 5 between positions 21 and 22, from base pair 112,118,468 to base pair 112,209,532. The APC gene has been shown to contain an internal ribosome entry site. This gene is expressed in a variety of fetal and adult tissues, including mammary and colorectal epithelium [8]. It encodes for a 312-kDa protein, 2,843 amino acids long [8]. Inactivation of the APC gene product constitutes the initial step in the development of colorectal cancer in Familial adenomatous polyposis (FAP). The protein sequence does not contain transmembrane regions or nuclear targeting signals, suggesting cytoplasmic localization. In researchers search for APC gene, Kinzler et al. identified several genes such as FER (176942), MCC (159350), SRP19 (182175), and TB2 (REEP5; ), in addition to the APC gene itself within a 5.5-Mb region of DNA linked to FAP. All genes were expressed in normal colonic mucosa [6]. The APC gene product was predicted to contain coiled-coil regions and was expressed in a wide variety of tissues. Joslyn et al. identified 3 genes within small deleted regions on chromosome 5q12 found in 2 unrelated patients with FAP [12]. One of these, termed DP2.5, was found to be the APC gene [7], and the other 2 genes were SRP19 and DP1 (REEP5) [12]. Northern blot analysis identified a 10-kb APC mrna [7]. Independently, Hampton et al. isolated 2 overlapping YACs containing the MCC gene; one of the YACs also included the complete APC gene [13]. In 1993, cdna clones representing transcripts expressed in human fetal brain and coding for the 5-prime end of the APC gene were isolated and sequence analyses revealed an alternative 5-prime untranslated region comprising at least 103 bp. This finding suggested that 2 APC-specific promoter elements exist, giving rise to 2 different untranslated regions (UTRs) [14]. Within the alternative UTR, 3 additional AUG codons was identified in the location of 5- prime to the intrinsic APC initiation site, suggesting that these codons may be relevant for the translational regulation of APC gene expression [14]. Horii et al. noted that transcriptional initiation of APC occurs at 3 sites in 2 distinct nontranslated exons at the 5-prime end of the gene [15]. Studies of transcripts from human colorectal tumor cell lines suggested the presence of mutations in the transcriptional control region. At least 5 different forms of 5-prime noncoding sequences, which were generated by alternative splicing, were detected [15]. Interestingly, the splicing mechanism appeared to be regulated in a tissue-specific fashion, and 1 transcript, expressed exclusively in brain, contained an extra exon. Gene Structure of APC As mentioned above, the APC gene contains 15 exons [7], including an oligomerization domain, an armadillo repeat-domain, a 15- or 20-residue repeat domain for binding to betacatenin, serine alanine methionine proline (SAMP) repeats for axin binding, a basic domain

261 Functions of the Tumor Suppressor Gene APC 255 for microtubule binding and a C-terminal domain binding to EB1 and DLG proteins [16]. Exon 15 is large and comprises more than three quarters of the coding region of the gene. Figure 1 reveals the schematic representation of APC protein and binding domains. The most important functional domains of the APC gene appear to be the first SAMP(axin binding) repeat at codon 1580 [17] and the first, second and third 20-amino acid repeats (20AARs) involved in beta-catenin binding and degradation. Sulekova and Ballhausen identified a novel coding exon of the APC gene, which was located 1.6-kb downstream from exon 10 [18]. To that point, this 54-bp exon was the smallest coding exon in the gene and termed exon 10A.. It is alternatively spliced and inserted inframe into mature transcripts; it gives an APC protein with an additional 18 amino acids. APC exon 10A flanking sequences were presented so that this exon could be included in mutation screening procedures [18]. Xia et al. described an alternatively spliced APC transcript which had not been reported previously [19]. Within this transcript, they found an evolutionarily conserved but previously unidentified exon between the known exons 10 and 11. The exon contains a heptad repeat motif [18]. Karagianni et al. identified an alternatively spliced APC transcript in mouse embryonic stem cells and colon tissue [20]. The transcript contains an untranslated exon, which was designated as exon N. Transcripts bearing exon N, which were spliced to either exon 1 or exon 2, were detected in all mouse tissues examined. A promoter region within exon N has features of a housekeeping gene, including high average GC content and lack of CAAT and TATA boxes. The researchers mapped the promoter about 40 kb upstream of the initiating methionine, and transient transfection experiments showed strong promoter activity [20]. Figure 1. Schematic presentation of APC protein. The 2,843 amino acid APC protein and its functional sites are indicated. The N-terminal region has a domain that regulates APC oligomerization. Repeated sequences with similarity to the Drosophila armadillo protein and its vertebrate homolog β-catenin (armadillo repeats) are localized in the N-terminal side of APC. Multiple independent 20 amino acid repeats that mediate binding to β-catenin and several binding sites for the Axin protein (SAMP repeats) are localized in the center of APC. The C-terminal side of APC has a basic region that is involved in microtubule (MT) binding and interactions with the proteins EB1 and hdlg. Modified from Reference 41. Most domains of this protein are solved structurally and exhibit high intrinsic disorder and flexibility as a monomer, and a low content of stable secondary structure. Thus it is a member of the intrinsically unstructured proteins.

262 256 Tomoki Yamatsuji, Munenori Takaoka, Takuya Fukazawa et al. Function of APC APC is expressed constitutively within the normal colonic epithelium. The APC gene product is a 310-kDa-homodimeric protein, which is localized in the cytoplasm and the nucleus. APC is a multifunctional protein involved in several cellular life processes [6, 21]. Wild-type APC are critical to cytoskeletal integrity, cellular adhesion and Wnt signaling. In in vitro and in vivo studies it has been shown that APC inhibits the canonical Wnt signal pathway, which is essential for tumorigenesis, cell multiplication and differentiation, development and homeostasis of a variety of tissues. Through intracellular mechanism, APC has a master role in the process of suppressing the classic Wnt signal pathway [22]. It is discovered that APC also has an important role in other basic vital activities, including the adhesion and migration of cells, organization of actin and microtubule skeleton network, spindle formation and chromosome segregation. Deactivation of APC make cell lose adhesion and cause the tumorigenesis. APC affects the cell adhesion through accommodating the distributions of β-catenin and E-cadherin between the cytoplasm and the cell membrane [23]. Wild-type APC interact with a tumor suppressor protein, DLG, which regulates cell cycle progression from the Go/G1 to the S phase of the cell cycle [24]. Recently it has been reported that APC human disc large complex is responsible for the above-mentioned effect in cell cycle in a manner without relying on the effect of β-catenin. In addition, APC may act as a negative regulator of β-catenin signaling. APC combines with GSK3-β and actin to form a complex that promotes β-catenin phosphorylated and consequently ubiquitin-dependent degradation of β-catenin. The β-catenin/tcf4 complex regulates the proto-oncogene and cell cycle regulator c-myc, the G1/S-regulating cyclin D1, the gene encoding the matrix-degrading metalloproteinase, matrilysin, the AP-1 transcription factors c-jun and fra-1 and the urokinase-type plasminogen activator receptor gene [25, 26]. Diseases Caused by APC Mutation It is clear that APC has a key role in number of vital life processes. Mutation of APC gene results in occurrences of various kinds of diseases and produces incomplete APC protein. The mutation is not only related to the familial adenomatous polyposis (FAP) but also to sporadic colorectal cancer [27]. Familial adenomatous polyposis (FAP) is one of the autosomal-dominant inherited diseases characterized by the development of small adenomatous polyps widely throughout the large intestine, which is associated with a very high risk of colorectal cancer. The number of polyps increases with the growing age accompanied by many characteristics of different extracolonic manifestations. Among them, benign tumors usually progress to carcinomas if they are not removed in time. FAP was first reported in 1925 and scientists had described in detail the pathology characteristics. FAP is traditionally diagnosed based on the presence of more than 100 adenomatous colorectal polyps. Genetic testing is now standard for the diagnosis of FAP. FAP accounts for less than 1% of all colorectal cancer cases seen in practice. Polyps often appear in teenagers or young people in their third decade of life. The risk of cancer usually depends on the number of adenomatous polyps. This leads to an almost 100% chance of malignant transformation in at least one of these polyps by the fifth decade.

263 Functions of the Tumor Suppressor Gene APC 257 At present, FAP is diagnosed by colonoscopy and the commonly used treatment method is surgery that involves total colectomy and proctomucosectomy with ileal pouch-anal anastomosis. The majority of FAP patients (over 70%) develop extra- colonic manifestations [28]. Most extracolonic manifestations have little clinical significance, but some lesions can cause serious complications and even lead to death. The most common extracolonic manifestation is congenital hypertrophy of the retinal pigment epithelium (CHRPE) found in 60 90% of FAP patients. Desmoids are benign tumors of the connective tissues that can lead to life-threatening complications through their sheer size and impingement on vital structures. They occur in 5 10% of FAP. It has been discovered that the CHRPE and desmoids are related to the mutation at definite region of the APC gene [29]. Moreover, in the mutation sites of APC database, more than 300 mutations in the APC gene have been identified in families with classic and attenuated types of familial adenomatous polyposis. Most are insertion, deletions and nonsense mutations that lead to frame shifts or premature stop codons resulting in truncation of the APC gene product. The most common mutation in familial adenomatous polyposis is a deletion of five bases in the gene [30]. The majority of the serious clinical symptom representations are caused by minor defect in codon Mutation at this site may cause the early development of colorectal polyps. The genetic basis of FAP relies on the genetic inheritance mutation of APC gene [31, 32]. Colon cancer is one of the most common malignancies in the USA and Western Europe, and is also one of the main factors of disease incidence and fatality caused by cancers throughout the world [33]. The great majority (80%) of patients with colorectal cancer have sporadic disease with no evidence of having inherited the disorder. In the remaining 20% a potentially definable genetic component exists. In the past decade germ line genetic mutations conferring high lifetime risk of colorectal cancer in carries have been found, accounting for 5 6% of all colorectal cases [34]. The most common mutation in colon cancer is inactivation of APC. Genetics studies using mouse model showed that mutation of APC gene has a close relationship with intestinal tumorigenesis. Somatic mutations in APC gene has been detected in the majority of colorectal cancers. At least four genetic changes appear sequentially to ensure colorectal cancer evolution, including an oncogene (KRAS) and three anti-oncogenes (APC, SMAD and p53) [35]. The functional defect of APC is precisely the triggering factor of these cascade changes, which ultimately lead to the malignant transformation of large intestine. The mutation of APC gene is one of the early events in the process of sporadic colorectal cancer. In all, in 100 of sporadic colorectal cancers carry APC gene mutation and a similar frequency was reported in colorectal adenomas [36]. Relationship between Wnt Signaling and the APC Tumor-Suppressor Gene in Activating the β-catenin APC is a classical tumor suppressor protein that plays a dominant role in Wnt signaling, in part by regulating the degradation of β-catenin. Wnt signals influence the stability of a protein complex containing β-catenin, conductin and GSK3 (glycogen synthase kinase 3).

264 258 Tomoki Yamatsuji, Munenori Takaoka, Takuya Fukazawa et al. In the absence of Wnt or the presence of wild-type APC protein, β-catenin is degraded. In the presence of Wnt, or the absence of APC, β-catenin target genes-including c-myc are expressed. Consequently, Myc expression induced the expression of the polyamine ornithine decarboxylase (ODC) which is a proto-oncogene. The APC gene product indirectly regulates transcription of several critical cell-proliferation genes, through its interaction with the transcription factor β-catenin [37]. APC binding to β-catenin leads to ubiquitin-mediated β- catenin destruction; loss of APC function increases transcription of β-catenin targets. When APC does not have an inactivating mutation, β-catenin does. These mutations can be inherited, or arise sporadically, often as the result of mutations in other genes that produce chromosomal instability. A mutation on APC or β-catenin must be followed by other mutations to lead to tumorigenesis; however, in carriers of an APC inactivating mutations, the risk of colorectal cancer by age 40 is almost 100% [25]. Gene Therapy and Targeted Therapy Hargest et al. have lipofected APC gene into the mouse colon and showed that transfection of APC gave prolonged high-level expression of the transgene, an important basis for gene therapy [38]. More, it has been reported that activation of PPAR inhibits APCdependent suppression of colon carcinogenesis and suppression of β-catenin inhibits the neoplastic growth of APC-mutant colon cancer [39, 40]. APC remains an attractive target for therapeutic intervention because its mutation is a common and early event in the continuum of colorectal tumor progression. Therefore, APC and its related genes are attractive targets for the design of therapeutic and chemopreventive strategies for colorectal cancer patients. Further investigation into the biology, biochemistry, and genetics of APC will no doubt result in the realization of these therapies Conclusion APC gene was found about twenty years ago classified as a tumor suppressor gene, those mutations are associated with most colon cancers although how these mutations affect the development of cancer was not fully understood. In this chapter, we have learned the history of the APC gene, cloning, location and structure of the gene. After that we described the protein function of APC, oncogenic mechanisms of its mutations, involvement in the Wnt signaling pathway, and we have discussed a novel target of gene therapy. APC is a traditional tumor suppressor gene. It would be involved in oncogenic signaling of the most colorectal cancers in the world. Therefore it should be a critical therapeutic target in the near future.

265 Functions of the Tumor Suppressor Gene APC 259 References [1] Bahmanyar S, Nelson WJ, Barth AI. Role of APC and its binding partners in regulating microtubules in mitosis. Adv. Exp. Med. Biol. 2009;656: [2] Spirio L, Otterud B, Stauffer D, Lynch H, Lynch P, Watson P, et al. Linkage of a variant or attenuated form of adenomatous polyposis coli to the adenomatous polyposis coli(apc) locus. Am. J. Hum. Genet. 1992; 51: [3] Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell 1996;87: [4] Fearon ER. Molecular genetics of colorectal cancer. Annu Rev. Pathol. 2011;6: [5] Bodmer WF, Bailey CJ, Bodmer J, et al. Localization of the gene for familial adenomatous polyposis on chromosome 5. Nature 1987;328: [6] Kinzler KW, Nilbert MC, Su LK, et al. Identification of FAP locus genes from chromosome 5q21. Science 1991;253: [7] Groden J, Thliveris A, Samowitz W, et al. Identification and characterization of the familial adenomatous polyposis coli gene. Cell 1991;66: [8] Van Es JH, Giles RH, Clevers HC. The many faces of the tumor suppressor gene APC. Exp. Cell Res. 2001;264: [9] Fearnhead NS, Britton MP, BodmerWF.TheABCof APC. Hum. Mol. Genet. 2001;10: [10] Foulkes WD. A tale of four syndromes: Familial adenomatous polyposis, Gardner syndrome, attenuated APC and Turcot syndrome. Q. J. Med. 1995;88: [11] Nishisho, I., Nakamura, Y., Miyoshi, Y., Miki, Y., Ando, H., Horii, A., Koyama, K., Utsunomiya, J., Baba, S., Hedge, P., Markham, A., Krush, A. J., Petersen, G., Hamilton, S. R., Nilbert, M. C., Levy, D. B., Bryan, T. M., Preisinger, A. C., Smith, K. J., Su, L.-K., Kinzler, K. W., Vogelstein, B.Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients. Science 253: , [12] Joslyn, G., Carlson, M., Thliveris, A., Albertsen, H., Gelbert, L., Samowitz, W., Groden, J., Stevens, J., Spirio, L., Robertson, M., Sargeant, L., Krapcho, K., Wolff, E., Burt, R., Hughes, J. P., Warrington, J., McPherson, J., Wasmuth, J., Le Paslier, D., Abderrahim, H., Cohen, D., Leppert, M., White, R. Identification of deletion mutations and three new genes at the familial polyposis locus. Cell 66: , [13] Hampton, G. M., Ward, J. R. T. J., Cottrell, S., Howe, K., Thomas, H. J. W., Ballhausen, W. G., Jones, T., Sheer, D., Solomon, E., Frischauf, A.-M., Bodmer, W. F.Yeast artificial chromosomes for the molecular analysis of the familial polyposis APC gene region. Proc. Nat. Acad. Sci. 89: , [14] Lambertz, S., Ballhausen, W. G. Identification of an alternative 5-prime untranslated region of the adenomatous polyposis coli gene. Hum. Genet. 90: , [15] Horii, A., Nakatsuru, S., Ichii, S., Nagase, H., Nakamura, Y.Multiple forms of the APC gene transcripts and their tissue-specific expression. Hum. Molec. Genet. 2: , [16] Polakis P. The adenomatous polyposis coli (APC) tumor suppressor. Biochim. Biophys. Acta Jun 7;1332(3):F [17] Smits R, Kielman MF, Breukel C, Zurcher C, Neufeld K, Jagmohan-Changur S, Hofland N, van Dijk J, White R, Edelmann W, Kucherlapati R, Khan PM, Fodde R.

