Mirnas and Cancer

Transcription

1 MINERVA BIOTEC 2008;20:13-21 Investigations on micrornas in human chronic lymphocytic leukemia and control of cancer-associated molecular pathways M. NEGRINI, A. VERONESE, M. FERRACIN, B. ZAGATTI, S. SABBIONI, A. CORALLINI, G. A. CALIN, C. M. CROCE A variety of studies on micrornas in human chronic lymphocytic leukemia (CLL) opened the way to the assessment of the importance of this large class of genes in human cancer. The first example of involvement of mirnas in a human neoplasm was provided by the discovery of mir-15a and mir-16-1 locus within the minimal region of deletion at chromosome 13q14 in human CLL. The proof that a large number of mirnas are deregulated in malignant cells was initially given by genomewide expression studies of mirnas in CLL. The finding of a prognostic mirna signature was discovered in human CLL. One of the first molecular links with cancer molecular pathways came by the discovery of the regulation of the BCL2 oncogene operated by mir-15a/mir- 16-1, which is aberrant in CLL cells. Finally, the potential cancer predisposing role of mirnas was suggested by the discovery of mir-15a/mir-16-1 germ-line mutations in families associated with an increased risk of CLL. Together with the CLL studies, many additional investigations increased the importance of the field as they proved that numerous mirnas are deregulated in human cancers and may act either as oncogenes or tumor suppressor genes. Aberrant mirna expression has been linked to cell cycle progression, loss of differentiation, increased survival, invasion and metastasis. The discovery that mirnas interact with oncogenes and tumor suppressor genes established multiple links with molecular pathways implicated in malignant transfor- Fundings. Supported by funding from Associazione Italiana per la Ricerca sul Cancro, Ministero dell Università e della Ricerca and Ministero della Salute. Received on April 21, Accepted for publication on May 29, Address reprint requests to: M. Negrini, Dipartimento di Medicina Sperimentale e Diagnostica, Centro Interdipartimentale per kla Ricerca sul Cancro, Università di Ferrara, Via Luigi Borsari 46, Ferrara, Italy. E- mail: ngm@unife.it Interdepartment Center for Cancer Research Department of Experimental and Diagnostic Medicine University of Ferrara, Ferrara, Italy mation. These findings hold the promise that mirnas could become important diagnostic and therapeutic tools. Key words: MicroRNAs - Neoplasms - Oncogenes - Genes, tumor suppressor. Malignant transformation is a process that involves genetic and epigenetic alterations that influence cell growth, death and differentiation. Most known cancer genes encodes for proteins. However, in recent years a class of genes that encode tiny RNAs called micrornas (mirnas), have been found to be altered in human cancer. Initially believed to play a regulatory role only in Caenorhabditis elegans, 1-3 the importance of mirnas increased in 2001 when they were identified and cloned from several organisms, including human, and their nucleotide sequences were found to be philogenetically conserved (4-7). Based on the most recent release of the mirbase Registry, different mirnas have been identified in animals, plants and viruses, and 678 mirnas are human. Computational algorithms predict that as many as mirnas may exist in the human genome. 9 In humans, mirna genes are located in all chromosomes, with the exception of the Y. Nearly 50% of known mirnas are found in clusters and are tran- Vol No 1. MINERVA BIOTECNOLOGICA 13

2 NEGRINI mirna AND CANCER scribed as polycistronic transcripts. The majority of mammalian mirna genes are located in introns of protein-coding genes or in intergenic non-coding transcriptional units; less often in exons and anti-sense orientation with the host gene. 10 Intergenic mirnas and, sometimes, intronic mirnas are transcribed by RNA polymerase II as independent units. The primary transcript (pri-mirna) is capped and polyadenylated. 11, 12 MicroRNA maturation begins in the nucleus, where the pri-mirna is processed by a protein complex known as Microprocessor, which contains the nuclear RNaseIII Drosha and its cofactor DGCR8/Pasha Microprocessor action generates a precursor mirna (pre-mirna), a nt long RNA with a stem-loop structure, that is rapidly exported to the cytoplasm by Exportin-5 in a Ran GTP-dependent manner. The mature mirna(s) may reside in the 5 arm or in the 3 arm of the pre-mirna stem; sometimes both arms generate mature mirnas. Once in the cytoplasm, a second RNaseIII, Dicer, acts on the pre-mirna to release a ~22 nt mirna duplex in which the mature mirna is partially paired to a mirna* present on the pre-mirna opposite stem strand. Usually, only the mirna (mature mirna) strand of the mirna::mirna* duplex is active and enters a protein complex, the RNA-induced silencing complex (RISC), to repress gene expression. 