THE HEDGEHOG PATHWAY IN MALIGNANT MESOTHELIOMA

THE HEDGEHOG PATHWAY IN MALIGNANT MESOTHELIOMA Chuan Bian Lim B.Sc. This thesis is presented for the degree of Doctor of Philosophy from the University of Western Australia School of Medicine and Pharmacology March 2015

ABSTRACT Malignant mesothelioma is an invasive, locally aggressive tumor, predominantly of the pleura and peritoneum, which is associated with asbestos exposure. Its incidence is increasing worldwide and it has an extremely poor prognosis, due to resistance to conventional treatment modalities, with a median survival of less than one year after diagnosis. Clearly, novel therapeutic strategies are required to improve survival of mesothelioma patients. Mounting evidence supports the aberrant hyperactivation of the Hedgehog (Hh) signaling pathway as crucial to the pathogenesis of certain cancers, including mesothelioma. The Hh pathway is critical for embryonic development and adult homeostasis but hyperactivation of the pathway, through mutations in the Hh pathway genes or overexpression of ligand or receptors, have been shown to drive tumorigenesis. This thesis examines the general hypothesis that targeting the Hh pathway can be a therapeutic strategy against mesothelioma. More specifically, this thesis aims to: 1. Identify mutations in Hh pathway genes in mesothelioma 2. Functionally characterize the mutations identified through in silico and in vitro analysis 3. Determine the preclinical efficacy of Hh pathway inhibitors, in particular the Gli inhibitor GANT61, on mesothelioma cells in vitro 4. Characterize the biological effects of GANT61 using the mesothelioma cell line LO68. Real-time PCR analysis of Hh pathway genes PTCH1, GLI1 and GLI2 were performed on seven human mesothelioma cell lines. Exon sequencing of 13 Hh pathway genes was also performed in cell lines and human mesothelioma tumors. In silico programs were used to predict the likelihood that an amino-acid substitution would have a functional effect. 2

GLI1, GLI2 and PTCH1 were highly expressed in mesothelioma cells, indicative of active Hh signaling. PTCH1, SMO and SUFU mutations were found in 2 of 11 mesothelioma cell lines examined. A non-synonymous missense SUFU mutation (p.t411m) was identified in LO68 cells. In silico characterization of the SUFU mutant suggested that the p.t411m mutation might alter protein function, however, no functional effect of this mutation on Gli activity was demonstrated. Deletion of exons of the PTCH1 gene and a 3- bp insertion (69_70insCTG) in SMO was also identified in JU77 cells and predicted to alter protein function. Although Hh pathway mutations are relatively rare in mesothelioma, these data suggest a possible role for a dysfunctional Hh pathway in the pathogenesis of a subgroup of mesothelioma patients and help rationalize the exploration of Hh pathway inhibitors for mesothelioma therapy. Recent studies link aberrant Hh signaling with mesothelioma growth and survival. Gli transcription factors, one of the critical and terminal elements of the Hh pathway, control cell growth and survival via the upregulation of genes related to proliferation and apoptosis. Overexpression of GLI1 has been reported to be significantly associated with worse overall survival in human mesothelioma. The Gli inhibitor GANT61 has been shown to inhibit the Hh signaling pathway by interfering with Gli transcription factors binding to DNA. In human mesothelioma LO68 cells displaying hyperactivated Hh signaling, GANT61 reduced the intracellular levels of GLI1 and GLI2. The reduction attenuated the expression of Gli target genes such as PTCH1 and Bcl-2. GANT61 was also found to suppress cell proliferation and clonogenic survival in LO68 cells. GANT61 treatment also led to induction of autophagy, as evidenced by the accumulation of autophagosomes observed by staining cells with acridine orange and CytoID Green autophagy detection reagent. Pharmacologic inhibition of GANT61-induced autophagy by bafilomycin A1, 3-methyladenine or chloroquine enhanced GANT61-induced 3

apoptotic cell death. Furthermore, exposure of LO68 cells to GANT61 led to G1 phase arrest and apoptotic cell death with increased annexin V staining. Notably, GANT61- induced apoptosis did not depend on its purported target - GLI1 or GLI2, but does involve reactive oxygen species (ROS). GANT61 triggered the generation of ROS and quenching of ROS by N-acetyl-cysteine, a ROS scavenger, which protected from GANT61-induced apoptosis in mesothelioma cells. Furthermore, it was demonstrated that mitochondria are important in GANT61-induced ROS production and apoptosis: (1) ROS production and apoptosis was significantly blocked by the mitochondrial complex I inhibitor rotenone; (2) GANT61 promoted superoxide formation in mitochondria and depolarization of mitochondrial membrane potential; and (3) mitochondrial DNA-deficient LO68 cells failed to induce superoxide compared to wild-type cells, and were more resistant than wild-type cells to apoptosis induced by GANT61. Taken together, the data demonstrate for the first time that GANT61 induces apoptosis in LO68 cells by promoting mitochondrial superoxide generation, independent of Gli inhibition. In conclusion, the data presented in this thesis suggest that mutations in the coding regions of Hh pathway genes are unlikely to be responsible for the hyperactivation of the pathway in mesothelioma. The driving mechanism of aberrant Hh signaling in mesothelioma is likely to be related to Hh-dependent activation of Gli transcription factors. This thesis also presented the first evidence that mitochondrial superoxide may be responsible for the anticancer effects of GANT61 and the concomitant inhibition of cytoprotective autophagy potentiates the anticancer effects of GANT61 in mesothelioma. Overall, this thesis provides a strong rationale for developing GANT61 as a novel treatment for mesothelioma. 4

DECLARATION The work presented in this thesis was performed solely by the candidate except where otherwise acknowledged and has been accomplished during enrolment. This thesis has not been previously accepted for any other degree in this or another institution. Chuan Bian Lim 5

ACKNOWLEDGEMENTS To everyone who has helped and encouraged me in this journey, my heartfelt thanks. First, I would like to thank my primary supervisor, Professor Steven Mutsaers for his unwavering support and confidence in my work and giving me the freedom to pursue my scientific interests. I would also like to acknowledge my co-supervisors, Associate Professor Cecilia Prêle, Professor Philip Thompson and Dr. Svetlana Baltic for their inputs and encouragement. My thanks are also to my labmates Faang Cheah, Huimin Cheah and Kimberly Birnie for their encouragement, support and importantly, friendship. I would like to thank Winthrop Professor Bruce Robinson, Professor Jenette Creaney and Professor Steven Albelda for mesothelioma cells; Associate Professor Brian McCaughan for patient samples; Professor Rune Toftgård for the Gli luciferase construct. I would also like to thank Associate Professor Paul Rigby, Associate Professor Matthew Linden, Ms Tracey Lee-Pullen and Ms Alysia Buckley (Centre for Microscopy, Characterisation and Analysis, University of Western Australia) for support with flow cytometry and confocal microscopy; Dr David Chandler, Dr Shane Herbert and Mr Matthew Davis (Australian Genome Research Facility) for support with sequencing. Last but not least, I like to thank Mom, Dad and Fatty for their patience, listening ears and love. 6

LIST OF PUBLICATIONS 1. Lim CB, Prêle CM, Cheah HM, Cheng YY, Klebe S, Reid G, Watkins DN, Baltic S, Thompson PJ & Mutsaers SE. Mutational analysis of Hedgehog signaling pathway genes in human malignant mesothelioma, PLoS ONE 8(6): e66685, 2013. 2. Lim CB, Prêle CM, Arthur PG, Creaney J, Watkins DN, Baltic S, Thompson PJ & Mutsaers SE. Mitochondria-derived reactive oxygen species drives GANT61- induced malignant mesothelioma cell apoptosis. Oncotarget, 6(3): 1519-1530, 2015. 7

LIST OF PUBLISHED ABSTRACTS 1. Lim CB, Cheah HM, Baltic S, Thompson PJ, Prêle CM & Mutsaers SE. Mutations in Hedgehog pathway genes PTCH1 and SMO are rare in malignant mesothelioma cells. Respirology, 17 (Supplement S1), TP-123, 2012. 2. Lim CB, Thompson PJ, Baltic S, Lee YCG, Watkins DN, Prêle CM & Mutsaers SE. Targeting of Hedgehog pathway via pharmacologic inhibition of Gli induces apoptosis in human malignant mesothelioma cells. Respirology, 18 (Supplement 2), O103, 2013. 3. Lim CB, Prêle CM, Arthur PG, Creaney J, Watkins DN, Baltic S, Thompson PJ & Mutsaers SE. Induction of mesothelioma cell apoptosis by GANT61, a small molecule inhibitor of Gli transcription factors: Evidence for redox-driven cytotoxicity. Respirology, 20 (Supplement 2), TP-227B, 2015. 8

TABLE OF CONTENTS Abstract 2 Declaration 5 Acknowledgements 6 List of publications 7 List of published abstracts 8 Table of contents 9 Abbreviations 15 Chapter 1 General introduction 17 1.1 Overview 18 1.2 Mesothelioma 18 1.2.1 Genetics and mesothelioma 22 1.2.2 Management of mesothelioma 23 1.2.3 Molecularly targeted therapy in mesothelioma 25 1.3 The Hedgehog signaling pathway 26 1.3.1 Hedgehog signaling in a nutshell 27 1.3.2 Hedgehog ligand 30 1.3.3 Hedgehog ligand reception at cell membrane surface 32 1.3.4 SMO as signal transducer 32 1.3.5 Gli as transcription factors 33 1.3.6 Regulation of Gli by SUFU 36 1.3.7 Primary cilium and Hedgehog signaling 36 1.4 Hedgehog signaling and cancer 37 1.4.1 Ligand-independent mutational activation of Hedgehog signaling 37 1.4.2 Autocrine, ligand-dependent Hedgehog 9

signaling 39 1.4.3 Paracrine, ligand-dependent Hedgehog signaling 40 1.4.4 Hedgehog signaling and the cell cycle 43 1.4.5 Hedgehog signaling and apoptosis 44 1.4.6 Hedgehog signaling and autophagy 45 1.5 Drugging the Hedgehog pathway 48 1.6 Reactive oxygen species and cancer therapy 50 1.7 Summary and scope of thesis 53 1.8 Thesis layout 55 Chapter 2 Materials and methods 58 2.1 Cell lines and culture conditions 59 2.2 Ethics statement 59 2.3 Patients and tumors 60 2.4 DNA isolation 60 2.5 PCR amplification 61 2.6 DNA sequence analysis 62 2.7 PCR assay for detection of PTCH1 exon deletions 63 2.8 In silico characterization of polymorphisms in exons 63 2.9 Drugs 63 2.10 Cell proliferation assay 64 2.11 Colony formation assay 64 2.12 Cell cycle analysis by DNA content 65 2.13 Detection of apoptosis 65 2.14 WST (Water-Soluble Tetrazolium)-1 assay 66 2.15 ROS detection 66 10

2.16 Mitochondrial membrane potential (ΔΨm) detection 66 2.17 OxyDNA assay 67 2.18 Detection of autophagy 67 2.19 Immunofluorescence analysis 68 2.20 RNA interference 68 2.21 Gli luciferase reporter assay 69 2.22 Western blot analysis 69 2.23 Antibodies 70 2.24 Generation of mitochondrial DNA-depleted LO68 cells 70 2.25 RNA isolation 71 2.26 cdna synthesis 71 2.27 Quantitative real-time PCR (qrt-pcr) analysis of gene expression 72 2.28 Drug combination analysis 72 2.29 Statistical analysis 73 Chapter 3 Mutational analysis of Hedgehog pathway genes in mesothelioma 74 3.1 Introduction 75 3.2 Materials and methods 77 3.2.1 RNA isolation, cdna synthesis and quantitative real-time PCR (qrt-pcr) analysis of gene expression 77 3.2.2 Gli luciferase reporter assay 77 3.3 Results 78 3.3.1 The canonical Hedgehog signaling pathway is active in human mesothelioma cell lines 78 3.3.2 PTCH1, SMO and SUFU mutations in mesothelioma cell lines 81 3.3.3 Hedgehog pathway gene variants in 11

mesothelioma cell lines 87 3.3.4 PTCH1, SMO and SUFU mutations in FFPE mesothelioma tumors 90 3.3.5 Functional characterisation of SUFU mutant 90 3.4 Discussion 92 Chapter 4 Targeting of Hedgehog pathway using Gli inhibitor GANT61 98 4.1 Introduction 99 4.2 Results 100 4.2.1 Gli inhibitor GANT61 is a more potent Hedgehog pathway inhibitor than Smo inhibitors (cyclopamine and GDC-0449) 100 4.2.2 GANT61 induced higher levels of apoptosis than Smo inhibitors (cyclopamine and GDC-0449) 104 4.2.3 GANT61 targets Gli transcription factors 106 4.2.4 GANT61 sensitivity correlates with GLI2 and GLI2 mrna expression 106 4.2.5 Depletion of GLI1, GLI2 and SMO reduces cell growth 110 4.2.6 GANT61-induced apoptosis and cell cycle are independent of Gli inhibition 110 4.2.7 GANT61 triggers induction of autophagy 114 4.2.8 Autophagy inhibition enhances GANT61-induced apoptosis 119 4.2.9 GANT61 sensitizes LO68 cells to standard mesothelioma chemotherapy-induced apoptosis 122 4.2.10 GANT61 mediates antagonistic to synergistic sensitization effects on standard chemotherapyinduced apoptosis 127 4.2.11 GANT61 triggers the production of reactive oxygen species 129 4.3 Discussion 135 Chapter 5 Mitochondria-derived ROS are critical in GANT61-12

induced apoptosis 146 5.1 Introduction 147 5.2 Results 148 5.2.1 ROS production does not appear to be a class phenomenon for Gli inhibitors 148 5.2.2 GANT61 kills LO68 cells through ROS -mediated DNA damage 151 5.2.3 GANT61 kils LO68 cells through ROS -mediated impairment of DNA repair 151 5.2.4 GANT61 downregulates GLI1, GLI2 and PTCH1 through ROS 154 5.2.5 GANT61-induced ROS is independent of Gli Inhibition 154 5.2.6 GANT61-induced apoptosis and ROS production are dependent on NADPH oxidase 159 5.2.7 GANT61-induced apoptosis and ROS production are dependent on mitochondria 161 5.2.8 Dissipation of mitochondrial membrane potential mediates GANT61-induced apoptosis 161 5.2.9 Mitochondrial superoxide is essential for GANT61 -induced apoptosis 170 5.3 Discussion 176 Chapter 6 General discussion 184 6.1 Future perspective 192 6.2 Conclusions 194 References 195 Appendices 228 Appendix A Intronic primer sequences for amplification of exons of Hedgehog pathway genes 229 Appendix B PCR protocols for amplification of exons of Hedgehog pathway genes 236 Appendix C Exonic primer sequences for PCR assay for detection 13

of PTCH1 exon deletions 240 Appendix D Buffers and solutions 241 14

ABBREVIATIONS 3-MA ABC ABL BafA1 BCC 3-methyladenine Adenosine triphosphate-binding cassette Abelson oncogene Bafilomycin A1 Basal cell carcinoma BCL2 B-cell CLL/lymphoma 2 Β-TrCP BCR BOC BSO β-transducin-repeat containing protein Breakpoint cluster region Brother of CDO Buthionine sulfoximine CDK1 Cyclin-dependent kinase 1 CDO Cell adhesion molecule-related/down-regulated by oncogenes CK1 Casein kinase 1 CML CI DHH DMSO ddh 2 O DPI FBS FOXM1 Chronic myelogenous leukemia Combination index Desert hedgehog Dimethyl sulfoxide double distilled H 2 O Diphenylene iodonium Fetal bovine serum Foxhead box M1 GAS1 Growth arrest specific 1 GPCR GSK3β H 2 O 2 G-protein-coupled receptor Glycogen synthase kinase 3β Hydrogen peroxide 15

Hh HHIP HPI-1 IHH GLI MDR mtor NOX PBS PI3K PKA PTCH Hedgehog Hedgehog interacting protein Hedgehog pathway inhibitor-1 Indian hedgehog GLI-Kruppel family member Multidrug resistance Mammalian target of rapamycin NADPH oxidase Phosphate buffered saline Phosphoinositide 3-kinase Protein kinase A Patched RFC1 Reduced folate carrier protein 1 SEM SHH sirna SMO SUFU Standard error of the mean Sonic hedgehog Small interfering RNA Smoothened Suppressor of fused SV40 Simian virus 40 TBS TGFβ Tris buffered saline Transforming growth factor β TGFBR1 Transforming growth factor β receptor 1 TGFBR2 Transforming growth factor β receptor 2 WST-1 Water-soluble tetrazolium-1 16

CHAPTER 1 GENERAL INTRODUCTION 17

1.1 OVERVIEW Malignant mesothelioma poses a significant public health threat worldwide, with one of the lowest survival rates of fewer than 10% of patients surviving more than five years (Yan et al, 2011b). This cancer is associated with asbestos exposure (Robinson & Chahinian, 2002) and originates predominantly from the pleura (73.1%), while other sites include peritoneum (23.7%) and pericardium (0.3%) (Suzuki, 2001). Despite advances in treatment modalities, survival rates remain disappointingly low (Yan et al, 2011b). Clearly new targets and novel therapeutic approaches need to be identified. Recent evidence has shown that the Hedgehog (Hh) signaling pathway is hyperactivated in mesothelioma, and thus therapeutic blockade of this pathway might be beneficial to a subset of mesothelioma patients in whom a deregulated Hh pathway seems to be driving the cancer (Shi et al, 2012). The thesis will test the hypothesis that targeting the Hh pathway can be a therapeutic strategy against mesothelioma. The thesis will identify mutations in Hh pathway genes in mesothelioma and characterize the mutations identified, in a bid to understand how genetic alteration might influence the pathogenesis of mesothelioma. This thesis will also examine the feasibility of targeting the Hh pathway in mesothelioma using small molecule inhibitors, and elucidate the mechanisms driving inhibitor responses. 1.2 MALIGNANT MESOTHELIOMA Malignant mesothelioma is an aggressive cancer that arises from mesothelial cells that line the serosal cavities of the pleura, peritoneum, pericardium and tunica vaginalis testis, of which pleural mesothelioma accounts for 70% of the cases (Suzuki, 2001). Mesothelioma can be classified into four main histologic types: epithelioid, sarcomatoid, desmoplastic and biphasic (Figure 1.1), according to the 2004 World Health Organisation 18

classification scheme for pleural tumors (Travis et al, 2004). Of the four histologic types, the epithelioid type is the most common (61%), followed by biphasic (22%), sarcomatous (16%) and desmoplastic (1-2%) cell type (Inai, ; Suzuki, 2001). However, the percentage of biphasic mesothelioma could increase to 63% when larger tumor specimens are taken via thoracoscopy, thoracotomy and autopsy for histologic diagnosis (van Gelder et al, 1991). Correct histologic classification has significant prognostic and therapeutic implications. A number of studies have demonstrated that epithelioid mesothelioma has a better prognosis than biphasic and sarcomatoid mesotheliomas (Haber & Haber, 2011; Neumann et al, 2004; Nojiri et al, 2011; Sugarbaker et al, 1993). However, despite the best clinical care, mesothelioma remains an invariably fatal cancer with a median survival of 5.9-12.6 months from the time of diagnosis (Curran et al, 1998; Edwards et al, 2000). Poorer prognostic factors include males, older age, weight loss, chest pain, poor performance status, low haemoglobin, leukocytosis, thrombocytosis and non-epithelioid histology (Edwards et al, 2000). Occupational asbestos exposure is the main risk factor for mesothelioma, accounting for 80% of the cases in men and 40% of the cases in women (Robinson & Chahinian, 2002). Intriguingly, 20-60% of people with mesothelioma in different studies do not have an obvious history of exposure to asbestos (Robinson & Chahinian, 2002). This clearly suggests that exposure to carcinogens other than asbestos could cause mesothelioma or that minimal environmental exposure to asbestos is sufficient in some people to trigger the disease. Exposure to erionite, an asbestos-like fibrous mineral with potent carcinogenic properties, has also been shown to cause mesothelioma in humans and rats (Baris et al, 1987; Baris et al, 1981; Wagner et al, 1985). In addition, ionizing radiation as well as Simian virus 40 (SV40) infection has been implicated in the genesis of mesothelioma (Baris et al, 1987; Carbone et al, 1994; Cicala et al, 1993; De Bruin et al, 19

2009; Wagner et al, 1985). 20

Figure 1.1 Mesothelioma. A) Epithelioid B) Biphasic C) Sarcomatoid D) Demosplastic. Source: Travis et al, 2004. 21

1.2.1 Genetics and mesothelioma Although occupational exposure to asbestos accounts for approximately 80% of mesothelioma cases, less than 5% of heavily exposed asbestos workers develop mesothelioma (Below et al, 2011). This finding suggests that, in addition to asbestos exposure, inter-individual genetic differences might influence mesothelioma risk. The strongest evidence comes from studies of a mesothelioma epidemic in three villages (Karain, Sarihidir and Tuzkoy) in the Cappadocian region of Turkey (Dogan et al, 2006). Exposure to environmental erionite was found to be responsible for the high incidence of mesothelioma observed in these villages. Erionite is a naturally occurring zeolite fibre with morphology that resembles amplibolic asbestos (IARC, 2012). Despite the similarity in appearance, erionite has been shown to be more carcinogenic than asbestos (Carthew et al, 1992; Wagner et al, 1985). Mesothelioma appears to be clustered in certain Turkish families, whereby more than 50% of all deaths were attributed to mesothelioma (Roushdy-Hammady et al, 2001). An extended pedigree analysis on six of these families consisting of 526 individuals suggests that mesothelioma was consistent with autosomal dominant transmission with incomplete penetrance (Roushdy-Hammady et al, 2001). Recently, Testa and co-workers were able to demonstrate through the use of arraycomparative genomic hybridisation and linkage analysis, that germline mutations in BAP1 (BRCA1 associated protein-1) gene predispose individuals to mesothelioma (Testa et al, 2011). In addition to mesothelioma, BAP1 germline mutations are also associated with breast cancer, lung adenocarcinoma, melanocytic tumors, meningioma, renal cancer, skin cancer and uveal melanoma (Abdel-Rahman et al, 2011; Njauw et al, 2012; Popova et al, 2013; Testa et al, 2011; Wiesner et al, 2011). The association of BAP1 germline mutations with the increased risk of developing a myriad of human cancers point to the important tumor suppressor role played by BAP1 in different tissues (Goldstein, 2011). 22

BAP1 is an 81-kDa nuclear ubiquitin carboxy-terminal hydrolase that is 729 amino acids in length and is located on chromosome 3p21.3 (Jensen et al, 1998). Notably, many cancers including mesotheliomas frequently display chromosomal loss of 3p31.3 (Hesson et al, 2007). BAP1 was first identified as an interacting partner of BRCA1 by direct binding to the RING finger domain of BRCA1, and plays an important role in proliferation and cell death (Jensen et al, 1998; Misaghi et al, 2009; Ventii et al, ). Mounting evidence suggests that BAP1 acts as a bona fide tumor suppressor via its deubiquitinase activity (Ventii et al, ). Co-expression of BAP1 and BRCA1 significantly reduced the colony forming ability of MCF7 breast cancer cells, compared to expression of BRCA1 alone (Jensen et al, 1998). This enhancement of BRCA1 growth suppression activity is dependent on the ubiquitin hydrolase activity of BAP1 (Jensen et al, 1998). Knockout of BAP1 was embryonic lethal in mice, suggesting that BAP1 is essential for normal embryonic development (Dey et al, 2012). Dey and colleagues found that conditional BAP1 knockout mice developed myelodysplastic syndrome that has features of human chronic myelomonocytic leukemia (Dey et al, 2012). It was further demonstrated that BAP1 regulates gene transcription by deubiquitinating and stabilizing host cell factor-1 and O-linked N-acetylglucosamine transferase, two proteins that are involved in chromatin modification and remodeling (Dey et al, 2012). In the context of mesothelioma, knockdown of BAP1 using small interfering RNA (sirna) led to growth inhibition, deubiquitinating host cell factor-1 inactivation and reduction in E2F-responsive gene expression (Bott et al, 2011). 1.2.2 Management of mesothelioma Despite years of research, mesothelioma remains an incurable cancer. At present, the therapeutic approaches to mesothelioma are classified either as life-prolonging or palliative (Favoni & Florio, 2011). Initial monotherapeutic approaches involving surgery, 23

radiation therapy or chemotherapy failed to provide clear survival benefit to the patients (Ceresoli et al, 2007; Kaufman & Flores, 2011; Steele & Klabatsa, 2005). Radiation therapy can only reduce the tumor burden and is therefore ineffective in extending the survival of mesothelioma patients (Baldini, 2009; McAleer et al, 2009). Due to the diffuse nature of the tumor, irradiation of the entire pleural surface is necessary which often results in fatal complications (Baldini, 2009; McAleer et al, 2009). In a study by Ball and Cruickshank (Ball & Cruickshank, 1990), out of 12 patients with pleural mesothelioma who were prescribed radical radiotherapy, two of them (17%) developed fatal hepatitis and myelopathy, respectively. Another study found a 46% rate of fatal pneumonitis after 13 patients were treated with intensity modulated radiation therapy after extrapleural pneumonectomy and adjuvant chemotherapy (Allen et al, 2006). These sequelae of radiation therapy led surgeons to propose radical surgical resection as an attempt to cure the disease. In a ground-breaking study conducted by Butchart and his co-workers in 1976, patients who are diagnosed with stage 1 epithelioid mesothelioma had a better prognosis after pleuropneumonectomy than those with biphasic and sarcomatoid mesothelioma that had progressed beyond stage 1 (Butchart et al, 1976). However, the rapid infiltration of the tumor throughout the hemithorax made it extremely difficult to achieve histologically negative margins and residual tumor left behind from the debulking procedure could seed tumor in the chest wall (Butchart, 1999; Favoni & Florio, 2011). For patients who are not suitable for radiotherapy and surgery, chemotherapy is the standard of care for treatment of mesothelioma (Metintas et al, 2001). To date, those chemotherapeutic regimens for mesothelioma only reduce tumor burden and offer 24

palliation of disease symptoms such as chest pain, breathlessness and chest wall masses (Sterman et al, 1999). Based on data from multicentre studies, two cisplatin-based combination regimens are recommended as first-line chemotherapy for mesothelioma. A multicentre phase III trial involving 448 patients reported a longer overall median survival of 12.1 months in patients treated with cisplatin/pemetrexed combination compared with 9 months in those treated with cisplatin alone (Vogelzang et al, 2003). The combination of cisplatin and gemcitabine has also been demonstrated in a multicentre phase II trial to produce an objective response rate of 47.6% with palliation of symptoms and improvement in quality of life (Nowak et al, 2002). However, no single approach is superior to supportive care alone in terms of overall survival in the treatment of mesothelioma (Jassem et al, ; Jenkins et al, 2011; Sharif et al, 2011; Zahid et al, 2011). Hence the nihilistic attitude toward mesothelioma harboured by many clinicians for many years are justified but a big change in the way mesothelioma is managed is to be expected with the advent of new therapies, which may bring hope to mesothelioma patients. 1.2.3 Molecularly targeted therapy in mesothelioma In the past few decades, drug discovery efforts have undergone a paradigm shift from predominantly cytotoxic agent-based treatment to therapy specifically directed at molecular gene targets. In 2001, the US Federal Drug Agency approved the small molecule inhibitor, Glivec (imatinib mesylate, Gleevec, Norvatis), for use in patients with chronic myelogenous leukemia (CML) and ushered in the era of molecularly targeted therapy in cancer. Glivec, a 2-phenylaminopyrimidine derivative, is a selective tyrosine kinase inhibitor of the chimeric BCR-ABL oncokinase (Stegmeier et al, 2010), which results from a chromosomal translocation involving a fusion of the Abelson oncogene 25

(ABL) from chromosome 9q34 with breakpoint cluster region (BCR) on 22q11 (Heisterkamp et al, 1983). Early in vitro studies with Glivec showed that it is able to suppress proliferation and colony formation and induce apoptosis of BCR-ABL positive cells obtained from CML patients (Deininger et al, 1997; Druker et al, 1996; Gambacorti- Passerini et al, 1997). The prospective, randomized, multicentre, open-label phase III International Randomized Study of Interferon and STI571 (IRIS) clinical trial of Glivec for the treatment of CML in newly diagnosed patients have demonstrated that Glivec can induce complete hematologic response in 95.3% of patients and superior progression-free survival compared to the combination of interferon and cytarabine (O'Brien et al, 2003). These landmark studies provided the proof-of-concept that therapy could be directed against specific molecular targets in cancer cells while leaving normal cells unharmed, and has inspired many studies to identify new therapeutic targets in mesothelioma. Several selective agents targeting epidermal growth factor, platelet derived growth factor, vascular endothelial growth factor, src kinase, histone deacetylase and the proteasome have been evaluated in clinical trials (Kindler, ). Although these agents only demonstrated modest clinical efficacy in Phase I/II trials, these results further highlighted the pressing need to discover novel targets to control mesothelioma cell growth. More recently, approaches combining conventional chemotherapy with targeted agents are being trialled. Evidence from in vitro studies show that many drugs do not have singleagent activity but combining them with other chemotherapeutic agents often produce synergistic activity (Favoni & Florio, 2011). 1.3 THE HEDGEHOG SIGNALING PATHWAY The Hh gene was first discovered in a large-scale genetic screen for mutations that disrupt the Drosophila larval body plan (Nusslein-Volhard and Wieschaus, 1980). The Hh 26

pathway is a highly conserved signaling pathway responsible for the regulation of proliferation, differentiation and pattern specification during embryonic development (Ingham & McMahon, 2001). It is responsible for the formation of vertebrate structures, including bone and cartilage, cerebellum, eye, gut, gonads, heart, limbs, lung, muscle, neural crest, pancreas, prostate, tooth and tongue (Ingham & McMahon, 2001). The pathway is also involved in the regulation of adult tissue homeostasis and stem cell maintenance in the gastrointestinal tract, brain and blood (Crompton et al, 2007; Ingham & McMahon, 2001; Jiang & Hui, ; Palma et al, 2005; van den Brink, 2007; Varjosalo & Taipale, ). Overall, the key components of the Hh pathway are highly conserved from fruit flies to humans, although additional pathway components have added complexity to the pathway in vertebrates (Varjosalo et al, 2006). This introduction, however, will focus on Hh signaling in vertebrates due to the broad and diverse nature of this pathway across species. 1.3.1 Hedgehog signaling in a nutshell Signaling begins with the Hh ligand; Sonic (SHH), Desert (DHH) or Indian (IHH) Hh, being released from the producing cell. The Hh ligand binds to Patched (Ptch) receptors; PTCH1 and PTCH2 (Carpenter et al, 1998), on target cells (Stone et al, 1996). This leads to endosomal internalization and degradation of Ptch protein, thereby relieving the repression of a transmembrane protein Smoothened (SMO) (Denef et al, 2000; Incardona et al, 2002; Ingham et al, 2000; Murone et al, 1999). SMO then enters the primary cilia where it promotes the dissociation of a Suppressor of fused (SUFU)-GLI family zinc finger (Gli) complex (Tukachinsky et al, 2010). SUFU is the main repressor of the mammalian Hh signaling pathway by sequestering Gli transcription factors in the cytoplasm and nucleus (Kogerman et al, 1999). This repressor is negatively regulated by serine/threonine kinase 36 (STK36), which in turn promotes activation and nuclear 27

accumulation of Gli (Murone et al, 2000). In addition to SUFU, the transmembrane Hhinteracting protein (HHIP) was identified as another negative regulator of the Hh pathway that acts by sequestering all three Hh homologs with similar affinity to that of PTCH1 protein (Chuang & McMahon, 1999). Recently, KIF7 was identified by sequence comparison as a Gli-interacting protein (Varjosalo et al, 2006). KIF7 physically binds to Gli, regulating their stability and degradation and controlled Gli-mediated transcription (Cheung et al, 2009). Subsequently, the Gli transcription factor translocates to the nucleus and becomes activated, where it stimulates the transcription of Hh pathway target genes, including PTCH1 and Gli (Katoh & Katoh, 2009; Ruiz i Altaba et al, 2007). A schematic diagram depicting the Hh signaling pathway in vertebrates is shown in Figure 1.2. 28