266 260 Tomoki Yamatsuji, Munenori Takaoka, Takuya Fukazawa et al. Apc1638T: a mouse model delineating critical domains of the adenomatous polyposis coli protein involved in tumorigenesis and development. Genes Dev May 15;13(10): [18] Suleková Z, Ballhausen WG. A novel coding exon of the human adenomatous polyposis coli gene. Hum. Genet Oct;96(4): [19] Xia, L., St. Denis, K. A., Bapat, B.Evidence for a novel exon in the coding region of the adenomatous polyposis coli (APC) gene. Genomics 28: , [20] Karagianni, N., Ly, M.-C., Psarras, S., Chlichlia, K., Schirrmacher, V., Gounari, F., Khazaie, K.Novel adenomatous polyposis coli gene promoter is located 40 kb upstream of the initiating methionine. Genomics 85: , [21] Goss KH, Groden J. Biology of the adenomatous polyposis coli tumor suppressor. J. Clin. Oncol. 2000;18: [22] Clevers H. Wnt/beta-catenin signaling in development and disease. Cell. 2006;127: [23] Ilyas M, Tomlinson IP. The interactions of APC, E-cadherin and beta-catenin in tumour development and progression. The Journal of pathology. 1997;182: [24] Ishidate T, Matsumine A, Toyoshima K, Akiyama T. The APC-hDLG complex negatively regulates cell cycle progression from the G0/G1 to S phase. Oncogene. 2000;19: [25] Fu Y, Zheng S, An N, Athanasopoulos T, Popplewell L, Liang A, et al. beta-catenin as a potential key target for tumor suppression. International journal of cancer Journal international du cancer. 2011;129: [26] Mann B, Gelos M, Siedow A, Hanski ML, Gratchev A, Ilyas M, et al. Target genes of beta-catenin-t cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas. Proc. Natl. Acad. Sci. U S A. 1999;96: [27] Rustgi AK. The genetics of hereditary colon cancer. Genes & development. 2007;21: [28] Lipton L, Tomlinson I. The genetics of FAP and FAP-like syndromes. Familial cancer. 2006;5: [29] Caspari R, Olschwang S, Friedl W, Mandl M, Boisson C, Boker T, et al. Familial adenomatous polyposis: desmoid tumours and lack of ophthalmic lesions (CHRPE) associated with APC mutations beyond codon Human molecular genetics. 1995;4: [30] Zeichner SB, Raj N, Cusnir M, Francavilla M, Hirzel A. A De Novo Germline APC Mutation (3927del5) in a Patient with Familial Adenomatous Polyposis: Case Report and Literature Review. Clinical Medicine Insights Oncology. 2012;6: [31] Caspari R, Friedl W, Mandl M, Moslein G, Kadmon M, Knapp M, et al. Familial adenomatous polyposis: mutation at codon 1309 and early onset of colon cancer. Lancet. 1994;343: [32] Wachsmannova-Matelova L, Stevurkova V, Adamcikova Z, Holec V, Zajac V. Different phenotype manifestation of familial adenomatous polyposis in families with APC mutation at codon Neoplasma. 2009;56: [33] Siegel R, Ward E, Brawley O, Jemal A. Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA: a cancer journal for clinicians. 2011;61:

267 Functions of the Tumor Suppressor Gene APC 261 [34] Dionigi G, Bianchi V, Rovera F, Boni L, Annoni M, Castano P, et al. Genetic alteration in hereditary colorectal cancer. Surgical oncology. 2007;16 Suppl 1:S11-5. [35] Senda T, Shimomura A, Iizuka-Kogo A. Adenomatous polyposis coli (Apc) tumor suppressor gene as a multifunctional gene. Anatomical science international. 2005;80: [36] Narayan S, Roy D. Role of APC and DNA mismatch repair genes in the development of colorectal cancers. Molecular cancer. 2003;2:41. [37] Myant K, Sansom OJ. Wnt/Myc interactions in intestinal cancer: partners in crime. Experimental cell research. 2011;317: [38] Hargest R, Eldin A, Williamson R. Gene therapy for familial adenomatous polyposis. Prolonged expression of the adenomatous polyposis coli gene after lipofection into mouse colon in vivo. Adv. Exp. Med. Biol. 1998;451: [39] Girnun GD, Smith WM, Drori S, Sarraf P, Mueller E, Eng C, et al. APC-dependent suppression of colon carcinogenesis by PPARgamma. Proc. Natl. Acad. Sci. U S A. 2002;99: [40] Foley PJ, Scheri RP, Smolock CJ, Pippin J, Green DW, Drebin JA. Targeted suppression of beta-catenin blocks intestinal adenoma formation in APC Min mice. J. Gastrointest. Surg. 2008;12: [41] Fearon ER. Molecular Genetics of Colorectal Cancer. Annu. Rev. Pathol. Mech. Dis :

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269 In: Tumor Suppressor Genes ISBN: Editors: Mehmet Gunduz and Esra Gunduz 2013 Chapter 13 Structure and Function of the Tumor Suppressor Gene p16 Zeynep Tarcan 1, Catherine Moroski Erkul 1, Bunyamin Isik 2, Esra Gunduz 1 and Mehmet Gunduz 1, Departments of 1 Medical Genetics and 3 Family Medicine, Faculty of Medicine, Turgut Ozal University, Istanbul, Turkey Abstract Since its identification in 1993, the P16 INK4a gene has been one of the most extensively studied tumor suppressors. As one of the three genes located in the unique INK4b-ARF-INK4a locus, its dysregulation is a common event in the initiation and progression of many different cancers. In some cancer types, such as pancreatic carcinoma, it is downregulated in up to 98% of cases. Thus it takes a place alongside P53 as an essential diagnostic tool and thereapeutic target in disease. The role of P16INK4a in cell cycle regulation, primarly through its inhibition of CDK4/6-pRb, is the most studied and best understood function of this mult-tasking molecule. It also has well documented roles in mechansisms of senescence, apoptosis, anoikis and the DNA damage response. Its role in the inhibition of inflammatory processes has also revealed a potential role in the suppression of rheumatoid arthritis. The aim of this review is to give a general overview of the various functions that P16 INK4a plays in oncogenic transformation and cancer progression and a brief look at its potential as a therapeutic target. 1. Introduction Cancer may be thought of as the culmination of a multitude of genetic and biochemical aberrations that result in unrestricted cell growth. Cellular proliferation is a complex and tightly controlled process that relies on the proper expression, function, and co-ordination of Corresponding author: Mehmet Gunduz, MD, PhD, Department of Medical Genetics, Faculty of Medicine, Turgut Ozal University, Turkey. Anadolu Bulvari 16A Gimat Ankara, Turkey. mgunduz@turgutozal.edu.tr.

270 264 Zeynep Tarcan, Catherine Moroski Erkul, Bunyamin Isik et al. many genes and proteins throughout the entire cell cycle. There are a number of cell cycle checkpoints that allow cells to pause, make sure that the current phase will be completed successfully and to ensure that the raw materials required to complete the next phase are available. This also allows replicating cells to repair any DNA damage that may have occurred or, in cases when damage is too ovewhelming, to undergo programmed cell death. These checkpoints are integral to maintaining proper cellular function and genomic integrity and for guarding against the proliferation of cells with serious genomic defects. The ability to escape these checks and balances and continue proliferating even in the presence of significant damamge is one of the hallmark features of cancer cells [1]. Cyclins are a group of proteins whose expression and activity are triggered by mitogenic stimuli and are necessary for entry into and progression through the cell cycle. They are regulated by both changes in gene expression and rates of degradation. There are several different cyclin proteins (Cyclin A, B, D and E) and each has unique characteristics and patterns of expression throughout the cell cycle. Another group of proteins integral to the cell cycle are cyclin dependent kinases (CDKs), a family of serine/threonine protein kinases whose concentrations remain constant but whose activity is regulated by cell cycle phasespecific changes in cyclin concentrations. Cyclins bind to specific CDK partners to form protein complexes. These cyclin-cdk complexes, which are activated by CDK-activating kinases (CAKs) and inhibited by cyclin dependent kinase inhibitors (CDKIs), together form functional holoenzymes that drive cells through cell cycle transitions [2]. In normal cells, these complexes contain two additional components, PCNA and P21 [3]. Among the cyclin family of proteins, Cyclin D1 has emerged as a gene with significant oncogenic potential. Depending on cell type, Cyclin D1 forms complexes with CDK4 and/or CDK6 in G 1 of the cell cycle. These complexes, as well as the cyclin E-CDK2 holoenzyme, are invovled in the transition from G1 to S phase, a critical regulatory point during the cell cycle [4]. Cyclin E and CDK2 are expressed ubiquitously and their expression increases at the later stages of G1 [5]. The G 1 to S phase transition is activated in part by the phosphorylation of retinoblastoma protein (prb) by the cyclin D-CDK4/6 and cyclin E-CDK2 complexes. The phosphorylation of prb results in the release of prb-bound E2F transcription factors. These transcription factors are then free to activate (via E2F-1, 2 and 3) or repress (via E2F-4 and 5) the expression of genes whose products are required for entry into S phase and commitment to mitosis [2, 6]. Also essential to cell cycle control, in this case for the negative regulation of growth, are CDK inhibitors. One CDK inhibitor in particular, P16 INK4a (inhibitor of CDK4 variant A) is the focus of this chapter. P16 INK4a and cyclin D competitively bind to CDK4/6. Thus when P16 INK4a is bound to CDK4/6, cyclin D cannot bind CDK4/6. In the absence of the cyclin D- CDK4/6 complex, E2F transcription factors remain bound to hypophosphorylated prb and together they are translocated to the cytoplasm. Cell cycle progression is thereby prevented. However, in the absence of prb, cyclin E expression is increased and the inhibition of cyclin D-CDK4/6 by P16 INK4a does not prevent progression to S phase [5]. Since these cyclins, CDKs and their regulators are central to cellular proliferation, it is not surprising that they play an important role in the process of tumor development. This also suggests they may have potential for use as clinical diagnostic markers and therapeutic targets in cancer [7]. The P16 INK4a gene is located on human chromosome 9p21 region [8-10]. In addition to its function in cell cycle regulation, P16 INK4a (also referred to as CDKN2A, MTS1, and INK4a)

271 Structure and Function of the Tumor Suppressor Gene p has a critical role in cellular senescence, anoikis, apoptosis, and aging. It is a member of the INK4 family of genes which also includes P15 INK4b, P18 INK4c, and P19 INK4d. All INK4 family members have structural similarity and can inhibit cyclin D kinase activity, but they also have unique biological functions [5, 11]. P16 INK4a has been identified as a tumor suppressor based on its germline and somatic inactivation and abberant expression (both downregulation and overexpression) in many tumor types [12]. Several types of P16 INK4a mutations have been identified in tumors including homozygous deletions, promoter hypermethylation, loss of heterozygosity, and point mutations; however it is mainly via homozygous deletion of the gene or promoter methylation that results in P16 INK4a inactivation [12-14]. Consequently, the clinical assessment of P16INK4a expression is an important aspect of cancer diagnosis, prognosis and therapy [15, 16]. 2. CDK Inhibitors There are a number of positive and negative regulators involved in cell cycle control. In addition to P16 INK4a, some of the important negative regulators in G 1 include the other INK4 family proteins P15 INK4b, P18 INK4c, and P19 INK4d as well as the Cip/Kip family proteins P21 (also known as WAF1, CAP20 and CIP1), P27 (KIP1), and P57 (KIP2). P21 and P27 can inhibit progression through G 1 in at least two points of the pathway; one is through steric inhibition of CAK-activation via binding to cyclin-cdk holoenzymes and another is by inhibiting the phosphorylation of prb by CAK-activated cyclin-cdk holoenzymes [2]. It should be noted that P21 is involved in both positive and negative regulation of cell cycle progression. Along with cyclin, CDK and PCNA, P21 is an integral component of G 1 to S phase transition-promoting complexes. When all of these components exist in the complex at a 1:1 ratio, the effect of P21 on growth is positive. If the ratio of P21 increases in relation to the other components in the complex, it then acts as a negative regulator [2]. In addition to cell cycle transition points, these two families of proteins have functions in transcription, the DNA damage response, apoptosis and other processes [17]. Among the positive regulators of the cell cycle are, of course, the cyclins and CDKs [16, 18]. 3. The INK4 Gene Family The INK4 family is made up of four proteins, P16 INK4a, P15 INK4b, P18 INK4c, and P19 INK4d. P16 INK4a and P15 INK4b are encoded by the INK4b-ARF-INK4a locus located on chromosome 9p21 [19] (Figure 1). This locus codes for three different proteins, P16 INK4a, P15 INK4b and P19 ARF (not to be confused with P19 INK4d ). P19 ARF /P14 ARF, or simply ARF, is also a tumor suppressor acting in the p53 pathway and is likely regulated by similar (or even the same) mechanisms as P16 INK4a and P15 INK4b (Figure 2). However, this protein is completely unrelated to the INK4 family of CDK inhibitors [20]. Also, while the INK4a nd INK4b proteins are highly conserved across many species this is not the case for ARF, despite the fact that they share the same genetic locus and have all been implicated in tumorigenesis to one degree or another [20]. The genetic locus of P18 INK4c is found at chromosome 1p32, and that of P19 INK4d is located at 19p13. The P15 INK4b protein contains 138 amino acids and has a

272 266 Zeynep Tarcan, Catherine Moroski Erkul, Bunyamin Isik et al. MW of 14.7-kDa. P18 INK4c is composed of 168 amino acids and has a MW of 18.1-kDa and P19 INK4d contains 166 amino acids and as a MW of 17.6-kDa (19) (Figure 3). Figure 1. The INK4b-ARF-INK4a locus. The same gene locus encodes three proteins, P15 INK4b, P16 INK4a, and P14 ARF. P15 INK4b has only two exons, whereas P16 INK4a and P14 ARF have three exons; the latter two genes share exons 2 and 3, but P16 INK4a is coded by exon 1α and P14 ARF is coded by exon 1β. Figure 2. Function of INK/ARF moleculer pathways in normal and tumor cells. P16 INK4a binds and inactivates CDK4/6 complexes. prb cannot be phosphorylated and this results in cell cycle arrest in normal cells. In tumor cells, P16 INK4a binds and activates CDK4/6-cyclin D. Thus, prb is phosphorylated and the E2F transcritption factor is released and cell cycle progression proceeds.

273 Structure and Function of the Tumor Suppressor Gene p Figure 3. INK4 Family members. Figure 4. Function of INK/ARF moleculer pathways in normal and tumor cells. P16 INK4a binds and inactivates CDK4/6 complexes. prb cannot be phosphorylated and this results in cell cycle arrest in normal cells. In tumor cells, P16 INK4a binds and activates CDK4/6-cyclin D. Thus, prb is phosphorylated and the E2F transcritption factor is released and cell cycle progression proceeds. Despite similar structure and inhibitory activity of CDK4 and CDK6, only P16 INK4a has clearly demonstrated a tumor suppressor capacity, primarily through its role in cell cycle control (Figure 4). Somatic mutations and altered protein expression (downregulation and overexpression) have been detected in a multitude of different cancers. In fact, after P53, P16 INK4a is one of the most frequently altered genes in human cancer. Thus, it is curious that the other INK4 family members have not been identified as having a more important role in the pathogenesis of cancer. While it is true that mutations in the other family members have been identified in scattered cases of cancer, they do not even begin to compare with the extent

274 268 Zeynep Tarcan, Catherine Moroski Erkul, Bunyamin Isik et al. of P16 INK4a aberrations in cancer. This suggests that one or more of P16 INK4a s other functions may be integral to its status as a tumor suppressor. 4. Discovery of P16 INK4a In 1993, Xiong et al. reported a 16-kDa protein associated with CDK4 in SV40 (simian virus 40) transformed human diploid fibroblasts [3]. It was given the name P16 INK4A by this group due to its inhibitory effect on CDK4. Around the same time, researchers were investigating the existence of a melanoma susceptibility gene at the 9p21 locus. Two different groups that isolated and studied P16 INK4A named it MTS1 (multiple tumor suppressor 1) and CDK4I (CDK4 inhibitor). It was given yet another name, CDKN2, for the human genome project [19]. Today, the gene is typically referred to either as P16INK4A (or simply INK4A) or CDKN2A. Since its initial discovery, P16 INK4a has become a widely studied gene in human cancer. P16 INK4a is one of the most frequently mutated genes in human cancers [21]. It is estimated that P16 INK4a is mutated in about 20% of breast cancer, 65% of non-small cell lung carcinoma, 30% of colorectal cancer, 60% of bladder cancer, melanoma, leukemia and multiple myleoma, 50-70% of head and neck squamous cell carcinoma, 70% of esophageal cancer and 85% of pancreatic carcinoma [22-24]. 5. Structure of P16 INK4a Gene and Protein The coding of the three genes located in the INK4b-ARF-INK4a locus is somewhat complex. P15 INK4a is coded by exons 1 and 2 and P16 INK4a is coded by exons 1 thorugh 3. However, between exons 2 and 3, there lies an alternate form of exon 1 that has its own promoter, and is referred to as exon 1β. The third gene in this locus, ARF, is coded by this alternate form of exon 1 along with exons 2 and 3 [6, 20]. Thus P16 INK4a and ARFshare a common exon 2 and exon 3. However, despite sharing these two exons, these proteins do not share any amino acid homology. This is because exon 2 is translated in a different reading frame in each protein (hence the name ARF, alternative reading frame, for P19/ ARF P14 ARF ) [20]. Approximately 100 kb downstream of this three-gene locus there is a highly conserved gene, MTAP. Looking at the organiziaton of these four genes in several organisms gives clues as to how this unique locus may have evolved. In the puffer fish, Fugu rubripes, a homolog of INK4b lies immediately next to MTAP. It is thus thought that P16 INK4a may have arisen from a gene duplication event. However, the presence of exon 1β physically separating these two genes complicates this simple explanation. It has been suggested that this phenomenon may be due to a need for common regulation of the genes within the INK4b-ARF-INK4a locus [20]. P16 INK4a is a 156 amino acid protein with a MW of approximately 16-kDa. The solution structure of full-length P16 INK4a was solved in 1998 by Byeon et al. [25]. Central to the P16 INK4a protein are its four ankyrin repeats [26, 27] which provide its structural scaffold and mediates protein-protein interactions [20]. Ankyrin repeats are 33 amino acids in length and are found in many different types of proteins. It is estimated that approximately 6% of