16 Mature mirna guides RNA-induced silencing complex (RISC) toward regions of partial complementarity in the 3 UTR of target mrnas, and triggers either their degradation or inhibition of translation depending on the degree of complementarity between the mirna and its target. In animal cells, post-transcriptional regulation by mirna requires a mrna sequence that is perfectly complementary to the seed sequence (positions 2-7 of the mature mirna). Various algorithms have been developed for predicting mirna targets interactions Based on predictive algorithms, each mirna may potentially regulate hundreds of target mrnas 20 and it seems plausible that most mrnas are post-transcriptionally regulated by mirnas. Recently, a deviation from the current view on mirnas acting as post-transcriptional repressors of gene expression has been brought in by the case of mir-369-3: this mirna was found to up-regulate translation of the tumor necrosis factor alpha (TNFalpha). 21 The study proved that the AU-rich element (ARE) found in the 3 untranslated regions of TNFalpha mrna recruits micro-ribonucleoprotein factors, including Argonaute and fragile X mental retardation-related protein 1, to activate translation upon cell cycle arrest. The same study proved that other mirnas, such as Let-7 and the synthetic microrna mircxcr4, can induce translation up-regulation of target mrnas on cell cycle arrest, yet they repress translation in proliferating cells. These findings suggest that activation is a common function of micrornps on cell cycle arrest, which leads the authors to propose that translation regulation by micrornps oscillates between repression and activation during the cell cycle. If confirmed, this finding will lead to a change in our way of thinking on the biological effects of mirnas. mir-15a and mir-16-1 as leukemic genes The first evidence of mirnas involvement in human cancer came from a study on chronic lymphocytic leukemia (CLL). 22 Human CLL is characterized by few chromosomal aberrations, the most common being the deletion at chromosome 13q14, which occurs in about 60% of the cases. By examining this recurring deletion in search for a tumor suppressor gene involved in CLL, this study found that two mirnas, mir-15a and mir-16-1, were located within the smallest minimal common region of deletion. These two mirnas are just few hundred nucleotides apart and share their primary transcript. Analysis of their expression in CLL samples and normal CD5+ lymphocytes, which represent the corresponding normal cells of malignant leukemic cells, revealed that down-regulation of both mirnas was consistently associated with leukemic cells and the deletion at chromosome 13q14. Thus, the narrowing down of the minimal common region of deletion to 30 kb, the presence of two mirnas within this region and their down-regulation in leukemic cells were the circumstantial evidences that lead to suggest a role for mirnas, mir-15a and mir-16-1, as tumor suppressor genes. Subsequent investigations confirmed the involvement of mir-15a and mir-16-1 in the pathogenesis of human CLL as tumor suppressor genes. First, transfection of mir-15a and mir-16-1 into MEG01 megakaryocytic human cells, which completely lack expression of these mirnas because of their homozygous deletion, resulted in induction of in vitro apoptotic cell death and in vivo suppression of tumorigenity. Second, it was proven that mir-15a and mir could negatively regulate the anti-apoptotic onco- 14 MINERVA BIOTECNOLOGICA March 2008

3 mirna AND CANCER NEGRINI gene BCL2. Third, an inverse correlation between the expression of BCL2 and the mirnas was detected in several human CLL samples. Thus, it was concluded that the loss or the reduction of mir-15a/mir-16-1 expression in CLL cells could lead to an accumulation of BCL2 protein, which in turn was responsible for cell survival of CLL cells. Because BCL2 is over-expressed in the majority of B cells of CLL 23 and, with the exception of less than 5% of cases in which BCL2 is juxtaposed to Ig loci, 24 no mechanism has been discovered, the lack of post-transcriptional control operated by mirnas represents at least one mechanism for the accumulation of BCL2 in CLL cells. A recent genome-wide screening based on microarray and proteomic analyses revealed that BCL2 is not the only target of mir-15a and mir Besides confirming BCL2 as a target, the study revealed various additional targets, which included MCL1, JUN, SCAP2, TRA1, PDCD6IP, RAD51C, and HSPA1A/1B, and others, such as CDK6, CDC27, and RAB11FIP2, that were already shown by another study. 25 Interestingly, the functions of genes regulated by these micrornas were most frequently associated with cell cycle control, such as G1-S and G2-M checkpoints, and antiapoptotic pathways, suggesting that mir-15a and mir are widely involved in the regulation of cancerassociated mechanisms. The putative tumor suppressor function of mir-15a and mir-16-1 in CLL was further supported by the discovery of two CLL patients carrying a germ-line point mutation that results in reduced levels of mature mir-15a and mir This finding also suggested that mirnas may have a role in familial cancer predisposition. Are mirnas involved in cancer predisposition? Germ-line mutation of tumor suppressor proteincoding genes have been associated with cancer-predisposing syndromes. Therefore, it is not illogical to think that germ-line alterations in mirna genes may potentially increase predisposition to cancer. Although this is not a firm conclusion, a first suggestion of this possibility was furnished by Calin et al., 26 who identified two cases of familial CLL in which a point mutation within the primary transcript