Figure 1.2 The Hh signaling pathway. Hh ligands (SHH, DHH or IHH) bind to the Ptch receptors (PTCH1 and PTCH2) and relieve the inhibition of SMO. HHIP, a negative inhibitor of Hh signaling, can compete with Ptch receptors to bind Hh ligand, resulting in the attenuation of Hh signaling. SMO then transduces signals through the cytoplasmic SUFU-Gli complex, resulting in the activation and nuclear translocation of the downstream Gli transcription factors (GLI1-3). STK36 further promotes activation and nuclear accumulation of Gli by antagonizing SUFU. KIF7 regulates Gli-mediated transcription both positively and negatively through physical interaction with Gli and regulating the stability and degradation of Gli proteins. 29

1.3.2 Hedgehog ligand The Hh ligand is synthesized as a 45-kDa precursor protein that is autoproteolytically cleaved to yield a 19-kDa N-terminal fragment and a 26-kDa C-terminal fragment. The N-terminal fragment is the active form of the ligand whereas the C-terminal fragment acts as both the endoprotease and a cholesterol transferase. In its active form, the N-terminus of the 19-kDa fragment is palmitoylated by Hh acyltransferase and its C-terminus modified by a cholesterol moiety after the full-length Hh protein is autoproteolytically cleaved at the C-terminus (Buglino & Resh, ; Lee et al, 1994; Pepinsky et al, 1998; Porter et al, 1996). A diagram of Hh ligand processing is shown in Figure 1.3. The lipid moieties are essential for the full activation of the Hh pathway (Chen et al, 2004a; Pepinsky et al, 1998). Several studies have demonstrated that lipidated Hh ligand is required for ligand trafficking and signal reception (Callejo et al, 2006; Grover et al, 2011). Palmitoylated Hh has also been shown to mediate long range signaling (Chen et al, 2004a). Three mammalian homologues of Hh have been identified by screening mouse genomic and cdna libraries, namely SHH, DHH and IHH, which can all signal through the same transduction pathway with SHH being the best characterized of the three (Chiang et al, 1996; Echelard et al, 1993; Pathi et al, 2001). SHH is the most widely expressed during development and appears to be most potent of the three Hh ligands (Ingham & McMahon, 2001; Pathi et al, 2001). It plays an important role in the development of many cell types (e.g. blood cells and neurons), tissues (e.g. neural crest) and organs (e.g. cerebellum, limbs, lung, muscle and pancreas) (Ingham & McMahon, 2001). IHH is responsible for stimulating hematopoietic and endothelial cell production in the yolk sac (Dyer et al, 2001). IHH is also required for the formation of bone, cartilage and gut (Chung et al, 2001; St-Jacques et al, 1999; van den Brink, 2007). DHH has been shown to be important 30

Figure 1.3 Posttranslational Hh processing. Endoprotease domain mediates autocleavage while Hh acyltransferase catalyzes the attachment of palmitate to the N-terminal of the signaling domain. Finally, the cholesterol transferase domain catalyzes the attachment of cholesterol to the C-terminal of the signaling domain. 31

for the regulation of spermatogenesis and nerve development (Bitgood et al, 1996; Parmantier et al, 1999). 1.3.3 Hedgehog ligand reception at the cell membrane surface The PTCH1 gene encodes a receptor for the Hh ligand (Marigo et al, 1996). There are two human PTCH homologues; PTCH1 is widely expressed in many cell types whereas PTCH2 is only expressed in the skin and testis (Carpenter et al, 1998). Structurally, the PTCH1 protein has two extracellular loops where Hh ligand binds, 12 membranespanning domains, a sterol-sensing domain and one intracellular loop (Carpenter et al, 1998). The Hh ligand serves to activate ligand-induced gene expression by antagonising PTCH activity (Chen & Struhl, 1996). In addition to PTCH1 and PTCH2, several other proteins such as BOC, CDO and GAS1 have been shown to bind Hh ligand with high affinity, which is thought to promote Hh signaling during craniofacial, neural tube and vertebral development (Allen et al, 2007; Tenzen et al, 2006). On the other hand, the membrane-associated Hedgehog-interacting protein (HHIP) provides negative regulatory feedback of Hh signaling by competing with PTCH1 for Hh binding (Chuang & McMahon, 1999). Ectopic overexpression of HHIP in the developing endochondral skeleton led to severe skeletal defects in the transgenic animals, which are similar to defects seen in IHH mutants (Chuang & McMahon, 1999; St-Jacques et al, 1999). 1.3.4 SMO as a signal transducer The Hh signal is transduced across the cell membrane to Gli transcription factors via SMO. SMO encodes a receptor-like protein that has seven transmembrane domains, an extracellular N-terminal domain and an intracellular C-terminal domain (Cohen, 2003). 32

Structurally, SMO resembles G-protein-coupled receptor (GPCR) and is most homologous to the Frizzled family of serpentine proteins (van den Heuvel & Ingham, 1996). In the absence of Hh ligand, PTCH inhibits the activity of SMO by preventing its entry into the primary cilium (Rohatgi et al, 2007; Taipale et al, 2002). The binding of Hh to PTCH1 causes activation of the Hh pathway, as the PTCH1-Hh complex is endocytosed followed by translocation of derepressed SMO to the primary cilium (Incardona et al, 2002; Rohatgi et al, 2007). However, recent studies indicate that ciliary translocation of SMO is not sufficient to activate SMO fully and suggest that a second activation step is required for its complete activation (Rohatgi et al, 2009). There is evidence to suggest that the GPCR kinase 2 (GRK2), at least in part, participates in the second activation step by phosphorylating the C-terminus of SMO, which promotes Hh signaling (Chen et al, 2004b; Meloni et al, 2006). Fully active SMO then initiates a signaling cascade that shifts the processing of the GLI transcription factors in favour of the activator form. 1.3.5 Gli as transcription factors The human GLI1 gene was first identified as an amplified gene in human glioblastomas (Kinzler et al, 1987; Wong et al, 1987). In vertebrates, there are three Gli transcription factors, namely GLI1, GLI2, and GLI3, which can regulate the transcription of Hh target genes (Kinzler et al, 1988; Ruppert et al, 1988; Yoon et al, 1998). They belong to the Kruppel family of zinc finger DNA-binding nuclear proteins (Kinzler et al, 1988). Gli proteins contain a central core of DNA-binding domain that consists of five zinc finger motifs of the C2-H2 class and can bind to the Gli-binding sequence 5'-GACCACCCA-3' (Kinzler & Vogelstein, 1990; Pavletich & Pabo, 1993). GLI1 protein contains a C-terminal transactivation domain while GLI2 and GLI3 possess 33

an N-terminal repressor domain in addition to the transactivation domain (Ruiz i Altaba, 1999; Wang et al, 2007). As GLI1 lacks the N-terminal repressor domain, it acts as a transcriptional activator (Bai et al, 2004; Dai et al, 1999). GLI2 can be processed into a transcriptional repressor when their C-terminal activation domains are cleaved but it is mostly an activator (Pan et al, 2006). GLI3 is the predominant repressor (Ruiz i Altaba, 1999; Shin et al, 1999; Wang et al, 2000), although it has been shown to act as an activator in some reports (Bai et al, 2004; Buttitta et al, 2003; Motoyama et al, 2003). When Hh ligand is absent, the Gli proteins are sequentially phosphorylated by protein kinase A (PKA), glycogen synthase 3β (GSK3β) and casein kinase 1 (CK1) (Jia et al, 2004; Tempe et al, 2006; Wang & Li, 2006; Zhang et al, 2006). After phosphorylation, the Gli proteins are ubiquitinated by an E3 ubiquitin ligase, β-transducin-repeat containing protein (β-trcp) and targeted for proteasomal degradation (Bhatia et al, 2006; Huntzicker et al, 2006; Tempe et al, 2006; Wang & Li, 2006). GLI1 and GLI2 are completely degraded by the proteasome whereas GLI3 is partially degraded to form a transcriptional repressor, which is responsible for turning off the Hh signaling pathway (Figure 1.4) (Huntzicker et al, 2006; Pan et al, 2006). In the presence of Hh ligand, both the proteasomal degradation of GLI2 and the proteolytic conversion of GLI3 to a repressor form are suppressed, which in turn leads to transcriptional induction of GLI1 and other Hh target genes (Dai et al, 1999; Lipinski et al, 2006). 34

Figure 1.4 Gli processing and degradation. In the absence of Hh ligands, Gli transcription factors are sequentially phosphorylated by PKA, GSK3β and CK1, and finally ubiquitinated by β-trcp before being targeted for proteasomal degradation. GLI1 and GLI2 proteolysis leads to complete degradation while GLI3 proteolysis leads to partial degradation and the generation of a transcriptional repressor. 35

1.3.6 Regulation of Gli by SUFU SUFU acts downstream of SMO as a negative regulator of Hh signaling (Ding et al, 1999; Kogerman et al, 1999; Stone et al, 1999). The importance of SUFU is demonstrated by the constitutive activation of Hh signaling when SUFU gene is deleted in mice (Svard et al, 2006). SUFU has been shown to be able to bind to all three Gli transcription factors (Dunaeva et al, 2003; Stone et al, 1999). SUFU mutant proteins are unable to bind and export GLI transcription factors from the nucleus (Taylor et al, 2002). SUFU can inhibit the transactivation of GLI1 through sequestration in the cytoplasm (Kogerman et al, 1999; Stone et al, 1999), and it can bind to a complex of DNA and GLI1, suggesting that SUFU can inhibit GLI1 through direct interaction in the nucleus. Furthermore, SUFU can inhibit the transcriptional activity of GLI by recruiting a SAP18-mSin3 corepressor complex (Cheng & Bishop, 2002). 1.3.7 Primary cilium and Hedgehog signaling The primary cilium is a ubiquitous microtubule-based organelle that projects from the apical surface of quiescent cells (Ishikawa & Marshall, 2011). Most vertebrate cells possess a primary cilium with the exception of myeloid and lymphoid cells (Wheatley et al, 1996). Primary cilia have a 9+0 axoneme structure, composed of nine outer doublet microtubules (Pazour & Witman, 2003). Unlike motile cilia, they lack axonemal dyneins and are thus immotile (Pazour & Witman, 2003). The primary cilium is essential in the regulation of vertebrate development (Goetz & Anderson, 2010). Huangfu and coworkers first demonstrated that cilia control embryonic patterning and survival in the mouse (Huangfu et al, 2003). Multiple lines of evidence highlight a key role for primary cilia in the Hh pathway (Roy, 2012). Mutations in ciliary genes have been shown to give rise to mice with phenotypes 36

that resemble those with loss-of-function mutation in Hh genes (Huangfu & Anderson, 2005; Huangfu et al, 2003). Many Hh pathway components reside within the primary cilium either under basal conditions or upon Hh stimulation (Oro, 2007). When Hh ligand is absent, PTCH1 migrates to the primary cilium and prevent SMO from entering it (Rohatgi et al, 2007). Upon Hh ligand binding to PTCH1, the inhibitory effect of PTCH1 on SMO is lifted as PTCH1 leaves the primary cilium, allowing SMO to enter the primary cilium (Tukachinsky et al, 2010). At the same time, Gli-SUFU complexes enter the primary cilium and accumulate at the ciliary tip (Tukachinsky et al, 2010). SMO in the primary cilium promotes the dissociation of SUFU from Gli, which then migrates to the nucleus in a microtubule-dependent fashion to activate transcription of Hh target genes (Tukachinsky et al, 2010). 1.4 HEDGEHOG SIGNALING AND CANCER 1.4.1 Ligand-independent, mutational activation of Hedgehog signaling Disruption in the Hh signaling pathway can give rise to cancer in humans. The first evidence that deregulated Hh signaling contributes to tumorigenesis came from the finding that GLI1 was overexpressed in glioblastoma (Kinzler et al, 1987). Inactivating mutations in PTCH1 gene were found in a rare hereditary form of basal cell carcinoma (BCC) (also known as basal cell nevus syndrome or Gorlin syndrome) (Hahn et al, 1996; Johnson et al, 1996). Gorlin syndrome is a hereditary disorder characterized by a number of developmental defects including skeletal abnormalities, macrocephaly, severe eye anomalies, cleft lip/palate, hyperkeratosis of hands and feet, jaw odontogenic keratocysts, calcification of falx cerebri and polydactyly or syndactyly and a predisposition to a wide variety of tumors other than BCC, which includes medulloblastoma, rhabdomyosarcoma and meningioma (Gorlin, 2004; Lo Muzio, ). Collectively, these are known as Type I Hh cancers, which are ligand-independent and mutation-driven (Rubin & de Sauvage, 37

2006). PTCH1 mutations have also been found in other sporadic malignancies, such as BCCs (Gailani et al, 1996), medulloblastomas (Xie et al, 1997), skin trichoepitheliomas (Vorechovsky et al, 1997), esophageal squamous cell carcinomas (Maesawa et al, 1998), skin squamous cell carcinomas (Ping et al, 2001), meningiomas (Xie et al, 1997), breast carcinomas (Xie et al, 1997) and bladder carcinomas (McGarvey et al, 1998), as well as in odontogenic keratocysts (Barreto et al, 2000; Ping et al, 2001; Song et al, 2006; Sun et al, ; Xie et al, 1997). In addition to primary tumors, missense mutations in PTCH1 have been identified in several oral squamous cell carcinoma and colon carcinoma cell lines (Michimukai et al, 2001; Xie et al, 1997). Thus far, more than 300 mutations have been reported in the Sanger Catalogue of Somatic Mutations in Cancer (COSMIC) database (http://www.sanger.ac.uk/genetics/cgp/cosmic/). However, neither mutation hot-spots nor genotype-phenotype correlations have been found so far (Wicking et al, 1997). Mutations in PTCH1 do not account for all cases of Gorlin syndrome as well as sporadic BCCs and medulloblastomas. Indeed, in these diseases, mutations in several genes in the Hh pathway have been found. A number of reports showed that a subset of patients with sporadic BCC possess mutations in SMO (Couve-Privat et al, 2002; Lam et al, 1999; Reifenberger et al, 2005; Reifenberger et al, 1998; Xie et al, 1998). Xie and colleagues further showed that expression of these mutant SMO proteins in mouse skin produced BCC-like tumors (Xie et al, 1998). PTCH2 mutations were also detected in some cases of sporadic BCCs and medulloblastomas (Smyth et al, 1999). Like PTCH1, PTCH2 is overexpressed in both familial and sporadic BCCs (Zaphiropoulos et al, 1999) and medulloblastomas (Lee et al, 2003), suggesting that PTCH2 is a direct gene target of Hh signaling and that PTCH2 may be negatively regulated by PTCH1 (Zaphiropoulos et al, 38

1999). Interestingly, Lee and co-workers recently reported that loss of PTCH2 contributes to enhanced tumorigenesis in PTCH1 haploinsufficient mice (Lee et al, 2006). Mutations in SUFU were detected in sporadic BCCs and seemed to predispose to medulloblastoma (Reifenberger et al, 2005; Taylor et al, 2002). Mutations in SHH have also been identified in a number of cancer types, including BCC (Oro et al, 1997), medulloblastoma (Oro et al, 1997), breast carcinoma (Oro et al, 1997), glioblastoma (McLendon R et al, ) and lung cancer (Kan et al, 2010). Oro and co-workers detected an identical somatic mutation in the putative zinc hydrolase site of SHH that caused a His to Tyr substitution at codon 133 of SHH. Notably, this mutation occurred in a sporadic BCC, a medulloblastoma and a breast carcinoma, hinting at the possible involvement of this nonsynonymous substitution in driving malignancies in these tumor types (Oro et al, 1997). The significance of mutations in Hh pathway genes has never been addressed in mesothelioma. It remains a possibility that, in patients with mesothelioma, Hh pathway gene mutations might confer selective growth advantage or chemoresistance on mesothelioma cells. To date, this hypothesis has not been tested. This thesis will be the first to: (1) determine the prevalence of Hh pathway gene mutations and (2) characterize the effect of these mutations on gene expression and function in mesothelioma cells. 1.4.2 Autocrine, ligand-dependent Hedgehog signaling Type II Hh cancers are autocrine and ligand-dependent in nature and usually involve Hh overexpression (Rubin & de Sauvage, 2006). These include lung, pancreatic, gastrointestinal tract, colorectal, prostate, breast and melanoma tumors. In autocrine signaling, the tumor cell secretes the Hh ligand, which acts upon itself. Kasperczyk and 39

co-workers provided the first clues to the mechanism behind Hh overexpression in cancer cells (Kasperczyk et al, 2009). It was found that in pancreatic cancer, the transcription factor NF-kB transactivates the expression of SHH, which leads to cell proliferation and resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis (Kasperczyk et al, 2009). Autocrine Hh signaling has also been demonstrated in gastric cancer. Kameda and co-workers reported the induction of SHH expression in estrogen receptor-alpha (ERα)-positive gastric cancer after activation of the ERα signaling pathway by exogenously applied 17-β-oestradiol (Kameda et al, 2010). This finding is consistent with immunohistochemical data from surgically resected gastric cancer specimens showing a positive correlation between ERα and SHH expression (Kameda et al, 2010). 1.4.3 Paracrine, ligand-dependent Hedgehog signaling In Type III Hh cancers, Hh signaling is activated in a ligand-dependent, paracrine manner, in which the ligand is produced by tumor cells and acts on surrounding stromal cells. The stromal cells respond by producing growth factors such as insulin-like growth factor and vascular endothelial growth factor and thereby promote tumor growth (Rubin & de Sauvage, 2006). Examples are pancreatic, lung, esophageal, gastric and prostate cancers. Fan and co-workers were the first to demonstrate the link between paracrine Hh signaling and prostate tumorigenesis (Fan et al, 2004). The overexpression of SHH in a human prostate LNCaP cell line resulted in cells that showed no difference in growth rate when compared to non-transfected LNCaP cells but grew faster when injected into nude mice, thereby pointing to the influence of host-derived stroma on the growth of tumor xenografts (Fan et al, 2004). Yauch and co-workers made a similar finding in colorectal and pancreatic cancers (Yauch et al, ). Yauch further demonstrated that the inhibition of stromal Hh signaling by Hh antagonists resulted in downregulation of Hh target genes 40

PTCH1, PTCH2 and GLI1 in the mouse stroma, which in turn led to the slowing of tumor growth in vivo (Yauch et al, ). The three modes of Hh signaling in cancer are summarized in Figure 1.5 (Gupta et al, 2010). 41

A B C Figure 1.5. Models of Hh pathway activation in cancer. A) Type I ligand-independent, mutation driven. B) Type II autocrine, ligand-dependent. C) Type III paracrine, ligand-dependent. 42

1.4.4 Hedgehog signaling and the cell cycle One of the hallmarks of cancer is uncontrolled cell proliferation (Hanahan & Weinberg, 2011). Activated Hh signaling modulates the expression of cell cycle regulators, which drive cell cycle progression and cell proliferation (Kenney & Rowitch, 2000; Regl et al, 2004a; Yoon et al, 2002). Examples of such regulators are cyclin-dependent kinase 1 (CDK1) and foxhead box M1 (FOXM1) (Regl et al, 2004a; Teh et al, 2002). CDK1 forms a complex with cyclin B and triggers the initiation of mitosis in cycling cells (Nurse, 2000). FOXM1 is a transcriptional activator that plays an important role in cell proliferation, and it is overexpressed in prostate, breast, lung, ovary, colon, pancreas, stomach, bladder, liver and kidney (Pilarsky et al, 2004; Wierstra & Alves, 2007). Teh and co-workers showed that GLI1 directly controls the expression of FOXM1 in BCC (Teh et al, 2002). Inhibition of Hh signaling in human colorectal cancer cells resulted in the downregulation of CDK1 and FOXM1 causing the cancer cells to undergo cell cycle arrest and apoptosis (Mazumdar et al, 2011a; Shi et al, 2010). Using a low-density microarray, Romagnoli and co-workers showed that CDK1 and FOXM1 were overexpressed in mesothelioma, hinting at the possibility that aberrant Hh signaling could induce CDK1 and FOXM1 expressions and contribute to the tumorigenesis of mesothelioma (Romagnoli et al, 2009). Hh signaling also upregulates osteopontin (Das et al, 2009). Osteopontin was first identified in osteoblasts (Oldberg et al, 1986) and was later found to be overexpressed in a variety of cancers, including mesothelioma, where its expression is deregulated (Fedarko et al, 2001; Pass et al, 2005). Osteopontin is a secreted phosphoprotein that promotes proliferation, adhesion, migration and survival in mesothelioma (Ivanov et al, 2009; Ohashi et al, 2009). Osteopontin has shown great promise as a diagnostic marker for mesothelioma with a sensitivity and specificity of 77% and 85%, respectively (Grigoriu et 43

al, 2007; Pass et al, 2005). The expression of osteopontin in melanoma cells could be reduced by knocking down endogenous Gli expression using RNA interference (Das et al, 2009). Silencing GLI1 was shown to abrogate the metastatic behaviour of melanoma cells, including cell motility, migration and invasion and the ability to form spontaneous metastases in vivo (Das et al, 2009). Intriguingly, the phenotype could be rescued in GLI1-knocked down cells with overexpression of osteopontin, suggesting that osteopontin is responsible for mediating the oncogenic action of GLI1 (Das et al, 2009). 1.4.5 Hedgehog signaling and apoptosis Apoptosis or Type I programmed cell death is responsible for timely removal of unwanted, damaged or infected cells in multicellular organisms. Disruption of apoptosis can give rise to a number of human diseases, ranging from cancer, neurodegenerative diseases, stroke, heart diseases and autoimmune diseases (Favaloro et al, 2012; Reed, 1999). In the case of cancer, key proteins in the apoptosis pathway are frequently mutated or overexpressed, resulting in cancer cells that are resistant to insults that can bring about the death of normal cells (Reed, 1999). As mentioned above, mesothelioma is particularly resistant to chemotherapy. A number of studies have demonstrated that the alteration of apoptotic pathway is a primary underlying reason for chemoresistance in mesothelioma (Barbone et al, 2011). Research within the fields of mesothelioma and apoptosis have been concentrated on the Bcl-2 family of pro-survival proteins because several members (Bcl-2, Bcl-xL and Mcl-1) are overexpressed in mesothelioma (O'Kane et al, 2006; Segers et al, 1994; Soini et al, 1999). Bcl-2, Bcl-xL and Mcl-1 mediate survival by putting a brake on cell death. This was demonstrated in a study by Kamada and colleagues, who demonstrated accelerated lymphocyte cell death in Bcl-2 knockout mice (Kamada et al, 1995). Similar acceleration 44

of apoptotic cell death was observed in various tissues and organs of Bcl-xl and Mcl-1 knockout mice (Opferman et al, 2005; Opferman et al, 2003; Wagner et al, 2000). Blockade of Bcl-2, Bcl-xl and Mcl-1 by pharmacologic or antisense means has been shown to restore normal function to the apoptotic machinery and thereby overcome chemoresistance in mesothelioma in vitro and in vivo (Cao et al, 2007; Hopkins- Donaldson et al, 2003; Ozvaran et al, 2004; Varin et al, 2010). Interestingly, Bcl-2 is also one of the best-known Hh target genes (Regl et al, 2004b). Numerous studies reported that aberrant Hh signaling contributes to tumor survival through the action of Bcl-2 in diffuse large B-cell lymphoma, gastric and pancreatic cancers (Bigelow et al, 2004; Han et al, 2009; Mazumdar et al, 2011a; Singh et al, 2010; Xu et al, 2009). Blockade of Hh signaling by a chemical inhibitor of GLI induced apoptotic cell death in colorectal cancer cells due to the downregulation of Bcl-2 expression (Mazumdar et al, 2011a). 1.4.6 Hedgehog signaling and autophagy In recent years, it is becoming appreciated that cells can undergo cell death via a number of mechanisms. These include autophagy, regulated necrosis, mitotic catastrophe, anoikis, entosis, parthanatos, pyroptosis, netosis and cornification (Galluzzi et al, 2012). For the purpose of this thesis, only autophagy will be elaborated. Autophagy was first described in mammalian cells by Nobel Laureate Christian de Duve in 1963 (Klionsky, 2007). Strictly speaking, autophagy refers to a catabolic process in all eukaryotic cells that involves degradation of intracellular components via lysosome-dependent pathways (Levine & Yuan, 2005). Autophagy is capable of large-scale degradation of long-lived proteins and entire organelles (Klionsky & Emr, 2000). Briefly, a double membrane structure, called the autophagosome or autophagic vacuole, sequesters cytoplasmic 45

constituents including organelles such as mitochondria and long-lived proteins such as glyceraldehydge-3-phosphate dehydrogenase (Aniento et al, 1993; Kundu et al, ; Mizushima, 2007; Twig et al, ). Studies have shown that the autophagosomal membrane could be assembled de novo or derived from the membranes of mitochondria, endoplasmic reticulum and Golgi apparatus (Tooze & Yoshimori, 2010). Following sequestration, the outer membrane of the autophagosome then fuses with a lysosome, forming an autolysosome, where the cargo is degraded (Mizushima, 2007). Finally, the resulting nucleotides, amino acids and fatty acids are reused in the synthesis of new macromolecules and ATP (Lum et al, 2005). The autophagy process is evolutionarily highly conserved from unicellular yeast to multicellular plant and animal cells (Klionsky & Emr, 2000). Autophagy plays a central role in mediating response to cellular stresses such as starvation, hypoxia and oxidative or genotoxic stress (Murrow & Debnath, 2013). Normally, it occurs at low levels to maintain cell homeostasis but can become upregulated by stresses such as nutrient deprivation and chemicals such as rapamycin (He & Klionsky, 2009). In addition to maintenance of normal cell physiology, autophagy is crucial for embryonic development and tissue remodeling (Mizushima & Levine, 2010). Thus disruption of autophagy has been implicated in the pathogenesis of a number of diseases, including cancer, neurological disorders and heart disease (Choi et al, 2013). Shintani and Klionsky described autophagy as a double-edged sword, in that it could promote cell survival or death, depending on the cellular context (Shintani & Klionsky, 2004). More than 30 years ago, Beaulaton and Lockshin made the first connection between autophagy and cell death, when they noted that mitochondria, glycogen particles 46

and ribosomes were degraded via autophagy during insect metamorphosis (Beaulaton & Lockshin, 1977). This mode of cell death is known as autophagic cell death or Type II programmed cell death (Clarke, 1990). Autophagic cell death is characterized by autophagic vacuolization of cytoplasm in the absence of chromatin condensation (Kroemer & Levine, ). It is now well documented that autophagic cell death occurs during development, in diseased mammalian tissues and in protozoan and mammalian cells treated with certain drugs (Levine & Yuan, 2005). Recently, the crosstalk between the Hh pathway and autophagy was described in a flurry of papers published independently and almost simultaneously (Jimenez-Sanchez et al, 2012; Li et al, 2012b; Petralia et al, 2013; Sun et al, 2014; Wang et al, 2013b; Xu et al, 2014). The findings from these studies provide evidence that Hh signaling regulates autophagy. Jimenez-Sanchez and colleagues also demonstrated that this crosstalk is highly conserved between mammals and Drosophila (Jimenez-Sanchez et al, 2012). Data reported so far show a discrepancy in how autophagy is regulated by the Hh pathway. Two of the earliest studies show that Hh signaling promotes autophagy in vascular smooth muscle cells (SMC) and hippocampal neurons (Li et al, 2012b; Petralia et al, 2013). Exposure of SMC and hippocampal neurons to SHH increased numbers of autophagosomes, the presence of which is indicative of active autophagy (Li et al, 2012b; Petralia et al, 2013). However, later studies, notably among these the contributions from Jimenez and colleagues, have clearly shown that Hh signaling blocks the formation of autophagosomes under basal and starvation conditions in vertebrates and invertebrates (Jimenez-Sanchez et al, 2012; Wang et al, 2013b; Xu et al, 2014). Furthermore, using genetic approaches, the study by Jimenez and colleagues has revealed that PTCH1, PTCH2, SMO and GLI2 are involved in autophagy regulation by Hh (Jimenez-Sanchez et al, 2012). 47