275 Structure and Function of the Tumor Suppressor Gene p proteins coded by human genes contain these motifs [28]. Different ankyrin repeats found in the same proteins can differ from one another in terms of amino acid sequence, allowing for structural and functional diversity. Ankyrin repeat-containing proteins are rather distinct from globular proteins in that they do not have a hydrophobic core and their tertiary structure does not include stabilizing contact between residues distant from one another on the polypeptide chain [27, 28]. Each of the four repeats found in P16 INK4a has a helix-turn-helix motif except for the first half of the second ankyrin repeat which only has one helical turn [25, 27]. The helices stack into bundles in a linear fashion facilitated by internal hydrophbic residues. The helix-turn-helix motifs are linked by loops that are positioned perpendicular to the helix. Each of the three loops have little amino acid sequence homology suggesting that each plays a unique role in different P16 INK4a functions [27]. Most of the hydrogen bonds that allow INK4 proteins to bind CDK6 are not found in other ankyrin repeat-containing proteins, again suggesting that sequence diversity is a mechanism that allows functional specificity [28]. 6. P16 INK4a as a Tumor Suppressor As a negative regulator of cellular proliferation, it has an important role in the prevention of tumor formation. Approximately 50% of all human cancers demonstrate P16 INK4a inactivation, with a range from about 25% to 70% [15]. Inactivation has been detected in the following types of cancer: head and neck, esophageal, biliary tract, liver, lung, bladder, colon and breast carcinomas; leukemia; lymphomas; and glioblastomas [4, 24, 29, 30]. Biological and biochemical analysis of P16 INK4a germ line mutations in some cancer types show abnormalities of P16 INK4a protein function [12, 31, 32] further evidence that P16 INK4a plays a role as a tumor suppressor. The P16 INK4a gene is inactivated in head and neck squamous cell carcinoma. Genetic and epigenetic analyses show that expression of P16 INK4a gene is lost in 74% of head and neck squamous cell carcinoma and promoter hypermethylation occurs in 27% [33]. P16 INK4a is inactivated most frequently in pancreatic carcinomas with 98% of cases demonstrating a loss of P16 INK4a function. Many mechanisms of P16 INK4a gene inactivation have been identified in these tumors, including homozygotic deletions, loss of heterozygosity, point mutations and promoter methylation [34, 35]. Other research has demonstrated germline mutations in P16 INK4a in familial melanoma [36]. For instance, in familial melanoma, the most prevalent changes (41% of cases) are associated with missense mutations or deletions in the INK4/ARF locus [15, 37]. 7. Specific Functions of P16 INK4a P16 INK4a is a tumor supprossor gene whose function in cell cycle regulation is extensively documented. P16 INK4a is involved in other cellular processes including (but not limited to) senscence, apoptosis/anoikis and DNA repair. In addition to its apparent role in the control of the G 1 to S transition, P16 INK4a may also regulate the cell cycle via its inhibition of cyclin dependent kinase 7 (CDK7)-carboxyl-terminal domain (CTD) kinase activity [38, 39]. Phosphorylation of this essential CTD, which is located in the largest subunit of RNA polymerase II, is involved in the regulation of transcription both in vitro and in vivo. In vitro

276 270 Zeynep Tarcan, Catherine Moroski Erkul, Bunyamin Isik et al. experiments have indicated that the CDK7 subunit of the general transcription factor TFIIH can carry out this phosphorylation. P16 INK4a has been found to associate with TFIIH as well as the CTD of RNA pol II. Furthermore, this association was observed to inhibit the phosphorylation of the CTD and contribute to cell cycle arrest [38, 39]. This function suggests a P16 INK4a -mediated connection between basal transcription mechanisms and the regulation of cell cycle progression. Experiments with chimeric INK4 family proteins indicate that this activity is specific to P16 INK4a, apparently carried out by residues on its aminoterminal end [39] Other Fuctions of P16 INK4a One of the first lines of research that led to the identification of P16 INK4a was the attempt to find a melanoma susceptibility gene near the 9p21 locus. P16 INK4a mutations are present in almost all human melanoma cell lines [40] and p16 INK4a knock-out mice are susceptible to tumor formation, including skin melanomas [41]. In families with germline mutations in P16 INK4a sunburn is a very high risk factor. One of the three major signalling cascades in higher organisms, collectively referred to as mitogen activated protein kinases (MAPKs), is the c-jun N-terminal kinases (JNKs). These signal transduction molecules are activated in response to extracellular stimuli, inflammatory cytokines, and environmental stress (including UV radiation) [42, 43]. They are involved in activation of the c-jun/activator protein-1 (AP-1) transcription factor complex. AP-1 transcription factors regulate gene expression in response to stress, growth factors and cytokines and thus is involved in controlling processes like proliferation, differentiation and apoptosis [44]. They also can cause neoplastic transformation and skin carcinogenesis in mice [45, 46]. Furthermore, tumor formation is inhibited in c-jun knock-out mice [47]. Choi et al. reported in 2005 that P16 INK4a, but not other INK4 proteins, binds JNK1 and JNK3 in vitro and in vivo with or without exposure to UVC. Upon exposure to 60J/m 2 UVC, this association increased 10-fold. After UV-exposure, its interaction with JNK proteins inhibits their ability to bind to c-jun, which in turn inhibits c-jun phosphorylation [42]. This function of P16 INK4a thereby contributes to the prevention of neoplastic transformation P16 INK4a in Apoptosis Apoptosis is a controlled form of cell death that is essential to growth and development of organisms and functions in actively proliferating cell types in adults such as skin and the gastrointestinal tract. Apoptosis is also essential in preventing the replication of cells that contain overwhelming amounts of DNA damage, thus ensuring that cells that harbor potentially deleterious mutations do not proliferate. Along with unrestrained proliferation, defects in apoptosis are another hallmark of cancer. The absence of proper apoptotic mechanisms aids proliferation beyond a cell s normal replicative lifespan. It also facilitates other mechanisms associated with cancer such as angiogenesis. Defective apoptosis presents a challenge for many chemotherapeutic agents that rely on apoptosis for their effect [48]. Evidence of P16 INK4a mediated P53-dependent apoptosis has been provided by studies in numerous cell lines and mouse models. Ectopic overexpression of P16 INK4a and P53 in tumor cell lines induces apoptosis. When delivered together to a mouse xenograft tumor model, their combined expression is capable of inhibiting tumor growth [49]. In several non-small cell

277 Structure and Function of the Tumor Suppressor Gene p lung cancer cell lines ectopic expression of P16 INK4a and P53 led to G1 arrest and several days later an induction of apoptosis. This effect was caused by direct downregulation and hypophosphorylation of Rb and indirect downregulation of the anti-apoptotic factor, bcl-2. The mechanism of apoptosis was later shown to involve caspase-3 activation followed by cleavage of PARP, MDM2 (a negative regulator of P53) and Rb [50]. A pan-caspase inhibitor significantly reduced the observed phenotype and in all experiments wild-type P53 was requisite for apoptosis to occur [50, 51]. P16 INK4a has also been reported to modulate apoptosis after DNA damage. After treatment of P16 INK4a -deficient U2OS cells (osteosarcoma cell line) and p16 null mouse embryonic fibroblasts (MEFs) with UV, apoptosis was triggered. However, in isogenic cell lines EH1 and EH2 which express P16 INK4a, very little apoptosis was observed after UV exposure. The same was true of p16 wild-type MEFs [48]. Conversely, after treatment of the same cell types with cisplatin, a chemotherapeutic agent that causes DNA damage and induces both p53-dependent and independent apoptosis, U2OS cells underwent minimal apoptosis while P16 INK4a proficient cells exhibited significant levels of apoptosis. Interestingly, while the MEFs exhibited an immediate response to cisplatin, the EH1 and EH2 cell lines underwent apoptosis only after being blocked at the G 2 /M checkpoint after 48h of treatment. The extent of the G 2 /M arrest was cisplatin dose-dependent. Cell synchronization experiments as well as experiments carried out in quiescent cells, suggested that cisplatin only causes apoptosis in proliferating cells [48] P16 INK4a in Tumor Cell Invasion As a tumor grows and acquires more aggressive proliferative capacity, changes take place both within the tumor and in surrounding stromal tissue that allows invasion and metastasis to take place. Invasion occurs when tumor cells acquire the ability to begin invading adjacent normal tissue. This is primarily caused by aberrant activation of cell motility and changes in adhesion properties. The latter is associated with matrix degradation and remodeling. Tumor cells must also harbor an ability to adapt to the new local tissue environment into which it invades. Metastasis requires yet more changes which allow cells that have managed to reach a conduit for travel to distant locations to survive in the blood/lymph circulation without a connection to supporting tissue [52]. One characteristic that invasion and metastasis appear to share is the activation of motility since metastasis has been observed to occur in tumors without any apparent invasive properties. P16 INK4a has been associated with the acquisition of the ability to invade surrounding tissue in serveral cancer types via its effect on factors directly involved in the processes of cellular motility. By comparing stromal fibroblasts in the tumor microenvironment with fibroblasts in cancer-free, histologically normal tissue isolated from the same patient, Al-Ansari et al. demonstrated that 83% of the cancer associated fibroblasts (CAFs) in the breast cancer cases they studied have lower P16 INK4a expression at both the mrna and protein level [53]. In a later study, they show, both in cell lines and in a mouse xenograft breast cancer model,that this downregulation results in enhanced invasion and VEGF-A-dependent angiogenesis via activation of Akt protein kinase. P16 INK4a normally represses the expression and secretion of VEGF-A via its suppression of the Akt/mTOR signalling pathway. Suppression of this pathway prevents HIF1-α, a downtstream effector of Akt/mTOR, from transactivating VEGF-A. Inhibition with a VEGF-A-specific inhibitor molecule suppressed the pro-angiogenic effect caused by this P16 INK4a deficiency [54].

278 272 Zeynep Tarcan, Catherine Moroski Erkul, Bunyamin Isik et al. Several studies over the past decade have found that P16 INK4a plays a role in inhibiting angiogenesis and cell migration via inhibition of the expression of cell surface receptor alpha(v)beta(3) integrins [55]. One study conducted in the malignant glioma cell line SNB19 found that adenoviral expression of P16 INK4a combined with knock-down of upar, a gene coregulated with alpha(v)beta(3), inhibited integrin-mediated cell adhesion, migration, and proliferation [56]. This combination was also found to be capable of inhibiting angiogenesis and activating cell death in the U251 glioma cell line [57]. Another study conducted in HUVEC cells came to similar conclusions, where P16INK4a was involved in a pathway that controls angiogenesis of endothelial cells via alpha(v)beta(3)-mediated migration [58]. The alpha(v)beta(3) integrins have also been implicated in invasion in melanoma [16] and pancreatic cancer [7]. The gamma2 chain of laminin 5 is associated with invasion in some types of carcinoma (Figure 5). P16INK4a and gamma2 chain of laminin 5 were co-expressed in regions of microinvasion and margins of squamous cell carcinoma of skin and oral cavity. P16 INK4a expression was, on the other hand, undetectable in benign hyperplastic lesions. Keratinocytes at the edges of wounds show a similar co-expression of P16 INK4a and gamma2, thus suggesting a possible role for P16 INK4a in wound healing [59]. This capacity may be commandeered by cancer cells to facilitate invasion. Beta-catenin is a well-established oncogene in colorectal cancer. P16 INK4a expression is regulated by beta-catenin (Figure 5) and is associated with low survival in colon cancers with an infiltrative front of invasion [60]. In a study investigating the expression and association between beta-catenin, P16 INK4a and c-myc in colorectal cancer at various stages of tumorigenesis and progression, there was an increase in nuclear expression of all three proteins correlated with severity of disease. Nuclear overexpression of beta-catenin and P16 INK4a were associated with tumors having lymph node metastasis but not distant metastasis [61]. Figure 5. The gamma2 chain of laminin 5 is associated with invasion in some types of carcinoma and p16 expression is regulated by beta catenin.

279 Structure and Function of the Tumor Suppressor Gene p In a study investigating the expression of cell cycle regulatory proteins in endometrial carcinomas, Horree et al. show, via immunohistochemical staining of tissue, that the invasive front of these tumors, as compared to central regions of the tumors, have higher rates of proliferation and lower rates of cell cycle regulatory proteins. P16 INK4a displayed higher expression in about 25% of the 39 cases included in their experiments [62]. In work published by Zhang et al. in 2012, the use of a P16 INK4a -specific artificial transcription factor (P16ATF) was able to re-activate P16 INK4a by demethylation of CpG islands that had inactivated P16 INK4a (and other tumor suppressor gene) expression. CpG islands are found in about 60% of human gene promoter regions and consist of many CpG dinucleotide repeats. Their methylation usually results in irreversible inhibition of gene expression [63]. Thus, the ability to re-activate gene expression via artificial transcription factors is a potentially powerful tool. Both the transient and stable expression of P16ATF not only led to re-activaiton of P16 INK4a but also successful inhibiton of cell migration and invasion. This targeted approach may be preferable to the global DNA methylation inhibitors currently used in cancer treatment as it may reduce or completely do away with the toxic side effects associated with such drugs [64] P16 INK4a in Anoikis Cells possess surface receptors on their membranes to which adhesion molecules such as integrins and cadherins can bind to. This allows them to maintain connections to the extracellular matrix and/or to neighboring cells. When these connections are lost, cells normally undergo a form of apoptosis referred to as anoikis. Cells that have the capacity to avoid anoikis are able to survive in suspension and thus can travel to distant sites. If these cells are cancerous, they can form new sites of tumor growth. Thus proper function of the biomolecular pathways involved in anoikis signalling is essential for the prevention of metastasis. In 2004, Douma et al. showed that repression of anoikis in vivo can induce metastasis. Using a retrovirally transfected cdna library screen, non-transformed rat intestinal epithelial cells, known to be highly sensitive to anoikis, were transferred from standard adhesive culture plates to ultra-low cluster plates containing a gel layer that prevents cell attachment. Most cells rapidly underwent apoptosis but some clones survived. The TrkB clone, in particular, was able to form large spheroid aggregates growing in suspension. When stimulated with BDNF, TrkB s primary ligand, these cells became even more resistant to apoptosis. To carry out experiments with these cells in mice, TrkB-expressing and TrkB/BDNF co-expressiong cells were engineered to also express luciferase, which would facilitate non-invasive in vivo imaging. Luciferase activity disappeared after 6 days in nude mice injected with control cells. However, TrkB-expressing cells were able to colonize the lungs and heart and form rapidly proliferating tumors. Even more striking was the ability of TrkB/BDNF co-expressing cells to cause metastasis throughout the body. This effect was determined to be caused by the activation of PI3K/Akt signaling pathways by TrkB [65]. TrkB is overexpressed in several cancer types including pancreatic, ovarian and neuoblastoma. And loss of P16 INK4a expression is known to activate Akt signalling [54]. More direct evidence of P16 INK4a involvement in anoikis comes from work performed in a number of different cancer cell lines. In 2000, P16 INK4a was reported to induce anoikis via transcriptional upregulation of the alpha(5) integrin chain of the alpha(5)beta(1) fibronectin receptor in the pancreatic cell line Capan-1, the hepatocellular carcinoma cell line SKHep1, and the melanoma cell line NKI4 [66]. When these P16 INK4a -deficient cells were transfected

280 274 Zeynep Tarcan, Catherine Moroski Erkul, Bunyamin Isik et al. with P16 INK4a expression vector, the cells ability to carry out anoikis was restored. sirna mediated knock-down of alpha(5) integrin chain in P16 INK4a -expressing cells did not undergo anoikis. A follow-up study carried out in pancreatic cell lines investigated the involvement of K-Ras in suppression of anoikis. Two common features of pancreatic cancer are activation of K-Ras and inactivation of P16 INK4a, P53 and SMAD4. Since K-Ras is known to regulate P16 INK4a, the authors wished to determine whether the converse was also true [67]. Capan-1 cells exhibit loss of P16 INK4a and an activating mutation in K-Ras. When P16 INK4a was reintroduced to Capan-1 cells, K-Ras protein was destabilized, its activity was suppressed, and anoikis mechanisms were restored [68]. Subsequent investigation into the role of alpha(5)beta(1) fibronectin receptor have confirmed previous findings of the role it plays in anoikis [69]. Andre et al. were able to restore epithelial tumor cell sensitivity to anoikis after stable transfection with a P16 INK4a expression vector. They found P16 INK4a to be implicated in the process of protein glycosylation. Glycosylation is simply the addition of a glycan (an oligosaccharide or polysaccharide) to a protein. These glycans have effects not only on protein structure but also are involved in cell adhesion and signalling mechanisms that can trigger apoptosis or proliferation. Abberant glycosylation is a characteristic observed in malignant transformation. The authors found that expression of P16 INK4a was associated with an increase in expression of fibronectin cell-surface receptors and that this was responsible for restoring sensitivity to cell death by anoikis [70]. P16 INK4a loss is a common occurrence in HCC and, like pancreatic cancer, HCC is very lethal. Restitution of P16 INK4a in HCC cell lines was reported to induce anoikis via decreased phosporylation of Akt and Survivin. After levels of survivin decreased, there was a limited supply of this protein for CDK4 to import into the nucleus. This caused cell cycle arrest and anoikis. The authors also demonstrated the ability of P16 INK4a to induce apoptosis via Akt/Survivin downregulation in vivo in a mouse xenograft model. P16 INK4a is thus a potentially useful therapeutic target in these difficult to treat cancers [71] P16 INK4a in Senescence Senescence is typically defined as irreversible cell cycle arrest. There are two types of senescence which are defined by the type of stimulus they are triggered by. Replicative senescence is caused by telomere shortening and dysfunction which is a normal part of the cellular and organismal aging process. Premature senescence is a stress-induced phenotype typically triggered by overwhelming DNA damage, replicative stress, oncogene activation and reactive oxygen species generation. The pathways involved in the cellular response to this stress are the DNA damage response (DDR), P16INK4a/pRb and ARF/P53/P21 [72]. While senescence pathways are active in pre-malignant tissue, they are bypassed and/or inhibited in cancerous cells and tissues. This is another mechanism whereby cancer cells attain their endless replicative capacity. Genes involved in the suppression of senescence are upregulated in cancer cells and genes involved in the induction of senscense are downregulated. Thus, reactivation of senescence pathways may be possible by activation of these senescence-inducing genes or inhibition of senescence suppressing genes [72]. Cells that have undergone senescence exhibit changes in organelle structure and function, many of which adversely affect mitochondria and mtdna [73]. P16 INK4a appears to be involved in both the initiation and maintenance of cellular senescence.