encoding for mir- 15a and mir-16-1 was responsible for reduced levels of mature mirnas. The development of animal model that mimics human diseases is the way that is usually employed to address the genetic influence on human diseases. A natural mouse strain became helpful in clarifying this point. In parallel to the above studies in human CLL, a genome-wide linkage study of the mouse strain NZB, which is predisposed to a CLL-like leukemic disease, was published. This study revealed three loci located on chromosomes 14, 18 and 19 linked to the development of CLL-like leukemias. Interestingly, the chromosome 14 locus D14mit160 is syntenic to human chromosome 13q14. Strikingly, the NZB mouse strain was found to carry a germ-line point mutation within the mir-15a/mir-16-1 locus, just 1 nucleotide from the germ-line mutation detected in human CLL samples. This nucleotide change is within a region conserved among human and mouse genomes and the same mutation was not found in any other mouse strain, including the closest neighbor NZW. Moreover, the authors report that a reduced level of these mirnas was associated with the mutation and that the delivery of mir-16 to NZB malignant B cells resulted in cell cycle alterations and increased apoptosis. These findings were strongly suggestive that the altered expression of mir-15a / mir-16-1 cluster caused by point mutations was responsible for the predisposition to the CLL-like disease in the NZB mice, 27 a situation presenting striking similarities with the human disease, supporting the hypothesis that germ-line mutations at the mir-15a/mir-16-1 locus could predispose to CLL in human. Numerous micrornas are involved in human cancer mir-15a and mir-16-1 were only the first mirnas to be linked to human malignancies. Evidence now indicates that the involvement of mirnas in cancer is much more extensive than initially expected. Initial clues came from the observation that about 50% of known mirna genes are located at sites of recurrent deletions or amplifications in human cancers. 28 More direct evidence for this has been provided by genomewide expression studies. Again, the first study was carried out in human CLL. By analyzing the expression of 190 mirnas, this study proved that several of them were differentially expressed in CLL cells versus the normal CD5+ lymphocytes, thus providing the proof of principle that malignant cells exhibit an extensive aberrant mirna Vol No 1. MINERVA BIOTECNOLOGICA 15

4 NEGRINI mirna AND CANCER expression. This study was followed by a number of investigations on several other types of human malignancies. All the studies revealed the existence of differences in mirna expression in neoplastic versus normal tissues. 26, These studies show that each neoplasm has a distinct mirna signature that differs from that of other neoplasms and that of each normal tissue counterpart. Moreover, it has become clear that some mirnas are recurrently deregulated in human cancer. In most cases, deregulation was consistently univocal, namely up-regulation or down-regulation, across the different types of cancers, suggesting a common mechanism of involvement in human tumorigenesis. Two large expression profiling studies have also been reported. In a large profiling analysis of 334 leukemias and solid cancers, Lu et al. 49 found that mirna-expression profiles classify human cancers based on developmental lineage and differentiation state of the tumour. This study also revealed a globally decreased mirna expression in tumors with respect to their normal counterpart. Volinia et al. 50 conducted a large-scale mirnome analysis on 540 samples representing six solid cancers (lung, breast, stomach, prostate, colon and pancreas) and corresponding normal tissues and have shown the existence of a tumor-specific mirna signature, 43 deregulated mirnas (26 up- and 17 down-regulated). These genome-wide studies indicate that members of the let-7 family, mir-145, mir-221, mir-21 and mir- 155, are deregulated in several cancers. Since a variety of other evidences has connected these mirnas to human cancer, the expression work indeed appears to identify mirnas relevant in cancer pathogenesis. Among the mirnas identified, several have not yet been thoroughly investigated; our knowledge of this aspect of human cancer is thus still at an early stage. mirnas as prognostic tools Given the findings discussed above, mirnas aberrant expression could influence cancer phenotype. If so, specific mirna expression signatures could reveal distinct subgroups of each cancer type. Indeed, this occurs in various cases. A specific expression signature consisting of 13 mirnas is linked to prognosis and disease progression in human CLL. 26 In human CLL, expression of the 70- kd zeta-associated protein (ZAP-70) and the mutational status of the immunoglobulin variable region (IgVH) are factors that can predict the clinical course of the disease. 2-6 Cases in which the leukemic cells have few or no mutations in the IgVH gene or a high level of expression of the 70-kD zeta-associated protein (ZAP-70) have an aggressive course, whereas cases involving mutated CLL clones or few ZAP-70 cells have an indolent course. 