In an attempt to modulate the Hh pathway for therapeutic intervention, two separate labs evaluated the cytotoxic effects of the Gli inhibitor GANT61 on hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC) cells (Wang et al, 2013b; Xu et al, 2014). GANT61 is a cell-permeable hexahydropyrimidine compound that was discovered in a cell-based screen for antagonists of Gli-mediated transcription (Lauth et al, 2007). GANT61 exposure showed induction of autophagy, as determined by an increase in microtubule-associated protein light chain 3 levels and a reduction in p62 levels (Wang et al, 2013b; Xu et al, 2014). Interestingly, inhibition of GANT61- induced autophagy via genetic or pharmacologic means resulted in decreased levels of apoptosis, which was mediated, at least in part, through Bnip3, a pro-apoptotic protein of the Bcl-2 family (Chen et al, 1997; Imazu et al, 1999), in HCC and PDAC cells (Wang et al, 2013b; Xu et al, 2014). More recently, Sun and colleagues demonstrated the importance of the role of GLI1 in apoptosis and autophagy induced by sirnamediated knockdown of GLI1 in chondrosarcoma cells (Sun et al, 2014). Furthermore, inhibition of autophagy rescued GLI1 sirna-mediated cell death and phosphorylation of mtor (mammalian target of rapamycin) appears to be linked to GLI1-dependent regulation of autophagic cell death in these cells (Sun et al, 2014). Apparent differences between these various studies may be related to the type of cells studied and the dual functions of autophagy serving as tumor promoter or suppressor in different tissue microenvironments. 1.5 DRUGGING THE HEDGEHOG PATHWAY Cyclopamine and jervine, steroidal alkaloids isolated from the poisonous plant Veratrum californicum, were the first Hh pathway inhibitors reported (Cooper et al, 1998; Incardona et al, 1998). Chen and colleagues showed that cyclopamine inhibit Hh signaling by 48

binding directly to the transmembrane α-helices of SMO receptor (Chen et al, 2002a), thereby establishing the inhibition of Hh pathway as an attractive strategy for cancer therapy and prevention. Cyclopamine was shown to inhibit the growth of a preclinical model of medulloblastoma, demonstrating the feasibility of treating this aggressive cancer with this new class of anticancer agent (Berman et al., 2002). Thereafter, cyclopamine was successfully shown to induce regression in several preclinical mouse models of human cancers that have been linked to aberrant Hh signaling, including those of glioma (Clement et al, 2007), gastrointestinal (Berman et al, 2003), haematopoietic (Zhao et al, 2009), melanoma (Stecca et al, 2007), pancreatic (Thayer et al, 2003), prostate (Karhadkar et al, 2004; Sanchez et al, 2005), and small cell lung (Watkins et al, 2003) origins. It was also demonstrated to enhance the cytotoxic effect of standard chemotherapeutic drugs such as paclitaxel, suggesting that the addition of cyclopamine to a standard chemotherapy regimen may prove an effective cancer therapy (Shafaee et al, 2006). Accumulating evidence suggests that Hh signaling might play a role to promote drug resistance in cancer by upregulating the expression of ABCB1 and ABCG2 (Sims- Mourtada et al, 2007). More recently, Lavie and colleagues discovered that the combination of cyclopamine with doxorubicin or vinblastine potently blocked MDR1- mediated drug efflux in multidrug-resistant cancer cells and in turn potentiates the cytotoxicity of exposure to doxorubicin and vinblastine (Lavie et al, 2001). Although cyclopamine has shown great potential as a Hh antagonist and anticancer agent, it displays low affinity, metabolic instability, suboptimal pharmacokinetics and limited bioavailability (Lipinski et al, ). As such, more stable and potent derivatives of cyclopamine, such as KAAD-cyclopamine and exo-cyclopamine, have been synthesized (Heretsch et al, 2011; Taipale et al, 2000). KAAD-cyclopamine and exo-cyclopamine displayed 10-20-fold greater inhibitory potency than cyclopamine in SHH-induced 49

pathway activity (Heretsch et al, 2011; Taipale et al, 2000). Other derivatives include carbohydrate-conjugated cyclopamine, which improves water solubility, where a particular cyclopamine derivative conjugated with L-rhamnose displayed better antiproliferative activity against the lung adenocarcinoma cell line A549 (Zhang et al., ). Since then, many small molecule compounds that have inhibitory effects on the Hh pathway have been reported. Notably, GDC-0449 (also known as vismodegib, Genentech-Curis), a SMO antagonist with nanomolar potency, has progressed to human clinical trials and yielded promising results in patients with BCC and medulloblastoma (LoRusso et al, 2011; Rudin et al, 2009; Von Hoff et al, 2009). Most recently, GDC-0449 was approved by the U.S. Food and Drug Administration for the treatment of metastatic BCC, locally advanced BCC that has relapsed following surgery or patients with BCC who are neither candidates for surgery nor radiotherapy (Axelson et al, 2013). Further efforts are currently underway to discover new druggable targets in the Hh pathway. Besides targeting SMO receptor, the SHH ligand and GLI1 transcription factor appear highly amenable to inhibition by small molecule inhibitors (Kim et al, 2010a; Lauth et al, 2007; Stanton et al, 2009). Of particular interest is the Gli inhibitor, GANT61, which was identified by screening a chemical library for Gli antagonists by using a firefly luciferase reporter under the control of Gli-binding site (Lauth et al, 2007). Studies have shown that by blocking Gli with GANT61, extensive cell death is induced in human colon carcinoma and B-cell chronic lymphocytic leukemia, suggesting that Gli could be a potential target for therapy in these cancers (Desch et al, 2010; Mazumdar et al, 2011a). 1.6 REACTIVE OXYGEN SPECIES AND CANCER THERAPY A reactive oxygen species (ROS), by definition, is simply a chemically reactive ion or molecule that contains oxygen, and can be further classified into free radicals and non- 50

radicals. Free radicals are defined as species that contain unpaired electrons in their outermost electron shell, which include superoxide, hydroxyl radical and nitric oxide. A non-radical ROS is a highly reactive species that lack unpaired electrons and can be converted to a free radical by reacting with free radicals. These include hydrogen peroxide (H 2 O 2 ), singlet oxygen and ozone. ROS can be present in the environment in the form of pollutants, pharmaceuticals, tobacco and smoke (Gupta et al, 2012). Within cells, ROS are produced as part of many enzymatic and non-enzymatic processes. ROS can be produced enzymatically via NADPH oxidases (NOX) complex found in cell membrane (Jiang et al, 2011), xanthine oxidase found in peroxisomes (Fransen et al, 2012), oxidoreductases found in endoplasmic reticulum (Santos et al, 2009), cytochrome P450- dependent oxygenases (Zangar et al, 2004), uncoupled endothelial nitric oxide synthase (Cai & Harrison, 2000; Montezano & Touyz, 2012) and arachidonic acid (Kim et al, ). The electron transport chain found within mitochondria is a non-enzymatic ROS generator (Turrens, 2003). In healthy cells, the levels of ROS are kept in check by complex antioxidant systems. Elimination of ROS from cells are mediated enzymatically via superoxide dismutases, glutathione peroxidase and catalase or by non-enzymatic molecules that scavenge ROS such as glutaredoxin, peroxiredoxin, thioredoxin, metal ion chelators and vitamins A, C and E (Manda et al, 2009). However, oxidative stress can result when this cellular state of equilibrium between ROS and antioxidants is disrupted and ROS predominate. Because of their reactive nature, ROS can disrupt normal cell physiology by reacting with DNA (Richter et al, 1988), proteins (Stadtman & Levine, 2000), lipids (Rubbo et al, 1994) and polysaccharides (Kaur & Halliwell, 1994), which in turn would lead to many human diseases, including cancer, if these damages are irreparable (Waris & Ahsan, 2006). 51

There is an increasing body of work demonstrating that ROS plays an important role in cell signaling (Janssen-Heininger et al, ). The first report about the production of H 2 O 2 by cancer cells was in 1991 (Szatrowski & Nathan, 1991) and it is now well accepted that cancer cells produce more ROS than normal cells. Moderate levels of ROS promote cell proliferation, differentiation and survival whereas excessive ROS damages cellular components that lead to cell cycle arrest and cell death (Chiu & Dawes, 2012; Trachootham et al, ). Homeostatic control of cellular ROS levels is thus of paramount importance. Since cancer cells have relatively higher levels of ROS, Kong and colleagues first theorized in 1998 that the balance of antioxidant/ros can be tipped to favour cancer cell killing through induction of ROS generation and suppression of antioxidant capacity, while sparing the normal cells. This is known as the threshold concept for cancer therapy (Kong et al, 2000; Kong & Lillehei, 1998). There are many anticancer drugs that are currently in use in the clinic that have been shown to mediate their cytotoxic effects through the promotion of ROS production (Gorrini et al, 2013). For example, paclitaxel induces cell death in various cancer cells including breast and lung cancer cells, through the production of ROS, which could be reversed by antioxidants and nitric oxide modulators (Alexandre et al, 2006; Alexandre et al, 2007; Ramanathan et al, 2005). Another example of a FDA-approved drug that works through generation of ROS is bortezomib. Bortezomib is an inhibitor of the β5 subunit of the proteasome that has been approved for the treatment of refractory multiple myeloma and mantle cell lymphoma (Chauhan et al, ; Richardson et al, 2007; Suh & Goy, ). It has been shown extensively in many tumor models, including myeloma, mantle cell lymphoma and colon cancer, that bortezomib-induced apoptosis is dependent on ROS production (Minami et al, 2005; Pei et al, 2004; Perez-Galan et al, 2006). 52

1.7 SUMMARY AND SCOPE OF THESIS Mesothelioma is an aggressive and extremely chemoresistant solid tumour of the mesothelial lining of the serosal cavities, with increasing incidence and an extremely poor prognosis (Robinson & Lake, 2005). Cisplatin in combination with pemetrexed or gemcitabine are the only drugs approved for the first-line treatment of mesothelioma (Nowak, 2012). However, treatment with these chemotherapeutic agents rarely produces a complete response. Consequently, the current median survival of patients from diagnosis with mesothelioma is less than 12 months (Carbone et al, 2012). Therefore the discovery of new treatments is a top research priority in the management of this disease. Previous studies in several human cancer models suggest that the Hh pathway presents a number of druggable targets, including SHH, SMO, GLI1/2 and primary cilium (Amakye et al, 2013). At present, all of the small molecule inhibitors of the Hh pathway in clinical trials act at the level of SMO, with Genentech s GDC-0449 being the first approved drug for advanced BCC (Lin & Matsui, 2012). Despite the clear benefits of SMO inhibitors in the clinical setting, patients treated with SMO inhibitors ultimately manifested disease progression because of acquired drug resistance (Rudin et al, 2009; Yauch et al, 2009). In addition, the clinical utility of SMO inhibitors does not extend beyond tumors harbouring PTCH1 or SMO mutations that confer sensitivity, as evident by the failures of clinical trials for chondrosarcoma, colorectal and ovarian cancers (Amakye et al, 2013; Berlin et al, 2013; Kaye et al, 2012). Several cancers have evolved non-canonical SMOindependent mechanisms for activating the Hh pathway, thus making SMO inhibitors useless against these cancers (Lauth & Toftgard, 2007). For example, mutations in SUFU gene or cross-talk with PI3K/AKT pathway have been shown to aberrantly hyperactivate this pathway (Buonamici et al, 2010; Riobo et al, 2006; Taylor et al, 2002). Thus, inhibitors of Gli transcription factors, the terminal step in the Hh pathway, 53

would have greater clinical utility against a broad range of cancers, and specifically against those cancers which are activated by mechanisms that act downstream of SMO. To date, a number of small molecule Gli inhibitors with therapeutic potential have been reported but none of these agents have made it to clinical trial (Mahindroo et al, 2009). Recently, it was found that the Hh signaling pathway was aberrantly reactivated in mesothelioma (Shi et al, 2012; Zhang et al, 2013). Shi and co-workers first demonstrated that human pleural mesothelioma tumors showed increased expression of SHH, PTCH1, HHIP and GLI1 compared to normal pleural tissue and that growth of primary mesothelioma cultures could be inhibited in vitro using HhAntag that blocks Hh signaling at the level of SMO (Shi et al, 2012). Furthermore, oral administration of HhAntag was effective in a nude mouse mesothelioma xenograft model (Shi et al, 2012). Professor David Jablons group has also developed a number of Hh inhibitors that have been shown to be efficacious against mesothelioma in vitro and in vivo (Bosco-Clement et al, 2014; Li et al, 2013). These observations led to the hypothesis that the Hh pathway can be targeted in mesothelioma as a therapeutic strategy. The specific aims of this thesis are: (1) To identify mutations in the Hh signaling pathway in mesothelioma. (2) To functionally characterize the mutations identified through in silico and in vitro analysis. (3) To determine the preclinical efficacy of Hh pathway inhibitors, in particular the Gli inhibitor GANT61, on mesothelioma cells in vitro. 54

(4) To characterize the biological effects of GANT61 in the mesothelioma cell line LO68. By addressing these aims, it is hoped that the research performed in this thesis will help to better elucidate the role of the Hh pathway and components of the pathway in mesothelioma growth, which in turn will assist in the development of improved antimesothelioma therapeutics. 1.8 THESIS LAYOUT Chapter 1: General introduction This chapter gives a review of the literature, providing background information on the concepts, hypothesis and aims that are outlined in the scope of this thesis. Chapter 2: Materials and methods This chapter gives a detailed description of the materials and methods used in this thesis. Chapter 3: Mutational analysis of Hedgehog pathway genes in mesothelioma This chapter explores the hypothesis that mutations in Hh pathway genes are responsible for the hyperactivation of the Hh pathway in mesothelioma cells. The mrna expression of molecular intermediates of the Hh pathway in mesothelioma cells was examined using qrt-pcr analysis and the mutation profile in these cells was evaluated by performing PCR amplification of coding regions of Hh pathway genes followed by sequencing analysis. Human mesothelioma tumors were genotyped and screened for the mutations identified from the in silico analysis. The mutations were then functionally characterized using in silico and in vitro approaches. 55

Chapter 4: Targeting of Hedgehog pathway in mesothelioma using Gli inhibitor GANT61 This chapter examines the effects of targeting the Hh pathway in mesothelioma cells using the Gli inhibitor GANT61. In vitro assays were used to examine GANT61 s effects on cell cycle, apoptosis, autophagy and oxidative stress. The feasibility of combining GANT61 with the standard mesothelioma chemotherapeutic agents, namely cisplatin, pemetrexed and gemcitabine, was also evaluated. Chapter 5: Mitochondria-derived ROS is a critical player in GANT61-induced apoptosis This chapter further investigates the ROS-inducing ability of GANT61. Specifically, the role of GANT61-induced ROS in the induction of apoptosis was studied. Using inhibitors of mitochondrial respiratory chain and NADPH oxidase, the involvement of mitochondria and NADPH oxidase in the generation of ROS following GANT61 treatment was examined. To confirm genetically that mitochondria are one of the major sites of GANT61-induced ROS production, LO68 cells were depleted of mitochondria DNA (ρ 0 cells), which render them functionally incapable of performing normal mitochondrial respiratory function. The ability of ρ 0 cells to generate ROS and undergo apoptosis following GANT61 treatment was compared to wild-type cells. Chapter 6: General discussion This chapter outlines how the data presented in this thesis addresses the hypothesis and aims that are laid out in Chapter 1. In addition, the implications of these findings and directions for future research are discussed. Appendix A: Intronic primer sequences for amplification of exons of Hedgehog pathway genes This chapter lists the sequences of the primers used for the PCR amplification and 56

sequencing of Hh pathway genes in Chapter 3. Appendix B: PCR protocols for amplification of exons of Hedgehog pathway genes This chapter lists the PCR thermal cycling parameters for each primer set that was used to amplify the coding regions of Hh pathway genes in Chapter 3 Appendix C: Exonic primer sequences for PCR assay for detection of PTCH1 exon deletions This chapter lists the sequences of the primers used for the PCR amplification and sequencing of exons 18-23 of PTCH1 gene in Chapter 3. Appendix D: Buffers and solutions This chapter lists the recipes of the buffers and solutions outlined in Chapter 2. 57

CHAPTER 2 MATERIALS AND METHODS 58

2.1 CELL LINES AND CULTURE CONDITIONS The human mesothelioma cell lines MSTO-211H (CRL-2081), NCI-H28 (CRL-5820), NCI-H226 (CRL-5826), NCI-H2052 (CRL-5915) and NCI-H2452 (CRL-5946) as well as the mouse embryonic fibroblasts C3H/10T1/2 (CCL-226) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The mesothelioma cell lines JU77, LO68, NO36, ONE58 and STY51 were kind gifts from Professor Bruce Robinson (University of Western Australia) (Manning et al, 1991) and REN cells were provided by Professor Steven Albelda (University of Pennsylvania). The cell lines GAY2911, OLD1612 and VGE were kind gifts from Professor Jenette Creaney (University of Western Australia). These cell lines were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Mulgrave, Australia) supplemented with 10% fetal bovine serum (FBS) (Serana, Bunbury, Australia), 4 mm L-glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin (all from Life Technologies) in a humidified incubator with 5% CO 2 at 37 o C. In addition, normal pericardial mesothelial cells were obtained from patients undergoing thoracic surgery as described previously (Holloway et al, 2006). 2.2 ETHICS STATEMENT Use of the archival tissue blocks in this retrospective study was approved by the Sydney Local Health District Human Research Ethics Committee (Concord), as part of a larger study to identify prognostic factors in malignant pleural mesothelioma. Patient consent for the use of these samples was waived by the ethics committee, consistent with the Human Tissue Act (1983) and the NHMRC National Statement on Ethical Conduct in Human Research (Commonwealth of Australia, 2007). 59

2.3 PATIENTS AND TUMORS The mesothelioma tumor DNA samples used in this study (Table 2.1) were kind gifts from Dr. Glen Reid (Asbestos Diseases Research Institute, Sydney, Australia). These samples were part of a series collected from patients who underwent extrapleural pneumonectomy at Royal Prince Alfred or Strathfield Private Hospitals (Sydney, Australia) between 1994 and 2009 (Kao et al, 2011). Table 2.1 Demographic and clinical characteristics of patients with mesothelioma Sample ID Subtype Gender Age Survival (months) 24 Epithelioid M 53 9.56 31 Biphasic M 60 5.42 32 Epithelioid M 58 41.40 48 Epithelioid F 48 44.16 50 Epithelioid M 54 72.90 51 Biphasic M 51 3.42 52 Epithelioid M 61 8.28 59 Epithelioid M 52 13.73 65 Epithelioid M 64 6.93 72 Epithelioid M 47 1.94 74 Epithelioid M 57 8.34 77 Biphasic M 56 2.56 78 Epithelioid F 59 14.52 79 Epithelioid M 56 10.32 2.4 DNA ISOLATION Genomic DNA was isolated from cells using the PureLink Genomic DNA kit (Life Technologies), according to the manufacturer s instructions. Briefly, cells were resuspended in 200 µl phosphate-buffered saline (PBS) and transferred to a 1.5 ml microfuge tube containing 20 µl of 20 mg/ml proteinase K and 20 µl of 20 mg/ml RNase A. Samples were vortexed and incubated at room temperature for 2 min. A 200 µl aliquot of PureLink Genomic Lysis/Binding Buffer was added to the cell suspension, vortexed and incubated at 55 o C for 10 minutes. A 200 µl solution of 96-100% ethanol was added to 60

the cell lysates, vortexed and transferred to a spin column, and centrifuged at 13,000 rpm for 1 min at room temperature. The spin column was placed into a clean collection tube, washed with 500 µl Wash Buffer 1 and centrifuged at 13,000 rpm for 1 min at room temperature. The spin column was placed into another clean collection tube, washed with 500 µl Wash Buffer 2 and centrifuged at 13,000 rpm for 3 min at room temperature. An aliquot of 50 µl nuclease-free water was added to the spin columns, which were then incubated at room temperature for 1 min. DNA was eluted from the column into a sterile microfuge tube by centrifuging at 13,000 rpm for 1.5 min at room temperature. The quantity and quality of extracted DNA was determined using a NanoDrop 2000c Spectrophotometer (Thermo Scientific). Genomic DNA was stored at -20 o C until required. 2.5 PCR AMPLIFICATION PCR primers that amplify the exons and flanking intronic sequences of 13 Hh pathway genes (Table 2.2) were either obtained from published literature (Jones et al, ; Putoux et al) or designed using GenBank sequences and the Vector NTI 11.0 software. The primer sequences are listed in Appendix A. Table 2.2 Genes sequenced in this study Gene Symbol Gene name NCBI Gene ID GLI1 GLI family zinc finger 1 2735 GLI2 GLI family zinc finger 2 2736 GLI3 GLI family zinc finger 3 2737 IHH Indian hedgehog 3549 PTCH1 patched 1 5727 SHH sonic hedgehog 6469 SMO smoothened, frizzled family receptor 6608 PTCH2 patched 2 8643 STK36 serine/threonine kinase 36 27148 DHH desert hedgehog 50846 SUFU suppressor of fused homolog (Drosophila) 51684 61

HHIP hedgehog interacting protein 64399 KIF7 kinesin family member 7 374654 PCR amplification of Hh pathway genes was performed as described previously with slight modifications (Sjoblom et al, 2006). PCR was carried out on a icycler thermal cycler (BioRad, Gladesville, Australia) in a 20 µl volume containing 10 ng genomic DNA, 1x GoTaq Flexi buffer (Promega, Alexandria, Australia), 1.5 mm MgCl 2 (Promega), 0.2 mm dntps (Life Technologies), 0.5 µm primers (Geneworks Pty Ltd, Hindmarsh, Australia), 6% dimethyl sulfoxide (DMSO) (Sigma Aldrich, Castle Hill, Australia) and 1.25 U GoTaq Flexi DNA polymerase (Promega). The optimized PCR conditions for each primer pair are listed in Appendix B. PCR products were separated on 1% agarose gels stained with 0.5 µg/ml ethidium bromide and visualized using a Molecular Imager Gel Doc XR system (BioRad). 2.6 DNA SEQUENCE ANALYSIS PCR products were sent to the Australian Genome Research Facility (Perth, Western Australia) and sequenced using a Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Mulgrave, Australia) and analysed on a 3730xl DNA Analyzer (Applied Biosystems). Base calling and quality assessment were carried out using the Sequence Scanner v1.0 software (Applied Biosystems) while sequence assembly was carried out using the Vector NTI v11.0 software (Life Technologies). All sequence variants found were confirmed by an independent PCR and sequencing reaction to exclude PCR artefacts. Seven mesothelioma cell lines (MSTO-211H, NCI-H28, JU77, LO68, NO36, ONE58 and STY51) were originally exon sequenced with a further four cell lines (NCI- H226, NCI-H2052, NCI-H2452 and REN) genotyped for identified mutations. 62

2.7 PCR ASSAY FOR DETECTION OF PTCH1 EXON DELETIONS To detect exon deletions of the PTCH1 gene in mesothelioma cell lines, a PCR assay was designed using exonic primers directed against exons 18, 19, 20, 21, 22, and 23 of PTCH1. The primer sequences are listed in Appendix C. The cycling conditions are: 1 cycle of 94 C for 1 min, 3 cycles of 94 C for 10 s, 64 C for 10 s, 72 C for 30 s, 3 cycles of 94 C for 10 s, 61 C for 10 s, 72 C for 30 s, 3 cycles of 94 C for 10 s, 58 C for 10 s, 72 C for 30 s, 40 cycles of 94 C for 10 s, 57 C for 10 s, 72 C for 30 s, 1 cycle of 72 C for 5 min. PCR products were separated on 4% agarose gels stained with 0.5 µg/ml ethidium bromide and visualized using a Molecular Imager Gel Doc XR system (BioRad). 2.8 IN SILICO CHARACTERIZATION OF POLYMORPHISMS IN EXONS The web-based programs SIFT (Ng & Henikoff, 2003) and PolyPhen2 (Adzhubei et al, 2010) were employed to predict the potential effect of non-synonymous amino acid substitutions resulting from the genetic alterations. The default settings were used for all parameters of each program. 2.9 DRUGS Chemical Abbreviation Concentration 3-methyladenine 3-MA 5 mm Arsenic trioxide As 2 O 3 20 µm Bafilomycin A1 BafA1 1-200 nm Chloroquine CQ 20 µm Cisplatin CPT 5-20 µm Cyclopamine 0.1-50 µm Cyclosporin A CsA 5 µm Diphenylene iodonium DPI 100 nm GANT61 1-30 µm GDC-0449 0.1-50 µm Gemcitabine GEM 30-60 µm Hedgehog pathway inhibitor-1 HPI-1 20 µm Hydrogen peroxide H 2 O 2 350 µm 63

L-glutathione GSH 10 mm Menadione 30 µm MitoTEMPO MT 100 µm N-acetyl-cysteine NAC 20 mm Pemetrexed PMX 50-100 µm Rotenone ROT 200 nm 2.10 CELL PROLIFERATION ASSAY Cell proliferation was determined using the methylene blue assay as previously described (Oliver et al, 1989). Briefly, cells were seeded in a 96-well flat bottom plate (Nunc, Scoresby, Australia) and treated the following day with 100% DMSO as vehicle or GANT61 (Tocris Bioscience, Bristol, UK) as described in the Results section. GANT61 was solubilised in DMSO to a stock concentration of 25 mm. Following treatment, cells were fixed with 4% paraformaldehyde (Sigma Aldrich, Castle Hill, Australia) for 10 min at 4 o C followed by staining with 2% methylene blue/0.01m borate (ph 8.5) solution (Sigma Aldrich, Castle Hill, Australia). Excess dye was then washed off using 0.01M borate buffer (ph 8.5) and the methylene blue dye eluted from cells with 1:1 (v/v) ethanol and 0.1 M hydrochloric acid and quantified at 650 nm on a Wallac 1420 VICTOR2 multilabel plate reader (Perkin Elmer, Glen Waverly, Australia). Half maximal inhibitory concentrations (IC 50 ) were determined using Graphpad Prism 4.03 software (Graphpad Software, Inc., La Jolla, CA). 2.11 COLONY FORMATION ASSAY Cells were counted and 3-5 x 10 3 cells were seeded in 6-well plates (Nunc, Scoresby, Australia) and after 24 h incubation in DMEM supplemented with 10% FBS, 4 mm L- glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin, treated with DMSO vehicle or GANT61 (5-20 µm). After three days, drugs were washed out with 2 ml PBS and cells were grown in 2 ml drug-free complete medium for 14-21 days. Cells were then washed with 1 ml 1x PBS (Sigma Aldrich), fixed for 30 min with 1 ml methanol and 64

finally stained with 0.5 ml 0.5% (v/v) crystal violet (Sigma Aldrich). After shaking on an orbital shaker for 15 min, excess crystal violet was washed off under running tap water. The plates were allowed to air dry overnight then images of the plates with stained cell colonies were taken using a multifunction device (Kyocera, North Ryde, Australia) and the images were analysed using Image J software (Rasband, 1997-2012). 2.12 CELL CYCLE ANALYSIS BY DNA CONTENT Cells were fixed with 2 ml ice-cold 70% ethanol overnight in 5 ml round bottom FACS tubes (Nunc, Scoresby, Australia) at -20 o C after GANT61 treatment. Fixed cells were centrifuged at 1,200 rpm for 5 min, ethanol was decanted and cells were washed twice with 2 ml PBS. Cells were stained with 500 µl PI/RNase staining buffer (50 µg/ml propidium iodide (Sigma Aldrich) and 100 µg/ml RNase A (Life Technologies) at room temperature in the dark for 30 min. Stained cells were analysed for DNA content using a FACSCalibur flow cytometer (BD, North Ryde, Australia) and quantified using the FlowJo software (Tree Star, Inc., Ashland, OR). 2.13 DETECTION OF APOPTOSIS Apoptotic cells were detected using the PE Annexin V Apoptosis Detection kit (BD, North Ryde, Australia) according to the manufacturer s protocol. Briefly, cells were harvested by incubation in a solution of 0.25% trypsin with 380 mg/l ethylenediaminetetraacetic acid (0.25% trypsin-edta) (Life Technologies) after drug treatment and washed twice with 2 ml ice-cold PBS. Cells were resuspended in 1x binding buffer containing 5 µl Annexin V PE conjugate and 5 µl 7-aminoactinomycin D (7AAD) and incubated at room temperature in the dark for 15 min. Stained cells were analysed within 1 h by flow cytometry and quantified using the FlowJo software. 65

2.14 WST (WATER-SOLUBLE TETRAZOLIUM)-1 ASSAY Cell proliferation was determined using the WST-1 assay (Roche, Castle Hill, Australia), according to the manufacturer s protocol. The WST-1 assay is based on the formation of a soluble formazan dye by extracellular reduction of the water-soluble tetrazolium salt WST-1 by viable cells. Cells (3 x 10 3 ) were seeded in 96-well flat bottom plates in a final volume of 100 µl/well culture medium. After incubation overnight, cells were treated with DMSO vehicle or 20 µm GANT61 for 48 h. WST-1 reagent (5 µl) were added to each well and the plates were returned to the 37 o C incubator. After 15 min incubation, absorbance of the samples was measured on a microplate reader at 450 nm. The reference wavelength was 690 nm. 2.15 ROS DETECTION Cells (1 x 10 5 ) were seeded in 6-well plates in a final volume of 2 ml/well culture medium. After incubation overnight, cells were treated with DMSO vehicle or 20 µm GANT61 for 48 h. Intracellular ROS production was determined by loading cells with 20 µm 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CH 2 DCFDA) (Life Technologies) at 37 o C for 45 min. Intramitochondrial superoxide production was determined by loading cells with 5 µm mitosox red mitochondrial superoxide indicator (Life Technologies, Mulgrave, Australia) at 37 o C for 45 min. Red and green fluorescence emissions were analysed by flow cytometry using excitation/emission wavelengths of 488/530 nm and 488/585 nm for CH 2 DCFDA and mitosox red, respectively. 2.16 MITOCHONDRIAL MEMBRANE POTENTIAL (ΔΨM) DETECTION Cells (1 x 10 5 ) were seeded in 6-well plates in a final volume of 2 ml/well culture medium. After incubation overnight, cells were treated with DMSO vehicle or 20 µm GANT61 for 48 h. ΔΨm was determined using the MitoProbe JC-1 Assay kit according 66

to manufacturer s instructions (Life Technologies, Mulgrave, Australia). Cells were trypsinized in 0.25% trypsin-edta and washed with 2 ml PBS. After resuspending in 1 ml of PBS, cells were incubated with 10 µg/ml JC-1 dye at 37 o C for 30 min. Stained cells were analyzed by flow cytometry with excitation set at 488 nm and observation wavelengths of 530 nm and 585 nm for green and red fluorescence, respectively. 2.17 OXYDNA ASSAY Oxidative DNA damage was determined by binding of fluorescein isothiocyanate (FITC)- protein conjugate to DNA adduct 8-oxoguanine using the OxyDNA kit (Calbiochem, Kilsyth, Australia). Briefly, 1 x 10 5 cells were seeded in 6-well plates in a final volume of 2 ml/well culture medium. After incubation overnight, cells were treated with DMSO vehicle or 20 µm GANT61. Following 48 h treatment, cells were trypsinized and washed with 2 ml PBS before they were fixed and permeabilized with 2 ml ice-cold 70% ethanol overnight at 4 o C. Fixed cells were then washed twice with 2 ml PBS and incubated with FITC-protein conjugate for 2 h at room temperature in the dark. After washing the cells twice with 2 ml 1x Tris buffered saline/tween-20, they were resuspended in 500 µl PBS and analysed by flow cytometry with excitation set at 488 nm and observation wavelengths of 530 nm for green fluorescence. 2.18 DETECTION OF AUTOPHAGY Autophagy was detected by staining cells with acridine orange as described previously (Lim et al, 2012) or with the Cyto-ID Green autophagy detection reagent (Enzo Life Sciences, Inc., Farmingdale, NY), according to the manufacturer s instructions. Briefly, 1 x 10 5 cells were seeded in 6-well plates in a final volume of 2 ml/well culture medium. After incubation overnight, cells were treated with DMSO vehicle or 20 µm GANT61. Following 48 h treatment, cells were trypsinized, washed with 2ml PBS and incubated 67

with acridine orange (1 µg/ml) or 1x CytoID Green autophagy detection reagent in a final volume of 0.5 ml/well PBS at 37 o C for 30 min. Bafilomycin A1 (200 nm) (BafA1) (Sigma Aldrich) was added to the cells 4 h before addition of acridine orange. Stained cells were analysed by flow cytometry using 488 nm excitation laser and quantified using FlowJo software. 2.19 IMMUNOFLUORESCENCE ANALYSIS Cells (2 x 10 5 ) were seeded in 35 mm glass bottom culture dishes (MatTek Corporation, Ashland, MA) in a final volume of 2 ml/well culture medium. After incubation overnight, cells were treated with DMSO vehicle or 20 µm GANT61. Following 24 h treatment, cells were co-stained with Cyto-ID Green reagent (Enzo Life Sciences, Inc.) and Hoechst 33342 (Enzo Life Sciences, Inc.) at 37 o C for 30 min, according to the manufacturer s instructions. Stained cells were then fixed in 4% paraformaldehyde (Santa Cruz Biotechnology, Inc., Dallas, TX) for 20 min at room temperature. Cellular localization of autophagolysosomes was visualized on the Nikon A1Si confocal microscope (Nikon Instruments Inc., Melville, NY). Data were further analysed using the Nikon NIS Elements software (Nikon Instruments Inc.). 2.20 RNA INTERFERENCE Cells (3 x 10 5 ) were seeded in 6-well plates in a final volume of 2 ml/well culture medium to obtain 80% confluency after overnight growth. Cells were then transfected with 100 nm GLI1 sirna (sigli1) (Santa Cruz Biotechnology, Inc.), GLI2 sirna (sigli2) (Sigma Aldrich) or negative control (NC) sirna (Santa Cruz Biotechnology, Inc.) using 15 µl Lipofectamine 2000 transfection reagent (Life Technologies) in a final volume of 2 ml/well antibiotic-free culture medium. The transfection medium was replaced with complete medium after 6 h to reduce cytotoxicity. After 24 h, cells were split 1:5, and 68

seeded in 6-well plates in a final volume of 2 ml/well culture medium. 2.21 GLI LUCIFERASE REPORTER ASSAY Gli transcriptional activity was measured using a Cignal Gli Reporter (luc) kit (SABiosciences, Chadstone, Australia), according to manufacturer s instruction. Briefly, 1 x 10 5 cells were seeded in 12-well plates 24 h before transfection. Cells were cotransfected with 500 ng of Gli luciferase reporter construct and a Renilla luciferase construct (40:1 ratio), using 2.5 µl Lipofectamine 2000 transfection reagent, with a 5:1 ratio (v/w) of Lipofectamine 2000 to DNA. After 24 h, cells were treated with DMSO vehicle or 20 µm GANT61. Cells were harvested using the Dual-Glo Luciferase assay system (Promega, Alexandria, Australia), 48 h after transfection according to the manufacturer s instruction. Luciferase activity was measured using a microplate reader. All reporter assays were normalized to Renilla luciferase activity. 2.22 WESTERN BLOT ANALYSIS Cells were harvested, lysed using CelLytic M mammalian cell lysis/extraction reagent (Sigma Aldrich), and the protein concentrations determined by NanoDrop 2000c Spectrophotometer (Thermo Scientific, Scoresby, Australia). Proteins (30 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose membrane (Millipore, Kilsyth, Australia), which was then blocked with 5% non-fat milk or 5% bovine serum albumin (Sigma Aldrich) in 1x Tris-buffered saline-0.1% Tween 20 for 1 h at room temperature. The membranes were incubated at 4 o C overnight with the indicated primary antibodies (Table 2.3). As a protein loading control, membranes were probed with a mouse anti-α-tubulin monoclonal antibody (Table 2.3). After repeated washing to remove unbound antibodies, the membranes were further incubated with horseradish peroxide-conjugated anti-rabbit or 69

anti-mouse secondary antibodies (Table 2.4) for 1 h at room temperature. Chemiluminescent detection of antibody binding was performed using the Immobilon Western HRP Substrate (Millipore, Kilsyth, Australia). Membranes were exposed to Amersham Hyperfilm ECL (GE Healthcare Australia Pty. Ltd., Rydalmere, Australia) and developed on an AGFA 1000 film processor (Raynostix, Louisville, KY). 2.23 ANTIBODIES The antibodies used in this thesis are listed in Table 2.3 and 2.4. Table 2.3 Primary antibodies Antibody Catalogue number Primary antibody dilution Secondary antibody dilution GLI1 #2534 1:1000 1:10000 GLI2 #2585 1:10000 1:15000 Bcl-2 #2870 1:1000 1:10000 Supplier Cell Signaling Technology α-tubulin T5168 1:10000 1:80000 Sigma Aldrich Table 2.4 Secondary antibodies Antibody Catalogue number Supplier Anti-rabbit HRP #7074 Cell Signaling Technology Anti-mouse HRP A4416 Sigma Aldrich 2.24 GENERATION OF MITOCHONDRIAL DNA-DEPLETED LO68 CELLS Mitochondrial DNA-depleted LO68 cells (termed ρ 0 ) were generated as described previously (King & Attardi, 1989). Briefly, 1 x 10 5 cells were seeded in 6-well plates in a final volume of 2 ml/well culture medium. After 24 h, cells were grown in complete medium supplemented with 100 ng/ml ethidium bromide (Sigma Aldrich), 50 µg/ml uridine (Sigma Aldrich) and 1 mm sodium pyruvate (Life Technologies) for 10 weeks. 70