281 Structure and Function of the Tumor Suppressor Gene p One of the side-effects of doxorubicin which limits its use in the clinic is cardiomyopathy and heart failure. This is caused by extensive apoptosis of mature cardiomyocytes and progenitor cells. This includes cardiac progenitor cells as well as endothelial progenitor cells (EPCs), that latter which are recruited from bone marrow. However, at lower doses of doxorubicin, cells experience telomeric dysfunction and enter a state of stress induced premature senescence (SIPS). P16 INK4a and JNKs are both implicated in this process. There is evidence that cellular senescence and aging are also induced by P16 INK4a expression in different progenitor cells such as neural progenitor cells [74], pancreas islet progenitor cells [75], and hematopoietic stem cells [76]. 8. Regulation of P16 INK4a Expression P16 INK4a gene expression is regulated by many different mechanisms at transcriptional, post-transcriptional and post-translational levels. Its physical location within the unique INK4b-ARF-INK4a locus means that its transcriptional regulation is, at least in some instances, intimately connected to the regulation of the ARF tumor suppressor. It also means that parsing the precise regulatory mechanisms associated with this gene has been a complicated endeavor. Finally, as is the case for many genes, the specific regulatory mechanisms involved are cell-type and context dependent. Several transcriptional activators of P16 INK4a have been identified. There is an ETS binding site in the P16 INK4a promoter that can be activated by Ets-1 and Ets-2 transcription factors (TFs). Conversely, other ETS family TFs, Pea3, Sap1 and Elk1, are capable of causing a reduction in promoter activity. PPAR gamma TF also binds the promoter and inhibits P16 upregulation. De-phosphorylation of PPAR gamma leads to it release from the promoter and upregulation of P16 INK4a [77]. Approximately 2 kb upstream of the P16 INK4 a promoter regions lies an area that canbe bound by H2A.Z protein. H2A.Z protein along with the transcription factor CTCF can modify the P16 INK4a locus in a manner that inhibits epigenetic silencing via chromatin remodelling of the P16 locus [78]. RAS-RAF-MEK signalling induces P16 INK4a expression (Figure 6). The precise mechanism is not clear but it may occur via binding of the P16 INK4a promoter by ETS2 TF or, alternatively, via secondary signalling through P38. MYC also appears capable of upregulating P16INK4a expression possibly through binding of the promoter. However in both of these cases, the kinetics of expression do not match up well with the proposed causative mechanism and thus more research is needed to clarify these ambiguities. And in the case of MYC, it can activate BMI-1 which is a Polycomb Group (PcG) gene and inhibitor of INK4a, further complicating the picture [20]. P16 INK4 a is also regulated by the AP-1 family of transcription factors. The overexpression of JUNB causes upregulation of p16 INK4a while another AP-1 family member, c-jun, represses P16 INK4a [20]. P16 INK4a expression can also be activated by interactions at the P16 INK4a locus between P300 and Sp1 which result in hypermethylation of histone H4 [79]. It has also been reported to be upregulated by inhibitor of growth 1 (ING1) [80]. Repressors of the locus include TAL1, ATM, EGR1, ZBT7B, and AML1 (RUNX1) [20].

282 276 Zeynep Tarcan, Catherine Moroski Erkul, Bunyamin Isik et al. Figure 6. RAS-RAF-MEK signalling in p16 expression. 9. Overexpression of P16 INK4a in Tumors Thus far, we have seen that P16 INK4a loss or downregulation is a common occurrence in many different cancer types. It plays an integral role in a host of cellular processes that guard against oncogenic transformation and its absence or aberrant expression allows a variety of pathological changes to occur. These changes contribute not only to oncogenic transformation but also to the invasive and angiogenic capacity of a tumor and the ability to metastasize. However, in some cancers, after malignant transformation takes place, the expression of P16 INK4a is reactivated [15]. Detection of the overexpression of P16 INK4a can be used as a diagnostic tool [29, 81] in some tumor types, such as cervical cancer and perianal lesions. Milde-Langosch et al. reported overexpression of P16 INK4a, as detected by Western blot (WB) and immunohistochemistry (IHC), in about 20% of 60 breast cancer tumor samples tested. Furthermore, this expression was associated with other markers indicating a poor prognosis [82]. In another study of gynecological cancer, Armes et al. found that invasive serous papillary ovarian cancer had strong P16 expression throughout the tumors. It was particularly common in grade 3 carcinomas [83]. A similar observation was reported by Chiesa-Vottero et al. in an examination of uterine serous carcinoma and ovarian high-grade serous carcinoma [84]. Overexpression has also been observed in high-grade ovarian serous carcinomas and uterine leiomyosarcomas [85, 86]. P16 INK4a overexpression can also be used as a marker of dysplasia and neoplasia in cervical epithelial biopsy samples. Using IHC, Klaes et al. found significant overexpression of P16 INK4a in cervical intraepithelial neoplasm (CIN) I lesions (n=47) that were associated with high-risk HPV types. Overexpression was also found in all CIN II lesions (n=32), CIN III lesions (n=60) and 58 of 60 invasive cervical cancers. This was in contrast to a lack of observable P16 staining in normal cervical epithelium (n=42), inflammatory lesions (n=48), and CIN I lesions associated with low-risk HPV types (n=7) [87].

283 Structure and Function of the Tumor Suppressor Gene p Both P16 INK4a downregulation and overexpression have been observed in colon cancer [88-90]. In colorectal cancer, P16 INK4a overexpression is associated with poor prognosis, and varies by case according to sex, distal location, and tumor grade and stage [15]. Zhao et al. found overexpression of P16 INK4a in 71.7% of Chinese patients with Hodgkin lymphoma. In some studies of pre-cancerous and cancerous lesions of the oral cavity, increasing P16 INK4a expression assayed by IHC has been associated with increasing level of malignancy [91]. However, in some studies of oral squamous cell carcinomas P16INK4a overexpression was correlated with favorable prognosis [92]. This apparent contradiction may be due to differences between the cancer sub-types that were studied or age-dependence. The latter study was conducted in young patients (age 18-39) and thus may reflect different mechanisms or germline versus somatic mutations. 10. Aberrant Subcellular Localization of P16 INK4a in Cancer In addition to downregulation and overexpression, aberrant subcellular localization of P16 INK4a has been reported in several cancer types. Other cell cycle regulatory proteins that have aberrant cytoplasmic expression in tumors are PTEN and P27. Zhao et al. used P16 INK4a and CDK4 IHC to examine changes in subcellular localization of these proteins in a series of colon tissue samples that ranged from normal histopathlogy to adenoma and carcinoma. While 95% of normal epithelia (n=42) had P16 INK4a positive staining localized to the nucleus, 25% of adenomas (n=43) had cytoplasm-only positive staining of P16 INK4a and 75% had cytoplasmic/nuclear staining. Out of 73 carcinomas, the proportion of samples exhibiting P16 localization in the cytoplasm increased to 62% and cytoplasmic/nuclear staining was observed in 37% of carcinomas. Furthermore, in cancer cells, the staining intensity of P16 INK4a was weak and sporadic [63]. Cytoplasmic P16INK4a expression has been described in astrocytomas and is associated with poor prognosis [29]. It has also been observed in terine leiomyosarcomas and in these it is typically associated with malignant tumors. In gastrointestinal stromal tumors (GISTs), Haller et al. describe P16INK4a cytoplasmic expression to be associated with poor prognosis but the precise mechanism involved in unclear. They also describe a set of GISTs in which low nuclear expression, also corrleated with poor prognosis, is associated with E2F1 transcription factor upregulation which causes increased proliferation [93]. The authors suggest that the proteins detected in cytoplasm and nucleus are actually two dinstict splice variants of P16INK4a, as had been previously reported by Lin et al. in 2007 [94]. 11. P16 INK4a as a Therapeutic Target Re-activation of P53 and P16 INK4A can reduce tumor burden via the induction of apoptosis and senscense, at least in murine models [95]. Promoter hypermethylation of P16 INK4a or cytoplasmic P16 Ink4a sequestration by anion exchanger 1 (AE1) has been identified as an alteration that contributes to malignant transformation. Hence, both p16 INK4A can be restored and premature senescence can be induced in cancer cells by demethylating therapies or

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293 In: Tumor Suppressor Genes ISBN: Editors: Mehmet Gunduz and Esra Gunduz 2013 The Functions and Roles of RB1 in Cancer Chapter 14 Erkan Koparir, 1 Asuman Koparir 1 and Mustafa Ozen 1,2, 1 Department of Medical Genetics, Istanbul University Cerrahpasa Medical School, Istanbul, Turkey 2 Department of Pathology & Immunology Baylor College of Medicine, Houston, TX, US Abstract The retinoblastoma tumor-suppressor gene (RB1) was cloned in 1986 and called as the prototype of tumor- suppressor genes. Up to now, we know there have been hundreds of reviews published and announced RB1 is most studied gene. Loss of RB1 gene function predispose childhood retinoblastoma tumor affecting approximately 1 in children, also many other human cancers. RB1 associate multiple pathways and has critical roles in any steps of cell cycle. It is also clear that RB1 regulates cell differentiation, apoptosis and genomic stability. RB1 acts in these processes through most especially regulating the function of E2F transcription factors by affecting G1 signaling pathway. prb is the product of RB1 and associates with p107 and p130 cellular proteins. All of these related proteins named as pocket protein family which have similar structures and functions. Three viral oncoprotein induce transformation by binding pocket domain of these proteins including prb, p107, p130. Therapeutic applications have improved for restore prb functions such as p53 inducer Topotecan, Mdm2 inhibitor Nutlin-3 and HDAC inhibitors. Keywords: RB1, retinoblastoma, tumor-suppressor genes, E2F, pocket proteins mozen@bcm.edu, mozen@istanbul.edu.tr.

294 288 Erkan Koparir, Asuman Koparir and Mustafa Ozen Introduction The retinoblastoma tumor-suppressor gene (RB1) was the first identified tumor suppressor, which has demonstrated to have a central role in cancer research [1, 2]. Since it regulates multiple cellular processes such as cell proliferation, cell differentiation, apoptosis and genomic stability, whose aberrancies result in oncogenesis, it has become one of the most studied proteins. Inactivation of RB1 as a result of deleterious mutations causes retinoblastoma, which develops during childhood period, however, deregulation of the pathways, where the protein product of RB1 (prb) functions as a primer regulator of E2F transcription factors, has shown to be involved in pathogenesis of several human cancers [3, 4]. This chapter summarizes the significance of RB1 in cellular functioning in relation to its tumor suppressive role through unraveling the recent insights into its expression, function and activity in normal and pathological states. 1. The RB1 Gene RB1, was cloned in 1986 [5] and found to be located at chromosome 13q14.2, consisting of 27 exons. This tumor suppressor gene has shown to be transcribed into a 4.7-kb messenger RNA (mrna) [6, 7]. It is a member of a large gene family, which includes two other RB-related genes called as retinoblastoma-like 1 (RBL1) and retinoblastoma-like 2 (RBL2). RB1, RBL1 and RBL2 genes encode for structurally related proteins; prb, p107 and p130, respectively [2]. 2. RB1 and Retinoblastoma RB1 mutations, which cause retinoblastoma, result from bi-allelic inactivation or loss of the retinoblastoma 1 gene (RB1) [8]. According to Knudson s two-hit hypothesis (9), which is originated from retinoblastoma gene, two mutational events or two hits are required for tumor onset. A patient may have inherited a germline mutation from a parent and that would constitute the first "hit" leading to the cancer, then a second mutational event, or second hit results in the development of the disease [10-12]. RB1 gene has shown to be heterozygous mutant in patients who are diagnosed with sporadic bilateral or familial retinoblastoma. These first hits were either inherited or developed de novo in parental germline cells or during embryonic development. In both cases, all cells of the affected individual turns to be carrier for RB1 mutation and a second mutation in the retina cells during the early childhood period results in the development of bilateral retinoblastoma. These heritable cases constitute approximately 40% of all cases, which are predisposed to retinoblastoma during childhood. Non-heritable RB cases made up of 60% of all cases and most of them develop unilaterally, with mutations occurring locally within the affected retina only [2, 13, 14].

295 The Functions and Roles of RB1 in Cancer The Pocket Protein Family prb and its related cellular proteins p107 and p130 comprise the family of pocket proteins that have structural and functional similarities (Figure 1). All three proteins contain a large pocket domain, which is required to mediate the binding site for many of viral and cellular proteins [15, 16]. Figure 1. Functional domains of the prb protein. The pocket proteins carry out overlapping functions such as binding E2Fs, restraining cell cycle progression, and serving as substrates for CDKs. It has been demonstrated in different studies that loss of p107 or p130 represents cell cycle defects similar to those lacking prb [17, 18], whereas in contrary to prb loss, mutational inactivation of p107 or p130 was rarely observed in human cancers [19]. The pocket proteins can bind DNA tumor virus oncoproteins as well as endogenous nuclear proteins, which promote cellular proliferation. These mitogenic oncoproteins, including adenovirus E1A protein, simian virus 40 (SV40) large T antigen and human papillomavirus E6 and E7 protein, inactivate the function of Rb and result in tumor formation [20-24]. 4. Functional Domains of the prb Protein Rb, p107, p130, as pocket proteins, share a structural element called the A/B pocket [25, 26]. The A/B pocket, which contains the LXCXE motif, is needed for binding of E2Fs, HDACs, and viral oncoproteins [27-30]. Cyclin E CDK2 complex inactivates prb through binding its phosphorylation site of the N terminus. Cyclin A CDK2 and cyclin D CDK4 binds phosphorylation site of the C terminus and inhibit function of prb. C-Abl protooncogene and the p53 inhibitor Mdm2 also have binding sites in the C terminus (Figure 1) [26, 30, 31]. prb protein have 16 CDK phosphorylation sites that regulate the prb activity throughout the cell cycle. Most of the RB1 gene mutations localize in the A/B pocket domain [33].

296 290 Erkan Koparir, Asuman Koparir and Mustafa Ozen 5. Rb in Cell Cycle Regulation The pocket protein family of cell cycle regulators is made up of prb and and its related cellular proteins p107 and p130. These proteins are primarily responsible for preventing the G1 S transition via regulation of E2F-responsive genes. In quiescent cells, RB proteins repress the transcriptional activity of E2Fs. When the cell growth is induced and the cell cycle entry is promoted, cyclin dependent kinases are activated. This leads to the phosphorylation and as a result deactivation of prb, which cause activation of E2F transcription factors and G1-S transition [1, 2, 16, 33] E2F and E2F Regulated Genes E2F transcription factors serve as targets for the Rb protein s growth-inhibitory action [22, 23, 34]. It has become clear that E2F1 is a member of sequence-specific DNA-binding transcription factors family and prb composes a complex with several members of this family, especially with transcriptional activators E2F1-3 and transcriptional repressors E2F4-5 [35, 39]. prb interacts with transcriptional activators E2F1-3 and contributes to active gene silencing through restraining the binding of transcriptional co-activators, and through recruitment of histone deacetylases (HDAC), ATPases, and DNA methyltransferases to the promoters of target genes (Table 1) [40, 41]. E2F transcription factors have reported to regulate multiple genes, which are responsible for cell cycle regulation, nucleotide synthesis and DNA replication. DNA polymerase-i and thymidine kinase, thymidylate synthase and dihyrofolate reductase, apoptosis regulators; caspases and apaf-1 and several proto-oncogenes are also shown to be regulated by these transcription factors. [42-47] RB Phosphorylation and CDK s/cdki s Phosphorylation and cyclin-dependent kinase (CDK) activation regulate prb-e2f interaction [48-50]. CDK enzymes consist of two components, which are catalytic CDK subunit and regulatory cyclin subunit.(kitap referans verilmeli ve başka yerlerde) Phosphorylation of prb was shown to be synchronized and it is hypophosphorylated during the cell cycle s G1 phase. Unphosphorylated form of prb, the active form, can directly bind to and inhibit activity of E2F activators. During the G1/S transition and G2/M phases of cell cycle, prb is phosphorylated. By the end of mitosis, prb is dephosphorylated and returns its active form (Figure 2) [16, 43, 45, 49, 51, 52]. When prb is hyperphosphorylated by CDKs induced by growth stimulation, E2F accumulates and thus causes S-phase entry [53-56]. After phosphorylation of prb, E2F gets free from it and elevates the expression of genes that are necessary for cell division [43]. Cyclin D and cdk4 functions are induced by cyclin E and cdk2 and promote inactivation of Rb. Cyclin D and cdk4 have been shown to phosphorylate prb in early G1, and whereas cyclin E and cdk2 phosphorylates prb in the late G1. Cyclin E and cdk2 are also regulated by E2F, having primary role for activation of replication [40, 54, 56]. Consequently, prb/e2f

297 The Functions and Roles of RB1 in Cancer 291 pathway controls both activation of DNA replication and the regulation of the G1-S transition. All these data proposed prb as a general cell cycle regulator (Figure 2). Regulation of prb by phosphorylation is a convoluted process in which, multiple CDKs are involved. They phosphorylate prb through 16 potential phosphorylation sites on prb [57]. CDK activity is regulated primarily by two parameters: the level of proper cyclins and cyclin-dependent kinase inhibitors (CDKI s) [58]. Two families of CDK inhibitors are used to control the G1 S transition and antagonize prb phosphorylation. The first family consists of INK4 proteins (p15, p16, p18, p19), which specifically inhibit kinases activated by D-type cyclins and the second one is Cip/Kip (p21, p27, p57) family, which inhibit D-, E-, and A- type cyclin CDK complexes [4, 59-63]. Figure 2. Regulation of prb in coordination with the cell cycle. Regulation of E2F-dependent promoters by Rb. Table 1. E2F transcription factors have reported to regulate multiple genes, which are responsible for cell cycle regulation, nucleotide synthesis and DNA replication [33] Cell cycle Regulation Cyclin A Cyclin E CDK1 E2F1 E2F2 E2f3 Skp2 HP1 DNA replication and Nucleotide synthesis DNA polymerase 1 Thymidylate synthase Thymidine kinase Dihyrofolate reductase Apoptosis Caspase- 3 Caspase- 7 Caspase- 8 Apaf1 Proto- oncogenes b- myb c-myb c- Myc Mutations or decreased expression of CDK inhibitors in many types of tumors has been attributed to genetic and epigenetic changes. Mutations of p16ink4a cyclin kinase inhibitor

298 292 Erkan Koparir, Asuman Koparir and Mustafa Ozen are the second most frequent mutations after RB1 in human cancers, disturbing the regulation of the prb/e2f pathway. The p16 protein controls the Cyclin D/cdk4 kinase activity. The absence of p16 activity is functionally equivalent to loss of RB1, since in absence of p16 Cyclin D/cdk4 kinase activity increases and leads to prb phosphorylation and subsequent E2F accumulation. Furthermore, loss of p16 function is relevant in a variety of sporadic cancers, but inherited p16ink4a mutation is observed in melanoma [64] RB1 and HDAC1 prb binds to E2F transcription activators and repress expression of their target genes through preventing the binding of transcriptional co-activators, and then leading to recruitment of HDACs (Figure 2). prb should have intact pocket domain in order to directly interact with HDAC1. This interaction has shown to be decreased as a result of RB1 mutations [40, 41, 65] RB1 and Skp2 Independent from E2F, prb protein can also modulate cell cycle through other mechanisms. One of these mechanisms involves interaction of prb with Skp2 that prevents degradation of the CDK inhibitor p27 and subsequently results in p27 accumulation. Inhibition of cyclin E-CDK activity by p27 is essential for normal progression of cell cycle. Besides, phosphorylation of prb by the cyclin E-CDK complex inhibits activity of prb itself and its downstream effectors, E2F transcription factors. Existence of mitotic signals results in binding of Skp2 to p27, which causes its subsequent degradation. When p27 is degraded, the inhibition of cyclin E-CDK activity decreases and phosphorylation of prb is allowed. This results in inhibition of prb and promotes G1 to S phase progression. In the absence of growth-promoting signals, prb binds to Skp2, and prevents it from binding to p27. In this case, free p27 inhibits the cyclin E-CDK activity and results in G1 cell cycle arrest [66, 67] RB1 and Viral oncoproteins The pocket proteins can bind DNA tumor viral oncoproteins in addition to endogenous nuclear proteins, which induce cell proliferation. These oncoproteins, such as adenovirus E1A, SV40 large T antigen and human papillomavirus E6 and E7 proteins, interact with prb and prevents prb/e2f binding and as a consequence cause activation of genes that are transcriptionally regulated by E2F [68].