7 By comparing the expression of 190 mirnas of 36 aggressive type CLLs versus 47 indolent type CLLs, it was shown that a signature consisting of 13 mirnas could distinguish the two groups. To validate the signature, a predictive analysis was carried out in an independent set of 50 CLL samples, which were all correctly classified. Interestingly, mir-15a and mir-16-1 were included in the prognostic signature. Low expression of these mirnas were correctly associated with the indolent disease, which is also associated indeed with the deletion at 13q14. Other examples of prognostic mirnas include the higher levels of mir-155 expression present in DLBCLs with an activated B cell phenotype than with the germinal center phenotype. Because patients with activated B cell-type DLBCL have a poorer clinical prognosis, quantification of this mirna may be clinically useful. 51 Similarly, let-7 down-regulation in non-small cell lung cancer is associated with poor prognosis and reduced post-operative survival. 52 Over-expression of mir-10b is present in about 50% of metastatic breast cancers. 53 mirnas as tumor suppressor genes and oncogenes As previously indicated, several examples of mirnas whose expression is deregulated in human cancer have been reported. Like mir-15a and mir-16-1, the mir-143 and mir-145 genes are significantly down-regulated in colon cancer tissue compared with colonic mucosa, 54 and let-7 family members are downregulated in more than 50% of lung cancers 52 as well as other neoplasms. 55 Because of their consistent down-regulation in neoplasms, these mirnas are believed to have a tumor suppressor function. Functional assays revealed that mir-145 can suppress cell growth of breast cancer cells of various human established cell lines. The tumor suppressor function of let-7 is supported by studies by Takamizawa et al. 52 and Akao et al., 56 who showed that let-7 can sup- 16 MINERVA BIOTECNOLOGICA March 2008

5 mirna AND CANCER NEGRINI Deletions Mutations DNA methylation mirna DOWN-REGULATION Let-7 mir-15/16 mir-125 mir-145 mir-127 mir-34a/b/c ONCOGENIC TARGETS RAS HMGA2 BCL2 CDK4/6 CCNE ERBB2/3 BCL6 Differentiation Apoptosis Proliferation Stress stimuli (HIF-1, p53) Angiogenesis Invasion Amplification Activated Transcription Factors (MYC, TWIST) mir-21 mir-155 mir-221/222 mir-10b mir mirna OVER-EXPRESSION P27 HOXD10 PTEN E2F SUPPRESSOR TARGETS Metastasis Figure 1. mirnas may act as oncogenes or tumor suppressor genes by repressing protein-coding genes with tumor suppressor or oncogenic functions. Deregulation of mirnas in cancer cells may occur by genetic (deletions, amplifications, point mutations) or epigenetic (DNA methylation) changes, by aberrant transcription factors expression (Myc, Twist, p53, Hif-1), and by abnormal responsiveness to various stimuli (differentiation, proliferation, hypoxia or other stress stimuli). Abnormally high or abnormally persistent, or lack of mirna expression may ultimately affect the expression of target genes. As a result, cells may not differentiate or undergo apoptosis, or they may increase their proliferation rate, motility and invasiveness, properties that represent hallmarks of invasive cancer. press the growth of A549 lung cancer cells and DLD- 1 colon cancer cells in vitro While loss of expression is the mechanism linking these tumor suppressor mirnas to human malignancies, mirna deregulation in cancer can also operate in the opposite direction, potentially recognizing oncogenic mirnas. The mir family is the most studied example. This family includes fourteen homologous mirnas, which are encoded by three gene clusters on chromosomes 7, 13 and X. 57 The cluster on chromosome 13 is amplified in human B-cell lymphomas, which lead to increased expression of various mirna members. Interestingly, enforced expression of the mir cluster acts together with MYC to accelerate tumour development in a mouse B-cell lymphoma model, 58 thus acting as an oncogene. Another important example of oncogenic mirna is mir-155. This mirna and its primary transcript BIC are over-expressed in Hodgkin s lymphoma, in pediatric Burkitt s lymphoma and in diffuse large B-cell lymphoma. 51, Direct evidence of its oncogenic activity comes from studies of a transgenic mouse model over-expressing this mirna in B lymphocytes. 62 As observed in human B-cell lymphomas, these mice exhibit preleukemic pre-b cell polyclonal expansion followed by B-cell malignancy, demonstrating the direct contribution of a mirna to malignant transformation. Vol No 1. MINERVA BIOTECNOLOGICA 17

6 NEGRINI mirna AND CANCER Another example of up-regulated mirna is mir- 21, a gene located at chromosome 17q23 in a chromosomal region frequently amplified in human cancer, 63, 64 which is up-regulated in human breast cancer and in glioblastoma. 38, 40, 41 Mechanisms of mirna deregulation in human cancer Genomic aberrations may be responsible for altering mirna expression. Indeed, mirna up-regulation has been associated with genomic amplification, 58, 65 and mirna down-regulation has been associated with chromosomal deletions, point mutations and aberrant promoter methylation 22, 26, 28, 66 (Figure 1). However, in many cases, genomic alterations do not seem to play a key role. Most frequently, the activation of oncogenic transcription factors, such as MYC, represents an important mechanism for altering mirna expression 65 (Figure 1). Similarly to c-myc, MYCN can up-regulate the mir cluster as well as other mirnas, including mir-221, which is up-regulated in several types of cancer. 