2.25 RNA ISOLATION Total RNA was extracted from cell lines using an RNeasy Mini kit (Qiagen, Chadstone, Australia), according to the manufacturer s instructions. Briefly, cells were lysed by addition of 600 µl of Buffer RLT. Cell lysates were collected into 1.5 ml microfuge tubes, vortexed and stored at -80 o C until ready for RNA extraction. After thawing the cell lysates at room temperature, 600 µl 70% ethanol was added to the cell lysates, mixed by pipetting gently and transferred to an RNeasy spin column, which was then centrifuged at 13,000 rpm for 30 s at room temperature. After discarding the flow-through, the spin column was washed with 350 µl Buffer RW1, which was then centrifuged at 13,000 rpm for 30 s at room temperature. On-column DNase digestion was performed by adding 80 µl reconstituted RNase-free DNase (Qiagen) to the spin column. After 15 min incubation at room temperature, the spin column was again washed with 350 µl Buffer RW1 and centrifuged at 13,000 rpm for 30 s at room temperature. The spin column was washed twice with 500 µl Buffer RPE and centrifuged at 13,000 rpm for 2 min at room temperature. Nuclease-free water (30-50 µl) was added to the spin columns and total RNA was eluted from the spin columns into 1.5 ml microfuge tube by centrifuging at 13,000 rpm for 1 min at room temperature. The quantity and quality of extracted RNA was determined using a NanoDrop 2000c Spectrophotometer (Thermo Scientific). RNA was stored at -80 o C until required. 2.26 CDNA SYNTHESIS Total RNA was reverse-transcribed into cdna using an Omniscript RT kit (Qiagen), according to the manufacturer s instructions. Briefly, cdna synthesis was carried out in a 20 µl volume containing 1-2 µg total RNA, 1x Buffer RT, 0.5 mm dntp mix, 0.6 µg random hexamers (Life Technologies), 10 units RNase inhibitor (Qiagen), 4 units 71

Omniscript Reverse Transcriptase. The mixture was vortexed briefly (less than 5 s) and incubated at 37 o C for 1 h. cdna was stored at -80 o C until required. 2.27 QUANTITATIVE REAL-TIME PCR (QRT-PCR) ANALYSIS OF GENE EXPRESSION qrt-pcr was performed according to the manufacturer s instructions. mrna expression of genes was quantified using the following TaqMan gene expression arrays (Applied Biosystems): DHH (hs00368306_m1), GLI1 (hs01110766_m1), GLI2 (hs01119974_m1), GLI3 (hs00609233_m1), SMO (hs01090242_m1), PTCH1 (hs00181117_m1), BRCA1 (hs01556193_m1), EXO1 (hs01116195_m1), RAD51 (hs00153418_m1 ), UNG (hs01037093_m1) and XRCC3 (hs00193725_m1). PGK1 (4326318E-1006008) was included as the endogenous control. Real-time PCR was carried out on the StepOne Plus Real-Time PCR System (Applied Biosystems, Mulgrave, Australia) in a 10 µl volume containing 1 µl cdna, 5 µl 2x QuantiFast Probe PCR Master Mix (Qiagen, Chadstone, Australia) and 0.5 µl Taqman probe (Applied Biosystems, Mulgrave, Australia). The thermal cycling parameters are as follows: two initial activation steps at 50 o C for 2 min and 95 o C for 10 min, followed by 40 cycles of 95 o C for 15 s and 60 o C for 1 min. The expression levels of each gene were normalized to PGK1 mrna and presented as fold change with respect to vehicle-treated cells for each gene as described previously (Livak & Schmittgen, 2001). 2.28 DRUG COMBINATION ANALYSIS The CompuSyn program (Combosyn, Inc., Paramus, NJ) was employed to determine the effects of drug combination. The program is based on the median effect equation of Chou and the combination index equation of Chou-Talalay (Chou & Talalay, 1984), which are 72

used to calculate the combination index (CI). CI range values are defined in Table 2.5 (Chou, 2006). Table 2.5 Description of CI values CI range Description < 0.1 Very strong synergism 0.1-0.3 Strong synergism 0.3-0.7 Synergism 0.7-0.85 Moderate synergism 0.85-0.9 Slight synergism 0.9-1.1 Nearly additive 1.1-1.2 Slight antagonism 1.2-1.45 Moderate antagonism 1.45-3.3 Antagonism 3.3-10 Strong antagonism >10 Very strong antagonism 2.29 STATISTICAL ANALYSIS Statistical calculations were performed using Graphpad Prism 4.03 software. Student's t- test was used to determine statistical differences between two groups. Statistical differences between multiple groups were calculated using one-way ANOVA analysis and Tukey s multiple comparison post-hoc test. Where percentage data were used, these were arcsin transformed before statistical analysis. For RT-PCR data, statistical analysis was performed using the data expressed as log2-fold change. P < 0.05 was considered statistically significant. 73

CHAPTER 3 MUTATIONAL ANALYSIS OF HEDGEHOG PATHWAY GENES IN MESOTHELIOMA 74

3.1 INTRODUCTION The long latency period (typically 20-40 years) between asbestos exposure and the development of mesothelioma suggests that multiple gene mutations are necessary to induce mesothelioma (Selikoff et al, 1980). Cytogenetic studies have revealed a number of recurrent chromosomal aberrations in mesothelioma, including monosomy 4 and 22 and deletions of chromosomal arms 1p, 3p, 6q, 9p, and 22q (Hicks, 2006; Murthy & Testa, 1999; Sandberg & Bridge, 2001). The tumor suppressor genes found within these regions i.e. BAP1 at 3p21.3, CDKN2A at 9p21.3 and NF2 (neurofibromatosis type 2) at 22q12.2, are frequently altered in mesothelioma (Bianchi et al, 1995; Bott et al, 2011; Illei et al, 2003; Sekido et al, 1995; Testa et al, 2011). The most common genetic alteration in mesothelioma is homozygous deletion of CDKN2A gene. In a cohort of pleural mesotheliomas, 70 out of 95 (74%) primary tumors had homozygous deletions of CDKN2A (Illei et al, 2003). The frequency of NF2 mutations observed in mesotheliomas is approximately 40% (Bianchi et al, 1995; Sekido et al, 1995). Recently, Bott and colleagues detected BAP1 loss, mutation or both in 22 out of 53 (42%) pleural mesothelioma tumors (Bott et al, 2011). Clearly, mutations and/or loss of these tumor suppressor genes only account for a subset of patients with mesothelioma. It is highly likely that there are mutations in genes that have not yet been associated with mesothelioma pathogenesis. The first connection between mutations in Hh pathway genes and cancer was made in the 1980s when germline mutations in the PTCH1 gene were discovered in patients with Gorlin syndrome (Hahn et al, 2002; Johnson et al, 1996). Soon after, mutations in SMO and SUFU were detected in BCC, medulloblastoma and meningioma (Brastianos et al, 2013; Clark et al, 2013; Lam et al, 1999; Reifenberger et al, 1998; Xie et al, 1998). However, genetic alterations in the Hh pathway in mesothelioma have not been 75

investigated, even though it has been shown that the Hh pathway is hyperactivated in this cancer (Li et al, 2013; Shi et al, 2012). The aim of this chapter is to: (1) Identify mutations in the Hedgehog pathway in mesothelioma (2) Functionally characterize the mutations identified through in silico and in vitro analysis. 76

3.2 MATERIALS AND METHODS 3.2.1 RNA isolation, cdna synthesis and quantitative real-time PCR (qrt-pcr) analysis of gene expression Total RNA was extracted from cell lines using an RNeasy Mini kit (Qiagen), as described in Section 2.24. Two micrograms of total RNA was reverse-transcribed into cdna using random primers (Invitrogen) and Omniscript RT kit (Qiagen). Taqman gene expression assays (Applied Biosystems) were used for quantifying the mrna expression levels of DHH, GLI1, GLI2, GLI3, SMO and PTCH1. PGK1 was included as the endogenous control. Real-time PCR was performed in duplicate on the StepOne Plus Real-Time PCR System (Applied Biosystems). Fold change in gene expression relative to PGK1 was calculated using the following formula: Fold change = 2 - Ct, where Ct = Ct (Target) Ct (PGK1). 3.2.2 Gli luciferase reporter assay C3H/10T1/2 cells were plated in triplicate on 24-well plates 24 h before transfection. Cells were cotransfected with 25 ng prl-tk (Promega), 0.1 µg 12XGLI1 luciferase reporter construct (a kind gift from Professor Rune Toftgård, Karolinska Institutet), 0.15 µg wild-type GLI1 construct (Origene) and 0.15 or 0.3 µg of the appropriate SUFU construct (wild-type SUFU or SUFU (p.t411m) (Blue Heron Bio) using FuGene 6 transfection reagent (Roche), with a 3:1 ratio (v/w) of FuGene 6 to DNA. Cells were harvested using the Dual-Glo Luciferase assay system (Promega) 48 hours after transfection according to the manufacturer s instructions. Luciferase activity was measured using a Wallac 1420 VICTOR2 multilabel plate reader (Perkin Elmer). All reporter assays were normalized to Renilla luciferase activity. All transfections were repeated in at least two independent experiments, which gave reproducible results. 77

3.3 RESULTS 3.3.1 The canonical Hedgehog signaling pathway is active in human mesothelioma cell lines qrt-pcr was used to measure the mrna expression of Hh pathway components, including the secretory Hh ligands (DHH, IHH and SHH), seven-transmembrane receptors (SMO and PTCH1) and the Gli transcription factors (GLI1, GLI2 and GLI3) in a panel of ten human mesothelioma cell lines that were derived from pleural fluids or tumors of mesothelioma patients. DHH, PTCH1, GLI1, GLI2 and GLI3 were highly expressed in all the human mesothelioma cell lines (Figure 3.1). In contrast, normal mesothelial cells expressed extremely low levels of DHH and GLI3 mrna while PTCH1, GLI1 and GLI2 were undetectable (Figure 3.1). Notably, none of the mesothelioma cell lines and primary mesothelial cells express SHH or IHH (data not shown). In addition, all the human mesothelioma cell lines and normal mesothelial cells expressed the seven-transmembrane receptor, SMO (Figure 3.1). These data suggest that the components necessary for active Hh signaling are expressed at significantly higher levels in all mesothelioma cell lines analysed, compared with controls. 78

A DHH B PTCH1 C SMO 1 0.1 0.01 0.001 79 NM1 NM2 MSTO-211H NCI-H28 JU77 LO68 NO36 ONE58 STY51 GAY2911 OLD1612 0.0001 Fold change relative to PGK1 1 0.1 0.01 NM1 NM2 MSTO-211H NCI-H28 JU77 LO68 NO36 ONE58 STY51 GAY2911 OLD1612 0.001 Fold change relative to PGK1 1.0 10 1.0 10 00 00 1.0 10 1.0 10-01 -01 1.0 10 1.0 10-02 -02 1.0 10 1.0 10-03 -03 1.0 10 1.0 10-04 -04 1.0 10 1.0 10-05 -05 1.0 10 1.0 10-06 -06 1.0 10 1.0 10-07 -07 VGE VGE NM1 NM2 MSTO-211H NCI-H28 JU77 LO68 NO36 ONE58 STY51 GAY2911 OLD1612 VGE D GLI1 E GLI2 F GLI3 1 0.1 0.01 0.001 NM1 NM2 MSTO-211H NCI-H28 JU77 LO68 NO36 ONE58 STY51 GAY2911 OLD1612 0.0001 1 0.1 0.01 Fold change relative to PGK1 NM1 NM2 MSTO-211H NCI-H28 JU77 LO68 NO36 ONE58 STY51 GAY2911 OLD1612 0.001 1 0.1 0.01 Fold change relative to PGK1 VGE NM1 NM2 MSTO-211H NCI-H28 JU77 LO68 NO36 ONE58 STY51 GAY2911 OLD1612 0.001 Fold change relative to PGK1 Fold change relative to PGK1 NM1 NM2 MSTO-211H NCI-H28 JU77 LO68 NO36 ONE58 STY51 GAY2911 OLD1612 VGE VGE VGE Fold change relative to PGK1

Figure 3.1 Relative expression (expressed in ΔCt) of the Hh pathway genes. For each gene, the relative expression of mrna was normalized to an endogenous PGK1 control. Values represent the mean ± standard error of the mean (SEM) of three independent experiments each performed in duplicates. A, DHH, B, PTCH1 and C, SMO, D, GLI1, E, GLI2 and F, GLI3 in ten mesothelioma cell lines and two primary cultures of normal mesothelial (NM1 and NM2) cells. 80

3.3.2 PTCH1, SMO and SUFU mutations in mesothelioma cell lines Through automated DNA sequencing, all the exons of 13 genes encoding components of the Hh signaling pathway were screened for mutations in a panel of seven mesothelioma cell lines. No mutation was found in the exonic regions of SHH, DHH, IHH, PTCH2, HHIP, KIF7, GLI1, GLI2 and GLI3 genes. However, one non-synonymous mutation in SUFU was identified (Table 3.3). This missense mutation, which was found in LO68, involved a C>T transition at nucleotide 1232 in exon 10 of SUFU, which substitutes threonine for methionine at position 411 (p.t411m) (Figure 3.2A). This mutation was characterized by SIFT (Ng & Henikoff, 2003) and PolyPhen2 (Adzhubei et al, 2010) and determined to be damaging. The data was also cross-checked with 1000 Genomes and confirmed that this insertion is not a germline variant. In addition, as shown in Figure 3.2B, amino acid sequence alignment from various species revealed that Thr411 of SUFU was evolutionarily highly conserved. Table 3.3 Mutations identified in mesothelioma cell lines Type of alteration Gene Exon Region Nucleotide change Amino acid change SIFT PolyPhen2 Cell line Point SUFU 10 Coding 1232 C>T T411M Damaging Possibly LO68 mutation damaging Deletion PTCH1 18 Coding - - Unknown Unknown JU77 PTCH1 19 Coding - - Unknown Unknown JU77 PTCH1 20 Coding - - Unknown Unknown JU77 PTCH1 21 Coding - - Unknown Unknown JU77 PTCH1 22 Coding - - Unknown Unknown JU77 PTCH1 23 Coding - - Unknown Unknown JU77 Insertion SMO 1 Coding 69_70insCTG 23L_24GinsL Unknown Unknown LO68 Although no point mutations were identified in the exonic region, homozygous deletion of exons 18, 19, 20, 21, 22 and 23 of PTCH1 in the JU77 cell line was observed and had been suspected by the absence of PCR product on agarose gel electrophoresis (Figure 3.3). Subsequently, this was confirmed by PCR using exonic primers and demonstrated complete absence of PCR amplicon in the cell line (Figure 3.4). In addition to the 81

identification and characterization of point mutations and exon deletions, a 3 base-pair insertion of CTG between nucleotides 69 and 70 in exon 1 of the SMO gene in LO68 cell line was identified. This 69_70insCTG mutation resulted in an in-frame addition of a leucine residue after amino acid 23 of the SMO protein (p.23l_24ginsl) with an unknown functional role (Figure 3.5). The same in-frame insertion was recently reported in two out of 39 gastric tumors (Wang et al, 2013a). 82

Figure 3.2 Identification of a SUFU T411M mutation in LO68 cell line. A, Electropherogram of SUFU gene. Left, the sequencing result of MSTO- 211H cell line, showing the wild-type SUFU gene. Right, the sequencing result of LO68 carrying the mutant allele. B, Amino acid sequence alignment of SUFU from various species. Shown in the red box is the T411 residue that is evolutionarily conserved among these different species. Numbers indicate the position of amino acid residue with the start codon (methionine) as number 1. 83

Figure 3.3 Agarose gel electrophoretic analysis of amplified exons 18 23 of PTCH1 gene from mesothelioma cell lines. JU77 has a homozygous deletion of all six exons. Lanes are labelled according to the cell lines, NTC = No template control. Exons assayed and size markers ( M ), in base pairs, are shown on left side of the gel image. 84

Figure 3.4 Confirmatory PCR. JU77 showing a deletion of exons 18 23 of the PTCH1 gene. Lanes are labelled according to the cell lines ( L = LO68, J = JU77 and 2 = NTC) and exons assayed and size markers ( M ), in base pairs, shown on both sides of the gel image. 85

A Wild-type B LO68 349_350insCTG CTG insertion Figure 3.5 Identification of a SMO 23insL mutation in LO68 cell line. A, Wild-type sequence in MSTO- 211H cell line. B, 3-bp CTG insertion in LO68 cell line. 86

3.3.3 Hedgehog pathway gene variants in mesothelioma cell lines Screening all the exons of each of the 13 Hh pathway genes revealed a total of 35 SNPs using our panel of seven mesothelioma cell lines (Table 3.4). Examination of the NCBI dbsnp database revealed that all the SNPs were previously reported variations. No polymorphism was found in the exonic regions of DHH and SHH genes. Among the 35 SNPs detected, all were located in the coding regions and 16 SNPs would cause substitution of an amino acid (Table 3.2). The most frequent SNPs include: (i) GLI2 rs2592595, ii) GLI2 rs3738880, iii) GLI2 rs10167980, iv) GLI2 rs12711538, v) KIF7 rs3803531, vi) KIF7 rs8037349, vii) KIF7 rs8179066 and SMO rs2228617, which were identified in all seven cell lines. In silico analysis of potential functional impact of the 16 non-synonymous SNPs were characterized by the SIFT and PolyPhen2 programs. There was an overlap between SIFT and PolyPhen2 predictions: only one SNP (KIF7 rs8179065) was predicted to be a non-deleterious substitution by both SIFT and PolyPhen2 (Table 3.2). The remaining five non-synonymous SNPs (PTCH1 rs357564, STK36 rs1344642 and rs1863704, KIF7 rs138354681 and GLI1 rs2228224) were predicted by PolyPhen2 as damaging but benign by SIFT (Table 3.2). 87

Table 3.4 SNPs identified in mesothelioma cell lines Amino Gene Exon Region Nucleotide change acid change dbsnp ID SIFT PolyPhen2 Cell line GLI1 5 Coding 576 G>A E192E rs2228225 Tolerated Benign JU77, LO68, NO36, STY51 GLI1 11 Coding 2798 G>A G933D rs2228224 Tolerated Damaging JU77, LO68, NO36, STY51 GLI1 11 Coding 3298 G>C E1100Q rs2228226 Tolerated Benign JU77, LO68, NO36, STY51 GLI2 5 Coding 801 G>A S267S rs2592595 Tolerated Benign MSTO- 211H, NCI- H28, JU77, LO68, NO36, ONE58, STY51 GLI2 13 Coding 3466 G>T A1156S rs3738880 Tolerated Benign MSTO- 211H, NCI- H28, JU77, LO68, NO36, ONE58, STY51 GLI2 13 Coding 3916 G>A D1306N rs12711538 Tolerated Benign MSTO- 211H, NCI- H28, JU77, LO68, NO36, ONE58, STY51 GLI2 13 Coding 3939 A>G P1313P rs10167980 Tolerated Benign MSTO- 211H, NCI- H28, JU77, LO68, NO36, ONE58, STY51 GLI3 4 Coding 547 A>G T183A rs846266 Tolerated Benign MSTO- 211H, NCI- H28, LO68, ONE58, STY51 GLI3 14 Coding 2993 C>T P998L rs929387 Tolerated Benign JU77, NO36, ONE58 HHIP 13 Coding 2058 T>C I686I rs11727676 Tolerated Benign MSTO- 211H, NCI- H28 IHH 3 Coding 600 G>A T200T rs3731878 Tolerated Benign STY51 IHH 3 Coding 753 T>C P251P rs3731881 Tolerated Benign MSTO- 211H, NCI- H28, JU77, LO68, ONE58 IHH 3 Coding 1128 T>C T376T rs394452 Tolerated Benign MSTO- 211H, NCI- H28, JU77, LO68, ONE58, STY51 KIF7 1 Coding 154 G>A D52N rs8179065 Tolerated Benign MSTO- 211H, NCI- H28, LO68 KIF7 1 Coding 195 G>C A65A rs8179066 Tolerated Benign MSTO- 211H, NCI- H28, JU77, LO68, NO36, ONE58, 88

STY51 KIF7 4 Coding 1102 A>G T368A rs8037349 Tolerated Benign MSTO- 211H, NCI- H28, JU77, LO68, NO36, ONE58, STY51 KIF7 11 Coding 2501 A>G Q834R rs138354681 Tolerated Damaging ONE58 KIF7 12 Coding 2658 A>C A886A rs3803531 Tolerated Benign MSTO- 211H, NCI- H28, JU77, LO68, NO36, ONE58, STY51 KIF7 13 Coding 2873 G>T S958I rs3803530 Tolerated Benign KIF7 14 Coding 3013 G>A G1005R rs12900805 Tolerated Benign JU77, ONE58, STY51 KIF7 14 Coding 3048 G>A S1016S rs9672286 Tolerated Benign JU77, ONE58, STY51 PTCH1 23 Coding 3944 C>T P1315L rs357564 Tolerated Damaging MSTO- 211H, NCI- H28, ONE58, STY51 PTCH2 2 Coding 90 G>A L30L rs45573433 Tolerated Benign MSTO- 211H, NCI- H28 PTCH2 8 Coding 1080 G>T V360V rs11573579 Tolerated Benign LO68 PTCH2 14 Coding 1821 A>G E607E rs2295997 Tolerated Benign NO36 PTCH2 14 Coding 2055 T>C A685A rs7525308 Tolerated Benign JU77, NO36 PTCH2 16 Coding 2487 C>T D829D rs2295996 Tolerated Benign JU77 SMO 1 Coding 74 A>G D25G rs41304185 Tolerated Unknown MSTO- 211H, NCI- H28, LO68, NO36 SMO 2 Coding 384 C>T A128A rs45571737 Tolerated Benign MSTO- 211H SMO 4 Coding 808 G>A V270I rs111694017 Tolerated Benign LO68 SMO 4 Coding 852 G>A Q284Q rs45445295 Tolerated Benign ONE58 SMO 6 Coding 1164 G>C G388G rs2228617 Tolerated Benign MSTO- 211H, NCI- H28, JU77, LO68, NO36, ONE58, STY51 STK36 13 Coding 1748 G>A R583Q rs1344642 Tolerated Damaging JU77 STK36 24 Coding 3008 G>A G1003D rs1863704 Tolerated Damaging JU77 SUFU 11 Coding 1299 T>C I433I rs17114803 Tolerated Benign NO36 89

3.3.4 PTCH1, SMO and SUFU mutations in FFPE mesothelioma tumors To validate the mutations that were identified in the cell lines, we analysed DNA from micro-dissected FFPE tumors from 14 patients with mesothelioma for PTCH1 exonic deletions, SMO insertion and SUFU point mutation. Sequencing results showed that one patient had a tumor harbouring the CTG insertion in the SMO gene. The patient was a male Caucasian who survived for 3.4 months after being diagnosed at age 51 years with biphasic mesothelioma. As blood was not collected from the patient, the germline vs. somatic origin of the mutation could not be determined. Lastly, we did not detect the presence of the PTCH1 exon deletions or the SUFU mutation in the patient cohort. 3.3.5 Functional characterization of SUFU mutant To corroborate the SIFT and PolyPhen2 predictions that the p.t411m mutation affects SUFU function, C3H10T1/2 cells were transiently transfected with a GLI1 luciferase reporter construct and expression constructs for wild-type GLI1, wild-type SUFU and the SUFU p.t411m mutant. Transfection of wild-type GLI1 resulted in increased GLI1 reporter activity whereas cotransfection of wild-type SUFU inhibited GLI1-induced reporter activity. However, the SUFU mutant failed to alter GLI1 reporter activity when compared with wild-type SUFU (p = 0.9262, unpaired t-test) (Figure 3.6). 90

Normalized luciferase activity 100 90 80 70 60 50 40 30 20 10 0 Gli1 Gli1+SUFU Figure 3.6 p.t411m mutation fails to disrupt the inhibitory function of SUFU. C3H10T1/2 cells were transfected with Gli-luciferase reporter construct with Renilla luciferase plasmid for 48 h before they were harvested for luciferase activity determination. Values represent the mean ± SEM of a representative experiment performed in triplicates. P value (NS, not significant) was determined by unpaired t-test. NS Gli1+SUFU T411M 91

3.4 DISCUSSION In this chapter, the expression profiles of the Hh pathway genes in mesothelioma cell lines and cultured primary mesothelial cells were examined. The Hh pathway components GLI1, GLI2 and PTCH1 were expressed at high levels in mesothelioma cell lines compared to normal mesothelial cells. This suggests that Hh signaling is active in mesothelioma, which corroborated findings from an earlier study (Li et al, 2013; Shi et al, 2012). This is consistent with recent findings of GLI1 and Hh interacting protein (HHIP) being elevated 2-6-fold in mesothelioma tissues using qrt-pcr analysis, relative to benign pleural tissue (Shi et al, 2012). In a similar study, the mrna and protein levels of GLI1, GLI2 and the signal transducer SMO in 46 mesothelioma tissues were found to be overexpressed as analysed by qrt-pcr and immunohistochemistry (Li et al, 2013). Notably, all the mesothelioma cell lines expressed the Hh ligand, DHH, which suggests that the Hh pathway is activated via autocrine mechanisms and is consistent with the study done by Shi and colleagues, which found that pleural mesothelioma tumors also had higher expression of DHH and SHH (Shi et al, 2012). Aberrant activation of Hh signaling in human cancers could result from genetic alterations in pathway components, including PTCH1, SMO, SUFU and GLI1 (Jiao et al, 2012; Scales & de Sauvage, 2009; Taylor et al, 2002; Xie et al, 1998). Utilizing automated sequencing of PCR-amplified DNA fragments, this study is the first time a mutational analysis of genes (13) encoding components of the Hh signaling pathway has been performed in mesothelioma cell lines (7). The rationale for screening Hh pathway genes for genetic alterations is based on several observations: 1) this pathway plays a critical role in development and growth, 2) high frequency of mutations in the Hh pathway genes screened to date, and 3) the Hh pathway components were found to be overexpressed in primary mesothelioma tumors and cell lines (Li et al, 2013; Shi et al, 2012). 92