299 6.1. RB1 and Apoptosis The Functions and Roles of RB1 in Cancer Other Roles for RB1 In addition to controlling cell proliferation and progression, RB1/E2F pathway is also involved in the control of apoptosis induction by regulating the function of E2F1 [69]. When E2F1 expression is elevated, it can activate apoptosis in certain cell types, and in the absence of RB1, E2F-1 inappropriately induces apoptotic signals. E2F1 s function of regulating apoptosis exists in multiple ways. First mechanism is controlling the accumulation of p53 through upregulation of ARF activity. The ARF protein is able to bind the Mdm2, which functions as a p53 ubiquitin ligase and destroys p53. Thus, loss of Rb function results in E2F1 activation that induce the ARF/Mdm2/p53 pathway, causing cell cycle exit and apoptosis [70-75]. Second mechanism involves the p53 homologue, p73. E2F1 stimulates p73 transcription that causes p53-independent apoptosis [76, 77]. Additionally, E2F1 triggers apoptosis by regulation of genes, which are important for apoptosis, such as apoptosis protease activating factor-1 gene (Apaf1) and pro-caspases [78, 79] RB1 and Differentiation prb has a role in the differentiation process of muscle cells, which is controlled by a set of four myogenic transcription factors: MyoD, myogenin, MRF4 and Myf-5. All of MyoD, myogenin, MRF4 and Myf-5 have similar functions, but their expression are detected at different stages of skeletal muscle development. MyoD and Myf-5 are expressed in the early stages of embryogenesis and are associated with muscle lineages. MRF4 acts in later stages and it is responsible for myotube maturation. prb affects this process in early stage by regulating transcription factor MyoD in two ways. Firstly, prb induces MyoD to interact with the coactivator myocyte enhancer factor 2 (MEF2). Secondly, prb blocks deacetylation of MyoD by directly binding histone deacetylase 1 (HDAC1), thus prb prevents MyoD- HDAC1 interaction and allows expression of MyoD transcriptional targets. p21 is a probable MyoD target and its expression is promoted by MyoD. It is included in differentiation program to cell cycle arrest by inhibiting CDK-mediated prb phosphorylation [33, 80-83]. 7. RB1 and Other Cancers The RB1/E2F pathway has critical role in controlling cell growth and prb affects some other pathways controlling replication, apoptosis as has been mentioned before. Thus, it is clearly known that the normal function of the pathway is destroyed by oncogenic mutations. In addition to retinoblastoma, RB1 gene mutations results in loss of Rb function and occurs in various types of human cancers, which are called as second primary malignancies including osteosarcomas, sarcomas, small cell lung carcinomas, breast, bladder, and prostate carcinomas, myelomas, and leukemias [84-87].

300 294 Erkan Koparir, Asuman Koparir and Mustafa Ozen 8. Therapeutic Applications RB1 pathway s damages are common feature of tumorigenesis, so restoration of Rb function initially seemed to be a useful way for correcting the disorders. Here are some challenges waiting to be solved. First, for not to let even rare cells with unrestored Rb function since any such cell can potentially give rise to tumors, a highly efficient dose of targeted drug delivery should be aimed. Second, as it is known that multiple mutations are associated with RB1 gene-related cancers, therefore, restoration of just one mutated function may not result in a fully functional restoration of the normal cell cycle [88, 89]. A novel therapeutic agent, Nutlin-3, is a Mdm2 inhibitor, which prevents association of Mdm2 with p53 [90]. Nutlin-3 treatment is currently in phase I clinical trial, and has demonstrated to restore the p53 pathway in RB cells, and thus induce p53-mediated apoptosis. Topotecan, another therapeutic agent, can inhibit topoisomerase-i and trigger DNA double-strand breaks, which promote apoptosis, through p53-dependent and -independent pathways. When p53 inducer Topotecan and Mdm2 inhibitor Nutlin-3 are used together, they function synergistically and 82-fold reduction in tumor burden was observed in mouse models after subconjunctival injection [33]. Unlike to systemic chemotherapeutic agents, no side effects were noted in animal models following Nutlin-3 and Topotecan combination therapy [91]. The HDAC inhibitors (HDACi) are another class of targeted therapies and currently they are in phase I clinical trial for treatment of RB. HDACi therapy has been found to be effective in cells with increased E2F1 activity by inducing expression of apoptotic factors [92]. Due to the loss of prb, E2F1 activity is known to elevate, thus RB-derived cell lines are sensitive to HDACi therapy and go apoptosis. Conclusion Since the discovery of RB1, remarkable progress has been made in the understanding of RB1 function. Although the critical roles of prb in regulating cell proliferation, differentiation, genome integrity and apoptosis have been clearly identified, there are several outstanding questions, where it acts in processes such as differentiation. As the prb and E2F deregulation leads to multiple cancer development, it is possible that the prb can have alternative tumor-promoting activities. There should be additional research projects to understand all mechanisms and to develop effective and applicable targeted treatment approaches. References [1] Goodrich DW. The retinoblastoma tumor-suppressor gene, the exception that proves the rule. Oncogene Aug 28;25(38): [2] Di Fiore R, D'Anneo A, Tesoriere G, Vento R. "RB1 in cancer: Different mechanisms of RB1 inactivation and alterations of prb pathway in tumorigenesis". J Cell Physiol Jan 28.

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307 In: Tumor Suppressor Genes ISBN: Editors: Mehmet Gunduz and Esra Gunduz 2013 Chapter 15 Endoplasmic Reticulum Protein 29 (ERp29) and Cancer: Molecular Functions, Mechanisms and Clinical Implication Daohai Zhang Discipline of Pathology, School of Medical Sciences, University of Sydney, Sydney, Australia Abstract The Endoplasmic reticulum (ER) is the primary subcellular organelle of synthesis and folding of secretory and membrane bound proteins. Disturbance of ER homeostasis results in accumulation of unfolded and/or misfolded proteins, leading to the ER stress that has been associated with disease progression including cancer. In the ER system, ERp29 belongs to the non-classical molecular chaperones and lacks its redox-active function due to the absence of an active motif consisting of double cysteines. While ERp29 plays a critical role in protein secretion as an escort carrier, accumulating evidence has demonstrated a potential role of ERp29 in cancer cell survival and carcinogenesis. However, its molecular function and pathological role in cancer cells have not been fully investigated. Recent studies demonstrated that ERp29 is an emerging tumor suppressive molecule by inducing cell growth arrest in breast cancer. This review will provide an overview of current understanding of ERp29 in modulating cell cycle arrest, resistance against genotoxic stress, epithelial-mesenthymal reverse transition and epithelial cell morphogenesis in breast cancer cells. The clinical importance of ERp29 in disease recurrence, as well as being a potential therapeutic target for preventing metastasis, is also discussed. dave6503@gmail.com.

308 302 Daohai Zhang Introduction The endoplasmic reticulum (ER), an intracellular organelle of all eukaryotic cells, is complex membrane system constituted of an extensively interlinked network of membranous tubules, sacs and cisternae. It is the main subcellular organelle that transports different molecules to their subcellular destinations or to the cell surface [1, 2]. The ER membrane spans from the nuclear envelope to the secretory vesicles and this structure is closely related to its multifacet cellular functions such as synthesis and sorting of secretory and membrane proteins, biosynthesis of lipids, degradation of glycogen, detoxification reactions and maintenance of intracellular calcium homeostasis and storage [2, 3]. In the ER system, protein folding is executed by a complex process that involves folding, assembly, modification, quality control, and recycling. Correctly folded and assembled proteins are packaged into membrane carriers and are transported from the ER to various cellular or extracellular destinations through the Golgi complex. These carriers are formed by the activity of cytosolic protein complexes involved COP II (coat complex II) machinery [4]. However, under certain conditions that affect proper protein folding, the inappropriately folded or assembled proteins are exported to proteasome and are subsequently degraded in the cytosol, a process known as ER-associated degradation [5]. When the misfolded or unfolded proteins are unable to be degraded and thereof accumulated in the ER, the unfolded protein response (UPR) [6, 7] is activated to relieve the ER stress. In response to UPR, X-box-binding protein 1 (XBP-1), a gene of ER stress sensor, is alternatively spliced by the activated endonuclease domain of inositol-requiring enzyme 1 (IRE1) [8]. Under these conditions, ER chaperones, including endoplasmic reticulum protein 29 (ERp29)/protein disulfide isomerase (PDI)-like proteins/glucose-response protein 78 (GRP78 or BiP), are up-regulated to bind to denatured or aggregated cellular proteins thereby alleviating their refolding, which facilitates cell survival and attenuates apoptotic stimuli. The ER contains a number of molecular chaperones physiologically involved in protein synthesis and maturation. Of the ER chaperones, PDI-like proteins are characterized by the presence of a thioredoxin domain and function as oxido-reductases, isomerases and chaperones [9]. Oxido-reductase activity is present in the chaperones with an active-site double-cysteine (CxxC) motif, such as PDI, ERp72 and ERp57. The CxxC motif allows the protein to cycle between a reduced and an oxidized state [10]. As such, PDI-related proteins have various functions, including noncovalent chaperone activity and disulfide bond formation and reduction [9, 11]. Furthermore, redox-inactive PDI-like proteins including ERp29, ERp27 and thioredoxin-related transmembrane protein 2 (TMX2) do not have the CxxC motif [12]. ERp29 is recognized as a characterized resident of the cellular ER, and it is expressed ubiquitously and abundantly in mammalian tissues [13]. ERp29 was proposed to be involved in the UPR as a factor facilitating transport of newly synthesized secretory proteins [14]. The increased expression of ERp29 was demonstrated in certain cell types both under the pharmacologically induced UPR and under the physiological conditions (e.g., lactation, differentiation of thyroid cells) [15, 16]. In most cases, ERp29 interacts with BiP/GRP78 to exert its function under ER stress [17]. Recently, it was reported that ERp29 was up-regulated under conditions that homocysteine or dopamine invokes ER stress [18, 19], or when cells were exposed to radiation [20]. Furthermore, ERp29 could potentiate resistance to doxorubicin (DOX) and

309 Endoplasmic Reticulum Protein 29 (ERp29) and Cancer 303 radiation by up-regulating Hsp27 in cancer cells through down-regulating the expression of eif2α [21, 22]. Hence, ERp29 is associated with resistance to oxidative and radiation stress and may play a potential protective role against stress. In cancer cells, the function of ERp29 has been actively addressed [23, 24], and several important roles have been reported. Recent studies have demonstrated significant involvements of ERp29 in: 1) cancer cell survival against genotoxic stress induced by DOX and radiation [21, 22, 25]; 2) cell growth arrest through modulating ER stress [26]; 3) mesenchymal-epithelial transition (MET), cell polarity and epithelial morphogenesis [24, 27]. The current review will focus on the functions of ERp29 as a novel tumor suppressive molecule and the key signaling pathways affected by ERp29 in cancer cells. ERp29: Structure and Biochemical Function ERp29 localizes in the luminal compartment of the ER and contains a PDI-like structure that consists of an N-terminal domain homologous to the thioredoxin-like domains in PDI, and a C-terminal domain with similarity to the P5 subfamily of PDI [12, 13, 28]. Despite the structural resemblance to PDI as manifested by the thioredoxin-like N-terminal domain, ERp29 has no characteristic disulfide isomerase or chaperone activity [12], but exhibits chaperone-like properties at both the biophysical and cellular levels [29, 30]. There is a single cysteine residue at the N-terminal domain (Cys157) indicating that ERp29 may present in either the reduced state or a mixed-disulfide bonded states. These two states may enable ERp29 to bind to substrates with different affinities. It was found that ERp29 can form dimers or higher oligomers with PDI [17]. In addition, ERp29 could change its conformation through noncovalent interactions with other ER factors to drive the substrate binding and release cycle [31]. Its N-terminal domain is critical for dimerization and this serves as a general mechanism to regulate its ER activities [32]. Moreover, both ERp29 N-terminal and C-terminal domains are essential for inducing the local unfolding of polyomavirus to initiate the ER membrane penetration process [32-34]. In addition to the ER chaperone functionality, ERp29 can act to escort secretory proteins through post-er compartments [12, 14, 35]. The existences of ERp29 in conditioned cell media [14] and in milk [36] are consistent with an escort role from the ER to the cell surface. It was found that, in rat spermatozoa, ERp29 is increased significantly on the sperm surface as well as in the cytoplasm of epididymal epithelia from caput to cauda during sperm epididymal maturation [37]. The present of ERp29 on mouse sperm membrane and upregulation as the sperm undergoes epididymal maturation may implicate other potential function in mammalian fertilization and sperm-oocyte fusion [38]. Generally, the ubiquitous expression of ERp29 in the secretory cells [14] and involvement in the secretion of thyroglobulin and other secretory proteins support ERp29 s function as a secretion factor/escort chaperone [14]. In fact, ERp29, as a bona fide molecular chaperone or cochaperone [30, 35], binds to thyroglobulin and mediates its secretion [39]. ER29 also plays a critical role in secretion and transport of membrane proteins. This is supported by a recent finding that ERp29 facilitates the appropriate processing and assembly of connexin43 hemichannels [35]. ERp29 regulates folding of newly synthesized integral membrane proteins like cystic fibrosis transmembrane conductance regulator CFTR [40],

310 304 Daohai Zhang indicting a key role of ERp29 in the regulation of CFTR biogenesis. Interestingly, the classical ER chaperones BiP/GRP78 and endoplasmin/grp94 do not interact with CFTR stably [41-43], and neither BiP nor calnexin has central roles in ER-associated degradation of CFTR [44-46]. Instead, ERp29 showed a robust interaction with ΔF508-CFTR in the absence of cross-linking. Hence, ERp29 could favor hydrophobic substrates such as integral membrane proteins [47]. Recent evidence also implicated the importance of ERp29 for polyomavirus infection through its C-terminal domain [34]. During this process, ERp29 can alter the conformation of polyomavirus' coat protein VP1 and internal protein VP2 through disruption of Py's disulfide bonds. Cooperation of ERp57 and PDI with ERp29 facilitates unfolding of the VP1 C- terminal arm [48]. This stimulates polyomavirus to bind to the surface of the ER membrane and then perforates ER membrane [31, 33]. Induction of a conformational change in the polyomavirus VP1 protein in turn facilitates passage of the virus across the ER membrane and successful infection [31]. This biophysical characterization suggests that ERp29 is functionally distinct from the classical ER chaperones [29, 49]. ERp29, ER Stress and Cell Growth Arrest 1. ER Stress-Related PERK/p-eIF2α Pro-Apoptotic Pathway The ER is a major signal transducing organelle that senses and responds to changes in cellular homeostasis. ER stress can be induced by UPR or by viral infection. The first response of ER stress involves up-regulation of genes encoding ER chaperones, which increase protein-folding/assembly activity and prevent protein aggregation in the ER. These ER resident molecular chaperones include chaperones of the heat shock protein family, including BiP/GRP78 and its co-chaperone partners, chaperone lectins and the foldase family of PDI and ERp29. Under normal conditions, Bip binds to stress transducers, such as double-stranded RNA (PKR)-like ER kinase (PERK) [50], activating transcription factor 6 (ATF6) [51], and IRE1 [52], to form complexes. However, at abnormal conditions, Bip dissociates from these transducers and binds to ER luminal un/misfolded proteins and activates the ER stress response [53] which mediates multiple molecular biological processes via ER stress sensors [54, 55] (Figure 1). Phosphorylation of eukaryotic translation-initiation factor 2α (eif2α) by PERK is a characteristic of PERK pathway activation. To reduce the risk of further accumulation of misfolded proteins in the ER, this pathway is activated to block the initiation of protein translation and attenuate global protein synthesis (Figure 1). Moreover, to overcome the ER stress, a subset of mrnas is able to bypass translational blockage and synthesize proteins that participate in the UPR/ER stress response. One of the critical downstream targets is activating transcription factor 4 (ATF4) which is upregulated and subsequently induces transcription and expression of a set of genes to cope with ER stress [50]. For instance, if misfolded protein levels are consistantly elevated, ATF4 stimulates transcription of CHOP gene (C/EBP homologous protein, also called GADD153) to support the ER stress-induced apoptotic program [56] (Figure 1).