67 In addition to up-regulate the oncogenic mir cluster, c-myc regulates a large set of mirnas that, unexpectedly, become repressed in consequence of Myc activation. Ectopic expression of repressed mirnas diminishes the tumorigenic potential of lymphoma cells, thus proving that the mirnas repressed by Myc also contribute to tumorigenesis. 68 Additional examples of transcription factors regulating mirnas involved in tumorigenesis are represented by the invasion and metastasis-associated factor Twist, which promotes transcription of mir-10b; 53 the tumor suppressor p53, which is altered in a large fraction of human neoplasms, induces the transcription of mir- 34a, -34b and 34c; the hypoxia induced factor 1 (HIF-1) is responsible for the activation of a number of mirnas in response to hypoxic environment. 75 Thus, either physiologically induced or aberrantly expressed, transcription factors are largely responsible for mirna up-regulation in human cancer (Figure 1). Molecular pathogenesis of cancer-associated mirnas We have already discussed the mir-15a/mir-16-1 example: in CLL, the down-regulation of the mirnas allows the accumulation of BCL2 and other oncogenic proteins, which confer a survival and proliferation advantage to leukemic cells. Several other examples exist. A molecular link between Let-7 mirna deregulation and RAS expression, crucial oncogenic element of the cascade of events that lead cells to proliferate, has been established. The 3 UTRs of the KRAS, NRAS and HRAS mrnas contain multiple complementary sites for binding of let-7 members, and forced expression of let-7 in human cancer cells reduces RAS protein levels. 55 Since let-7 is generally down-regulated in several human cancers, this mechanism could lead to the activation of the RAS pathway. mir-221, which was recently shown to be induced by MYCN 67 and repressed by p53, 69 emerged as a significantly up-regulated mirna in glioblastoma, pancreatic, hepatocellular, kidney, bladder, prostate and thyroid cancer, 33, 34, 38, 39, 44, 76, 77 thus suggesting an oncogenic role in several human neoplasms. Its oncogenic function was substantiated by the discovery of its ability to modulate the expression of the cyclindependent kinase inhibitors CDKN1B/p27 and CDKN1C/p57, key controllers of cell cycle progression (Fornari F, Gramantieri L, Ferracin M, Veronese A, Sabbioni S, Calin GA et al. MiR-221 controls CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma. Oncogene submitted). 77, 78 It was shown that the tumor suppressor PTEN is a direct target of mir-21, 79 a mirna that is frequently over-expressed in most of human cancers. Thus, PTEN could be repressed by over-expression of mir-21, which would lead to cell survival through PI3K-AKT pathway activation. mir-21 can also down-regulate the tumor suppressor Programmed Cell Death 4 (Pdcd4). 80, 81 Pdcd4 is believed to have a role in TGFbeta induced apoptosis. Another important example linking mirnas to apoptosis and cell proliferation pathways is given by the mir cluster that acts together with MYC to accelerate tumor development in a mouse B-cell lymphoma 58 (Figure 2). These lymphomas, differently from those arising in the MYC-only system, are characterized by the absence of apoptosis, which suggests that various mir family members regulate a pro-apoptotic gene. Interestingly, two mirnas encoded by the cluster, mir-17-5p and mir-20a, negatively regulate the expression of E2F1, a transcription factor that promotes cell cycle progression but is also a strong inducer of apoptosis. The absence of apoptosis might thus be linked to the tight control of E2F1 18 MINERVA BIOTECNOLOGICA March 2008

7 mirna AND CANCER NEGRINI Myc E2F1 Abnormal cell cycle progression high apoptosis Myc E2F1 mir Abnormal cell cycle progression controlled by mir low apoptosis oncogenes or loss-of function of tumor suppressor genes, which would result in the aberrant post-trascriptional regulation of other oncogenes or tumor suppressor genes. Thus, bidirectional relationships between protein-coding cancer genes and mirna exist. By inhibiting oncogenes or functioning as their effectors, mirnas could themselves act as tumor suppressors or oncogenes (Figure 1). Lymphomas in 3 months high apoptosis Lymphomas in 2 months low apoptosis Figure 2. mir cluster cooperates with Myc in lymphomagenesis. Myc activates the transcription of E2F1-3 and viceversa, thus creating a positive loop. MiR cluster, which is transcriptionally activated by both factors, negatively regulates E2Fs and establish a negative feed-back loop, thus balancing E2Fs activity.66, 84 This mechanism allows physiological cell cycle progression without the risk of E2Finduced apoptosis. Oncogenic activity of mir requires its overexpression, which lets cells to escape apoptosis even in case cell cycle progression is aberrant. Therefore, in the mouse model described by He et al., 59 the mir cluster cooperates with Myc to accelerate the appearance of B-cell lymphomas characterized by the absence of apoptosis, otherwise common in Myc-induced lymphomas. by the mir-17 family. A more recent report, reveals that E2F1, E2F2, and E2F3 directly bind the promoter of the mir cluster, activating its transcription, and mir- 20a, a member of the mir cluster, modulates the translation of the E2F2 and E2F3 mrnas. 