In the present study, only three mutations were found in three out of 13 genes screened and they were detected in only two cell lines. The first mutation that we have identified is a novel multiple-exon deletion in the PTCH1 gene in JU77 cell line, whereby exons 18-23 were deleted. This mutation would result in a putative truncated PTCH1 protein, in which one of two extracellular loops and the cytoplasmic C-terminal domain are lost, or could undergo nonsense-mediated mrna decay. However, this multi-exonic deletion was not detected in the cohort of tumor samples from patients with mesothelioma, but this may be due to the small sample size of the patient cohort. The tumor suppressor gene PTCH1 was cloned in 1996 and subsequently shown to be involved in repression of the Hh pathway (Hahn et al, 1996; Johnson et al, 1996; Taipale et al, 2002). As expected for a classical tumor suppressor gene, PTCH1 was found to be mutated in Gorlin syndrome and many other cancers (Lindstrom et al, 2006). Previously published studies on mutations in PTCH1 have found more than 300 mutations, most of which appeared to be clustered in the large extracellular and intracellular loops of the PTCH1 protein (Lee et al, 1994; Lindstrom et al, 2006). Although the functional impact of the deletion of exons 18-23 in PTCH1 has yet to be elucidated, the key role of the extracellular loops in mediating Hh ligand binding and the role of the cytoplasmic C- terminal domain in mediating subcellular localization and turnover of PTCH1 as well as inhibition of Hh gene targets, suggest that the receptor lacking exons 18-23 may lead to an aberrantly activated Hh pathway (Johnson et al, 2000; Lu et al, 2006; Marigo et al, 1996). Recent studies also suggest that the C-terminal fragment of PTCH1 could be localized to the nuclear region of the cell where it represses the transcriptional activity of GLI1, even though the canonical view of PTCH1 as a transmembrane protein is deeply entrenched in the literature (Kagawa et al, 2011). In addition to the exonic deletion, we 93

found a SNP (c.3944c>t) in exon 23 of PTCH1, which was also reported in cancers of the skin, bone and vulva (Evans et al, 2000; Hafner et al, 2007; Michimukai et al, 2001; Ohki et al, 2004; Wolter et al, 1997). This non-synonymous SNP leads to substitution of leucine for proline at position 1315 in the C-terminal domain of the PTCH1. Modelling analysis suggested that the Leu 1315 substitution might alter the secondary structure of PTCH1 resulting in constitutive pathway activation (Asplund et al, 2005). Intriguingly, this biallelic SNP has recently been associated with a higher risk for development of breast and nonmelanoma skin cancers (Asplund et al, 2005; Chang-Claude et al, 2003; Liboutet et al, 2006). The significance of this SNP with respect to etiopathogenesis of mesothelioma, however, requires further study. We also detected a SMO mutation in one mesothelioma cell line, which was also present in a tumor sample. SMO acts as a signal transducer of the Hh pathway, mediating communication between transmembrane PTCH1 receptor and transcriptional activators GLI1 and GLI2. Xie et al. found activating somatic mutations in SMO in three out of 47 (6.4%) patients with sporadic BCC (Xie et al, 1998). Furthermore, they showed that overexpression of these mutant SMO proteins in mouse skin produced BCC-like tumors (Xie et al, 1998). In this study, the identified mutation, 23L_24GinsL, lies in the signal peptide region of SMO and has been detected in two cases of human gastric cancer (Wang et al, 2013a). This mutation might affect the processing of the SMO precursor and in turn potentially interfere with protein targeting to the cell membrane. One of the most intriguing results of this analysis is the identification of a mutation in SUFU. SUFU was originally identified as a negative regulator of the Hh pathway in early embryonic development (Stone et al, 1999). Taylor et al. first implicated SUFU in the tumorigenesis of childhood MB in 2002 (Taylor et al, 2002). They identified germline 94

and somatic SUFU nonsense mutations in 8.7% (4 of 46) of desmoplastic MBs. Subsequently, SUFU mutations were found in 4.8% (2 of 42) and 2.5% (2 of 83) of sporadic BCCs and MBs, respectively (Reifenberger et al, 2005; Slade et al, 2011). I found a missense mutation affecting Thr 411 in SUFU in one of the 11 cell lines. Importantly, the same mutation was recently detected in a patient with colorectal cancer as part of the Cancer Genome Atlas (Cancer Genome Atlas, 2012), supporting a role of this mutation in tumorigenesis. This mutation was predicted by two web-based programs, SIFT and PolyPhen2, that predict the potential functional impact of altered amino acid sequences on encoded protein function, to result in a major change in amino acid class (from small, polar to large, hydrophobic) in the N-terminal region located close to the GLI1 binding domain, thereby implying a negative impact on protein function. SUFU acts as a classic tumor suppressor gene, with mutations leading to the inability of SUFU to transport GLI1 out of the nucleus to the cytoplasm, thereby resulting in aberrant activation of Hh signaling in MB (Taylor et al, 2002). However, functional characterization showed that the p.t411m mutation does not alter the negative regulatory function of SUFU in a GLI1 luciferase reporter assay. In addition to PTCH1, SUFU and SMO, the mesothelioma cell line panel was screened for mutations in nine other Hh pathway genes PTCH2, DHH, IHH, SHH, GLI1, GLI2, GLI3, KIF7, STK36 and HHIP. No evidence for mutations was found in any of these genes in those cell lines. However, there were a number of previously reported SNPs, of which 16 were shown to result in non-synonymous codon substitution. To explore the functional significance of these SNPs, all those that are non-synonymous were analysed by SIFT and PolyPhen2. There was considerable difference between the predictions from different algorithms: a KIF7 SNP rs8179065 was predicted by SIFT to have altered protein function whereas the PolyPhen2 program predicted the PTCH1 SNP rs357564, 95

STK36 SNPs rs1344642 and rs1863704, KIF7 SNP rs138354681 and GLI1 SNP rs2228224 to be deleterious amino acid substitution. Algorithmic differences aside, the real functional significance of these non-synonymous SNPs on Hh signaling need to be validated by biochemical studies. Recently, SNPs in SHH, GLI2 and GLI3 genes have been reported to be associated with clinical outcome of trans-urethral resection and Bacillus Calmette-Guerin intravesical therapy for non-muscle-invasive bladder cancer patients (NMIBC) (Chen et al, 2010). The GLI2 SNP rs11685068 and SHH SNP rs1233560 demonstrated significant associations with recurrence in NMIBC patients who received trans-urethral resection. However, the NMIBC patients receiving Bacillus Calmette-Guerin treatment who carried the GLI3 SNPs rs3801192 and rs6463089 were found to have a higher cancer recurrence rate and shorter recurrence-free survival time compared to those carrying the wild-type genotype. In a separate genome-wide association study, common variants in the SHH, BTRC and HHIP genes have been associated with the risk of pancreatic cancer (Li et al, 2012a). These studies demonstrated a link between common genetic variations in the Hh pathway and cancer risk, thus highlighting the importance of examining the association between SNPs in Hh pathway genes and the risk of mesothelioma. Clearly, the frequency of mutations in key Hh pathway genes in mesothelioma is less than in BCC and MB where the mutations lead to hyperactivation of the pathway. However, the importance of these and other unidentified mutations in Hh pathway genes in the pathogenesis of a subset of mesothelioma tumors cannot be discounted and needs further investigation in a larger sample set. In summary, we report mutations in SMO and SUFU and a novel multi-exonic deletion in 96

PTCH1 in mesothelioma cell lines and tumors. Our data suggest that unlike BCC, MB and rhabdomyosarcoma in Gorlin syndrome, aberrant activation of Hh signaling in mesothelioma is unlikely to be driven by mutations in the majority of tumors but instead activated through autocrine signaling as suggested by Shi and colleagues. (Shi et al, 2012) This pathway may represent a novel therapeutic target in mesothelioma for the recently developed Hh pathway inhibitors. 97

CHAPTER 4 TARGETING OF HEDGEHOG PATHWAY USING GLI INHIBITOR GANT61 98

4.1. INTRODUCTION Several small molecule compounds with inhibitory effects on the Hh pathway have been reported. Notably, US Food and Drug Administration have approved GDC-0449, a SMO antagonist, for the treatment of patients with advanced stages of basal cell carcinoma (FDA, 2012). Besides targeting the SMO receptor, the Hh ligand and Gli transcription factors appear highly amenable to inhibition by small molecule inhibitors. Of particular interest is a cell-permeable compound, GANT61, discovered in a cell-based screen for antagonists of Gli-mediated transcription (Lauth et al, 2007). Studies have shown that blocking Gli with GANT61 induces extensive cell death in human colon carcinoma (Mazumdar et al, 2011a), rhabdomyosarcoma (Kawabata et al, 2011) and B-cell chronic lymphocytic leukemia (Desch et al, 2010) cells and suppresses the growth of human prostate carcinoma (Lauth et al, 2007) and neuroblastoma (Wickstrom et al, 2013) in mice, suggesting that Gli transcription factors could be a target for therapy in these cancers. The cytotoxic effect of GANT61 was previously shown to be attributed to the inhibition of Gli binding to DNA (Lauth et al, 2007), thus preventing the transcription of Hh target genes such as Bcl-2 (Mazumdar et al, 2011b), which is involved in cell proliferation and apoptosis. In addition, GANT61 was also reported to cause DNA damage (Mazumdar et al, 2011a). However, the mechanism by which this occurs is still unclear. Furthermore, upregulation of death receptor family proteins, such as TRAIL- R1/DR4, TRAIL-R2/DR5 and Fas were also implicated in GANT61-induced apoptosis (Fu et al, 2013; Mazumdar et al, 2011b). Recent studies also revealed that GANT61 could induce autophagic cell death in human hepatocellular carcinoma (HCC) cells through upregulation of Bnip3 (Wang et al, 2013b). At present, however, it is unclear whether GANT61 could have effects that are independent of Hh/Gli signaling. 99

4.2 RESULTS 4.2.1 Gli inhibitor GANT61 is a more potent Hedgehog pathway inhibitor than SMO inhibitors (cyclopamine and GDC-0449) To determine the antiproliferative activity of GANT61 in mesothelioma cells, a methylene blue cell proliferation assay (Oliver et al, 1989) was used to generate doseresponse curves and evaluate cell proliferation following treatment with varying concentrations (0.2 to 50 µm) of GANT61 for 72 h. As shown in Figure 4.1, GANT61 inhibited cell proliferation in all mesothelioma cell lines in a dose-dependent manner. Among the cell lines tested, JU77 cells showed highest sensitivity to GANT61 treatment (IC 50 = 4.02 µm) whereas NO36 cells appeared to be the most resistant (IC 50 = 31.8 µm) (Figure 1A). In contrast, two SMO antagonists, cyclopamine and GDC-0449, demonstrated poor antiproliferative activity against these mesothelioma cell lines (Figures 4.2 and 4.3). The average IC 50 value for cyclopamine for all ten cell lines was 26.2 µm whereas the average IC 50 value for GDC-0449 was more than 50 µm. Clonogenicity describes the colony-forming ability of cells where a single cell grows and expands into a clonal cell population. To determine the anti-clonogenic activity of GANT61, a clonogenic assay (Franken et al, 2006) was used to evaluate the cellular reproductive potential following GANT61 treatment. Clonogenicity of mesothelioma cells was also reduced in a dose-dependent manner after exposure to GANT61 for 72 h (Figure 4.4), consistent with the proliferation assay. As shown in Figure 4.5, concomitant with growth inhibitory effect, GANT61 induced G1 cell cycle arrest as indicated by the increased percentage of LO68 cells in G1 cell cycle phase at 24 h compared to vehicletreated cells. There was also an accumulation of cells in the sub-g1 fraction at 48-72 h compared to vehicle-treated cells, suggesting that apoptosis is involved (Figure 4.6). 100

Figure 4.1 Dose-response curves for GANT61 cytotoxicity following treatment for 72 hr. IC 50 values are shown in brackets for each cell line. Values are the mean of three independent experiments each with six replicates. Data are represented as (mean ± SEM; n = 3). Figure 4.2 Dose Dose-response curves for cyclopamine cytotoxicity following treatment for 72 hr. Values are the mean of two independent experiments each with six replicates. 101

Figure 4.3 Dose-response curves for GDC-0449 cytotoxicity following treatment for 72 hr. Values are the mean of two independent experiments each with six replicates. Effective concentration (µm) 20 15 10 5 0 JU77 ONE58 MSTO-211H NCI-H28 STY51 GAY2911 OLD1612 LO68 VGE NO36 Vehicle 5 GANT61 (µm) Figure 4.4 Clonogenic survival assays were 10 15 20 JU77 LO68 NO36 performed in cell cultures exposed to 5, 10, 15 and 20 µm GANT61 and the concentration at which the number of colonies is reduced by more than 90% for each cell line is shown. Data represent the mean ± SEM of three independent experiments. Representative cell lines with differential sensitivity to GANT61 are shown. 102

Vehicle Relative cell number GANT61 DNA content Figure 4.5 Cell cycle analysis of LO68 cells treated for 24 h with either 20 µm GANT61 (open graph) or vehicle (grey graph). The inset shows the percentage of cells at different phases of the cell cycle (G1, S and G2/M) of GANT61 and vehicle-treated cells. The data shown represent one of three independent experiments with comparable outcomes. % Cell count 100 80 60 40 20 SubG1 G1 S G2M 0 0h 24h 32h 40h 48h 72h Figure 4.6 Cell cycle analysis of LO68 cells treated for 0-72 h with 20 µm GANT61. The data shown represent one of three independent experiments with comparable outcomes. 103

4.2.2 GANT61 induces higher levels of apoptosis than SMO inhibitors (cyclopamine and GDC-0449) To understand the cell death response to GANT61 in LO68 cells, cells were exposed to GANT61 and the level of phosphatidylserine, a marker of early apoptosis was measured. GANT61 exposure induced an increase in apoptosis. FACS analysis of annexin V- binding demonstrated that this increase in apoptosis occurred in a dose-dependent manner (Figure 4.7). As a comparison, the level of apoptotic cells after treatment with cyclopamine and GDC-0449 was quantified. Treatment of LO68 cells with cyclopamine increased the number of apoptotic cells in a dose-dependent manner (Figure 4.8). Surprisingly, when the level of apoptotic cells was quantified after treatment with GDC- 0449, no difference could be observed between cells treated with GDC-0449 or with vehicle (Figure 4.8). 104

% Annexin V+ cells 100 80 60 40 20 24h 48h * * *** *** *** 0 Vehicle 10µM 20µM 30µM Figure 4.7 Apoptosis (as assessed by the annexin V/7AAD assay) was quantified in LO68 cells treated with 10, 20 or 30 µm GANT61 or vehicle for 24-48 h. Data represent the mean ± SEM of three independent experiments. *, p < 0.05 or ***, p < 0.001, compared to vehicle-treated cells. % Annexin V+ cells 60 50 40 30 20 10 Cyclopamine GDC-0449 ** 0 Vehicle 10µM 20µM 30µM Figure 4.8 Apoptosis (as assessed by the annexin V/7AAD assay) was quantified in LO68 cells treated with 10, 20 or 30 µm cyclopamine, GDC- 0449 or vehicle for 48 h. Data represent the mean ± SEM of three independent experiments. 105

4.2.3 GANT61 targets Gli transcription factors As expected, qpcr analysis showed that GANT61 reduced mrna expression of GLI1 and GLI2 following treatment with 20 µm GANT61 for up to 72 h (Figure 4.9). The expression of the Gli downstream target gene PTCH1 was also downregulated by treatment with GANT61 (Figure 4.9). A similar downregulation of GLI1 and GLI2 proteins was observed after 24 h exposure to different concentrations of GANT61 (10-30 µm) (Figure 4.10). The protein level of Bcl-2, a Gli downstream target protein, was also reduced after GANT61 treatment (Figure 4.10). The specificity of inhibition of GLI1 and GLI2 by GANT61 was confirmed by examining its efficacy in a Gli luciferase reporter assay. Consistent with previous findings, GANT61 inhibited the Gli reporter activity in LO68 cells (Figure 4.11). 4.2.4 GANT61 sensitivity correlates with GLI1 and GLI2 mrna expressions As GANT61 is a GLI1 and GLI2 antagonist (Lauth et al, 2007), I hypothesized that the sensitivity of mesothelioma cell lines to GANT61 (as indicated by IC 50 ) might relate to the mrna expression levels of GLI1 and GLI2. A positive correlation between GLI1 (r = 0.68, p<0.005) (Figure 4.12A) and GLI2 (r = 0.49, p<0.05) (Figure 4.12B) expression and sensitivity to GANT61 was found. These findings point to GANT61 being a specific inhibitor of GLI1 and GLI2 (Lauth et al, 2007). 106

Fold change in gene expression 1.2 1.0 0.8 0.6 0.4 0.2 0.0 ** *** 0 24 48 72 *** *** *** ** ** 0 24 48 72 0 24 48 72 GLI1 GLI2 PTCH1 Hour post treatment Figure 4.9 qpcr analysis of the Hh pathway genes GLI1, GLI2 and PTCH1 (expressed as fold change) was performed on LO68 cells treated with 20 µm GANT61 or vehicle for 24-72 h. The expression levels of each gene were normalized using PGK1 mrna as an endogenous control and are indicated as fold change with respect to the vehicle-treated LO68 cells. Error bars represent the mean ± SEM of three independent experiments each performed in duplicate. **, p < 0.01 or ***, p < 0.001, compared to vehicle-treated cells. Vehicle 10 20 30 GANT61 (µm) GLI GLI2 Bcl-2! β-actin! Figure 4.10 Western blot analysis of GLI1, GLI2, Bcl-2 and β-actin on LO68 cells treated with 10, 20 or 30 µm GANT61 or vehicle for 24 h. 107

1.0 Fold change (Luciferase activity) 0.8 0.6 0.4 0.2 ** *** 0.0 Vehicle 10 20 GANT61 (µm) (24h) 30 Figure 4.11 Gli transcriptional activity was determined by transfecting LO68 cells with a Gliresponsive luciferase reporter plasmid. Cells were treated with either 10, 20 or 30 µm GANT61 or vehicle for 24 h. Luciferase activity of cell lysates was measured and normalized to Renilla luciferase activity obtained by co-transfection with a constitutively expressed Renilla luciferase internal control plasmid. Data represent the mean ± SEM of three independent experiments. **, p < 0.01 or ***, p < 0.001, compared to vehicletreated cells. 108

A 0 GLI1 expression (relative to PGK1) -1-2 -3 r 2 = 0.68 p < 0.005-4 0.4 0.6 0.8 1.0 1.2 1.4 1.6 GANT61 IC 50 (log10) B GLI2 expression (relative to PGK1) -0.5-1.5 r 2 = 0.49 p < 0.05-2.5 0.4 0.6 0.8 1.0 1.2 1.4 1.6 GANT61 IC 50 (log10) Figure 4.12 Relative expression of A) GLI1 and B) GLI2 mrna, shown as log10 2 -ΔCt values, versus log IC 50 values (from methylene blue proliferation assay). 109

4.2.5 Depletion of GLI1, GLI2 and SMO reduces LO68 cell growth Because GLI1 and GLI2 are upregulated in various cancers (Teglund & Toftgard, 2010; Yang et al, 2010), including mesothelioma (Li et al, 2013; Shi et al, 2012) and has been shown to regulate cell proliferation (Sanchez et al, 2004), the effect of depleting GLI1 and GLI2 using small interfering RNAs (sirnas) in LO68 cells on cell proliferation was examined using a WST-1 cell-viability assay. qrt-pcr analyses demonstrated knockdowns of GLI1 and GLI2 mrnas in LO68 cells (Figure 4.13) and this knockdown reduced cell proliferation compared with NC sirna control (Figure 4.14). 4.2.6 GANT61-induced apoptosis and cell cycle arrest are independent of Gli inhibition Because GANT61 is an inhibitor of Gli transcription factors, I examined whether GANT61-induced G1 cell cycle arrest and apoptosis are dependent on Gli inhibition. sirna-mediated depletion of GLI1 and GLI2, either individually or combined, surprisingly had no effect on cell death as indicated by the small sub-g1 apoptotic population (~5% in sub-g1 phase in GLI1- and/or GLI2-depleted cells compared to ~5% cells in sub-g1 phase in LO68 cells transiently transfected with NC sirna), indicating that the pro-apoptotic activity of the drug is likely to be independent of Gli inhibition (Figure 4.15). LO68 cells were then quantified in various cell cycle phases following transient transfection with sigli1 and/or sigli2. The results from FACS analysis showed that the knockdowns of GLI1 and GLI2 by sirna did not result in G1 cell cycle arrest (~60% in G1 phase in GLI1- and/or GLI2-depleted cells compared to ~60% cells in G1 phase in LO68 cells transiently transfected with NC sirna) (Figure 4.16). Taken together, the data suggest that induction of apoptosis and cell cycle arrest in response to GANT61 is independent of Hh/Gli s signaling in LO68 cells. 110

Fold change in gene expression 1.2 1.0 0.8 0.6 0.4 0.2 0.0 *** ** sinc sigli1 sigli2 sigli1+sigli2 *** ** * sinc sigli1 sigli2 sigli1+sigli2 Gli1 Gli2 Figure 4.13 Expression of GLI1 and GLI2 mrnas after sirna transfection, determined by qrt-pcr. LO68 cells were transfected with negative control (sinc), GLI1 (sigli1) or GLI2 (sigli2) sirna for 96 h, then GLI1 mrna expression analysed by qrt-pcr. Error bars represent the mean ± SEM of three independent experiments each performed in duplicates. *, p < 0.05 or ***, p < 0.001, compared to NC sirna-transfected cells. 111

A Abs (450 nm) 0.5 0.4 0.3 0.2 0.1 NC sirna GLI1 sirna 0.0 24 48 72 96 Hour post transfection B Abs (450 nm) 0.4 0.3 0.2 0.1 NC sirna GLI2 sirna 0.0 24 48 72 96 Hour post transfection Figure 4.14 Depletion of GLI1 and GLI2 reduce cell growth. sirna-mediated depletion of A) GLI1 and B) GLI2 inhibited proliferation of LO68 cells compared with NC sirna. Proliferation was measured using the WST-1 assay. The graphs display the mean ± SD of two or three experiments. 112

20 SubG1 fraction (%) 15 10 5 0 sinc sigli1 sigli2 sigli1+sigli2 Figure 4.15 Cell death (subg1 fraction) was measured by flow cytometry 96 h after transfection. Data represent the mean ± SEM of three independent experiments. Results are not statistically significant when compared to cells transfected with NC sirna. % Cell count 100 80 60 40 20 G1 S G2M 0 sinc sigli1 sigli2 sigli1+sigli2 Figure 4.16 The cell cycle was analysed by flow cytometry 96 h following transfection. Histogram profiles of flow-cytometric analysis show the cell cycle distribution of the cell population. Data represent the mean ± SEM of three independent experiments. 113

4.2.7 GANT61 triggers induction of autophagy LO68 cells Because recent studies have shown that inhibition of the Hh pathway can activate autophagy (Jimenez-Sanchez et al, 2012; Wang et al, 2013b), the effect of inhibiting Gli on autophagy was assessed in LO68 cells. The presence of acidic vesicular organelles (AVOs) in the cytoplasm is a marker of autophagy, which can be detected by vital staining of cells with the lysosomotropic agent acridine orange (Paglin et al, 2001; Traganos & Darzynkiewicz, 1994). Acridine orange is a cell-permeable weak base that becomes protonated when it enters the cell and accumulates in acidic subcellular compartments to form red fluorescent aggregates. As shown in Figure 4.17A, GANT61 treatment increased the percentage of cells with AVOs in a dose-dependent manner. There was also a dose-dependent increase in the red fluorescence intensity following GANT61 treatment, indicative of autophagy induction (Figure 4.17B). The effect of bafilomycin A1, an autophagy inhibitor (Yamamoto et al, 1998), was next investigated on GANT61-induced AVOs. As shown in Figure 4.18, bafilomycin A1 reduced the red fluorescence intensity from 22.58 ± 2.97 to 13.23 ± 0.41 in GANT61-treated cells. Since acridine orange primarily stains lysosomes (Allison & Young, 1964; Zelenin, 1966), the induction of autophagy after GANT61 treatment was confirmed by Cyto-ID Green staining and quantification by flow cytometry in LO68 cells. Cyto-ID Green dye has been shown to label only autophagosomes (Oeste et al, 2013). As shown in Figure 4.19, GANT61 treatment induces an increase in Cyto-ID green fluorescence 24 h after treatment in LO68 cells and this effect is abolished by pretreatment with bafilomycin A1. 114

A % Cells with AVOs 0 10 20 30 40 50 60 70 B Mean Fluorescence Intensity 15 10 5 0 *** ** Vehicle 5µM 10µM 20µM 30µM GANT61 concentration *** *** * Vehicle 5µM 10µM 20µM 30µM GANT61 concentration Figure 4.17 GANT61 treatment in LO68 cells is associated with an increase in AVOs in a dosedependent manner. LO68 cells were treated with 10-30 µm GANT61 or vehicle for 24 h. The cells were stained with acridine orange, and the fluorescence was measured by flow cytometry. Data are expressed as A) percentage of cells with AVOs and B) mean fluorescence intensity of cells with AVOs measured in each experimental condition. Data represent the mean ± SEM of three independent experiments. *, p < 0.05, **, p < 0.01 or ***, p < 0.001, compared to vehicle-treated cells. 115

Mean Fluorescence Intensity 30 20 10 0 * Vehicle 20µM GANT61 200nM BafA1 + 20µM GANT61 Figure 4.18 GANT61-induced AVO formation in LO68 cells is inhibited by bafilomycin A1 (BafA1). LO68 cells were treated with 20 µm GANT61 or vehicle for 24 h. The cells were further treated with BafA1 for 4 h before staining with acridine orange, and the fluorescence was measured by flow cytometry. Data are expressed as mean fluorescence intensity of cells with AVOs measured in each experimental condition. Data represent the mean ± SEM of three independent experiments. *, p < 0.05, compared to GANT61-treated cells. 116

A Vehicle GANT61 Transmitted light Hoechst 33342 Cyto-ID Merge 117

B 80 % Fluorescent cells C Mean Fluorescence Intensity *** 70 *** 60 50 ** 40 30 20 10 0 Vehicle 5µM 10µM 20µM 30µM GANT61 concentration 17.5 *** 15.0 *** 12.5 *** 10.0 ** 7.5 5.0 2.5 0.0 Vehicle 5µM 10µM 20µM 30µM GANT61 concentration Figure 4.19 Confirmation of GANT61-induced autophagy using Cyto-ID Green reagent. LO68 cells were treated with 5-30 µm GANT61 or vehicle for 24 h before they were stained with Cyto-ID Green reagent. A) Stained cells were imaged by confocal microscopy while B-C) fluorescence was measured by flow cytometry. Data are expressed as B) percentage of fluorescent cells and C) mean fluorescence intensity of fluorescent cells measured in each experimental condition. Data represent the mean ± SEM of three independent experiments. **, p < 0.01 or ***, p < 0.001, compared to GANT61-treated cells. 118

4.2.8 Autophagy inhibition enhances GANT61-induced apoptosis A number of studies have shown that chemotherapeutic agents are capable of inducing either protective autophagy or autophagic cell death, depending on the cellular context (Yang et al, 2011). LO68 cells were exposed for 48 h to a sublethal concentration of bafilomycin A1 (1 nm) in conjunction with sublethal concentration of GANT61 (5 µμ), after which annexin V/7AAD assay was performed. Coadministration of bafilomycin A1 with GANT61 resulted in a dramatic increase in the percentage of annexin V + cells (Figure 4.20). Consistent with these results, the exposure of LO68 cells to two other autophagy inhibitors, 3-methyladenine (5 mm) and chloroquine (20 µμ) together with GANT61, produced similar results (Figure 4.21). 119

40 ** % Annexin V+ cells 30 20 10 0 Vehicle 1nM BafA1 5µM GANT61 BafA1+GANT61 Figure 4.20 Pharmacological inhibition of autophagy with BafA1 significantly increases the cytotoxic effects of GANT61 on LO68 cells. LO68 cells were pretreated with 1 nm BafA1, then with 5 µm GANT61 or vehicle for 48 h. Apoptosis was quantified using annexin V/7AAD assay. Data represent the mean ± SEM of three independent experiments. **, p < 0.01, compared to GANT61-treated cells. 120

% Annexin V+ cells 80 70 60 50 40 30 20 10 0 Vehicle 5µM GANT61 5mM 3MA 3MA+GANT61 20µM CQ+GANT61 Figure 4.21 Pharmacological inhibition of autophagy with chloroquine (CQ) or 3- methyladenine (3MA) significantly increased the cytotoxic effects of GANT61 on LO68 cells. LO68 cells were pretreated with 5 mm 3MA or 20 µm CQ for 1 h, then with 5 µm GANT61 or vehicle for 48 h. Apoptosis was quantified using annexin V/7AAD assay. Data represent the mean ± SEM of three independent experiments. **, p < 0.01 or ***, p < 0.001, compared to GANT61-treated cells. ** *** 121

4.2.9 GANT61 sensitizes LO68 cells to standard mesothelioma chemotherapyinduced apoptosis Cisplatin, gemcitabine and pemetrexed are three established anticancer drugs. Cisplatin together with either gemcitabine or pemetrexed constitute effective combinations for treating mesothelioma (Nowak et al, 2002; Vogelzang et al, 2003). Unfortunately, resistance to chemotherapy is a common feature of mesothelioma (Mujoomdar et al, 2010). Addition of GANT61 to standard chemotherapeutic agents, namely doxorubicin, vincristine, cisplatin and irinotecan, has been shown to increase in vitro efficacy in neuroblastoma (Wickstrom et al, 2013). As shown in Figure 4.7, GANT61 was able to induce apoptosis in LO68 cells, a cell line that is resistant to cisplatin-induced cell death (Cregan et al, 2013). Combined treatment of LO68 cells with GANT61 and cisplatin, gemcitabine or pemetrexed was then assessed. As shown in Figure 4.22, combined treatment with GANT61 and cisplatin sensitized LO68 cells to cisplatin-induced apoptosis. Treatment with GANT61 alone only induced apoptosis in ~10% cells. Similarly, treatment with cisplatin alone at C max concentration and below induced apoptosis in 30% cells. However, the level of apoptotic cells further increased by combined administration of GANT61 and cisplatin in 3 out of 4 dose regimes tested (Figure 4.22). Increased cytotoxicity of the combination was dependent on drugconcentration (Figure 4.22). While the combination of 2 µm GANT61 and 5 µm cisplatin did not increase the cytotoxic efficacy of cisplatin, combining 4 µm GANT61 with either 5 or 10 µm cisplatin significantly induced apoptosis yielding up to 58% cell kill at 4 µm GANT61 combined with 10 µm cisplatin (p < 0.001) (Figure 4.22). Similar to results obtained with GANT61-cisplatin combination, combined treatment of LO68 cells with GANT61 and gemcitabine or pemetrexed for 48 h clearly increased the levels of chemotherapy-induced apoptosis (ANOVA, p < 0.0001). Annexin V analysis 122

showed that enhanced apoptotic induction 48 h after combined treatment compared to either treatment alone occurred in a dose-dependent manner yielding maximum levels of apoptosis in the presence of 4 µm GANT61 and 60 µm gemcitabine or 100 µm pemetrexed (Figure 4.23-24). Moreover, at all dose regimes tested, efficacy of combined treatment depended on the chemotherapy type with most pronounced efficacy observed with the GANT61-gemcitabine combination (Figure 4.23). 123

% Annexin V+ cells 70 60 50 40 30 20 10 0 *** *** Vehicle 2µM GANT61 4µM GANT61 5µM CPT 10µM CPT 2µM GANT61+5µM CPT 2µM GANT61+10µM CPT 4µM GANT61+5µM CPT 4µM GANT61+10µM CPT Figure 4.22 GANT61 and cisplatin cooperate to induce apoptosis of LO68 cells. LO68 cells were treated with cisplatin, together with GANT61. Induction of apoptosis was quantified 48 h after treatment using annexin V/7AAD assay. Data represent the mean ± SEM of three independent experiments. ***, p < 0.001. 124

*** *** * *** % Annexin V+ cells 90 80 70 60 50 40 30 20 10 0 Vehicle 2µM GANT61 4µM GANT61 30µM GEM 60µM GEM 2µM GANT61+30µM GEM 2µM GANT61+60µM GEM 4µM GANT61+30µM GEM 4µM GANT61+60µM GEM Figure 4.23 GANT61 and gemcitabine cooperate to induce apoptosis of LO68 cells. LO68 were treated with gemcitabine together with GANT61 as indicated. Induction of apoptosis was quantified 48 h after treatment using annexin V/7AAD assay. Data represent the mean ± SEM of three independent experiments. *, p < 0.05, **, p < 0.01 or ***, p < 0.001. * *** 125