311 Endoplasmic Reticulum Protein 29 (ERp29) and Cancer 305 Figure 1. Depiction of ER stress and the regulatory role of ERp29 in ER stress signaling. Under ER stress, the ER-associated transmembrane sensors PERK, IRE-1α and ATF6 are activated by homodimerization and autophosphorylation (for PERK and IRE1α) and proteolytic cleavage at the Golgi (for ATF6). Activation of PERK leads to phosphorylation of eif2α, resulting in termination of global translation and selectively translation of ER stress-associated proteins, such as ATF4. Prolonged ER stress consistently activates p-perk/p-eif2α pathway to induce cell apoptosis. The CHOPregulated GADD34 inhibits eif2α phosphorylation via PP1 to restore translational function of eif2α and ER homeostatsis. Active ATF6 translocates into nucleus to promote transcription of XBP-1 and ER chaperones. XBP-1 mrna is spliced by activated IRE-1 to generate a shorter spliced variant (XBP-1s) which encodes an active XBP-1s transcription factor. p58 IPK is one of the targets regulated by XBP-1s, and prevents eif2α phosphorylation by binding to PERK. ERp29 enhances XBP-1s/p58 IPK to antagonize cell apoptosis regulated by p-perk/p-eif2α pathway. On the other hand, CHOP also stimulates the expression of growth arrest and DNA damage inducible protein 34 (GADD34), a feedback inhibitor on eif2α phosphorylation by protein phosphatase type 1 (PP1) [57]. This feedback regulation on translation ensures general translational function of eif2α and ER homeostasis. 2. ER Stress-Related XBP-1 Cell Survival Signaling IRE1 is a bifunctional molecule with serine/threonine protein kinase and endoribonuclease (RNase) activity in its cytosolic domain [58]. Dissociation from GRP78 triggers its dimerization and autophosphorylation to activate its downstream process [55]. Activation of IRE1 involves its oligomerization and trans-autophosphorylation of the

312 306 Daohai Zhang kinase domains. Activated IRE1 removes an intron from unspliced XBP-1 (XBP-1u) mrna to generate spliced and activated XBP-1 (XBP1s) [8] (Figure 1). XBP-1s is a highly active transcription factor and is one of the key regulators of ER folding capacity [59, 60]. XBP-1s activates genes that enhance ER protein-folding capacity and degradation of misfolded ER proteins [61] and phospholipid synthesis for the expansion of ER membranes under ER stress [52, 59]. Recent studies showed that prolongation of IRE1 signaling during ER stress can promote cell survival [62, 63]. It was also shown that heat shock protein 72 (HSP72) physically interacts with the cytoplasmic part of IRE1 to protect cells from ER stressinduced apoptosis by prolonging XBP-1 splicing [64]. Thus, IRE1-activated XBP-1s serves as an important adaptive mechanism under stress. Interestingly, the unspliced form of Xbp-1u mrna encodes a rapid-turnover protein that can function as a dominant negative factor to inhibit XBP-1s activities [65, 66] and causes increased apoptosis of tumor cells [67]. In contrast, high expression level of XBP-1s was shown to associate with increased tumor survival [67]. ATF6 is a transcription factor with basic leucine zipper (bzip) motif and localizes on ER membrane. GRP78 binds to ATF6 and prevents its translocation to the Golgi apparatus. Under ER stress, ATF6 dissociates from GRP78 and is transported to the Golgi apparatus where it is cleaved by Golgi-resident site-1 and site-2 proteases (S1P and S2P) (Figure 1). In general, two isoforms of ATF6, ATF6α and ATF6β, are activated during ER stress by the same mechanism [68]. Upon migrates to the nucleus, activated ATF6α stimulates transcription of unspliced XBP-1 gene [55] and the genes that participate in ER-associated degradation [69] (Figure 1). XBP-1s is essential for the regulation of several UPR target genes including p58 IPK [61]. Of interest, p58 IPK can directly bind to PERK to inhibit PERK phosphorylation, resulting in inhibition of phosphorylation of eif2α [70, 71]. As such, ATF6 activation helps the cell to cope with ER stress. 3. ERp29 Inhibits eif2α /CyclinD 1/2 to Induce G 0 /G 1 Arrest via Activating p38 Phosphorylation Tumor cell growth arrest and survival have also been mechanistically linked to ER stress signaling [72]. In particular, PERK autophosphorylation phosphorylates eif2α at Ser-51 upon stress and consequently attenuates global protein translation and induces G 0 /G 1 arrest [73]. The role of p38 activation in inducing cell growth arrest by regulating the PERK/p-eIF2α pathway has been established in human squamous carcinoma cells [72, 74]. The mechanistic link between ER stress and tumor cell growth arrest through p38 activation suggests an important role of p38-regulated networks in modulating tumor cell quiescence, survival and apoptosis [72, 74, 75]. Given that ERp29 induces G 0 /G 1 arrest in breast cancer cells [24, 26], activation of p38 may play a critical role in this aspect. Indeed, studies from the gene expression microarray revealed a significant reduction of urokinase plasminogen activator receptor (upar), β1-integrin and epidermal growth factor receptor (EGFR) [24], an important upstream regulatory complex modulating extracellular signal-regulated kinase (ERK) and p38

313 Endoplasmic Reticulum Protein 29 (ERp29) and Cancer 307 activity [76] (Figure 2). Activation of ERK facilitates cell proliferation and tumorigenesis, whereas activation of p38 promotes G 0 /G 1 arrest [76]. Figure 2. ERp29 induces cancer cell growth arrest via p38 activation. Over-expression of ERp29 in MDA-MB-231 cells decreases expression of the upar β1-integrin EGFR ternary complex, leading to activation of p38 and suppression of phosphorylated extracellular signal-regulated kinase (p-erk). Phosphorylation of p38 inhibits the expression of basal eif2α and cyclin D 2 and increases the expression of cyclin-dependent kinase inhibitors (p15, p16 and p21), thus causing the G 0 /G 1 arrest. When ERp29 was over-expressed in MDA-MB-231 cells, the basal level of p38 was reduced, while its phosphorylation was remarkably enhanced [77]. Meanwhile, ERK activation was concomitantly inhibited by ERp29 in these cells [24] (Figure 2). Interestingly, the enhanced p38 phosphorylation by ERp29 in MDA-MB-231 cells inhibited the expression of basal eif2α, while the relative phosphorylation of eif2α was not significantly changed. This may suggest a novel regulatory mechanism of ERp29 in modulating eif2α activity and protein translation. In general, inactivation of eif2α by phosphorylation induces G 0 /G 1 arrest and cell survival by blocking cyclin D 1 /D 2 translation/stability [73]. The ERp29-reduced expression of basal eif2α causes repression of cyclin D 1 /D 2 expression, in particular of cyclin D 2 level [77]. Knockdown of ERp29 by shrna in MCF-7 cells resulted in up-regulation of cyclin D 2, with no significant effect on cyclin D 1 expression. These results further suggest ERp29 induces G 0 /G 1 arrest, to a greater extent, via suppressing cyclin D 2 expression. On the other hand, ERp29 expression increases the levels of cyclin-dependent kinase inhibitors (e.g., p15, p21 and p27) at both mrna and protein levels [24, 77], thereby leading to G 0 /G 1 arrest (Figure 2). The role of p38 activation in regulating cyclin D 2 and p15 was experimentally verified by

314 308 Daohai Zhang gene knockdown or pharmacological inhibition of p38 activity [77]. Therefore, increased expression of ERp29 can induce growth arrest through modulating p38 phosphorylation and eif2α expression. 4. ERp29 Activates XBP-1s/P58 IPK Cell Survival Pathway XBP-1s is the transcription factor that controls p58 IPK expression during the UPR [61]. The protein p58 IPK can directly bind to PERK to inhibit PERK phosphorylation, resulting in inhibition of eif2α phosphorylation [70]. Consequently, up-regulation of p58 IPK upon ER stress may relieve eif2α phosphorylation and restore general protein translation. ERp29 over-expression in MDA-MB-231 cells led to activation of XBP-1 by stimulating splicing [78] and a significant up-regulation of p58 IPK [77]. Activation of XBP-1/p58 IPK by ERp29 may play a key role in suppressing eif2α phosphorylation induced by p38- phosphorylation (Figure 1). Indeed, in our cell models, silencing of p58 IPK stimulated eif2α phosphorylation and activated expression of ATF4/CHOP and cleavage of caspase-3 [26]. Enhanced phosphorylation of eif2α is a mechanism leading to attenuation of general protein synthesis and activation of ATF4/CHOP under ER stress [79]. Hence, up-regulation of p58 IPK may facilitate cell survival under stress by repressing eif2α phosphorylation (Figure 1). The interplay between p38 phosphorylation and p58 IPK up-regulation has key roles in modulating ERp29-induced cell growth arrest and survival. This new knowledge could lead to novel and effective therapies against drug-resistant cancer cells, e.g., by targeting p58 IPK. ERp29 and Mesenchymal-Epithelial Transition 1. Epithelial-Mesenchymal Transition (EMT) and Mesenchymal-Epithelial Transition (MET) The epithelial-mesenchymal transition (EMT) is an essential process during embryogenesis [80] and its pathological activation during tumor development increases primary tumor cells to metastasize [81, 82]. The pathological conditions such as inflammation, organ fibrosis and cancer progression facilitate EMT [83]. The EMT is characterized as: (1) loss of their junctions, for example, adherens junctions (AJs) and tight junctions (TJs); (2) loss of apical basal polarity; (3) reorganization of their cytoskeletal components and distribution; and (4) gain of migration and invasion abilities [84]. The involvement of EMT in cancer progression has been considered to be an important process for the tumor cells to escape from the primary site and to metastasize [85]. E-cadherin is considered to be a key molecule that provides the physical structure for both cell cell attachment and the recruitment of signaling complexes [86]. Loss of E-cadherin is a hallmark of EMT and the EMT-inducing factors can impair the expression or function of E-cadherin to initiate epithelial reorganization [87]. Therefore, characterizing transcriptional regulators of E-cadherin expression during EMT has provided important insights into the molecular mechanisms underlying the loss of cell cell adhesion and the acquisition of migratory properties during carcinoma progression [88].

315 Endoplasmic Reticulum Protein 29 (ERp29) and Cancer 309 Clinically, loss of E-cadherin is widely observed in invasive and metastatic carcinoma and correlates with the aggressiveness of tumors and poor survival rates in cancer patients [89]. This observation is consistent with that the enhanced expression of the transcriptional repressors of E-cadherin is associated with tumor progression and lymph node metastasis [90]. While the epithelial cancer cells can be transited to mesenchymal-like cells to initiate metastasis, mesenchymal cells can also regain a fully differentiated epithelial phenotype via the mesenchymal epithelial transition (MET) [80, 91]. The MET may also have a role in carcinogenesis by mediating the establishment of distant metastatic tumors at secondary sites [92]. Indeed, recent studies demonstrated that distant metastases in breast cancer expressed an equal or stronger E-cadherin signal than the respective primary tumors and the re-expression of E-cadherin was independent of the E-cadherin status of the primary tumors [93]. Similar findings were also reported in which E-cadherin is re-expressed in bone metastasis or distant metastatic tumors arising from E-cadherin-negative poorly differentiated primary breast carcinoma [94], or from E-cadherin-low primary tumors [95]. The gain of E-cadherin expression during metastasis has been well studied in MDA-MB-468 breast cancer cell line [96]. Analysis of MDA-MB-468 xenografts revealed that some tumor cells exist a metastable phenotype [97, 98], characterized by the expression of both vimentin and E-cadherin [96], The cells at the invasive front showed a positive expression for vimentin and negative expression for E-cadherin, consistent with an EMT. On the other hand, the lymphovascularinvaded tumor cells showed a gradual transition of invaded tumor cells from mesenchymal to metastable and then to the epithelial phenotype, indicating that a MET process occurs as an early event in the metastatic process [97, 98]. The interaction of nonmetastatic mesenchymallike cells and metastatic epithelial-like cells accelerates their metastatic colonization in prostate and bladder cancer cells [99]. Therefore, the EMT and MET process may co-exist and work co-operatively in driving metastasis. 2. Molecular Regulation of EMT Regulation of E-cadherin expression is mediated via transcriptional repressors including zinc-finger proteins (e.g., Snai1 and Slug), zinc-finger E-box-binding proteins (e.g., ZEB1/2), the basic helix-loop-helix proteins (e.g., Twist) and Ets-1 [ ] (Figure 3). Importantly, knockdown of these repressors induces the re-expression of E-cadherin [103] and restores the epithelial phenotype [104]. In addition, several known signaling pathways, such as those involving transforming growth factor-β (TGF-β), Notch, fibroblast growth factor and Wnt signaling pathways, have been shown to trigger epithelial dedifferentiation and EMT [105, 106]. These signals exert their action through modulating transcription factors that repress transcription of epithelial genes, such as those encoding E-cadherin and cytokeratins, and that activate transcription programs that facilitate fibroblast-like motility and an invasive phenotype [88, 106]. Recently, the involvement of micrornas (mirnas) in controlling EMT has been emphasized [ ]. mirnas are small non-coding RNAs (~23 nt) that silence gene expression by pairing to the 3 UTR of target mrnas to cause their posttranscriptional repression [110]. To date, the mir-200 family has been shown to be major regulators of EMT through silencing the EMT-transcriptional inducers ZEB1 and ZEB2, which in turn repress

316 310 Daohai Zhang mir-200f in a double-negative feedback loop [111, 112]. MiR-200f opposes EMT by directly targeting genes involved in motility and invasion [113, 114]. Therefore, expression of mir- 200f in normal and cancer cells promotes the maintenance of an epithelial phenotype [115]. mir-200 and mir-205 RNAs are negative regulators of EMT and their expression is decreased in cells that have undergone a full EMT under different stimuli [116, 117]. Figure 3. ERp29 regulates mesenchymal-epithelial transition. Exogenous expression of ERp29 in mesenchymal MDA-MB-231 breast cancer cells suppresses transcription and protein expression of E- cadherin transcription repressors (e.g., ZEB2, SNAI1 and Twist), resulting in re-expression of E- cadherin and re-establishment of epithelial cell phenotype. ERp29 over-expression also inhibits expression of mesenchymal cell markers (e.g., vimetin, N-cadherin and fibronectin) and increases expression of differentiation markers (e.g., cytokeratin 19) in this cell model. Recent studies have shown that mir-200f members including mir-200h, mir-200a, mir- 200c and mir-141 are epigenetically regulated in cancer and normal tissue [118, 119]. Indeed, epigenetic regulation is a critical mechanism affecting mir-200f expression to modulate EMT-MET [120]. Recent studies also indicated a significant role of mirnas as a prodisposed factors for cancer cell metastasis. For instance, the elevated levels of the epithelial mir-200 family in primary breast tumors associate with poorer outcomes and metastasis [121]. The enhanced E- cadherin re-expression by the mir-200 family members was driven via the repression of ZEB family genes [122, 123]. These findings support a potential role of epithelial mirs in MET to promote metastatic colonization [124].

317 Endoplasmic Reticulum Protein 29 (ERp29) and Cancer ERp29 Promotes MET in Breast Cancer The role of ERp29 in regulating MET has been established in breast cancer cells (Figure 3). When ERp29 was over-expressed in mesenchymal MDA-MB-231 cells, the spindle-like fibroblastic morphology was remarkably changed to a cobble-stone-like phenotype, which is identical to that observed in epithelial MCF-7 cells [24] (Figure 3). Accompanying the phenotypic change, over-expression of ERp29 resulted in cytoskeletal reorganization with loss of filamentous stress fibers and cortical actin formation. Reorganization of the actin cytoskeleton is tightly linked to myosin-driven contraction initiated by MLC phosphorylation [125, 126]. ERp29 expression markedly reduced the level of MLC phosphorylation, suggesting its critical role in ERp29-regulated cortical actin formation and epithelial phenotype [127]. The extracellular matrix (ECM) component fibronectin, which is involved in cell transition from quiescence to proliferation [128], was concomitantly decreased by ERp29 over-expression. Importantly, ERp29 expression reactivated both transcription and protein expression of epithelial cell marker E-cadherin and regulated its membranous localization. Meanwhile, the mesenchymal cell marker vimentin was highly reduced and the epithelial differentiation marker cytokeratin 19 was increased [127] (Figure 3). The ERp29 s role in inducing MET was further substantiated in epithelial MCF-7 cells where knockdown of ERp29 led to a fibroblast-like cellular phenotype, enhanced cell spreading, decreased expression of E-cadherin and increased expression of vimentin [24, 127]. Given that E- cadherin expression inhibits the EMT process [129], reactivation of E-cadherin by ERp29 supports its role in modulating MET in breast cancer cells. 4. ERp29 Targets E-Cadherin Transcription Repressor The transcription repressors such as Snai1, Slug, ZEB1/2 and Twist [ ] have been considered to be the main regulators for E-cadherin expression. Mechanistic studies revealed that ERp29 expression significantly down-regulated transcription of these repressors, leading to their reduced nuclear expression in MDA-MB-231 cells [24, 127] (Figure 3). Consistent with this, the extracellular signal-regulated kinase (ERK) pathway which is an important upstream regulator of Slug and Ets1 was highly inhibited [24]. Apparently, ERp29 up-regulates the expressions of E-cadherin transcription repressors through repressing ERK pathway. Interestingly, ERp29 over-expression in basal-like BT549 cells resulted in incomplete MET and did not significantly affect the mrna or protein expression of Snai1, ZEB2 and Twist, but increased the protein expression of Slug [127]. The differential regulation of these transcriptional repressors of E-cadherin by ERp29 in these two cell-types may occur in a cellcontext-dependent manner. 5. ERp29 Antagonizes Wnt/ β-catenin Signaling Wnt proteins are a family of highly conserved secreted cysteine-rich glycoproteins. The Wnt pathway is activated via a binding of a family member, such as Wnt1, Wnt3, Wnt3a, Wnt7A, or Wnt10B, to a frizzled receptor (Fzd) and the LDL-Receptor-related protein co-

318 312 Daohai Zhang receptor (LRP5/6). There are three different cascades that are activated by Wnt proteins: namely canonical/β-catenin-dependent pathway and two non-canonical/β-catenin-independent pathways that include Wnt/Ca 2+ and planar cell polarity [130]. Of note, the Wnt/β-catenin pathway has been extensively studied, due to its important role in cancer initiation and progression [131]. β-catenin binds to E-cadherin as a part of adherens junctions (AJs) in the absence of Wnt. The presence of Wnt promotes formation of a Wnt Fzd LRP complex, recruitment of the cytoplasmic protein Disheveled (Dvl) to Fzd and the LRP phosphorylationdependent recruitment of Axin to the membrane, thereby leading to release of β-catenin from membrane and accumulation in cytoplasm and nuclei. Figure 4. ERp29 over-expression turns-off activated Wnt/β-catenin signaling. In mesenchymal MDA- MB-231 cells, high expression of nuclear β-catenin activates its downstream signaling involved in cell cycles and cancer stem cell self-renewal. When ERp29 is over-expressed in this cell model, nuclear β- catenin is relocated at the membrane where it binds to E-cadherin, and Wnt/β-catenin signaling is switched off. Meanwhile, over-expression of ERp29 results in up-regulation of TCF3 and increases expression of genes involved in differentiation. N: Nucleus. In the cytoplasm, the unbound β-catenin is constantly degraded by a complex composed of the scaffold proteins Axin and adenomatous polyposis coli (APC), the casein kinase 1 (CK1) and glycogen synthase kinase 3 beta (GSK3β) [132]. β-catenin is sequentially phosphorylated by CK1 and GSK3β, ubiquitinated by β-trcp ubiquitin ligase and degraded by proteasome [133]. However, mutations of APC and Axin, or β-catenin cause constitutive Wnt activation [134]. When β-catenin is absent in nuclear, the transcription factors T-cell factor/lymphoid enhancer factors (TCF/LEF) recruits co-repressors of the TLE/Groucho family and function as transcriptional repressors. However, nuclear β-catenin replaces TLE/Groucho co-repressors and recruits co-activators to activate expression of Wnt target