82 These results suggest the existence of a feed-back regulatory loop involving mir-20a and E2F that protects cells from apoptosis induced by excessive E2F expression (Figure 2). In human solid tumors, expression of the mir-17 cluster at chromosome 13 is up-regulated in small-cell lung cancer, and ectopic over-expression of this cluster enhances lung cancer cell growth. 83, 84 Importantly, mir up-regulation leads to increased tumor angiogenesis, which is mediated by down-regulation of the anti-angiogenic factors thrombospondin-1 (Tsp1) and connective tissue growth factor (CTGF), both predicted targets of the mir micrornas. 84 Many additional important examples could be provided. Taken together, these known examples teach us two main lessons: first, the aberrant expression of mirnas can potentially affect all the known molecular and biological functions associated with cancer initiation and progression; second, aberrant mirna expression could be induced by the activation of Conclusions MiRNAs clearly play a pivotal role in human tumorigenesis. Strong evidence of their consistent deregulation in human neoplasms indicate that they may act as oncogenes or tumor suppressor genes. Several lines of evidence support their role in biological processes that are aberrant in cancer, such as cell growth, differentiation and apoptosis, which may confer increased invasive and metastatic properties to cancer cells. In addition, the demonstration of the interactions with the cancer-associated gene products P53, BCL2, MYC and RAS establishes a basis for understanding mechanisms linking mirna deregulation to specific molecular pathways. Besides gene expression studies, which have been methodically applied to a variety of human malignancies, all the other studies need to be extended to define in full the role of mirnas in cancer and the mechanisms connected to their deregulation. Additional studies are needed to identify stimuli that induce changes in mirna expression, to reveal new mirnas gene targets and establish the relationships with the molecular pathways involved in cancer. Finally, assessment of the potential for mirnas as diagnostic markers or therapeutic molecules or targets is still only beginning. The development of animal models will certainly help us to establish the role of mirnas in tumorigenesis and develop tools useful for in vivo testing of anti-cancer AMOs and mirnas. References 1. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin- 14. Cell 1993;75: Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 1993;75: Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE et al. The 21-nucleotide let-7 RNA regulates devel- Vol No 1. MINERVA BIOTECNOLOGICA 19

8 NEGRINI mirna AND CANCER opmental timing in Caenorhabditis elegans. Nature 2000;403: Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Maller B et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 2000;408: Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science 2001;294: Lau NC, Lim LP, Weinstein EG, Bartel DP. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 2001;294: Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science 2001;294: Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ. mirbase: microrna sequences, targets and gene nomenclature. Nucleic Acids Res 2006;34:D Berezikov E, Guryev V, van de Belt J, Wienholds E, Plasterk RH, Cuppen E. Phylogenetic shadowing and computational identification of human microrna genes. Cell 2005;120: Kim VN, Nam JW. Genomics of microrna. Trends Genet 2006;22: Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH et al. MicroRNA genes are transcribed by RNA polymerase II. Embo J 2004;23: Cai X, Hagedorn CH, Cullen BR. Human micrornas are processed from capped, polyadenylated transcripts that can also function as mrnas. RNA 2004;10: Han J, Lee Y, Yeom KH, Nam JW, Heo I, Rhee JK et al. Molecular basis for the recognition of primary micrornas by the Drosha- DGCR8 complex. Cell 2006;125: Han J, Lee Y, Yeom KH, Kim YK, Jin H, Kim VN. The Drosha- DGCR8 complex in primary microrna processing. Genes Dev 2004;18: Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J et al. The nuclear RNase III Drosha initiates microrna processing. Nature 2003;425: Tang G. sirna and mirna: an insight into RISCs. Trends Biochem Sci 2005;30: Microrna.org [homepage on the Internet]. New York: Memorial Sloan-Kettering Cancer Center [last update 2008 Jan 8]. Available from: htpp://www-microrna.org/. 18. TargetScan [homepage on the Internet]. Cambridge, MA: Whitehead Institute for Biomedical Research [Release 4.2: April 2008]. Available from: htpp:// 19. Pic Tar [homepage on the Internet]. New York: Center for Comparative Functional Genomics [last update 2007 March 26]. Available from: htpp://pictar.bio.nyu.edu/. 20. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microrna targets. Cell 2005;120: Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation: micrornas can up-regulate translation. Science 2007;318: Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E et al. Frequent deletions and down-regulation of microrna genes mir15 and mir16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2002;99: Kitada S, Andersen J, Akar S, Zapata JM, Takayama S, Krajewski S et al. Expression of apoptosis-regulating proteins in chronic lymphocytic leukemia: correlations with in vitro and in vivo chemoresponses. Blood 1998;91: Adachi M, Tefferi A, Greipp PR, Kipps TJ, Tsujimoto Y. Preferential linkage of bcl-2 to immunoglobulin light chain gene in chronic lymphocytic leukemia. J Exp Med 1990;171: Linsley PS, Schelter J, Burchard J, Kibukawa M, Martin MM, Bartz SR et al. Transcripts targeted by the microrna-16 family cooperatively regulate cell cycle progression. Mol Cell Biol 2007;27: Calin GA, Ferracin M, Cimmino A, Di Leva G, Shimizu M, Wojcik SE et al. A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med 2005;353: Raveche ES, Salerno E, Scaglione BJ, Manohar V, Abbasi F, Lin YC et al. Abnormal microrna-16 locus with synteny to human 13q14 linked to CLL in NZB mice. Blood 2007;109: Calin GA, Sevignani C, Dumitru CD, Hyslop T, Noch E, Yendamuri S et al. Human microrna genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci USA 2004;101: Wang T, Zhang X, Obijuru L, Laser J, Aris V, Lee P et al. A micro- RNA signature associated with race, tumor size, and target gene activity in human uterine leiomyomas. Genes Chromosomes Cancer 2007;46: Subramanian S, Lui WO, Lee CH, Espinosa I, Nielsen TO, Heinrich MC et al. MicroRNA expression signature of human sarcomas. Oncogene 2008;27: Tetzlaff MT, Liu A, Xu X, Master SR, Baldwin DA, Tobias JW et al. Differential expression of mirnas in papillary thyroid carcinoma compared to multinodular goiter using formalin fixed paraffin embedded tissues. Endocr Pathol 2007;18: Martinez I, Gardiner AS, Board KF, Monzon FA, Edwards RP, Khan SA. Human papillomavirus type 16 reduces the expression of microrna-218 in cervical carcinoma cells. Oncogene 2008;27: Gramantieri L, Ferracin M, Fornari F, Veronese A, Sabbioni S, Liu CG et al. Cyclin G1 is a target of mir-122a, a microrna frequently down-regulated in human hepatocellular carcinoma. Cancer Res 2007;67: Gottardo F, Liu CG, Ferracin M, Calin GA, Fassan M, Bassi P et al. Micro-RNA profiling in kidney and bladder cancers. Urol Oncol 2007;25: Mattie MD, Benz CC, Bowers J, Sensinger K, Wong L, Scott GK et al. Optimized high-throughput microrna expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies. Mol Cancer 2006;5: Calin GA, Liu CG, Sevignani C, Ferracin M, Felli N, Dumitru CD et al. MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad Sci USA 2004;101: Liu CG, Calin GA, Meloon B, Gamliel N, Sevignani C, Ferracin M et al. An oligonucleotide microchip for genome-wide microrna profiling in human and mouse tissues. Proc Natl Acad Sci USA 2004;101: Ciafre SA, Galardi S, Mangiola A, Ferracin M, Liu CG, Sabatino G et al. Extensive modulation of a set of micrornas in primary glioblastoma. Biochem Biophys Res Commun 2005;334: He H, Jazdzewski K, Li W, Liyanarachchi S, Nagy R, Volinia S et al. The role of microrna genes in papillary thyroid carcinoma. Proc Natl Acad Sci USA 2005;102: Chan JA, Krichevsky AM, Kosik KS. MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res 2005;65: Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S et al. MicroRNA gene expression deregulation in human breast cancer. Cancer Res 2005;65: Kutay H, Bai S, Datta J, Motiwala T, Pogribny I, Frankel W et al. Downregulation of mir-122 in the rodent and human hepatocellular carcinomas. J Cell Biochem 2006;99: Murakami Y, Yasuda T, Saigo K, Urashima T, Toyoda H, Okanoue T et al. Comprehensive analysis of microrna expression patterns in hepatocellular carcinoma and non-tumorous tissues. Oncogene 2006;25: Pallante P, Visone R, Ferracin M, Ferraro A, Berlingieri MT, Troncone G et al. MicroRNA deregulation in human thyroid papillary carcinomas. Endocr Relat Cancer 2006;13: Roldo C, Missiaglia E, Hagan JP, Falconi M, Capelli P, Bersani S et al. MicroRNA expression abnormalities in pancreatic endocrine and acinar tumors are associated with distinctive pathologic features and clinical behavior. J Clin Oncol 2006;24: MINERVA BIOTECNOLOGICA March 2008

9 mirna AND CANCER NEGRINI 46. Weber F, Teresi RE, Broelsch CE, Frilling A, Eng C. A limited set of human MicroRNA is deregulated in follicular thyroid carcinoma. J Clin Endocrinol Metab 2006;91: Yanaihara N, Caplen N, Bowman E, Seike M, Kumamoto K, Yi M et al. Unique microrna molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell 2006;9: Bottoni A, Zatelli MC, Ferracin M, Tagliati F, Piccin D et al. Identification of differentially expressed micrornas by microarray: a possible role for microrna genes in pituitary adenomas. J Cell Physiol 2007;210: Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D et al. MicroRNA expression profiles classify human cancers. Nature 2005;435: Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M et al. A microrna expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A 2006; 103: Eis PS, Tam W, Sun L, Chadburn A, Li Z, Gomez MF et al. Accumulation of mir-155 and BIC RNA in human B cell lymphomas. Proc Natl Acad Sci USA 2005;102: Takamizawa J, Konishi H, Yanagisawa K, Tomida S, Osada H, Endoh H et al. Reduced expression of the let-7 micrornas in human lung cancers in association with shortened postoperative survival. Cancer Res 2004;64: Ma L, Teruya-Feldstein J, Weinberg RA. Tumour invasion