% Annexin V+ cells 60 50 40 30 20 10 0 Vehicle 2µM GANT61 4µM GANT61 50µM PMX 100µM PMX 2µM GANT61+50µM PMX 2µM GANT61+100µM PMX 4µM GANT61+50µM PMX 4µM GANT61+100µM PMX Figure 4.24 GANT61 and pemetrexed cooperate to induce apoptosis of LO68 cells. LO68 were treated with pemetrexed together with GANT61 as indicated. Induction of apoptosis was quantified 48 h after treatment using annexin V/7AAD assay. Data represent the mean ± SEM of three independent experiments. **, p < 0.01 or ***, p < 0.001. ** *** ** *** 126

4.2.10 GANT61 mediates antagonistic to synergistic sensitization effects on standard chemotherapy-induced apoptosis To determine whether the interactions between GANT61 and chemotherapy in LO68 cells were additive, synergistic or antagonistic, quantitative assessments were performed using the Chou-Talalay method (Chou, 2006; Chou & Talalay, 1984). In this method, the term Combination Index (CI) is used to quantitatively represent synergism (CI < 1), additivity (CI =1) and antagonism (CI > 1) (Chou, 2006). As shown in Table 4.1, LO68 cells were most responsive to combined treatment with GANT61 and gemcitabine, showing strong to very strong synergistic effects (CI = 0.08 0.1) 48 h after treatment. Similarly, in all dose regimes tested, the GANT61-cisplatin combination revealed synergistic effects (CI = 0.2 0.4) 48 h after treatment, except the combination of 2 µm GANT61 and 5 µm cisplatin that displayed antagonistic effects (CI = 1.6) (Table 4.1). Interestingly, combinations of GANT61 with pemetrexed were found to be very strongly antagonistic in all dose regimes tested (Table 4.1). 127

Table 4.1 Quantitative assessments of drug combinations using Chou-Talalay Combination Index method GANT61 and Cisplatin (CPT) GANT61 and Gemcitabine (GEM) GANT61 (µm) CPT (µm) Combination index Effect 2 5 1.56906 Antagonism 2 10 0.29709 Strong synergism 4 5 0.4108 Synergism 4 10 0.19229 Strong synergism GANT61 (µm) GEM (µm) Combination index Effect 2 30 0.08634 Very strong synergism 2 60 0.10141 Strong synergism 4 30 0.10251 Strong synergism 4 60 0.08509 Very strong synergism GANT61 and Pemetrexed (PMX) GANT61 (µm) PMX (µm) Combination index 2 50 2.17E+59 2 100 5.11E+61 4 50 7.10E+134 4 100 3.90E+139 Effect Very strong antagonism Very strong antagonism Very strong antagonism Very strong antagonism 128

4.2.11 GANT61 triggers the production of reactive oxygen species Previous studies showed that GANT61 can induce DNA damage in colorectal cancer cells (Mazumdar et al, 2011a). I hypothesized that GANT61 triggers the production of reactive oxygen species (ROS), which in turn damages DNA. To test this hypothesis, ROS levels were measured using the carboxy derivative of fluorescein, CH 2 DCFDA. As shown in Figure 4.25, ROS levels increased significantly in LO68 cells treated with GANT61 in a dose- and time-dependent manner. GANT61 also triggered ROS generation in HCT116 and HT29 colorectal cancer cell lines, suggesting that the production of ROS could be a general effect of GANT61 exposure (Figure 4.26). Moreover, pretreatment of LO68 cells with N-acetylcysteine (NAC) and reduced L- glutathione (GSH), two potent ROS scavengers, completely attenuated this accumulation in ROS (Figure 4.27). As shown in Figure 4.28, the neutralization of ROS by NAC in GANT61-treated cells restores cell viability, suggesting that ROS is responsible for GANT61 cytotoxicity. In agreement with this data, annexin V/7AAD assays showed that NAC pretreatment rescued LO68 cells from GANT61-induced apoptosis (Figure 4.29). 129

Mean Fluorescence Intensity 120 24h 100 48h 80 60 * * 40 20 0 *** Vehicle 10µM GANT61 20µM GANT61 Figure 4.25 GANT61 treatment in LO68 cells is associated with an increase in ROS in a doseand time-dependent manner. LO68 cells were treated with 10 and 20 µm GANT61 or vehicle for 24 or 48 h. The cells were stained with the fluorescent probe CH 2 DCFDA, and the fluorescence was measured by flow cytometry. Data are expressed as mean fluorescence intensity of positive cells measured in each experimental condition. Data represent the mean ± SEM of three independent experiments. *, p < 0.05 or ***, p < 0.001, compared to vehicletreated cells. 130

Mean Fluorescence Intensity 100 80 60 40 20 0 Vehicle 10 µm GANT61 ** ** HCT116 HT29 Figure 4.26 ROS is induced in GANT61-treated colon cancer cells. HCT116 and HT29 cells were treated with vehicle or GANT61 (10 µm) for 48 h before subjected to CH 2 DCFDA flow cytometric analysis. Data represent the mean ± SEM of three independent experiments. **, p < 0.01 or ***, p < 0.001, compared to vehicletreated cells. 131

Mean Fluorescence Intensity 100 80 60 40 20 0 Vehicle 20µM GANT61 20mM NAC+GANT61 ** *** 10mM GSH+GANT61 Figure 4.27 LO68 cells were treated with vehicle or 20 µm GANT61 for 48 h with or without antioxidants NAC (20 mm) and GSH (10 mm). Bar graph shows the increase in the mean fluorescence intensity of CH 2 DCFDApositive cells measured in each experimental condition. Data represent the mean ± SEM of three independent experiments. **, p < 0.01 or ***, p < 0.001, compared to vehicletreated cells. 132

Vehicle 20mM NAC 20µM GANT61 NAC+GANT61 Figure 4.28 Representative light micrographs showing the effects of 20 µm GANT61 or vehicle after 48 h with or without 20 mm antioxidant NAC on the morphology of LO68 cells. Note the reduced cell growth and altered cell morphology in GANT61-treated LO68 cells, which were not seen in vehicle-treated cells. Pretreatment with the antioxidant NAC restores cell growth and abrogates apoptosis in LO68 cells treated with 20 µm GANT61 for 24 h. 133

% Annexin V+ cells 100 90 80 70 60 50 40 30 20 10 0 Vehicle 20µM GANT61 20mM NAC+GANT61 Figure 4.29 Apoptosis (as assessed by the annexin V/7AAD assay) was quantified in LO68 cells treated with vehicle or GANT61 (20 µm) with or without 20 mm antioxidant NAC for 48 h. Data represent the mean ± SEM of three independent experiments. **, p < 0.01, compared to GANT61-treated cells. ** 134

4.3 DISCUSSION The combination of cisplatin and pemetrexed is considered the gold-standard treatment in patients with mesothelioma. This was based on a multicentre phase III trial that reported a longer overall median survival of 12.1 months in patients treated with cisplatin/pemetrexed compared with 9 months in those treated with cisplatin alone, with a response rate of 41% (Vogelzang et al, 2003). The modest survival benefits conferred by the cisplatin/pemetrexed combination have spurred research programs in the past decade into identifying oncogenic signaling pathways that play critical roles in the pathogenesis of mesothelioma. These efforts have led to clinical trials of targeted therapies in this population, particularly inhibitors that block epidermal growth factor receptor, plateletderived growth factor receptor, vascular endothelial growth factor receptor, raf kinase, src kinase, mammalian target of rapamycin, histone deacetylase, proteasome, and mesothelin (van Meerbeeck et al, 2011). Unfortunately, compared to other cancers, there is no effective molecular target for mesothelioma to date. Thus, exploration for additional novel drug targets for mesothelioma is urgently needed. It is now becoming evident that aberration of the Hh pathway may be involved in the formation and maintenance of many cancers (Amakye et al, 2013). More recently, it has been demonstrated that the Hh pathway is deregulated in mesothelioma (Li et al, 2013; Shi et al, 2012). The data presented in Chapter 3 suggest that mesothelioma is not a cancer that is driven by mutations in the Hh pathway, even though mutations and amplifications of Hh pathway genes are a feature of many Hh-dependent cancers, including BCC, medulloblastoma and rhabdomyosarcoma (Teglund & Toftgard, 2010). The activating mechanism in mesothelioma is at present unclear. However, it has been suggested that overexpression of GLI1 and GLI2 mediated through SHH/DHH-induced activation of autocrine Hh signaling may play a critical role in the tumorigenesis and/or 135

maintenance of mesothelioma (Shi et al, 2012). The central role played by the Hh pathway in the formation and maintenance of multiple human cancers makes Hh pathway inhibitors an increasingly important drug target class. Since the discovery of the prototypical SMO inhibitor cyclopamine, many SMO inhibitors with diverse chemical scaffolds have been developed and are being tested in clinical trials at various phases involving patients with a wide range of cancers (Amakye et al, 2013). Notably, the first-in-human, first-in-class Hh pathway inhibitor is GDC-0449 (vismodegib; Erivedge), discovered by Genentech, Inc. in a high-throughput screen in 2004, was granted approval by the U.S. Food and Drug Administration on 30 January 2012. Similar to cyclopamine, vismodegib inhibits the Hh pathway by binding to the SMO transmembrane protein (Chen et al, 2002a; Chen et al, 2002b; Robarge et al, 2009) and is indicated for patients with metastatic BCC or for those with locally advanced BCC who are not candidates for surgery or radiotherapy (FDA, 2012). In the landmark international, single-arm, multi-centre, two-cohort, non-randomised Phase II ERIVANCE BCC/SHH4476g trial, 104 patients with metastatic or locally advanced BCC received daily oral dosing of 150 mg vismodegib until tumor progression or unacceptable toxicities were reported (Sekulic et al, 2012). Overall, the independently assessed objective response rate was 30% and 43% for patients with metastatic and locally advanced BCC, respectively (Sekulic et al, 2012). Aside from BCC, vismodegib, given alone or in combination with other chemotherapeutic drugs, is currently being trialled for advanced medulloblastoma, multiple myeloma, pancreatic, breast, colorectal, ovarian and gastric cancers (Amakye et al, 2013). Despite these encouraging trial results, there have been reports of emergence of acquired resistance to GDC-0449 in a medulloblastoma patient (Rudin et al, 2009; Yauch et al, 2009) and failure of GDC- 0449 phase II trials in patients with colorectal and ovarian cancers (Berlin et al, 2013; Kaye et al, 2012). Because Gli transcription factors are not solely activated by the 136

canonical Hh pathway but also by other signaling pathways that are downstream of SMO such as Ras, c-myc, Akt and TGFβ (Lauth & Toftgard, 2007), targeting Gli transcription factors might provide an attractive way for treating cancers, irrespective of the signaling pathways that activate Gli signaling. Targeting the Hh pathway either through RNA-interference knockdown of GLI1 and GLI2 or using Gli inhibitors, has been shown to induce growth inhibition and cell death in mesothelioma cells and xenograft tumors in vivo (Li et al, 2013; Shi et al, 2012; You et al, 2013; Zhang et al, 2013). A particularly promising anticancer agent GANT61, with Gli inhibitory activity, displayed potent cytotoxic activity against diverse human cancer types (Mazumdar et al, 2011b; Nagao et al, 2011; Pan et al, 2012; Tostar et al, 2010; Wickstrom et al, 2013; You et al, 2013). There is evidence that GANT61 appears to have a novel anticancer mechanism that differs from other Hh antagonists. Recent studies have reported that GANT61 triggers apoptosis via induction of DNA double strand breaks and activation of ATM-Chk2 DNA damage response in colorectal cancer cells (Mazumdar et al, 2011a). Two recent studies have attempted to block SMO using cyclopamine in human mesothelioma cells but only achieved cytotoxicity at high doses (> 10 µm) (Li et al, 2013; Zhang et al, 2013), which is likely to be off-target effects of the drug. In the present study, using a panel of mesothelioma cell lines, GANT61 induced greater antiproliferative effects compared to the SMO antagonists cyclopamine and GDC-0449, which suggests that Hh activation in mesothelioma is likely to be independent of SMO. A similar effect has been observed for human neuroblastoma and colorectal cancer cell models, whereby targeting the Gli transcription factors exhibited a higher level of cytotoxicity (Mazumdar et al, 2011b; Wickstrom et al, 2013). Furthermore, GANT61- induced anti-proliferative effects were found to be related to the inhibition of cell 137

growth, as confirmed by reduction of clonogenic survival and induction of G1 cell cycle arrest. The data presented also showed that GANT61 induced apoptotic cell death in LO68 cells in a concentration- and time-dependent manner. Similar to the cell growth inhibitory data, however, the level of apoptotic cells in cyclopamine-treated cells was only evident at high doses. On the other hand, treatment of mesothelioma cells with the more specific and potent SMO inhibitor GDC-0449 did not result in an increase in the level of apoptosis, which further strengthens the argument that cyclopamine is a nonspecific SMO inhibitor at doses above 1 µm (Romer et al, 2004; Yauch et al, ). Such off-target effects of cyclopamine are not unique to mesothelioma cells. In fact there have been reports of SMO-independent mechanisms of cytotoxicity in human medulloblastoma and breast cancer cells (Meyers-Needham et al, 2012; Zhang et al, 2009a). With regard to mechanisms of action, Meyers-Needham and colleagues have demonstrated that exposure of Daoy human medulloblastoma cells to cyclopamine caused a marked increase in apoptosis relative to vehicle-treated cells that was mediated by nitric oxide-dependent induction of neutral sphingomyelinase 2/ceramide and is independent of Hh/Gli signaling (Meyers-Needham et al, 2012). These findings raise important questions regarding the role that SMO may play in the pathogenesis of mesothelioma, and also pose a serious question whether SMO inhibitors such as GDC- 0449, although efficacious against BCC and medulloblastoma, can be used more widely in the treatment of patients with other types of cancers. Data presented in this chapter show that GANT61 specifically acts on the Hh-Gli pathway, as demonstrated by a reduction in the expression levels of downstream pathway effectors GLI1 and GLI2 and Gli target gene PTCH1. GANT61 also significantly 138

decreased the Gli-luciferase reporter activity in a dose-dependent manner. These results are consistent with previous reports in HEK294 cells transiently overexpressing GLI1, and colon cancer cells with constitutively active Hh signaling (Lauth et al, 2007; Mazumdar et al, 2011b). Additionally, a positive correlation between the log IC 50 values of GANT61 and GLI1 and GLI2 mrna expression was found. The JU77 cell line, expressing the lowest levels of GLI1 and PTCH1 was the most sensitive to GANT61 treatment whereas the NO36 cell line, expressing high levels of GLI1, GLI2 and PTCH1, was the least sensitive. In addition, sirna-mediated depletion of GLI1 and GLI2 significantly reduced mesothelioma cell proliferation. This data corroborate previous findings that GANT61 inhibits Hh signaling at the level of Gli transcription factors. However, silencing GLI1 and GLI2, individually or together, were not sufficient to induce cell death in LO68 cells, strongly suggesting that GANT61-induced apoptosis was not associated with Gli inhibition. These data are in contrast to previous sirna experiments, where silencing of GLI1 resulted in increased apoptosis and reduced level of antiapoptotic Bcl-2 in HCC, glioma and breast cancer cells (Chen et al, ; Thomas et al, 2011; Wang et al, 2010). Thus, it is possible that GANT61-induced apoptosis in mesothelioma cells may be initiated by another factor other than Gli inhibition. An alternative explanation for caspase- and Gli-independent induction of apoptosis by GANT61 is production of ROS. Previous reports have shown that certain chemotherapeutic drugs can induce apoptosis by the production of ROS and this is independent of caspase activation (Rosato et al, 2003; Ruefli et al, 2001). The present studies showed that ROS were generated concomitantly with apoptosis in a dose- and time-dependent manner in LO68 cells upon treatment of cells with GANT61. An intriguing observation, similar to GANT61-induced apoptosis, is that Gli silencing by 139

sirna showed that although GLI1 and GLI2 were downregulated in LO68 cells, they did not appear to be involved in induction of ROS generation. Furthermore, the apoptogenic role of ROS production was supported by the ability of two general antioxidants, NAC and GSH, to rescue cells from GANT61-induced apoptosis. Despite the encouraging in vitro data on GANT61 in several cancer cell types (Fu et al, 2013; Mazumdar et al, 2011b; Pan et al, 2012; Wickstrom et al, 2013; You et al, 2013), the proof of preclinical efficacy in animal models remains lacking. So far, there have been two reports on the in vivo efficacy of GANT61 (Lauth et al, 2007; Wickstrom et al, 2013). In the first study, Lauth and colleagues showed that GANT61 induced significant tumor regression in a human tumor xenograft model of prostate cancer (Lauth et al, 2007). However, GANT61 was administered subcutaneously proximal to the tumor (Lauth et al, 2007), which is not feasible in the clinic setting for prostate cancer. The other study by Wickstrom and colleagues reported tumor growth delay but not regression after oral administration of GANT61 in a human tumor xenograft model of neuroblastoma (Wickstrom et al, 2013). This route of administration, in contrast, is clinically relevant in the treatment of neuroblastoma. Thus, GANT61 might benefit from being combined with standard chemotherapeutic agents used in treatment of mesothelioma. This strategy is also supported by the observation that chemotherapeutic drugs demonstrate most activity in mesothelioma patients when given as a cocktail containing cisplatin plus pemetrexed or gemcitabine compared with single agents alone and how anticancer drugs invariably lose efficacy in the clinic due to the emergence of resistance (Al- Lazikani et al, 2012; Chabner & Roberts, 2005; Ellis et al, 2006). In this study, by using the CompuSyn program based on the median effect equation of Chou and the combination index equation of Chou-Talalay (Chou, 2006), the 140

quantitation of additivity, synergism or antagonism between GANT61 and three standard mesothelioma chemotherapeutic drugs yielded interesting results. The majority of mesothelioma patients receive a first-line combination regimen that comprises a platinum-based agent (cisplatin or carboplatin) and pemetrexed or gemcitabine (Tsao et al, 2009). The combination effect of GANT61 was first investigated with cisplatin, since this agent is a widely used first-line treatment of mesothelioma, and GANT61 was recently found to be active against this cancer in an in vitro study (You et al, 2013). Cisplatin, a small molecule platinum compound, is the standard of care for first-line chemotherapy for patients with various cancer types, including cervix, ovary, head and neck and testes (Prestayko et al, 1979). Cisplatin exerts its cytotoxic effects by forming intra-strand crosslinks in DNA (Zwelling et al, 1979). Several studies have shown that enhanced repair of these platinum-dna lesions is the major mechanism for acquired cisplatin resistance in cancer cells (Bedford et al, 1988; Eastman & Schulte, 1988; Lai et al, 1988; Parker et al, 1991). In this study, GANT61 was found to interact synergistically with cisplatin in 3 out of 4 dose regimens tested. However, the GANT61-cisplatin combination has been shown to have an additive effect in neuroblastoma in vitro. It is important to test this combination in five or more mesothelioma cell lines to rule out that the synergistic effect observed in this study is cell line-specific. Moreover, the results showed that when combined with cisplatin, GANT61 produced superior cytotoxic activity compared to the single agents alone. Such enhancement in therapeutic efficacy could be related to the ability of GANT61 to block GLI1-mediated c-jun activation and expression of three vital DNA repair genes ERCC1, XPD and XRCC1 in response to cisplatin treatment, which ultimately leads to reduced repair of DNA damage induced by cisplatin and GANT61 (Kudo et al, 2012; Mazumdar et al, 2011a). 141

Gemcitabine, an antifolate, in combination with cisplatin, has been shown to be an effective treatment option for patients with mesothelioma. Six Phase II trials to date have reported objective response rates of 16 50 % and a median overall survival of 10 41 months (Byrne et al, 1999; Castagneto et al, 2005; Kalmadi et al, ; Kovac et al, 2012; Nowak et al, 2002; van Haarst et al, 2002). This drug regimen is generally well tolerated, with hematologic and gastroenterologic toxicities being the most common severe adverse effects (Byrne et al, 1999; Castagneto et al, 2005; Kalmadi et al, ; Kovac et al, 2012; Nowak et al, 2002; van Haarst et al, 2002). In this study, when combined with GANT61, gemcitabine produced superior cytotoxic activity over singleagent alone. In fact, the GANT61-gemcitabine combination is the most synergistic out of the three combination regimens tested in this study. The extremely strong synergistic interaction between GANT61 and gemcitabine could be attributed to the ability of GANT61 to target cancer cells that express multidrug transporters. Previous reports found that GANT61 can inhibit the expression of two particular multidrug transporters p-glycoprotein and breast cancer resistance protein (Das et al, 2013; Tang et al, 2012) and thus reverse chemoresistance (Queiroz et al, 2010). P-glycoprotein has been shown to be overexpressed in many human cancers, including cancers of the brain, colon, kidney and liver as well as hematologic malignancies such leukemia, lymphomas and multiple myeloma (Goldstein et al, 1989). It was thus no surprise when mesothelioma was found to express p-glycoprotein (Ramael et al, 1992). Notably, sirna-mediated knockdown of p-glycoprotein has been shown to restore gemcitabine sensitivity in gemcitabine-resistant pancreatic cancer cells (Song et al, 2013a). Together, this suggests that increased gemcitabine sensitivity may be related, at least in part, to GANT61- mediated reduced expression of p-glycoprotein. Pemetrexed is an antifolate inhibitor of thymidylate synthase, dihydrofolate reductase 142

and glycinamide ribonucleotide transferase that exerts its cytotoxic effects by blocking DNA synthesis and folate metabolism (Chattopadhyay et al, 2007; Hanauske et al, 2001). The combination of cisplatin plus pemetrexed was reported to show survival benefits over cisplatin alone in the largest multicenter, randomized, single-blind Phase III clinical trial conducted in mesothelioma patients (Vogelzang et al, 2003). This trial showed that the response rate with combination therapy (41.3%) was found to be superior to the cisplatin monotherapy control arm (16.7%) (Vogelzang et al, 2003). Thus, the combination of cisplatin plus pemetrexed is now recognized as the standard of care first-line chemotherapy for mesothelioma (Tsao et al, 2009). In this study, the combination of GANT61 and pemetrexed was highly antagonistic in all dose regimens tested. Although the reason for this strong antagonistic effect is not understood, the observed antagonism could be attributed to the modulation of SLC19A1 expression by GANT61. Based on data from microarray-based cdna expression profiles, SLC19A1 expression was found to be significantly downregulated in human colon carcinoma cells treated with GANT61 compared to vehicle-treated cells (Shi et al, 2010). SLC19A1 encodes the reduced folate carrier (RFC) that transports reduced folates and antifolate chemotherapeutic agents such as pemetrexed (Matherly et al, 2007). Previous studies suggest that reduced expression or mutation of RFC in cancer cells results in defective transport of antifolates and are major mechanisms of acquired resistance to pemetrexed and other antifolates (Gorlick et al, 1997; Wang et al, 2003). Further study into this drug combination might be warranted as it has clinical implications for antifolate class of chemotherapeutic agents in general. In a highly cited review article, Shintani and Klionsky were the first to describe autophagy as a double-edged sword, referring to the role played by autophagy in both tumor formation and suppression, depending on the cellular context (Shintani & 143

Klionsky, 2004). Although much work have been done on elucidating the mechanistic action of GANT61 in the induction of apoptosis (Agyeman et al, 2012; Mazumdar et al, 2011a; Mazumdar et al, 2011b), very little is known about the role played by autophagy in GANT61-induced cell death. Recent studies showed that beyond apoptotic cell death, GANT61 could also induce autophagy-dependent cell death in both hepatocellular carcinoma and pancreatic ductal adenocarcinoma cells (Wang et al, 2013b; Xu et al, 2014). This contrasts to my data in mesothelioma LO68 cells based on studies of inhibitors of autophagy, which suggested cytoprotective effects of autophagy in GANT61-induced cell death. In this regard, my data are consistent with a recent study by Battisti and colleagues showing that autophagy is highly activated in mesothelioma cells and is important for cell survival under nutrient-deficient conditions (Battisti et al, 2012). In this thesis, it has been clearly demonstrated that mesothelioma cells treated with GANT61 underwent apoptosis by dual staining with annexin V and 7AAD. On the other hand, FACS analysis showed that GANT61 induced dose-dependent cytoprotective autophagy in mesothelioma cells that is characterized by autophagosome formation as detected by acridine orange staining, which was confirmed by Cyto-ID Green staining and confocal imaging. Further evidence that autophagy protects mesothelioma cells from the apoptogenic effects of GANT61 was the observation that BafA1, which inhibits autophagy by inhibiting autophagosomes maturation through blocking vacuolar H + ATPase-mediated acidification of autophagosome (Yamamoto et al, 1998), rescued cells from GANT61-induced apoptosis. After exposure to a combination of sublethal doses of GANT61 and bafilomycin A1, autophagy was inhibited and higher levels of apoptosis occurred. Finally, similar results were obtained with two other autophagy inhibitors, chloroquine, which acts by causing an elevation in lysosomal ph and in turn inhibits fusion of autophagosome and lysosome (Iwai-Kanai et al, ), and 3-methyladenine, 144

which acts via inhibition of class III phosphoinositide 3-kinase (PI3K) that plays a critical role in the biogenesis of autophagosomes (Petiot et al, 2000; Seglen & Gordon, 1982). The findings of this chapter, implicating autophagy as a cytoprotective mechanism during the induction of the cytotoxic effects of GANT61 on mesothelioma cells, raise the possibility that drugs that block autophagy could act as sensitizers of mesothelioma cells to the anticancer effects of GANT61. Importantly, already-approved drugs such as chloroquine could be repurposed and used in combination with GANT61 as a novel anti-mesothelioma therapy. 145

CHAPTER 5 MITOCHONDRIA-DERIVED ROS ARE CRITICAL IN GANT61-INDUCED APOPTOSIS 146

5.1 INTRODUCTION Reactive oxygen species (ROS) are traditionally viewed as toxic molecules that could potentially damage cellular DNA, proteins and lipids (Fang et al, 2009). However, relatively recent findings point out that ROS could also serve important physiological signaling functions (Sena & Chandel, 2012). ROS is produced in several subcellular compartments, with the majority generated via an enzymatic pathway by NADPH oxidase or via a nonenzymatic process through the mitochondrial electron transport chain (Turrens, 2003). Generation of ROS in response to chemotherapy and radiation therapy has been reported to induce cell cycle arrest and/or cell death in various cancer models (Wondrak, 2009). In Chapter 4, I demonstrated for the first time that GANT61 induces ROS generation. At present, however, it is unclear whether GANT61 could have effects that are independent of Hh/Gli signaling. Therefore, I sought to examine the role of ROS in mediating GANT61-induced apoptosis and elucidate whether this occurs via Hh/Gli signaling. 147

5.2 RESULTS 5.2.1. ROS production does not appear to be a class phenomenon for Gli inhibitors In Chapter 4, the data showed that GANT61 could trigger the production of ROS in cancer cells. However, it is unclear whether the induction of ROS production is a feature of this class of Gli inhibitors. To determine whether ROS generation is a class phenomenon for Gli inhibitors, the effects of exposing LO68 cells to another two Gli inhibitors, Hh pathway inhibitor-1 (HPI-1) and arsenic trioxide (As 2 O 3 ) were examined. HPI-1 has been described to reduce SHH-induced accumulation of SMO in the primary cilium and block the increase in GLI2 full length/repressor ratio upon SHH stimulation (Hyman et al, 2009). As 2 O 3 inhibits Gli transcriptional activity and in turn inhibits transcription of Hh target genes by blocking ciliary accumulation of GLI2 as well as reducing GLI2 protein levels (Kim et al, 2010b). As shown in Figure 5.1, ROS levels were increased in LO68 cells after treatment with 20 µm HPI-1 but not with 20 µm As 2 O 3. To confirm that these two Gli inhibitors mediate their effects through inhibition of Hh signal transduction, the transcript levels of GLI1 were measured by qpcr in LO68 cells following treatment. As expected, there was a marked decrease in GLI1 expression (Figure 5.2). Taken together, these results suggest that ROS production does not appear to be a class phenomenon for Gli inhibitors of different chemical structures. 148

Mean fluorescence Intensity 60 50 40 30 20 10 0 * Vehicle 20µM HPI-1 20µM As 2 O 3 Figure 5.1 ROS generation is not a class phenomenon for Gli inhibitors. Intracellular ROS levels were quantified in LO68 cells treated with vehicle and Gli inhibitors HPI-1 (20 µm) or As 2 O 3 (20 µm) for 48 h. Data represent the mean ± SEM of three independent experiments. *, p < 0.05, compared to vehicle-treated cells. 149

Fold change in gene expression 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 *** Vehicle 20µM HPI-1 20µM As 2 O 3 Figure 5.2 Expression of GLI1 mrna in LO68 cells treated with HPI-1 or As 2 O 3. Data are presented as fold change in gene expression relative to vehicle-treated cells and normalized to PGK1. Error bars represent the mean ± SEM of three independent experiments each performed in duplicate. ***, p < 0.001, compared to vehicle-treated cells. * 150

5.2.2. GANT61 kills LO68 cells through ROS-mediated DNA damage To further investigate whether GANT61-induced ROS generation is linked to DNA damage and subsequent apoptotic cell death in response to treatment with GANT61, LO68 cells were exposed to 10-20 µm GANT61 for 48h, and the level of 8-oxoguanine, a biomarker of oxidative DNA damage (Helbock et al, 1999) was monitored using a FITCconjugated binding protein that has strong affinity for 8-oxoguanine in DNA. As shown in Figure 5.3, exposure of LO68 cells to GANT61 resulted in increased levels of 8- oxoguanine in DNA of treated cells. Notably, the radical scavenger NAC blocked the accumulation of 8-oxoguanine following exposure to GANT61, indicating that oxidative DNA damage plays a critical role in GANT61 cytotoxicity (Figure 5.3). 5.2.3. GANT61 kills LO68 cells through ROS-mediated impairment of DNA repair GANT61-mediated apoptosis was then examined to determine if it is attributed to impaired DNA repair, as a result of decreased expression of DNA repair-related genes. qpcr analysis revealed that BRCA1, EXO1, RAD51, UNG and XRCC3, genes involved in DNA damage response and repair pathway (Ronen & Glickman, 2001), were downregulated upon GANT61 treatment (Figure 5.4). Interestingly, GANT61-mediated downregulation of these five DNA repair-related genes were abolished by pretreatment of cells with NAC, suggesting that GANT61-induced ROS is responsible for suppression of expression of those genes (Figure 5.4). 151

Vehicle 20mM NAC 20µM GANT61 NAC + GANT61 Counts Mean Fluorescence intensity Figure 5.3 Oxidative DNA damage was assessed by OxyDNA assay. Cells were pretreated with NAC for 1 h, then with 20 µm GANT61 or vehicle for 48 h. The cells were stained with the FITC-protein conjugate and fluorescence measured by flow cytometry. Data are presented as histogram profiles of vehicle-, GANT61- and GANT61+NAC-treated cells that were measured by flow cytometry upon FITCprotein conjugate binding. The data shown represent one of three independent 152