319 Endoplasmic Reticulum Protein 29 (ERp29) and Cancer 313 genes. The most important genes regulated are those related to proliferation, such as Cyclin D 1 and c-myc [135, 136] (Figure 4), which are over-expressed in most β-catenin-dependent tumors. Wnt pathway is required for driving the stem cell/progenitor compartment. This pathway is altered together with other stem cell-regulating pathways such as Hedgehog and Notch signaling, which supports the cancer stem cells (CSC) model [137]. In fact, nuclear expression of β-catenin and/or mutation in this gene or in other genes of the pathway such as Axin1 are frequently found in poorly and undifferentiated carcinomas, and the Wnt/β-catenin pathway is necessary for the maintenance of CSCs. In breast cancer, the Wnt pathway is upregulated in CSCs by Wnt ligands secreted by the tumor microenviroment [138]. Thus, repressing this pathway by increasing the stability of β-catenin-degrading complex [139] is an alternative therapeutic strategy for the treatment of β-catenin-dependent tumors. As a novel tumor suppressive molecule, ERp29 significantly decreased the expression of cyclin D 1/2 [77], one of the downstream targets of activated Wnt/ β-catenin signaling [136], indicating an inhibitory effect of ERp29 on this pathway. Indeed, when ERp29 was overexpressed in mesenchymal MDA-MB-231 breast cancer cells, nuclear β-catenin was translocated from nucleus to membrane where it forms complex with E-cadherin [27] (Figure 4). This causes a disruption of β-catenin/tcf/lef complex and abolishes its transcription activity. Meanwhile, expression of ERp29 in this cell type increased the nuclear expression of TCF3, a transcription factor regulating cancer cell differentiation while inhibiting selfrenewal of cancer stem cells [140, 141]. Hence, ERp29 may play dual functions in mesenchymal MDA-MB-231 breast cancer cells by: 1) suppressing activated Wnt/β-catenin signaling via β-catenin translocation; and 2) promoting cell differentiation via activating TCF3 (Figure 4). Because β-catenin serves as a signaling hub for the Wnt pathway, it is particularly important to focus on β-catenin as the target of choice in Wnt-driven cancers. Though the mechanism by which ERp29 expression promotes the disassociation of β- catenin/tcf/lef complex in MDA-MB-231 cells remains elusive, activating ERp29 expression may be a promising therapeutic intervention for the poorly differentiated and Wntdriven tumors. ERp29 and Epithelial Cell Integrity 1. Cell Adherens and Tight Junctions Adherens junctions (AJs) and tight junctions (TJs) are composed of transmembrane proteins that adhere to similar proteins in the adjacent cell [142]. The transmembrane region of the TJs is composed mainly of claudins, tetraspan proteins with two extracellular loops [143]. AJs are mediated by Ca 2+ -dependent homophilic interactions of cadherins [144] which interact with cytoplasmic catenins that link the cadherin/catenin complex to the actin cytoskeleton [145]. The cytoplasmic domain of claudins in TJs interacts with occludin and several zona occludens (ZO) proteins (ZO1-3) to form the plaque that associates with the cytoskeleton [146]. The AJs form and maintain intercellular adhesion, whereas the TJs serve as a diffusion

320 314 Daohai Zhang barrier for solutes and define the boundary between apical and basolateral membrane domains [147]. The AJs and TJs are required for integrity of the epithelial phenotype, as well as for epithelial cells to function as a tissue [86]. Especially, the TJs are closely linked to the proper polarization of cells for the establishment of epithelial architecture [148]. During EMT, the expression of proteins that are responsible for the formation of AJs, TJs and apical basal polarity is affected, resulting in loss of cell polarity in epithelial cells [149]. In fact, cancer development is frequently associated with the failure of epithelial cells to form TJs and to establish correct apico basal polarity [150]. For instance, alterations of TJs protein expression and distribution cause the loss of contact inhibition of cell growth [151]. In addition, reduction of ZO-1 and occludin were found to be correlated with poorly defined differentiation, higher metastatic frequency and lower survival rates [152, 153]. Hence, TJs proteins have a tumor suppressive function in cancer formation and progression. 2. Apical Basal Cell Polarity Epithelial cells maintain two types of cell polarity, planar and apical basal polarity [154]. The apical basal polarity of epithelial cells in an epithelium is characterized by the presence of two specialized plasma membrane domains: namely, the apical surface and basolateral surface [154]. The asymmetrical distribution of lipids and proteins between both apical and basal domains is caused by polarized trafficking and the presence of a physical frontier established by the apical junctional complex. In this complex, TJs provide a tight seal while AJs maintain the adhesion between neighboring cells [142, 155]. In general, apical basal cell polarity of epithelial cells is determined by three core interacting protein complexes that influence the assembly and localization of the junctional complexes. These protein complexes include: (1) the partitioning-defective (PAR) complex; (2) the Crumbs (CRB) complex; and (3) the Scribble complex [154, 156, 157]. In mammals, PAR complex is composed of two scaffold proteins (PAR6 and PAR3) and an atypical protein kinase C (apkc), and is localized to the apical junction domain for the assembly of TJs [158, 159]. The Crumbs complex is formed by the transmembrane protein Crumbs (Crb) and the cytoplasmic scaffolding proteins such as the homologue of Drosophila Stardust (Pals1) and Pals-associated tight junction protein (Patj) and localizes to the apical [160]. The Scribble complex is comprised of three proteins, Scribble (Scrib), Disc large (Dlg) and Lethal giant larvae (Lgl) and is localized in the basolateral domain of epithelial cells [161]. Decreased expression or mis-localization of these core polarity proteins may have a causal link to suppression of mammalian tumorigenesis [ ]. Accumulating evidence supports that apical basal cell polarity is established as the result of mutually antagonistic interactions between the PAR, Crumbs and Scribble complexes, thereby leading to the distribution of proteins in a polarized manner [165]. The PAR and Crumbs complexes cooperate to establish the apical domain and the assembly of TJs, whereas the Scribble complex has a key role in the definition of the basolateral plasma membrane domain [148, 160]. The Par6/Par3/aPKC in PAR complex has a pivotal function in polarization and this process is triggered by protein kinases such as Rac1/Cdc42 GTPases [166, 167].

321 Endoplasmic Reticulum Protein 29 (ERp29) and Cancer 315 Indeed, activated Cdc42 is recruited by Par6 to the PAR complex where it causes the activation of apkc and phosphorylation of Par3. Phosphorylated Par3, in turn, promotes the formation of an active PAR complex at the apical domain and the assembly of the junctional structure. At this stage, Crumbs complex is critical to stabilize active PAR complexes. On the other hand, Lgl proteins of the Scribble complex compete with Par3 for binding to the PAR complex, thus sequestering the active PAR complex away from the apical junction domain [168]. Conversely, Lgl phosphorylation by apkc inactivates the Scribble complex [156]. Therefore, the basolaterally located Scribble complex functions as an antagonist of the apical localization of the active PAR complex. 3. Molecular Regulation of AJs, TJs and Cell Polarity Accumulating evidence indicates that EMT inducers concomitantly regulate the expression of genes involved in the formation of AJs and TJs. Apart from E-cadherin, Snail and ZEB factors down-regulated the components of the TJs, including occludin and several members of the claudin family [169, 170]. Mechanistically, Snai1/Snai2 can directly bind to the conserved E-box elements in the corresponding promoters of occludin, claudin-1 and claudin-7 [170, 171]. Gene profiling studies showed that expression of Snai1, Snai2, E47 or other EMT regulators promotes EMT through down-regulating claudin-4, the junctional adhesion molecule-1 (JAM-1/JAM-A) and Dlg3 in carcinoma cells and in Madin-Darby canine kidney (MDCK) cells [172, 173] All these E-cadherin repressors are recognized as key inducers of EMT. Indeed, expression of these EMT inducers results in genetic EMT programs including genes regulating epithelial and mesenchymal phenotypes and genes involved in cytoskeletal reorganization, cell movements and cell survival [88]. Recently, it has been reported that EMT inducers can directly target members of the Crumbs and the Scribble complexes to regulate their expression in different cell systems. For instance, ZEB1 binds at specific proximal E-box sequences of CRB3 and PATJ gene promoter to repress their transcription as demonstrated by promoter analysis and chromatin immunoprecipitation assays [174]. Similarly, ZEB1 silencing also targets polarity gene LGL2 promoter to upregulate its expression in colorectal carcinoma cells [174]. By the similar mechanism, ZEB1 silencing leads to enhanced transcription of CRB3, PATJ and the human homolog of lethal giant larvae 2 (HUGl2/LGL2), as well as genes for TJs components (JAM-1; occludin, claudin-7) [174]. In breast and colorectal carcinoma cells, silencing of ZEB1 leads to partial reversion of the epithelial phenotype and the relocalization of CRB3 and/or LGL2 to the membrane to restore apical basal polarity [175]. Snai1 shows a similar function by acting through distal E-box sequences of the CRB3 promoter in breast carcinoma cells [174]. Apparently, EMT transcriptional inducers act cooperatively to repress polarity proteins to reinforce EMT.

322 316 Daohai Zhang 4. ERp29 Restores AJs and TJs ERp29 involves the establishment of the apical junctional complex, which is formed by AJs and TJs [27] (Figure 5). These complexes are located in the upper portion of a polarized epithelial cell and are composed of trans-membrane proteins that interact with molecules in adjacent cells [142]. In MDA-MB-231 cells, β-catenin is expressed and localized in nuclear. ERp29 over-expression resulted in an increased expression and membrane localization of E- cadherin and translocation of β-catenin from the nucleus to the cell membrane [27] (Figure 4). The ERp29-mediated membrane localization of β-catenin facilitates the assembly of E- cadherin/β-catenin complex and formation of AJs [155] (Figure 5). Intriguingly, ERp29 over-expression led to an increase of TJ components such as ZO-1 and occludin at the membrane and cell cell junctions in breast cancer cells (Figure 5). The increased expression of ZO-1 and occludin is regulated at translational level, as ERp29 overexpression did not affect their mrna levels [27]. The role of ERp29 in ZO-1 protein expression and trafficking was further demonstrated in the ERp29-knockdown MCF-7 cells. Figure 5. ERp29 regulates epithelial cell integrity. Exogenous expression of ERp29 in mesenchymal MDA-MB-231 breast cancer cells increases expression and membrane distribution of E-cadherin and translocation of nuclear β-catenin to the membrane to form E-cadherin/β-catenin complex. The levels of tight junctions proteins ZO-1 and occludin are increased by ERp29 over-expression and their membrane distribution enhances cell-cell contact of epithelial cells. In addition, ERp29 over-expression in this cell model up-regulates expression of apical protein Par3 and basolateral protein Scribble, leading to formation of apical-basolateral cell polarity. Translational up-regulation of ZO-1 and occludin by ERp29 in these cell models may provide a mechanism of how ERp29 induces tumor suppression in breast cancer [24]. Additionally, the formation of cortical actin filaments is critical for the establishment of AJs and TJs and the regulation of epithelial cell apical basal polarity [86]. Reorganization of the actin cytoskeleton induces recruitment of ZO-1 to cell periphery before the assembly of junctional complexes between adjacent cells [176]. The ERp29-induced restoration of ZO-1

323 Endoplasmic Reticulum Protein 29 (ERp29) and Cancer 317 expression may be associated with actin reorganization. Hence, ERp29 plays a critical role in restoration of an epithelial-like phenotype by establishing cell cell contact. 5. ERp29 Restores Cell Polarity In line with the role of ERp29 in regulating MET and re-establishment of the epitheliallike phenotype, ERp29 over-expression can restore epithelial polarity [27] (Figure 5). In mesenchymal MDA-MB-231 and basal-like BT549 cells, ERp29 expression did not affect mrna levels of Par3 and Scribble, but increased their protein translation and membrane distribution. It was reported that Cdc42, a small GTPase, is one of the key regulators modulating the expression of Par6 and apkc [177, 178]. and has a critical role in establishing cell polarity in epithelial cells [179]. However, ERp29 over-expression did not affect both the expression and localization of Cdc42, Par6 and apkc, indicating these PAR complex members are not involved in ERp29-regulated apical polarity. Thus, ERp29 selectively upregulates Par3 protein expression during epithelial morphogenesis. These studies indicate that in the apical Par complex, Par3, but not the Cdc42/Par6/aPKC, is a downstream target that is specifically regulated by ERp29. In addition, ERp29 over-expression did not markedly alter the expression and distribution of Crumb1, a member of the Crumbs complex [180] (Figure 5), Similar to that observed for Par3, ERp29 over-expression resulted in a significant increase of protein expression, but not the mrna level, of Scribble in both MDA-MB-231 and BT549 cells. Suppression of ERp29 by shrna in epithelial MCF-7 cells resulted in reduction of these core polarity proteins, leading to the disruption of cell cell contact and increased cell spreading. Previous studies demonstrated that polarity proteins are synthesized in the endoplasmic reticulum, transported to the Golgi complex and sorted at the trans-golgi network into distinct apical and basolateral vesicular routes [181]. Given that ERp29 mediates the folding and secretion of newly synthesized proteins in the ER system [39], it is plausible that, in addition to increased protein expression of TJs and the core polarity complex, ERp29 may also have a critical role in protein trafficking and the maintenance of protein stability to modulate epithelial cell integrity. In agreement with this, the ERp29-induced tumor suppression in breast cancer cells is linked to the integrity of apical basal polarity that is crucial for the prevention of tumor development [164, 182]. Increased ERp29 expression up-regulates expression and localization of AJs (E-cadherin and β-catenin), TJs (ZO-1 and occludin) and core polarity proteins (Par3 and Scribble) at cell cell junctions. Restoration of these proteins at cell cell junctions leads to close contact of cells and establishment of epithelial-like cell features, a phenotypic characteristic of MET (Figure 5). Consequently, ERp29 has a pivotal role in regulating MET and establishing epithelial cell integrity in breast cancer. ERp29 and Resistance to Genotoxic Stress Recent studies have demonstrated that ERp29 is a novel molecule protecting cells from the genotoxic stress-induced cell apoptosis [78, 183]. To survive from the stress environment,

324 318 Daohai Zhang cells have developed a variety of responsive mechanisms to cope with the stress-induced cell death, such as cell cycle arrest and activation of the DNA repair. In an early study, when cells were exposed to ionization radiation, ERp29 expression was elevated in several types of cultured cells [20]. Concomitantly, splicing of XBP-1 mrna under radiation was increased, suggesting the involvement of UPR sensor might be a reason to induce ERp29 gene expression [20]. In nasopharyngeal carcinoma (NPC) cells, ERp29 knockdown attenuated radio-resistance of NPC CNE-1 cells, whereas ERp29 over-expression enhanced radioresistance of NPC CNE-2 cells. This was reflected by the experimental data showing that ERp29 knockdown in CNE-1 cells increased radiation-induced cell apoptosis, while ERp29 over-expression in CNE-2 cells reduced radiation-induced cell apoptosis. Clearly, ERp29 could potentiate resistance to radiation in NPC cells [183]. Doxorubicin (DOX) is one of the conventional chemotherapeutic drugs for cancer intervention via the intercalation of DNA and subsequent activation of the tumor suppressor p53 [184]. While most of cancer cells are sensitive to DOX and eventually killed by this drug, some cells develop an adaptive response to DOX-induced genotoxic stress and survive from stress. Clinically, chemo-resistance of cancer cells is a predominant cause of cancer recurrence after long-term treatment. It has been found that DOX induced ERp29 expression and ERp29 expression is causally linked to resistance against DOX by a mechanism that requires PERK [185]. PERK activation promotes the phosphorylation of a general translation factor eif2α and attenuates translation of global proteins including cyclin D 1 [73], thereby resulting in inhibition of cell cycle. Apparently, the DOX-induced ERp29 facilitates cell's response to genotoxic stress that ultimately results in an resistance against chemotherapy by DOX. Indeed, recent studies further supported ERp29 s role for cell survival under genotoxic stress condition. When ERp29 was over-expressed in MDA-MB-231 cells, these cells showed a significant resistant to DOX treatment, whereas knockdown of ERp29 in MCF-7 cells led to an enhanced sensitivity of these cells to DOX [78]. Mechanistic studies revealed a critical role of up-regulated Hsp27 in the ERp29-induced DOX resistance in these cell models. In addition, the ERp29-induced activation of ER stress-related XBP-1/p58 IPK cell survival pathway also plays a pivotal role in this aspect [77]. In support of this, silencing of p58 IPK in MCF-7 cells and ERp29-overexpressing MDA-MB-231 clones re-sensitizes them to DOX by activating ATF4/CHOP/caspase-3 pro-apoptotic signaling [77]. Interestingly, in addition to these identified downstream molecules that involve in ERp29-induced DOX resistance, ERp29 over-expression in MDA-MB-231 cells significantly up-regulated the expression of O 6 -methylguanine-dna methyltransferase (MGMT), a DNA repair protein through facilitating dissociation of p53/msin3a/hdac1 transcription repressors via suppressing p53 (unpublished data). MGMT repairs the mutagenic and cytotoxic interstrand DNA cross-links via rapidly reversing alkylation, including methylation, at the O 6 position of guanine by transferring the alkyl group to the active site of the enzyme [186]. In addition to DNA repair function, MGMT plays a role in integrating DNA damage/repair-related signals with replication, cell cycle progression and genomic stability [187, 188]. Hence, MGMT is also an important factor in ERp29-induced anti-genotoxic stress and cell survival. The ERp29-upregulated DNA repair pathway cause resistance to chemoand radio- therapy, and thus targeting this pathway might have a potential to develop alternative strategy for efficient treatment of chemo- and /or radio-resistant cancer cells.