and metastasis initiated by microrna-10b in breast cancer. Nature 2007;449: Michael MZ, O Connor SM, van Holst Pellekaan NG, Young GP, James RJ. Reduced accumulation of specific micrornas in colorectal neoplasia. Mol Cancer Res 2003;1: Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A et al. RAS is regulated by the let-7 microrna family. Cell 2005;120: Akao Y, Nakagawa Y, Naoe T. let-7 microrna functions as a potential growth suppressor in human colon cancer cells. Biol Pharm Bull 2006;29: Tanzer A, Stadler PF. Molecular evolution of a microrna cluster. J Mol Biol 2004;339: He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Goodson S et al. A microrna polycistron as a potential human oncogene. Nature 2005;435: van den Berg A, Kroesen BJ, Kooistra K, de Jong D, Briggs J, Blokzijl T et al. High expression of B-cell receptor inducible gene BIC in all subtypes of Hodgkin lymphoma. Genes Chromosomes Cancer 2003;37: Metzler M, Wilda M, Busch K, Viehmann S, Borkhardt A. High expression of precursor microrna-155/bic RNA in children with Burkitt lymphoma. Genes Chromosomes Cancer 2004;39: Kluiver J, Poppema S, de Jong D, Blokzijl T, Harms G, Jacobs S et al. BIC and mir-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas. J Pathol 2005;207: Costinean S, Zanesi N, Pekarsky Y, Tili E, Volinia S, Heerema N et al. Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice. Proc Natl Acad Sci USA 2006;103: Ehrbrecht A, Muller U, Wolter M, Hoischen A, Koch A, Radlwimmer B et al. Comprehensive genomic analysis of desmoplastic medulloblastomas: identification of novel amplified genes and separate evaluation of the different histological components. J Pathol 2006;208: Sinclair CS, Rowley M, Naderi A, Couch FJ. The 17q23 amplicon and breast cancer. Breast Cancer Res Treat 2003;78: O Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c- Myc-regulated micrornas modulate E2F1 expression. Nature 2005;435: Saito Y, Liang G, Egger G, Friedman JM, Chuang JC, Coetzee GA et al. Specific activation of microrna-127 with downregulation of the proto-oncogene BCL6 by chromatin-modifying drugs in human cancer cells. Cancer Cell 2006;9: Schulte JH, Horn S, Otto T, Samans B, Heukamp LC, Eilers UC et al. MYCN regulates oncogenic MicroRNAs in neuroblastoma. Int J Cancer 2008;122: Chang TC, Yu D, Lee YS, Wentzel EA, Arking DE, West KM et al. Widespread microrna repression by Myc contributes to tumorigenesis. Nat Genet 2008;40: Tarasov V, Jung P, Verdoodt B, Lodygin D, Epanchintsev A, Menssen A et al. Differential regulation of micrornas by p53 revealed by massively parallel sequencing: mir-34a is a p53 target that induces apoptosis and G1-arrest. Cell Cycle 2007;6: Raver-Shapira N, Marciano E, Meiri E, Spector Y, Rosenfeld N, Moskovits N et al. Transcriptional activation of mir-34a contributes to p53-mediated apoptosis. Mol Cell 2007;26: Corney DC, Flesken-Nikitin A, Godwin AK, Wang W, Nikitin AY. MicroRNA-34b and MicroRNA-34c are targets of p53 and cooperate in control of cell proliferation and adhesion-independent growth. Cancer Res 2007;67: Tazawa H, Tsuchiya N, Izumiya M, Nakagama H. Tumor-suppressive mir-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells. Proc Natl Acad Sci USA 2007;104: He L, He X, Lim LP, de Stanchina E, Xuan Z, Liang Y et al. A microrna component of the p53 tumour suppressor network. Nature 2007; 447: Chang TC, Wentzel EA, Kent OA, Ramachandran K, Mullendore M, Lee KH et al. Transactivation of mir-34a by p53 broadly influences gene expression and promotes apoptosis. Mol Cell 2007;26: Kulshreshtha R, Ferracin M, Wojcik SE, Garzon R, Alder H, Agosto- Perez FJ et al. A microrna signature of hypoxia. Mol Cell Biol 2007;27: Lee EJ, Gusev Y, Jiang J, Nuovo GJ, Lerner MR, Frankel WL et al. Expression profiling identifies microrna signature in pancreatic cancer. Int J Cancer 2007;120: Galardi S, Mercatelli N, Giorda E, Massalini S, Frajese GV, Ciafre SA et al. mir-221 and mir-222 expression affects the proliferation potential of human prostate carcinoma cell lines by targeting p27kip1. J Biol Chem 2007;282: Le Sage C, Nagel R, Egan DA, Schrier M, Mesman E, Mangiola A et al. Regulation of the p27(kip1) tumor suppressor by mir-221 and mir-222 promotes cancer cell proliferation. Embo J 2007;26: Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST, Patel T. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology 2007;133: Asangani IA, Rasheed SA, Nikolova DA, Leupold JH, Colburn NH, Post S et al. MicroRNA-21 (mir-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene 2008;27: Frankel LB, Christoffersen NR, Jacobsen A, Lindow M, Krogh A, Lund AH. Programmed cell death 4 (PDCD4) is an important functional target of the microrna mir-21 in breast cancer cells. J Biol Chem 2008;283: Sylvestre Y, De Guire V, Querido E, Mukhopadhyay UK, Bourdeau V, Major F et al. An E2F/miR-20a auto-regulatory feed-back loop. J Biol Chem 2007;282: Hayashita Y, Osada H, Tatematsu Y, Yamada H, Yanagisawa K, Tomida S et al. A polycistronic microrna cluster, mir-17-92, is overexpressed in human lung cancers and enhances cell proliferation. Cancer Res 2005;65: Dews M, Homayouni A, Yu D, Murphy D, Sevignani C, Wentzel E et al. Augmentation of tumor angiogenesis by a Myc-activated microrna cluster. Nat Genet 2006; 38: Vol No 1. MINERVA BIOTECNOLOGICA 21