1.2 BRCA1 RAD51 1.0 0.8 0.6 XRCC3 EXO1 UNG 0.4 0.2 Figure 5.4 Analyses of genes involved in DNA repair (BRCA1, EXO1, RAD51, UNG and XRCC3) by qrt-pcr. LO68 cells were treated with 20 µm GANT61 for 48h. Data are presented as fold change in gene expression relative to vehicle-treated cells and normalized to PGK1. Error bars represent the mean ± SEM of three independent experiments each performed in duplicate. ***, p < 0.001, compared to GANT61-treated cells. 153 Vehicle 20µM GANT61 0.0 NAC+GANT61 Fold change in gene expression

5.2.4. GANT61 downregulates GLI1, GLI2 and PTCH1 through ROS The effect of NAC on GANT61-mediated GLI1, GLI2 and PTCH1 expression was examined to determine if it is attributed to ROS-mediated downregulation, as a result of ROS generation induced by GANT61. As shown in Figure 5.5, the blockade of ROS accumulation by pretreating cells with NAC, prevented reduction of GLI1, GLI2 and PTCH1 mrna levels indicating the involvement of ROS in modulation of the Hh pathway. To determine if ROS could impact on the Hh pathway, the effect of exposing LO68 cells to menadione, a ROS generator, and hydrogen peroxide (H 2 O 2 ), a mimic of oxidative stress, on the Hh pathway was examined. FACS analysis of intracellular ROS production indicated that exposure of LO68 cells to menadione and H 2 O 2 resulted in a significant increase in ROS production as measured by the fluorescent CH 2 DCFDA probe (Figure 5.6A and B). Furthermore, qpcr analysis of gene expression in LO68 cells following treatment clearly indicated the ability of menadione and H 2 O 2 to downregulate the expression of GLI1, a marker of Hh pathway activity (Figure 5.7A and B). Together, these findings suggest that ROS plays a critical role in the suppression of GLI1, GLI2 and PTCH1 expression by GANT61. 5.2.5. GANT61-induced ROS is independent of Gli inhibition Because GANT61-induced apoptosis and cell cycle arrest are independent of Gli inhibition, it might be expected that the ROS generation observed after GANT61 administration occur independently of Gli signaling. Indeed, depletion of GLI1 and GLI2, individually or together using sirnas did not result in ROS generation in LO68 cells (Figure 5.8). 154

1.8 GLI1 1.6 1.4 *** *** GLI2 PTCH1 1.2 1.0 0.8 0.6 0.4 0.2 0.0 *** Vehicle 20µM GANT61 NAC+GANT61 Figure 5.5 Analyses of Hh pathway genes (GLI1, GLI2 and PTCH1) by qrt-pcr. LO68 cells were pretreated with NAC for 1 h and then treated with 20 µm GANT61 or vehicle for 48 h. Data are presented as fold change in gene expression relative to vehicle-treated cells and normalized to PGK1. Error bars represent the mean ± SEM of three independent experiments each performed in duplicate. ***, p < 0.001, compared to GANT61-treated cells. 155 Fold change in gene expression

A Mean Fluorescence Intensity 70 60 50 40 30 20 10 0 ** Vehicle 350µM H 2 O 2 B Mean Fluorescence Intensity 50 40 30 20 10 0 Vehicle *** 30µM Menadione Figure 5.6 LO68 cells were treated with vehicle or (A) 350 µm H 2 O 2 or (B) 30 µm menadione for 24 h. The cells were stained with CH 2 DCFDA and the fluorescence measured by flow cytometry. Bar graph represents the increase in mean fluorescence intensity of positive cells measured in each experimental condition. Values represent the mean ± SEM of three independent experiments. **, p < 0.01 or ***, p < 0.001, compared to vehicle-treated cells. 156

A B Fold change in gene expression 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Vehicle 350µM H 2 O 2 * Fold change in gene expression 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Vehicle * 30µM Menadione Figure 5.7 Analysis of GLI1 mrna expression by qrt-pcr. LO68 cells were treated with vehicle or (A) 350 µm H 2 O 2 or (B) 30 µm menadione for 24 h. Data are presented as fold change in gene expression relative to vehicle-treated cells and normalized to PGK1. Error bars represent the mean ± SEM of three independent experiments each performed in duplicates. *, p < 0.05, compared to vehicle-treated cells. 157

Mean fluorescence intensity 40 30 20 10 0 sinc sigli1 sigli2 sigli1+sigli2 Figure 5.8 The level of intracellular ROS was monitored using CH 2 DCFDA and the fluorescence measured by flow cytometry. Data represent the mean ± SEM of three independent experiments. 158

5.2.6. GANT61-induced apoptosis and ROS production are dependent on NADPH oxidase NADPH oxidase (NOX) is an important site of ROS generation within cells. To determine the site of ROS production in response to GANT61, LO68 cells were treated with GANT61 in the absence or presence of diphenylene iodonium (DPI), a NOX inhibitor, and the effects on GANT61-induced ROS generation and apoptosis assessed. As shown in Figure 5.9, GANT61-induced ROS production and apoptosis were significantly blocked by the addition of DPI, indicating that NOX is one of the sites of ROS generation. Because the blockade of GANT61-induced ROS production and apoptosis by DPI was partial, this suggests there could be other sites of ROS production. 159

A B Mean Fluorescence Intensity 100 80 60 40 20 0 Vehicle 20µM GANT61 ** 100nM DPI+GANT61 100 % Annexin V+ cells 80 60 40 20 *** 0 Vehicle 20µM GANT61 100nM DPI+GANT61 Figure 5.9 LO68 cells were pretreated with 100 nm DPI for 1 h, then with 20 µm GANT61 or vehicle for 48 h. (A) The cells were stained with CH 2 DCFDA and the fluorescence measured by flow cytometry. (B) Apoptosis (as assessed by the annexin V/7AAD assay) was quantified. Bar graphs show the mean ± SEM of three independent experiments. **, p < 0.01 or ***, p < 0.001, compared to GANT61-treated cells. 160

5.2.7. GANT61-induced apoptosis and ROS production is dependent on mitochondria The mitochondrion is a major site of ROS generation in mammalian cells (Sena & Chandel, 2012). To determine the site of ROS production in response to GANT61, LO68 cells were treated with GANT61 in the absence or presence of rotenone, a mitochondrial complex I inhibitor, and the effects on GANT61-induced ROS generation and apoptosis were assessed. GANT61-induced ROS production (Figure 5.10A) and apoptosis (Figure 5.10B) were significantly blocked by the addition of rotenone, indicating that the ROS produced in response to GANT61 was of mitochondrial origin. The addition of a mitochondria-targeted antioxidant, MitoTEMPO, also blocked the increase in apoptosis induced by GANT61 (Figure 5.11). To further confirm the mitochondrial origin of ROS, the level of superoxide within mitochondria after exposure to GANT61 was measured using the fluorescent probe mitosox red. As shown in Figure 5.12, exposure to 20 µm GANT61 for 24 h induced an increase in intramitochondrial superoxide levels. In addition, this increase in intramitochondrial superoxide was attenuated by pretreating cells for 1 h with 20 mm NAC before co-treating with 20 µm GANT61 (Figure 5.12). 5.2.8. Dissipation of mitochondrial membrane potential mediates GANT61-induced apoptosis Studies have shown that there is a tight connection between ROS and collapse of the ΔΨ m (Kroemer & Reed, 2000; Zamzami et al, 1995). It has been suggested that the dissipation of ΔΨ m involves the activation of mitochondrial permeability transition (MPT) caused by opening of a large conductance channel in the inner mitochondrial membrane, known as the MPT pore (Crompton, 1999). ΔΨ m was first examined as an indicator of mitochondrial integrity and function, by staining cells with JC-1 dye, which accumulates 161

A Mean fluorescence intensity 100 80 60 40 20 0 Vehicle 20µM GANT61 *** 200nM ROT+GANT61 B 80 % Annexin V+ cells 70 60 50 40 30 20 10 *** 0 Vehicle 20µM GANT61 200nM ROT+GANT61 Figure 5.10 LO68 cells were pretreated with 200 nm rotenone (ROT) for 1 h, then with 20 µm GANT61 or vehicle for 48 h. (A) The cells were stained with CH 2 DCFDA and the fluorescence measured by flow cytometry. (B) Apoptosis (as assessed by the annexin V/7AAD assay) was quantified. Bar graphs show the mean ± SEM of three independent experiments. ***, p < 0.001, compared to vehicle-treated cells. 162

% Annexin V+ cells 80 70 60 50 40 30 20 10 0 Vehicle 20µM GANT61 *** 100µM MT+GANT61 Figure 5.11 Mitochondria-targeted superoxide dismutase mimetic mitotempo attenuates GANT61-induced apoptosis. LO68 cells were pretreated with mitotempo (100 µm) for 1 h and further incubated with 20 µm GANT61 for 48 h. Apoptosis was measured by annexin- V/7AAD assay. Data represent the mean ± SEM of three independent experiments. ***, p < 0.001, compared to GANT61-treated cells. 163

A DMSO 20µM GANT61 GANT61+NAC Counts B MitoSOX Red Mean fluorescence intensity 90 80 70 60 50 40 30 20 10 0 Vehicle 20µM GANT61 *** NAC+GANT61 Figure 5.12 GANT61 induces mitochondrial superoxide production in LO68 cells. Cells were pretreated with 20 mm NAC for 1 h and further treated with 20 µm GANT61 or vehicle for 48 h before subjected to mitosox red flow cytometric analysis. A. Histogram profiles of GANT61- and vehicle-treated cells measured by flow cytometry upon mitosox red staining. B. Bar graph represents the increase in the mean fluorescence intensity of mitosox red-positive cells measured in each experimental condition. Data represent the mean ± SEM of three independent experiments. ***, p < 0.001, compared to GANT61-treated cells. 164

within mitochondria and yields red fluorescence. A significant reduction of ΔΨ m was observed in LO68 cells treated with GANT61 (Figure 5.13). When the MPT pore opening was blocked using cyclosporine A (CsA), which in turn preserved mitochondrial integrity and function, the dissipation of ΔΨ m was significantly blocked (Figure 5.13). The proton ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) served as a positive control that caused depolarization of the ΔΨ m (Figure 5.13). This result shows that the loss of mitochondrial integrity and function plays an important role in ROS generation. In addition, pretreatment with CsA also rescued cells from GANT61-induced apoptosis by 50% relative to LO68 cells treated with 20 µm GANT61 (Figure 5.14). Finally, the addition of NAC prior to GANT61 exposure prevented the loss of ΔΨ m, suggesting that GANT61-induced ROS is critical for the induction of MPT due to increased MPT pore opening (Figure 5.13). CsA was used to further investigate if the MPT pore is involved in GANT61-induced superoxide production. As shown in Figure 5.15, pre-treatment of cells with CsA before exposure to GANT61 resulted in a significant decrease in mitosox red fluorescence, suggesting that MPT pore opening was necessary for mitochondrial superoxide production following GANT61 treatment. I similarly found that GANT61- treated cells had higher levels of intracellular ROS, as determined using the ROSsensitive fluorophore CH 2 DCFDA, which was reduced by addition of CsA (Figure 5.16). Taken together, these results suggest that mitochondria are responsible for ROS production after GANT61 treatment. 165

Red/Green fluorescence ratio 2.5 2.0 1.5 1.0 0.5 0.0 Vehicle 20µM GANT61 *** * NAC+GANT61 CsA+GANT61 CCCP Figure 5.13 NAC and CsA prevent loss of mitochondrial membrane potential. Cells were pretreated with 20 mm NAC or 5 µm CsA and then after 1 h, 20 µm GANT61 or vehicle was added for 48 h. CCCP served as a positive control for disruption of mitochondrial membrane potential. The cells were then stained with the fluorescent probe JC-1, and fluorescence measured by flow cytometry. Bar graph represents the change in the mean fluorescence intensity of JC-1-positive cells measured in each experimental condition. Data represent the mean ± SEM of three independent experiments. *, p < 0.05 or ***, p < 0.001, compared to GANT61-treated cells. 166

Mean Fluorescence Intensity 100 90 80 70 60 50 40 30 20 10 0 *** Vehicle 20µM GANT61 CsA+GANT61 Figure 5.14 CsA inhibits GANT61-induced mitochondrial superoxide production in LO68 cells. Cells were incubated with 5 µm CsA for 1 h and then, 20 µm GANT61 added for 48 h. The cells were then stained with mitosox red and the fluorescence was measured by flow cytometry. Bar graph represents the changes in the mean fluorescence intensity of mitosox red-positive cells measured in each experimental condition. Data represent the mean ± SEM of three independent experiments. ***, p < 0.001, compared to GANT61-treated cells. 167

% Annexin V+ cells 80 70 60 50 40 30 20 10 0 *** Vehicle 20µM GANT61 CsA+GANT61 Figure 5.15 CsA inhibits GANT61-induced apoptosis in LO68 cells. Cells were treated with 5 µm CsA then 20 µm GANT61 was added after 1 h for a further 48 h. Apoptosis was then measured by annexin V/7AAD assay. Data represent the mean ± SEM of three independent experiments. ***, p < 0.001, compared to GANT61-treated cells. 168

Mean fluorescence intensity 100 90 80 70 60 50 40 30 20 10 0 Vehicle 20µM GANT61 * CsA + GANT61 Figure 5.16 CsA inhibits GANT61-induced intracellular ROS production in LO68 cells. Cells were treated with 5 µm CsA for 1 h and 20 µm GANT61 was then added for 48 h. The cells were stained with CH 2 DCFDA and the fluorescence was measured by flow cytometry. Bar graph represents the mean fluorescence intensity of CH 2 DCFDApositive cells measured in each experimental condition. Data represent the mean ± SEM of three independent experiments. *, p < 0.05, compared to GANT61-treated cells. 169

5.2.9. Mitochondrial superoxide is essential for GANT61-induced apoptosis To genetically confirm involvement of mitochondrial superoxide in GANT61-induced apoptosis, LO68 ρ 0 cells were generated, which lack mitochondrial DNA, by exposing cells to a low concentration of ethidium bromide. Mitochondrial DNA depletion was verified in LO68 ρ 0 cells by amplifying three mitochondria-encoded genes, cytochrome c oxidase subunit 1 (COX1), D-loop and NADH dehydrogenase 6 (ND6), by PCR. In addition, glyceraldehyde-3-phosphate dehydrogenase (GADPH) was amplified to serve as a control for nuclear-encoded genes. As shown in Figure 5.17, EtBr treatment resulted in a marked reduction of PCR products for COX1, D-loop and ND6. There was no difference in the levels of GAPDH PCR product between LO68 and LO68 ρ 0 cells, indicating that nuclear DNA was not depleted in the process of establishing LO68 ρ 0 cells (Figure 5.17). Microscopically, there was no significant morphological difference between LO68 and LO68 ρ 0 cells (data not shown). Next, LO68 and LO68 ρ 0 cells were treated with 10-20 µm GANT61 for 48 h before staining with mitosox red. In LO68 cells, exposure to GANT61 for 48 h resulted in an induction of mitochondrial superoxide formation. MitoSOX red oxidation was significantly reduced in LO68 ρ 0 cells, indicating that a functional respiratory chain is required for the GANT61 induction of superoxide formation (Figure 5.18). To determine whether functional mitochondrial respiratory chain is important for mediating GANT61-induced apoptosis, apoptosis in LO68 and LO68 ρ 0 cells exposed to 10-20 µm GANT61 for 48 h was compared. As shown in Figure 5.19 GANT61 increased apoptosis in a dose-dependent manner in LO68 cells as assessed by annexin V staining. In contrast, GANT61 induced negligible apoptosis in LO68 ρ 0 cells after exposure to GANT61. Furthermore, to exclude the possibility that LO68 ρ 0 cells were resistant to apoptosis because of their loss of a functional mitochondrial respiratory chain, LO68 and LO68 ρ 0 cells were treated with cisplatin 170

WT ρ 0 NTC COX1 D-Loop ND6 GAPDH Figure 5.17 Depletion of mtdna in LO68 cells. Total DNA was isolated from wild-type (WT) and mtdna-depleted (ρ 0 ) LO68 cells and 50 ng total DNA was subjected to PCR amplification using primers for the mtdna regions COX1, D- loop and ND6. The nuclear DNAencoded gene GAPDH was also amplified as control. The PCR products were visualized on 1.5% agarose gel stained with EtBr. M, molecular size marker. 171

Mean fluorescence intensity 100 80 60 40 20 0 WT p0 Vehicle 10µM 20µM GANT61 concentration Figure 5.18 Detection of mitochondrial superoxide on GANT61 treatment. WT or ρ 0 LO68 cells were treated with 10-20 µm GANT61 or vehicle for 48 h. The cells were then stained with mitosox red, and the fluorescence was measured by flow cytometry. Bar graph represents the increase in the mean fluorescence intensity of mitosox red-positive cells measured in each experimental condition. Data represent the mean ± SEM of three independent experiments. 172

% Annexin V+ cells 100 90 80 70 60 50 40 30 20 10 0 WT p0 Vehicle 10µM 20µM GANT61 concentration Figure 5.19 ρ 0 LO68 cells are resistant to GANT61-induced apoptosis. WT or ρ 0 LO68 cells were treated with 10-20 µm GANT61 for 48 h. Apoptosis was then measured by annexin- V/7AAD assay. Data represent the mean ± SEM of three independent experiments. 173

(10 and 20 µm), a standard chemotherapeutic drug known to induce apoptosis in LO68 cells, for 48 h, and apoptosis was assessed by annexin V staining. As shown in Figure 5.20 LO68 and LO68 ρ 0 cells undergo apoptosis when treated with cisplatin. Also there was no statistical difference in the level of apoptosis between LO68 and LO68 ρ 0 cells as demonstrated by annexin V assay (p > 0.05). 174

% Annexin V+ cells 70 60 50 40 30 20 10 0 WT p0 NS NS Vehicle 10µM 20µM Cisplatin concentration Figure 5.20 No apparent difference was observed in the sensitivity to apoptosis by cisplatin in WT or ρ 0 LO68 cells. Cells were treated with 10-20 µm Cisplatin for 48 h. Apoptosis was then measured by annexin- V/7AAD assay. Data represent the mean ± SEM of three independent experiments. NS, not significantly different from untreated control cells. 175

5.3. DISCUSSION Considerable improvements in cancer treatments have led to increased five-year relative survival rate for all cancers from 49% in 1975-1977 to 68% in 2002- (Society, 2013). However, mesothelioma remains an almost fatal cancer with rapid progression and limited treatment options that are mostly palliative and results in death within 12 months even with the best standard of care (Vogelzang et al, 2003). One way to improve patient outcome may be to explore the use of new agents as part of a multimodality approach to mesothelioma treatment, which includes surgery, radiotherapy, chemotherapy and targeted therapy. Recently, a number of laboratories have started exploring novel agents that are able to increase levels of intracellular ROS as treatment for mesothelioma. Studies show that targeting cancer cells through redox modulation might be a realistic approach to completely eradicate these cells (Gorrini et al, 2013). To date, taurolidine (Aceto et al, 2009), 3-bromopyruvate (Zhang et al, 2009c), citrate (Zhang et al, 2009b), ascorbate (Ranzato et al, 2011), selenite (Nilsonne et al, 2006), alphatocopheryl succinate (Stapelberg et al, 2005) and epigallocathechin-3-gallate (Satoh et al, 2013) have shown promise in mesothelioma. In reality, generation of ROS is a common phenomenon of many chemotherapeutic agents that form the armamentarium of modern cancer treatment. These include doxorubicin (Tsang et al, 2003), paclitaxel (Alexandre et al, 2006), pemetrexed (Buque et al, 2012) and cisplatin (Berndtsson et al, 2007) as well as a long list of molecularly targeted small molecules, for examples, bortezomib (Ling et al, 2003), sorafenib (Coriat et al, 2012) and vorinostat (Ruefli et al, 2001). In chapter 4, the data show that GANT61 increases the levels of ROS, as indicated by CH 2 DCFDA fluorescence, in both mesothelioma and colorectal cancer cells. Moreover, GANT61-induced ROS accumulation was abrogated by two antioxidants, NAC and GSH, confirming oxidative stress caused by GANT61. Finally, the results indicate that 176

apoptosis mediated by GANT61 is reduced in the presence of NAC, thereby pointing to the importance of ROS in GANT61-induced apoptosis. However, the origin and nature of ROS induced by GANT61 is unknown. NAC and GSH are two general antioxidants while CH 2 DCFDA is a general oxidative stress indicator, all of which provide no information on the identity or source of ROS. In this chapter, the data showed that ROS generation induced by Gli inhibitors does not appear to be a class phenomenon because this has been observed for only 2 out 3 Gli inhibitors (including GANT61) tested. Moreover, sirna knockdown of GLI1 and GLI2, individually or together, was not sufficient to induce ROS production. Therefore, the ability of GANT61 and HPI-1 to induce ROS was probably not a direct result of Gli inhibition. These results are consistent with the observation of the SMO-independent induction of nitric oxide production by cyclopamine in Daoy medulloblastoma cells (Meyers-Needham et al, 2012). Taken together, the induction of ROS production by Hh pathway inhibitors appear to be largely independent of Hh pathway blockade. The data presented suggest that GANT61 has off-target effects, which are mediated via ROS. So, what effect does ROS have on the Hh pathway? Mechanistic studies of GANT61 indicate this small molecule can induce DNA damage in colorectal cancer cells (Mazumdar et al, 2011a). The observation of an increased abundance of 8-oxoguanine in LO68 cells treated with GANT61 validated the finding of Mazumdar and colleagues (Mazumdar et al, 2011a). Furthermore, quenching of ROS using NAC reduced the level of bound 8-oxoguanine in cells, implying a link between GANT61-induced ROS and DNA damage. It is also possible that GANT61 induces DNA damage through the disruption of DNA repair machinery. This concept is supported by the finding that GANT61 suppressed the expression of DNA repair genes (BRCA1, EXO1, RAD51, UNG 177

and XRCC3) in GANT61-treated cells, whereas NAC coadministration significantly abolished the downregulation of these genes, which again implicated ROS in mediating the effect of GANT61. In addition to the five DNA repair genes, NAC pretreatment also abolished the downregulation of Hh pathway genes GLI1, GLI2 and PTCH1, suggesting that ROS could act as an upstream regulator of the Hh pathway. To better understand the significance of ROS production on the Hh pathway, exogenous H 2 O 2 and menadione were added to LO68 cells to mimic the increase in ROS induced by GANT61. The effects of ROS in cells have been studied extensively by exposing cells to well-known ROS species such as hydrogen peroxide or redox cycling drugs that generate ROS within cells such as menadione (Comporti, 1989). In this chapter, I have demonstrated that increasing intracellular ROS using exogenous H 2 O 2 or menadione significantly increased the production of ROS in cells in a manner similar to GANT61. Consistent with a potential role of ROS in Hh signaling, I also found significant reduction of GLI1 mrna expression in LO68 cells. Collectively, the data presented identify ROS as a novel upstream regulator of the Hh pathway. It is hard to determine the specific ROS species that are generated from GANT61 exposure in LO68 cells. It is likely that the superoxide produced in the mitochondria might play an important role in the induction of apoptosis. Approximately 1-2% of electrons can leak to oxygen to form superoxide in a reaction mediated mainly by complex I and III of the mitochondrial respiratory chain (Orrenius et al, 2007). To identify the source of ROS, rotenone, a complex I inhibitor, was able to reduce GANT61-induced ROS production and rescue LO68 cells from GANT61-induced apoptosis when it was added to cells prior to GANT61 exposure. This result implies that GANT61-induced ROS might come from mitochondria. 178

Mitochondrial DNA-depleted ρ cells lack a working respiratory chain and are not able to generate ATP and ROS within mitochondria (Chandel & Schumacker, 1999). The nonfunctional mitochondria of LO68 ρ cells would likely affect ROS production and in turn apoptosis after treatment with GANT61. Consistent with my hypothesis, LO68 ρ cells showed resistance to GANT61, and lower mitochondrial superoxide levels were also observed after GANT61 treatment. The resistance of LO68 ρ cells to GANT61 was not associated with a decreased susceptibility to apoptosis, as indicated by the equal sensitivity to cisplatin of wild-type and ρ cells. Overall, my results support the hypothesis that sensitivity to GANT61 stems from enhanced production of ROS from the mitochondrial respiratory chain. This study is the first to reveal the role of mitochondria in ROS production and apoptosis mediated by GANT61 in LO68 cells. Because the blockade of GANT61-induced apoptosis by rotenone was partial, there could also be other sites of ROS production. Another major site of ROS generation is plasma membrane NOX (Jiang et al, 2011). In this thesis, the NOX inhibitor DPI was found to attenuate the induction of ROS mediated by GANT61, implicating NOX as the other major site of ROS production in response to GANT61. In addition, the induction of apoptosis by GANT61 is inhibited by DPI, implicating NOX in GANT61-induced cell death. While DPI is a potent inhibitor of NOX, it also inhibits the iron-sulphur clusters of complex I of the mitochondrial electron transport chain (Majander et al, 1994). Further work including genetic knockdown of NOX in LO68 cells is necessary to clarify the contribution of NOX in GANT61-induced ROS production and apoptosis. An important event that precedes apoptosis is MPT, which is mediated by the opening of nonselective multiprotein complex pores in the inner membrane of the mitochondria. The 179

induction of MPT leads to ΔΨm depolarization, uncoupling of oxidative phosphorylation, release of mitochondrial solutes and pro-apoptotic factors and mitochondrial swelling (Lemasters et al, 1998). In LO68 cultures, GANT61 exposure caused collapse of the ΔΨm, which is the first report of GANT61-induced changes of the MPT in human mesothelioma cells. The ΔΨm depolarization caused by GANT61 is probably due to induction of the PTP, as CsA, a specific inhibitor of the PTP (Crompton et al, 1988), abolished the GANT61-elicited ΔΨm depolarization. Previous studies showed that ΔΨm depolarization that reflects opening of the MTP pore could be induced by the action of ROS (Vercesi et al, 1997), which was consistent with my findings, as NAC did prevent ΔΨm depolarization. Because CsA and NAC inhibited GANT61-induced ΔΨm depolarization, I examined whether these two compounds protected LO68 cells from GANT61-induced apoptosis. Both CsA and NAC inhibited GANT61-induced apoptosis, as evidenced by reduced annexin V staining, suggesting that MPT induction and ROS are major contributors to the cell death caused by GANT61. Overall, these data suggest that MPT and ROS are involved in the depolarization of the mitochondrial membrane and apoptosis induced by GANT61. The oxidative capacity of GANT61 shown in this thesis is probably attributed to its chemical structure: the presence of dimethylaniline (Figure 5.21). It has been reported that dimethylaniline has an affinity to react with molecular oxygen to generate hydrogen peroxide (Graham & Mesrobian, 1963). Thus, such chemical configuration gives GANT61 a similar affinity to react with molecular oxygen. Furthermore, a recent study showed that dimethylaniline is likely to undergo cytochrome P450 hydroxylation in cells to form aminophenol or quinone imine structures that generate ROS through redox cycling (Chao et al, 2012). A hypothetical mechanism for GANT61 cytotoxicity is thus described whereby GANT61 generates ROS through redox cycling following 180

cytochrome P450 oxidation. GANT61-derived ROS induces MPT in a small number of mitochondria and this further triggers ROS production in neighboring mitochondria, which ultimately leads to the induction of apoptosis. This phenomenon is known as ROS-induced ROS release (Zorov et al, 2006). In conclusion, the present study not only demonstrates the therapeutic potential of GANT61 in mesothelioma, but also reveals a novel mechanistic action of GANT61 in inducing G1 cell cycle arrest and apoptosis. This is the first report of apoptosis induced by GANT61 via generation of mitochondrial ROS. Based on the findings in this thesis, I proposed a model showing the relationship of GANT61, ROS and mitochondria in the induction of growth suppression and apoptosis of LO68 cells (Figure 5.22). My results offer an initial proof-of-concept that mitochondrial ROS-mediated anticancer mechanisms may be exploited for therapeutic benefits in mesothelioma. 181

Dimethylaniline Dimethylaniline Figure 5.21 Chemical structure of GANT61 182

Figure 5.22 Schematic representation of the proposed mechanism of GANT61-induced apoptosis in mesothelioma cells. GANT61 triggers the production of mitochondrial ROS independent of Hh/Gli signaling. GANT61-induced ROS causes DNA damage and reduced DNA repair, which leads to G1 cell cycle arrest and apoptosis. Pretreatment of cells with either NAC or rotenone can reverse this ROSinduced apoptosis. 183