325 1. ERp29 and Tumorigenesis Endoplasmic Reticulum Protein 29 (ERp29) and Cancer 319 Clinical Implication The role of ERp29 in carcinogenesis has been studied with inconsistent accounts. For example, ERp29 was found to be highly expressed in some primary tumors, e.g., basal cell carcinoma and lung cancer [23, 24, 189, 190]. In lung tumors, ERp29 expression varied within and between tumor stages and inversely correlated with tumor progression [23]. Similarly, a tissue array study in 98 breast tumors showed that ERp29 expression was found to be down-regulated in tumors with a more aggressive phenotype [24]. These results indicate a negative association of ERp29 expression with tumor progression, at least in breast and lung tumors. However, the correlation of abnormal ERp29 expression to tumor progression in epithelial cancers needs to be extensively assessed in large cohort of clinical specimens. Shnyder et al reported a significant importance of ERp29 during the histogenetic stage of tumorigenesis in which ERp29 was noticeably over-expressed in all epithelial cancers investigated, and its expression correlated with the rates of cancer growth and lactogenesis [23]. As such, ERp29 expression in epithelial cells could be a factor leading to tumor histogenesis. Recent studies revealed that ERp29 was significantly expressed in radioresistant nasopharyngeal carcinoma (NPC) tissues compared to radio-sensitive NPC tissues, indicating a potential role ERp29 in radio-resistance in NPC tumors [191]. ERp29 s role in resistance to radiotherapy has been well established in cell lines [22]. 2. ERp29 and Metastasis Recent studies showed that increased ERp29 expression has been found in highly metastatic cancer cells. A proteomics study identified that ERp29 was significantly increased in the highly metastatic variant of parental MDA-MB-231 cells compared to the parental cells [192]. Similarly, ERp29 was found to be one of the proteins that were highly expressed in the metastatic tissues compared to the primary uveal melanoma tissues [189]. These results may implicate an important role of ERp29 in cancer cell metastasis and disease recurrence. Indeed, high expression of ERp29 in breast tumors strongly associated with reduced relapse time of disease and short survival time of patients (unpublished data). The ERp29-enhanced metastasis is probably related to its role in driving MET in cancer cells [24]. The role of MET in facilitating distant metastasis has been clinically recognized by the observation that MET is able to reversibly convert the disseminated mesenchymal cancer cells to an epithelial cell state [193]. ERp29 may have a critical role in promoting distant metastasis during cancer progression, although this needs to be investigated further. Consequently, understanding the association of ERp29 with disease recurrence and distant metastasis is of significance in assessing its prognostic value in clinical applications.

326 320 Daohai Zhang 3. Tumor Microenvironment Affects MET and Metastasis Tumor microenvironment is an important factor in regulating cancer metastasis via MET [194, 195]. The interplays between tumor cells, host cells, and the extracellular matrix in tumor ecosystem endow cancer cells with malignant properties, leading to metastatic dissemination. Interestingly, Shyder et al reported that the expression of ERp29 was significantly affected by the culture mode [23], where ERp29 expression was significantly increased in xenografts compared with the same cell types cultured in monolayer or spheroid condition. This indicates that ERp29 could be physiologically regulated in tumor ecosystem. However, it is uncertain whether the increased expression of ERp29 in tumor ecosystem could facilitate MET process, similar to that observed in exogenously ERp29-overexpressed MDA-MB-231 cells [24]. It should be noted that PC3 prostate cancer cells underwent a MET in a three-dimensional culture system [196]. In another study, it was reported that DU145 and PC3 prostate cancer cells expressed high level of E-cadherin when co-cultured with hepatocytes and these carcinoma cells bound to hepatocytes in an E-cadherin-dependent manner [197]. Similarly, when MDA-MB-231 cells were co-cultured with hepatocytes, E- cadherin was re-expressed, resulting in an increased chemo-resistance [198]. In vivo studies demonstrated that MDA-MB-231 cells formed E-cadherin-negative primary tumors, but showed a re-activated E-cadherin expression in lung metastatic site via MET, suggesting an effect of the microenvironment on cells at the metastatic site [199]. Although the tumor microenvironment-induced MET and metastasis is a complex process, investigating the involvement of ERp29 in MET and metastasis may enhance our knowledge and understanding of its biological and pathological functions in cancer progression. Conclusion Accumulating evidence supports a critical role of ERp29 in cancer cell survival and metastasis. However, the current controversy regarding the role of ERp29 in different cancers further emphasizes its importance in understanding its pathological regulation in tumorigenesis. Therefore, it is important to carefully examine the role of ERp29 in each tumor type. The current data from breast cancer cells supports that ERp29 can function as a tumor suppressive protein, in terms of suppression of cell growth and primary tumor formation and inhibition of signaling pathways that facilitate EMT. Nevertheless, the significant role of ERp29 in cell survival against drugs, induction of cell differentiation and potential promotion of metastasis may lead us to re-assess its function in cancer progression, particularly in metastasis. It is of importance to explore in detail the ERp29 s role in cancer as a friend or foe and elucidate its clinical significance in breast cancer and other epithelial cancers. Targeting ERp29 and/or its downstream molecules might be an alternative molecular therapeutics for metastatic cancer treatment.

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339 In: Tumor Suppressor Genes ISBN: Editors: Mehmet Gunduz and Esra Gunduz 2013 Chapter 16 Divergent Roles for Tumor Suppressor Genes in Cancer Marina Trombetta-Lima 1, Thiago Jacomasso 2, Sheila Maria Brochado Winnischofer 2 and Mari Cleide Sogayar 1 1 Biochemistry Department, Chemistry Institute, University of São Paulo, São Paulo, Brazil 2 Biochemistry and Molecular Biology Department, Federal University of Paraná, Paraná, Brazil Abstract Tumor suppressor genes or anti-oncogenes are defined as genes which control cell growth and may lead to the development of cancer upon mutation or deregulated expression. Several tumor suppressor genes that were initially taken as great promises for the clinic, such as the tissue inhibitor of matrix metalloproteinases TIMP-1, have been shown to have more complex roles and even, in some models, to be constitutive markers of tumor aggressiveness. Contrasting roles of a certain gene associated with the same molecular pathway might be explained by the multifuncionality of its products through: (i) epigenetic modifications; (ii) balance between different isoforms displaying different properties, especially since alternative splicing is observed in more than 90% of the human genes; (iii) post-translational modifications, such as methylation, phosphorylation and glycosylation; and/or (iv) differences in the microenvironment and molecular context under different physiological and pathological conditions. Here, we focus on analyzing cases of tumor suppressor genes multifunctionality acting to promote divergent roles of these genes in tumorigenesis, tumor progression and metastasis, thereby imposing a great challenge to translation of these genes as clinical tools. Support: FAPESP, CNPq, BNDES, MS-DECIT, MCTI. Tumor suppressor genes, also known as anti-oncogenes, are wild type alleles playing roles in processes such as: modulation of cell proliferation, differentiation, angiogenesis, invasion, and apoptosis, among others. Classically, the hallmark of these functionally distinct

340 334 M. Trombetta-Lima, Th. Jacomasso, Sh. M. B. Winnischofe et al. genes is the fact that their loss or functional inactivation has an oncogenic outcome. This class of regulatory genes was first identified using different approaches such as: in vitro phenotypic reversion from malignant to normal of tumor cell lines that were transfected with normal cell transcripts; fusion of normal and malignant cells; and induction of terminal differentiation of malignant cells [1-4]. Since their first characterization, tumor suppressor genes became a great promise to the clinic, but in several cases the use of these genes as therapeutic tools revealed to be a great challenge. Here, we discuss the factors which influence tumor suppressor genes dual function and role in tumorigenesis and tumor progression, namely: (i) epigenetic modifications; (ii) alternative splicing; (iii) post-translational modifications; and (iv) differences in the microenvironment and molecular context under different physiological and pathological conditions. I. Epigenetic Modifications Alterations in the epigenetic state of a cell lead to hereditable changes in gene expression without involving modifications in the DNA sequence. Epigenetic mechanisms comprise changes in micrornas expression and affinity, DNA methylation status, and histone modifications. Taken together, these tools compose an orchestrated control of gene expression leading to different physiological and pathological processes such as cancer [5]. MicroRNAs (mirnas, mirs) are short non-coding RNA sequences (18-25nt when maturated), which integrate the RNA-Induced Silencing Complex (RISC) cascade resulting in silencing of mrnas which display a complementary sequence to the seed portion of the mir, 2-7 nt long near the 5 extremity, that usually targets the mrna at its 3 -UTR [6,7]. As coding genes, mirs can either act as tumor suppressor or display a pro-tumoral activity. In this context, Single Nucleotide Polymorphism (SNP) may have an important impact not only in mir expression, but, also, in mirs target sequence specificity and affinity, and, thus, in its function [8] (Figure 1A). This is the case of the Rs polymorphism in hsa-mir- 196a2, shown to alter mature microrna expression and function [9] and described to suppress proliferation and invasion capacity of breast cancer cells, having among its targets, the tumor protein p63 [10]. The Rs polymorphism is associated to susceptibility to breast and lung cancer and to severe toxicity in lung cancer patients submitted to a platinumbased treatment [9,11], highlighting the importance of SNPs to mirs structures and target recognition. The transcriptional activity of a DNA region is considered to be a reflection of its methylation status. Methylation occurs in the carbon 5 position of cytosine residues, generally followed by a guanine residue, being mediated by methyltransferases. Highly methylated regions are found in the heterochromatin and in transcriptionally silenced regions, frequently occurring in genes displaying promoter regions rich in CpG dinucleotides, the so called CpG Islands [5]. In tumors, promoter regions of tumor suppressor genes which contain CpG Islands are found to be hypermethylated (Figure 1B). It is important to highlight that different tumors display different patterns of CpG methylation aberrations, suggesting specific and fine tuned controlling mechanisms that upon disruption may give rise to different alteration profiles [5,12]. Several genes involved in chemosensitivity have been described to display an aberrant methylation profile in different types of cancer, as for example: the O6-

341 Divergent Roles for Tumor Suppressor Genes in Cancer 335 methylguanine-dna Methyltransferase (MGMT,) gene, involved in DNA repair, reduces the toxicity of alkylating agents, the methylation of its promoter is associated with better response to temozolomide [5,13-17]; on the other hand, promoter hypermethylation of the Transforming Growth Factor-Beta-Inducible gene-h3 (TGFBI) gene, involved in microtubule stabilization through integrin signaling, is associated with resistance to paclitaxel in ovarian cancer [18,19]; also associated with chemoresistence is the promoter hypermetylation of Transcription Factor AP-2 Epsilon (TFAP2E) in colorectal cancer [20,21]. Therefore, Methyltransferase family members cannot be classified as neither tumor suppressors nor oncogenes. A Precursor mirna Figure 1. (Continued). DICER Processing SNP Target mrna Target mrna Degradation of the target mrna Target mrna

342 336 M. Trombetta-Lima, Th. Jacomasso, Sh. M. B. Winnischofe et al. Figure 1. Epigenetic mechanisms influencing genes fate. mirnas, which may act as tumor suppressors due to the fact that their mrna targets are subject to SNPs which may alter their target recognition efficiency and specificity, thereby altering their function (A). In cancer, many tumor suppressor genes containing CpG Islands in their promoters are hypermethylated, silencing their expression (B). At the same time, hypomethylation in repeated DNA regions causes opening of the chromatin and DNA breakage, accounting for many of the chromosomal aberrations observed in cancer (C). Histones are subject to modifications of different natures in their N-terminal, which may be acetylated, methylated, ubiquitinilated, ribosylated and phosphorylated [5]. While acetylation in the surroundings of the transcription start sites leads to an open chromatin configuration and is often correlated to an active transcriptional state, histone modifications have a complex pattern that may contribute to activate or silence transcription of a gene displaying a crucial impact in cancer progression. Examples of such phenomenon are: the inhibition of histone deacetylation, resulting in a higher level of MGMT transcription in glioblastoma xenographs, with similar MGMT promoter methylation status, results in a higher chemoresistance to temozolomide [13]; similarly, in the Multidrug Resistance 1 (MDR1), gene of the ATP-Binding Cassette (ABC) family, which is closely related to chemoresistance by effluxing the drug molecules from the cells, promoter methylation is dependent on the Mixed Lineage Leukemia 1 (MLL1) protein, a histone methyltransferase specific for H3K4 [22]. DNA methylation in mammals, combined with histone modifications such as H3K9, is predominant in repeated regions, such as transposable elements and satellite DNA, present in the heterochromatin. Methylation, and thus transcriptional silencing, of these regions is thought to be involved in maintenance of genomic stability and integrity as a host defense mechanism[5]. Global methylation levels are diminished in tumor cells, when compared to healthy tissues. Hypomethylation in repeated DNA regions causes opening of the chromatin and DNA breakage, accounting for many of the chromosomal aberrations observed in cancer [5,23] (Figure 1C).

343 Divergent Roles for Tumor Suppressor Genes in Cancer 337 The use of therapies that explore epigenetic mechanisms is of extreme interest due to the importance of altered epigenetic profiles to the onset of cancer. Inhibitors of methyltransferases, such as 5-aza-2'deoxycytidine (Decitabine) and 5-Azacytidine (Vidaza), have shown promising clinical results and their use has been approved by the Food and Drug Administration (FDA). These Methytransferases inhibitors may be used either as a single drug for treatment, as, for instance, in clinical trials targeting myelodysplastic syndrome and acute myeloid leukemia in older patients or patients that are not prone to a more intensive treatment, which have shown significant improvement [24-31]; or synergistically, when combined with other drugs, such as romiplostim, aclacinomycin/cytarabine and vorinostat, showing prognostic improvement for different tumor types [32-35]. However, the promiscuous activity of methyltransferases represents a challenge to their successful implementation. Genome wide hypomethylation promoted by 5-aza-2'deoxycytidine was shown to induce trinucleotide CAG CTG repeat instability in hamster CHO cells and in human cells from myotonic dystrophy patients. In the latter, destabilization of repeat tracks in the Dystrophia-Myotonica Protein Kinase (DMPK) gene was observed [36,37]. This kind of genomic instability is observed in several neurodegenerative diseases, such as Huntington s disease, type 1 myotonic dystrophy and different spinocerebellar ataxias [36-41]. The potentiality of genomic instability, as a consequence of treatment with Methyltransferases inhibitors, is controversial. In animal models, hypomethylation has been associated to chromosomal instability and tumor induction, and different types of human tumors are known to be associated with global hypomethylation and chromosomal instability, such as colorectal cancer and leukemias [42-48]. At the same time, it has been reported that patients with myelodysplastic syndrome treated with 5-aza-2'deoxycytidine did not display higher chromosomal instability, when compared to the cohorts that did not received this specific treatment [49]. Yang et al. and Lengauer highlighted that caution should be taken to transpose the data obtained upon 5-aza-2'deoxycytidine treatment consequences in humans, since the different results may be explained by the different models used. Nevertheless, some aspects to be taken into consideration are that methylation status of the treatment appears to be transient and according to tumor type the resulting genomic instability could either promote tumor progression and/or induce tumorigenesis in a second site; or, in the case of excessive chromosomal instability, contribute to the anti-tumoral efficacy of the treatment, leading the tumor cells to death [12,49-51]. Epigenetic manipulation revealed to be a useful tool for the clinic and further studies aiming to access its side effects both at short and long term and to refine its specificity will amplify the range of tumor types which may be susceptible to this approach. II. Alternatively-Spliced Tumor Suppressor Genes Pre-mRNA alternative splicing is a major source of proteomic diversity in eukaryotic cells. It is estimated that more than 90% of human multiexon genes generate at least two different transcripts [52]. Alternative selection of exon-intron boundaries during pre-mrna maturation may cause the protein products to present different or even antagonizing functions (Figure 2). Splicing events associated with cancer and other diseases have been reported and extensively reviewed [18,53-57].

344 338 M. Trombetta-Lima, Th. Jacomasso, Sh. M. B. Winnischofe et al. Figure 2. Alternative splicing influencing gene fate. Isolated or combined: exon skipping, inclusion and alternative selection of exon-intron boundaries during pre-mrna maturation can cause the protein products to present different or even antagonizing functions (A). The balance between the alternative isoforms may influence the outcome of different pathways, thereby leading to a pathological condition, such as cancer (B). Regarding cancer biology, alternative splicing plays a crucial role in determining whether some genes will ultimately generate an oncoprotein or a tumor suppressor protein. The set of exons present in the mature mrna population through alternative splicing is affected by oncogenic signaling pathways, such as Ras/PI3K/Akt [58,59] Ras/Raf/MEK/ERK [60] and c- Myc [61], which activate downstream non-specific splice regulators, promoting changes in the splice pattern of several genes to activate global splicing programs [53]. The most studied splicing regulators are those of the serine-arginine (SR) class of proteins. These factors bind to exonic splicing enhancer (ESE) elements in the pre-mrna to favor either exon inclusion or exclusion during splicing. One member of this family in particular, namely, SRSF1, has been widely implicated in pro-tumoral splicing events, appearing to be a point of convergence of oncogenic signaling cascades [18]. Overexpression of SRSF1 alone was shown to be sufficient to transform normal cells, whereas its RNAi-mediated knockdown attenuated the aggressive phenotype of tumor cells [62,63]. Despite its oncogene-like behavior, SRSF1- induced splicing shows great functional variety, producing either pro- or anti-tumoral isoforms, depending on the gene. The mrna variants population from a given gene may also be affected by SNPs and point mutations in splice sites or cis-acting elements on the premrna, disrupting their recognition by trans-regulatory factors [56]. Alternatively spliced isoforms of tumor suppressors may either lack tumor suppressor activity or generate antagonistic, pro-tumoral products. One example of antagonistic splice variants is the Krüppel-like zinc finger transcription factor 6 gene (Klf6). KLF6 promotes growth suppression by several mechanisms (reviewed in [64]), including up-regulation of p21 in a p53-independent manner [65,66] and inhibition of Cyclin D1 binding to CDK4 [67]. Its