CHAPTER 6 GENERAL DISCUSSION 184

Malignant mesothelioma remains a significant clinical problem worldwide. It is now accepted that prolonged exposure to asbestos, particularly crocidolite (also known as blue asbestos), predisposes individuals to mesothelioma, which has a 30-50 year latency period (Bianchi et al, 1997; Burdorf et al, 2003; Mossman et al, 1983). Occupational asbestos exposure is the main risk factor for mesothelioma, accounting for 80% of the cases in men and 40% of the cases in women (Robinson & Chahinian, 2002). The global incidence of mesothelioma has been on the rise since the 1960s and is projected to increase until at least 2050 due to the widespread use of asbestos during the past decades (Moolgavkar et al, 2009; Peto et al, 1999). In addition, due to the lack of regulations on the use of asbestos in developing countries such China and India, an epidemic of mesothelioma is likely to happen in these countries in the coming decades (Joshi & Gupta, 2004; Le et al, 2011). Despite years of research, the majority of mesothelioma patients experience disease progression and 90% die within five years of diagnosis (Yan et al, 2011b). Thus there is an urgent need to develop novel therapeutic strategies to improve the outcome of patients with advanced stage mesothelioma. Targeting the Hh signaling pathway has recently emerged as a novel therapeutic paradigm based on numerous preclinical and clinical data, demonstrating potent anticancer activity of SMO inhibitors in various tumor models (Lin & Matsui, 2012). The Hh pathway simplistically comprises Hh ligand itself, followed by PTCH1, then SMO and finally Gli transcription factors. Hh signaling begins with Hh ligand binding the cell surface PTCH1 receptor. This results in inactivation of PTCH1, which lifts the repression imposed by PTCH1 on SMO, which localizes to the primary cilium and hence activate Gli transcription factors, resulting in transcriptional activation or repression of Hh target genes. As the Hh pathway has been validated as a potential target in mesothelioma (Shi et al, 2012), studies by a number of laboratories have 185

confirmed that SMO and Gli inhibitors have the ability to hamper the growth of mesothelioma in vitro and in vivo (Li et al, 2013; You et al, 2013; Zhang et al, 2013). However, it is unknown whether mesothelioma behaves like a Hh-independent cancer, such as BCC and medulloblastoma, such that it is driven by mutations in Hh pathway genes, or like a Hh-dependent cancer, such as breast, lung and prostate cancers, in which overexpression of Hh ligand drives the activation of the Hh pathway (Amakye et al, 2013). In Chapter 3, I hypothesized that common mutations in Hh pathway genes could drive mesothelioma tumorigenesis. Using an automated DNA sequencing approach to identify mutations across all exons in 13 Hh pathway genes in 11 mesothelioma cell lines and 14 human mesothelioma tumors, only three mutations were revealed. Out of a panel of 11 mesothelioma cell lines, an insertion mutation in SMO, a missense mutation in SUFU and a multi-exon deletion in PTCH1 were identified in two of the cell lines screened for alterations. Notably, the 3-bp insertion mutation in SMO (69_70insCTG) was also present in the patient cohort, being detected in 1 out of 14 mesothelioma tumors. This SMO mutation would lead to the insertion of an additional leucine residue in the signal peptide region of SMO, which could potentially alter SMO precursor processing and disrupt SMO localization to the cell surface. The importance of this mutation is further underscored by the report that two gastric tumors have been found to harbor this mutation (Wang et al, 2013a). However, these SMO mutant gastric tumors did not display higher Hh pathway activity compared to SMO wild-type tumors and, therefore, was not considered to be a driver mutation (Wang et al, 2013a). PTCH1 was first linked to cancer development in 1996 when some patients with Gorlin syndrome were found to have germline PTCH1 mutations (Hahn et al, 1996; Johnson et 186

al, 1996). This syndrome is characterized by familial clustering of BCC, medulloblastoma, rhabdomyosarcoma and ovarian fibroma (High & Zedan, 2005). In this study, I report a novel multi-exon deletion in PTCH1 in a single cell line (i.e. JU77). In keeping with other PTCH1 mutations, it is predicted that this mutation causes the loss of the extracellular loops and cytoplasmic C-terminal domain, which may lead to hyperactivation of Hh signaling (Johnson et al, 2000; Lu et al, 2006; Marigo et al, 1996). Unfortunately, the PTCH1 exonic deletion was not found in the patient cohort. The low frequency of recurrent mutations in the patient cohort might be related to the small sample size. Further studies need to be conducted on a larger patient cohort (n 100) to establish the role of mutations in Hh pathway genes in the pathogenesis of mesothelioma. Of the three mutations identified in this study, the most likely mutation to have a role in tumorigenesis was the SUFU missense mutation. This is because 1) the SUFU missense mutation was predicted in silico to be phenotypically damaging by both SIFT and PolyPhen2 programs and 2) the SUFU missense mutation has also recently been detected in a colorectal cancer patient as part of the Cancer Genome Atlas Project. Hence, it was logical to functionally characterize the SUFU missense mutation. Using a Gli-luciferase reporter plasmid and overexpressing wild-type GLI1, wild-type SUFU and T411M SUFU mutant in mouse embryonic fibroblast cells, I did not find any functional alteration in the T411M mutant. This finding was not surprising, as a previous study looking for PTCH1 and SMO mutations in gastric tumors found no link between hyperactivated Hh signaling and mutations in PTCH1 and SMO (Wang et al, 2013a). In a similar study by Guleng and colleagues, SMO mutations identified in bile duct, colon and pancreatic cancer cell lines were not found to impact Hh pathway activity (Guleng et al, 2006). This seems to further corroborate the findings of previous 187

studies, that a great number of cancer types appear to be activated by Hh ligandindependent mechanisms that are downstream of SMO (Lauth & Toftgard, 2007). Therefore, mesothelioma is unlikely to be driven by mutation in the Hh pathway but instead behaves like a Type II or III Hh cancer, where the activation of the pathway is autocrine or paracrine in nature. This also would imply that drug development strategies should focus not only on SMO inhibitors but more promising targets that are downstream of SMO, such as the Gli transcription factors. Therefore in Chapter 4 I investigated targeting the terminal Gli transcription factors in mesothelioma, using GANT61, a small molecule inhibitor of GLI1 and GLI2. GANT61 was discovered in a cellular screen to identify compounds that could block Gli transcriptional activity (Lauth et al, 2007). I investigated the in vitro effects of this Hh pathway inhibitor on cell growth inhibition and cell death mechanisms in mesothelioma cells. Inhibition of GLI1 and GLI2 by GANT61 led to a dose-dependent reduction in cell growth and clonogenic survival in the panel of 10 mesothelioma cell lines. In contrast, the SMO inhibitors, cyclopamine and GDC-0449, showed minimal cytotoxicity in mesothelioma cells. As discussed earlier, the absence of pathogenic mutations in PTCH1 and SMO in mesothelioma cells suggest that hyperactivation of the Hh pathway in mesothelioma is likely to be mediated by mechanisms downstream of SMO that converge and trigger Gli activation. It is perhaps not surprising then that inhibition of SMO in mesothelioma cell lines has shown little therapeutic response, an observation made in vitro in this study and by others (Li et al, 2013; Zhang et al, 2013). Similar observations have been reported in colorectal and neuroblastoma cancer models (Mazumdar et al, 2011b; Wickstrom et al, 2013). GANT61 induced G1 cell-cycle arrest and apoptosis in LO68 cells with increased Annexin V staining. The expression of GLI1 and GLI2 mrna was significantly reduced in GANT61-treated cells in a time-dependent 188

fashion. Furthermore, GANT61 downregulated PTCH1 mrna and Bcl-2 protein expression, which are direct targets of GLI1, suggesting that the antitumor action of GANT61 is mediated through blockade of GLI1. Additionally, median dose-effect analysis revealed that co-treatment with GANT61 and standard anti-mesothelioma chemotherapeutic agents (cisplatin and gemcitabine) induced synergistic cytotoxic effects against mesothelioma cells. It is important to note that only LO68 cells were used to investigate the mechanism of GANT61-induced apoptosis and although tantalising to think that this may be the action in all mesothelioma cells, more studies have to be performed on more mesothelioma cell lines before this can be definitively stated. While GANT61 as an inducer of apoptosis was shown extensively in several human cancer models (Desch et al, 2010; Kawabata et al, 2011; Mazumdar et al, 2011a; Pan et al, 2012; Tostar et al, 2010; Wickstrom et al, 2013; Yan et al, 2011a; Yan et al, 2013; You et al, 2013), the induction of autophagy, also known as Type II programmed cell death, has also been investigated. In a study using HCC cells, Wang and colleagues demonstrated the ability of GANT61 to kill cancer cells by inducing autophagy (Wang et al, 2013b). This finding was further expanded in a series of studies that demonstrated the ability of the Hh pathway to regulate autophagy in a wide range of cell types, including hippocampal neurons (Jimenez-Sanchez et al, 2012; Li et al, 2012b; Petralia et al, 2013). Interestingly, GANT61 was found to exert a similar autophagic response in LO68 cells, as shown by vital dye staining of LO68 cells with acridine orange followed by the accumulation of acidic vesicular organelles in the cytoplasm of cells. This was inhibited by addition of BafA1, an autophagy inhibitor. Furthermore, combination of GANT61 with BafA1 potentiated the anticancer effect of GANT61 through induction of apoptosis, unravelling the cytoprotective role of GANT61-induced autophagy in LO68 cells. Although this novel finding is in sharp contrast to what has been published 189

previously on GANT61 s impact on autophagy, it does not contradict the double-edged sword nature of autophagy where it can be cytotoxic or cytoprotective depending on the cell type (Shintani & Klionsky, 2004). Surprisingly, silencing GLI1 and GLI2 using sirna did not result in cell cycle arrest and apoptosis, as would be expected if GANT61 was indeed mediating its effects through GLI1 and GLI2. Clearly, this suggests that the cytotoxic effects of GANT61 are independent of the Hh/Gli axis, indicating that alternative mechanisms might mediate GANT61 cytotoxic activity. It is well known that many chemotherapeutic agents, including cisplatin and doxorubicin, kill cancer cells via the induction of oxidative stress. Notably, the classical SMO inhibitor, cyclopamine, was reported to induce apoptosis in Daoy medulloblastoma cells through a nitric oxide-dependent mechanism (Meyers- Needham et al, 2012). Consistent with my hypothesis, GANT61 triggered the generation of ROS and quenching of ROS by antioxidants protected the cells from GANT61-induced cytotoxicity, supporting ROS-mediated cytotoxic effects of GANT61 in LO68 cells. Furthermore, using two colorectal cancer cell lines, I demonstrated that this novel ability of GANT61 to induce apoptosis in a ROS-dependent manner is not restricted to LO68 cells. This finding provides an arguable case for ROS generation as a general phenomenon of GANT61 action in cancer cells in general. Studies were then performed in Chapter 5 to further delineate the type of ROS being generated and the subcellular site of production. First, using sirna to silence endogenous GLI1 and GLI2, individually or together, I was unable to generate ROS. This finding was expected, as similar silencing experiments in Chapter 4 did not result in induction of apoptosis and cell cycle arrest. Instead, blocking NADPH oxidase and mitochondrial respiratory chain activity using DPI and rotenone, respectively, protected 190

cells from GANT61-induced apoptosis and inhibited ROS generation. This demonstrates that NADPH oxidase and mitochondria but not Gli1 and Gli2 are involved in GANT61-induced apoptotic cell death and ROS generation. While the treatment of cells with DPI reduced GANT61-induced cell death and ROS generation, it is important to note that DPI has been reported to inhibit respiratory chain activity (Majander et al, 1994). Therefore, the focus was placed on understanding the role of mitochondria in GANT61-induced ROS generation. The production of mitochondrial ROS, specifically superoxide, was investigated using a redox-sensitive dye, mitosox red. Upon GANT61 administration, the fluorescence intensity of mitosox red increased dramatically, suggesting that GANT61 effectively induced the generation of superoxide that originates from the mitochondria. In addition, the inability of GANT61 to induce a significant increase of mitochondrial superoxide and apoptosis in LO68 cells that have been depleted of mitochondrial DNA, further confirms that the generation of mitochondrial superoxide is important for the action of GANT61. The results together indicate that the effect of GANT61 on cell death is through the cytotoxic activity of mitochondrial superoxide. Preliminary data also highlighted a novel role for ROS in the regulation of the Hh pathway. Given that ROS can regulate many cell functions through redox modification of proteins, it was not surprising that ROS could regulate the Hh pathway. LO68 mesothelioma cells and exogenous H 2 O 2 were used to elucidate the involvement of ROS in regulation of the Hh pathway. GLI1 mrna levels, a surrogate marker for Hh pathway activity, in LO68 cells, were significantly reduced at 24 h after H 2 O 2 treatment, demonstrating that ROS are involved in Hh regulation. However, the specifics of this interaction are still unclear. It was previously reported that ROS induces T cell 191

apoptosis through downregulation of Bcl-2 expression (Hildeman et al, 2003). I speculate that ROS induces cells to undergo apoptosis by reducing Bcl-2 expression via downregulation of Hh signaling. 6.1 FUTURE PERSPECTIVE In this thesis, I have demonstrated that mutations in the coding regions of Hh pathway genes are rare in mesothelioma and hence it is unlikely that mutations in this pathway drive the activation of Gli transcription factors that is widely observed in this cancer. However, the exon-intron junctions were not sequenced in this thesis and mutations in these regions have been shown to affect mrna splicing (Cartegni et al, 2002). Further DNA sequencing of the exon-intron junctions of Hh pathway genes should be performed. In addition, other forms of genetic variations including SNP and copy number variation may also be responsible for the hyperactivation of Hh signaling in mesothelioma. In total, 35 SNPs were detected in 11 out of 13 Hh pathway genes, of which 16 were nonsynonymous SNPs. Notably, five of these non-synonymous SNPs are predicted by PolyPhen2 to be damaging. It would be of interest to determine the functional importance of these SNPs in Hh signaling. Recent studies reported that synonymous mutations, traditionally viewed as silent or evolutionarily neutral mutations, can adversely affect protein expression, structure and function (Sauna & Kimchi-Sarfaty, 2011). Hence, it would be equally important to functionally characterize the synonymous SNPs that were identified in this thesis. More recently, the promoter regions of Hh pathway genes, including PTCH1 and HHIP, have been shown to be aberrantly methylated in cancers (Martin et al, 2005; Shahi et al, 2011; Song et al, 2013b; Tada et al, ; Wang et al, 2006). The methylation status of these genes should be investigated in mesothelioma, which in turn might help to explain the hyperactivity of the Hh signaling pathway in this cancer. 192

There are a number of unanswered questions with regard to Hh signaling and GANT61 in mesothelioma. From the work of others and data in this thesis, it is not clear from gene silencing experiments whether the Hh pathway is important for mesothelioma cell survival. This could be answered by generating mesothelioma cell lines that have GLI1 and/or GLI2 inactivated by zinc finger nuclease technology. Having demonstrated that ROS could impact on Hh signaling, further in vitro studies could be performed in an Hh-dependent cancer model such as medulloblastoma, to confirm the cross-talk between ROS and Hh signaling. In this thesis, I have also demonstrated that GANT61 can potentiate the in vitro efficacy of first-line mesothelioma chemotherapeutic agents. The preclinical efficacy of these combinations needs to be further investigated in mouse tumor xenograft models. I have also demonstrated that GANT61-induced apoptosis is mediated through the generation of mitochondrial superoxide. Based on the pharmacologic inhibition data, NADPH oxidase appears to be as another site of ROS production in response to GANT61 treatment. The role of NADPH oxidase in GANT61-induced ROS production could be investigated by silencing various NADPH oxidase isoforms. Although GANT61 has been tested in several cancer xenograft models, including prostate cancer and neuroblastoma (Lauth et al, 2007; Wickstrom et al, 2013), preclinical efficacy data on mesothelioma is lacking. The potential of GANT61 as a novel mesothelioma therapy could be evaluated by testing GANT61 in the MexTAg mouse model of asbestosinduced mesothelioma developed by Professor Bruce Robinson s group (Robinson et al, 193

2011). Although GANT61 shows great therapeutic promise as an anti-mesothelioma drug, it suffers from serious drawbacks such as poor aqueous solubility and low potency (micromolar range). More efficacious agents with improved water solubility, pharmacokinetic and potency can be developed through structure-activity relationship studies of GANT61. 6.2 CONCLUSIONS In conclusion, this thesis has presented evidence that in mesothelioma, mutations identified in the coding region of Hh pathway genes are unlikely to be responsible for the hyperactivation of the Hh pathway. Future work should focus on the characterization of SNPs identified in Hh pathway genes. Similarly, the role of other forms of genetic variants requires further examination. Deregulation of Hh signaling in mesothelioma appears to be driven by activation of signaling pathways that converge on Gli transcription factors to promote tumor formation and maintenance. The data presented also suggest that GANT61 could be a potential chemotherapeutic drug for the treatment of mesothelioma although the mode of action is unlikely to be through specifically blocking the Hh pathway. GANT61 suppresses the growth and clonogenicity of LO68 cells by inducing apoptosis through generation of mitochondrial superoxide. In vitro combination treatment with GANT61 and standard anti-mesothelioma drugs also demonstrated synergism against LO68 cells. Collectively, this work is the first to show that mitochondrial ROS-mediated anticancer mechanisms may be developed as a novel therapy for mesothelioma. 194

REFERENCES 195

Abdel-Rahman M. H., Pilarski R., Cebulla C. M., Massengill J. B., Christopher B. N., Boru G., Hovland P., Davidorf F. H. (2011) Germline BAP1 mutation predisposes to uveal melanoma, lung adenocarcinoma, meningioma, and other cancers. J Med Genet 48: 856-859 Aceto N., Bertino P., Barbone D., Tassi G., Manzo L., Porta C., Mutti L., Gaudino G. (2009) Taurolidine and oxidative stress: a rationale for local treatment of mesothelioma. Eur Respir J 34: 1399-1407 Adzhubei I. A., Schmidt S., Peshkin L., Ramensky V. E., Gerasimova A., Bork P., Kondrashov A. S., Sunyaev S. R. (2010) A method and server for predicting damaging missense mutations. Nat Methods 7: 248-249 Agyeman A., Mazumdar T., Houghton J. A. (2012) Regulation of DNA damage following termination of Hedgehog (HH) survival signaling at the level of the GLI genes in human colon cancer. Oncotarget 3: 854-868 Al-Lazikani B., Banerji U., Workman P. (2012) Combinatorial drug therapy for cancer in the post-genomic era. Nat Biotechnol 30: 679-692 Alexandre J., Batteux F., Nicco C., Chereau C., Laurent A., Guillevin L., Weill B., Goldwasser F. (2006) Accumulation of hydrogen peroxide is an early and crucial step for paclitaxel-induced cancer cell death both in vitro and in vivo. Int J Cancer 119: 41-48 Alexandre J., Hu Y., Lu W., Pelicano H., Huang P. (2007) Novel action of paclitaxel against cancer cells: bystander effect mediated by reactive oxygen species. Cancer Res 67: 3512-3517 Allen AM, Czerminska M, Janne PA, Sugarbaker DJ, Bueno R, Harris JR, Court L, Baldini EH (2006) Fatal pneumonitis associated with intensity-modulated radiation therapy for mesothelioma. Int J Radiat Oncol Biol Phys 65: 640-645 Allen B. L., Tenzen T., McMahon A. P. (2007) The Hedgehog-binding proteins Gas1 and Cdo cooperate to positively regulate Shh signaling during mouse development. Genes Dev 21: 1244-1257 Allison A. C., Young M. R. (1964) Uptake of Dyes and Drugs by Living Cells in Culture. Life Sci 3: 1407-1414 Amakye D., Jagani Z., Dorsch M. (2013) Unraveling the therapeutic potential of the Hedgehog pathway in cancer. Nat Med 19: 1410-1422 Aniento F., Roche E., Cuervo A. M., Knecht E. (1993) Uptake and degradation of glyceraldehyde-3-phosphate dehydrogenase by rat liver lysosomes. J Biol Chem 268: 10463-10470 Asplund A., Gustafsson A. C., Wikonkal N. M., Sela A., Leffell D. J., Kidd K., Lundeberg J., Brash D. E., Ponten F. (2005) PTCH codon 1315 polymorphism and risk for nonmelanoma skin cancer. Br J Dermatol 152: 868-873 Axelson M., Liu K., Jiang X., He K., Wang J., Zhao H., Kufrin D., Palmby T., Dong Z., Russell A. M., Miksinski S., Keegan P., Pazdur R. (2013) U.S. Food and Drug Administration approval: vismodegib for recurrent, locally advanced, or metastatic basal cell carcinoma. Clin Cancer Res 19: 2289-2293 Bai C. B., Stephen D., Joyner A. L. (2004) All mouse ventral spinal cord patterning by hedgehog is Gli dependent and involves an activator function of Gli3. Dev Cell 6: 103-115 196

Baldini E. H. (2009) Radiation therapy options for malignant pleural mesothelioma. Semin Thorac Cardiovasc Surg 21: 159-163 Ball DL, Cruickshank DG (1990) The treatment of malignant mesothelioma of the pleura: review of a 5-year experience, with special reference to radiotherapy. Am J Clin Oncol 13: 4-9 Barbone D., Ryan J. A., Kolhatkar N., Chacko A. D., Jablons D. M., Sugarbaker D. J., Bueno R., Letai A. G., Coussens L. M., Fennell D. A., Broaddus V. C. (2011) The Bcl-2 repertoire of mesothelioma spheroids underlies acquired apoptotic multicellular resistance. Cell Death Dis 2: e174 Baris I., Simonato L., Artvinli M., Pooley F., Saracci R., Skidmore J., Wagner C. (1987) Epidemiological and environmental evidence of the health effects of exposure to erionite fibres: a four-year study in the Cappadocian region of Turkey. Int J Cancer 39: 10-17 Baris Y. I., Saracci R., Simonato L., Skidmore J. W., Artvinli M. (1981) Malignant mesothelioma and radiological chest abnormalities in two villages in Central Turkey. An epidemiological and environmental investigation. Lancet 1: 984-987 Barreto D. C., Gomez R. S., Bale A. E., Boson W. L., De Marco L. (2000) PTCH gene mutations in odontogenic keratocysts. J Dent Res 79: 1418-1422 Battisti S., Valente D., Albonici L., Bei R., Modesti A., Palumbo C. (2012) Nutritional stress and arginine auxotrophy confer high sensitivity to chloroquine toxicity in mesothelioma cells. Am J Respir Cell Mol Biol 46: 498-506 Beaulaton J., Lockshin R. A. (1977) Ultrastructural study of the normal degeneration of the intersegmental muscles of Anthereae polyphemus and Manduca sexta (Insecta, Lepidoptera) with particular reference of cellular autophagy. J Morphol 154: 39-57 Bedford P., Fichtinger-Schepman A. M., Shellard S. A., Walker M. C., Masters J. R., Hill B. T. (1988) Differential repair of platinum-dna adducts in human bladder and testicular tumor continuous cell lines. Cancer Res 48: 3019-3024 Below J. E., Cox N. J., Fukagawa N. K., Hirvonen A., Testa J. R. (2011) Factors that impact susceptibility to fiber-induced health effects. J Toxicol Environ Health B Crit Rev 14: 246-266 Berlin J., Bendell J. C., Hart L. L., Firdaus I., Gore I., Hermann R. C., Mulcahy M. F., Zalupski M. M., Mackey H. M., Yauch R. L., Graham R. A., Bray G. L., Low J. A. (2013) A randomized phase II trial of vismodegib versus placebo with FOLFOX or FOLFIRI and bevacizumab in patients with previously untreated metastatic colorectal cancer. Clin Cancer Res 19: 258-267 Berman D. M., Karhadkar S. S., Maitra A., Montes De Oca R., Gerstenblith M. R., Briggs K., Parker A. R., Shimada Y., Eshleman J. R., Watkins D. N., Beachy P. A. (2003) Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours. Nature 425: 846-851 Berndtsson M., Hagg M., Panaretakis T., Havelka A. M., Shoshan M. C., Linder S. (2007) Acute apoptosis by cisplatin requires induction of reactive oxygen species but is not associated with damage to nuclear DNA. Int J Cancer 120: 175-180 Bhatia N., Thiyagarajan S., Elcheva I., Saleem M., Dlugosz A., Mukhtar H., Spiegelman V. S. (2006) Gli2 is targeted for ubiquitination and degradation by beta-trcp ubiquitin ligase. J Biol Chem 281: 19320-19326 Bianchi A. B., Mitsunaga S. I., Cheng J. Q., Klein W. M., Jhanwar S. C., Seizinger B., Kley N., Kleinszanto A. J. P., Testa J. R. (1995) High-Frequency of Inactivating 197

Mutations in the Neurofibromatosis Type-2 Gene (Nf2) in Primary Malignant Mesotheliomas. Proc Natl Acad Sci U S A 92: 10854-10858 Bianchi C., Giarelli L., Grandi G., Brollo A., Ramani L., Zuch C. (1997) Latency periods in asbestos-related mesothelioma of the pleura. Eur J Cancer Prev 6: 162-166 Bigelow R. L., Chari N. S., Unden A. B., Spurgers K. B., Lee S., Roop D. R., Toftgard R., McDonnell T. J. (2004) Transcriptional regulation of bcl-2 mediated by the sonic hedgehog signaling pathway through gli-1. J Biol Chem 279: 1197-1205 Bitgood M. J., Shen L., McMahon A. P. (1996) Sertoli cell signaling by Desert hedgehog regulates the male germline. Curr Biol 6: 298-304 Bosco-Clement G., Zhang F., Chen Z., Zhou H. M., Li H., Mikami I., Hirata T., Yagui- Beltran A., Lui N., Do H. T., Cheng T., Tseng H. H., Choi H., Fang L. T., Kim I. J., Yue D., Wang C., Zheng Q., Fujii N., Mann M., Jablons D. M., He B. (2014) Targeting Gli transcription activation by small molecule suppresses tumor growth. Oncogene 33: 2087-2097 Bott M., Brevet M., Taylor B. S., Shimizu S., Ito T., Wang L., Creaney J., Lake R. A., Zakowski M. F., Reva B., Sander C., Delsite R., Powell S., Zhou Q., Shen R. L., Olshen A., Rusch V., Ladanyi M. (2011) The nuclear deubiquitinase BAP1 is commonly inactivated by somatic mutations and 3p21.1 losses in malignant pleural mesothelioma. Nat Genet 43: 668-U681 Brastianos P. K., Horowitz P. M., Santagata S., Jones R. T., McKenna A., Getz G., Ligon K. L., Palescandolo E., Van Hummelen P., Ducar M. D., Raza A., Sunkavalli A., Macconaill L. E., Stemmer-Rachamimov A. O., Louis D. N., Hahn W. C., Dunn I. F., Beroukhim R. (2013) Genomic sequencing of meningiomas identifies oncogenic SMO and AKT1 mutations. Nat Genet 45: 285-289 Buglino J. A., Resh M. D. () Hhat is a palmitoylacyltransferase with specificity for N-palmitoylation of Sonic Hedgehog. J Biol Chem 283: 22076-22088 Buonamici S., Williams J., Morrissey M., Wang A., Guo R., Vattay A., Hsiao K., Yuan J., Green J., Ospina B., Yu Q., Ostrom L., Fordjour P., Anderson D. L., Monahan J. E., Kelleher J. F., Peukert S., Pan S., Wu X., Maira S. M., Garcia-Echeverria C., Briggs K. J., Watkins D. N., Yao Y. M., Lengauer C., Warmuth M., Sellers W. R., Dorsch M. (2010) Interfering with resistance to smoothened antagonists by inhibition of the PI3K pathway in medulloblastoma. Sci Transl Med 2: 51ra70 Buque A., Muhialdin JSh, Munoz A., Calvo B., Carrera S., Aresti U., Sancho A., Rubio I., Lopez-Vivanco G. (2012) Molecular mechanism implicated in Pemetrexed-induced apoptosis in human melanoma cells. Mol Cancer 11: 25 Burdorf A., Dahhan M., Swuste P. (2003) Occupational characteristics of cases with asbestos-related diseases in The Netherlands. Ann Occup Hyg 47: 485-492 Butchart E. G. (1999) Contemporary management of malignant pleural mesothelioma. Oncologist 4: 488-500 Butchart E. G., Ashcroft T., Barnsley W. C., Holden M. P. (1976) Pleuropneumonectomy in the management of diffuse malignant mesothelioma of the pleura. Experience with 29 patients. Thorax 31: 15-24 Buttitta L., Mo R., Hui C. C., Fan C. M. (2003) Interplays of Gli2 and Gli3 and their requirement in mediating Shh-dependent sclerotome induction. Development 130: 6233-6243 198

Byrne M. J., Davidson J. A., Musk A. W., Dewar J., van Hazel G., Buck M., de Klerk N. H., Robinson B. W. (1999) Cisplatin and gemcitabine treatment for malignant mesothelioma: a phase II study. J Clin Oncol 17: 25-30 Cai H., Harrison D. G. (2000) Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 87: 840-844 Callejo A., Torroja C., Quijada L., Guerrero I. (2006) Hedgehog lipid modifications are required for Hedgehog stabilization in the extracellular matrix. Development 133: 471-483 Cancer Genome Atlas Network (2012) Comprehensive molecular characterization of human colon and rectal cancer. Nature 487: 330-337 Cao X., Rodarte C., Zhang L., Morgan C. D., Littlejohn J., Smythe W. R. (2007) Bcl2/bcl-xL inhibitor engenders apoptosis and increases chemosensitivity in mesothelioma. Cancer Biol Ther 6: 246-252 Carbone M, Pass HI, Rizzo P, Marinetti M, Di Muzio M, Mew DJ, Levine AS, Procopio A (1994) Simian virus 40-like DNA sequences in human pleural mesothelioma. Oncogene 9: 1781-1790 Carbone M., Ly B. H., Dodson R. F., Pagano I., Morris P. T., Dogan U. A., Gazdar A. F., Pass H. I., Yang H. (2012) Malignant mesothelioma: facts, myths, and hypotheses. J Cell Physiol 227: 44-58 Carpenter D., Stone D. M., Brush J., Ryan A., Armanini M., Frantz G., Rosenthal A., de Sauvage F. J. (1998) Characterization of two patched receptors for the vertebrate hedgehog protein family. Proc Natl Acad Sci U S A 95: 13630-13634 Cartegni L., Chew S. L., Krainer A. R. (2002) Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet 3: 285-298 Carthew P., Hill R. J., Edwards R. E., Lee P. N. (1992) Intrapleural Administration of Fibers Induces Mesothelioma in Rats in the Same Relative Order of Hazard as Occurs in Man after Exposure. Hum Exp Toxicol 11: 530-534 Castagneto B., Zai S., Dongiovanni D., Muzio A., Bretti S., Numico G., Botta M., Sinaccio G. (2005) Cisplatin and gemcitabine in malignant pleural mesothelioma: a phase II study. Am J Clin Oncol 28: 223-226 Ceresoli G. L., Gridelli C., Santoro A. (2007) Multidisciplinary treatment of malignant pleural mesothelioma. Oncologist 12: 850-863 Chabner B. A., Roberts T. G., Jr. (2005) Timeline: Chemotherapy and the war on cancer. Nat Rev Cancer 5: 65-72 Chandel N. S., Schumacker P. T. (1999) Cells depleted of mitochondrial DNA (rho0) yield insight into physiological mechanisms. FEBS Lett 454: 173-176 Chang-Claude J., Dunning A., Schnitzbauer U., Galmbacher P., Tee L., Wjst M., Chalmers J., Zemzoum I., Harbeck N., Pharoah P. D., Hahn H. (2003) The patched polymorphism Pro1315Leu (C3944T) may modulate the association between use of oral contraceptives and breast cancer risk. Int J Cancer 103: 779-783 Chao M. W., Kim M. Y., Ye W., Ge J., Trudel L. J., Belanger C. L., Skipper P. L., Engelward B. P., Tannenbaum S. R., Wogan G. N. (2012) Genotoxicity of 2,6- and 3,5- dimethylaniline in cultured mammalian cells: the role of reactive oxygen species. Toxicol Sci 130: 48-59 199

Chattopadhyay S., Moran R. G., Goldman I. D. (2007) Pemetrexed: biochemical and cellular pharmacology, mechanisms, and clinical applications. Mol Cancer Ther 6: 404-417 Chauhan D., Hideshima T., Anderson K. C. () Targeting proteasomes as therapy in multiple myeloma. Adv Exp Med Biol 615: 251-260 Chen G., Ray R., Dubik D., Shi L., Cizeau J., Bleackley R. C., Saxena S., Gietz R. D., Greenberg A. H. (1997) The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis. J Exp Med 186: 1975-1983 Chen J. K., Taipale J., Cooper M. K., Beachy P. A. (2002a) Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev 16: 2743-2748 Chen J. K., Taipale J., Young K. E., Maiti T., Beachy P. A. (2002b) Small molecule modulation of Smoothened activity. Proc Natl Acad Sci U S A 99: 14071-14076 Chen M. H., Li Y. J., Kawakami T., Xu S. M., Chuang P. T. (2004a) Palmitoylation is required for the production of a soluble multimeric Hedgehog protein complex and longrange signaling in vertebrates. Genes Dev 18: 641-659 Chen M., Hildebrandt M. A., Clague J., Kamat A. M., Picornell A., Chang J., Zhang X., Izzo J., Yang H., Lin J., Gu J., Chanock S., Kogevinas M., Rothman N., Silverman D. T., Garcia-Closas M., Grossman H. B., Dinney C. P., Malats N., Wu X. (2010) Genetic variations in the sonic hedgehog pathway affect clinical outcomes in non-muscle-invasive bladder cancer. Cancer Prev Res (Phila) 3: 1235-1245 Chen W., Ren X. R., Nelson C. D., Barak L. S., Chen J. K., Beachy P. A., de Sauvage F., Lefkowitz R. J. (2004b) Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2. Science 306: 2257-2260 Chen X. L., Cao L. Q., She M. R., Wang Q., Huang X. H., Fu X. H. () Gli-1 sirna induced apoptosis in Huh7 cells. World J Gastroenterol 14: 582-589 Chen Y., Struhl G. (1996) Dual roles for patched in sequestering and transducing Hedgehog. Cell 87: 553-563 Cheng S. Y., Bishop J. M. (2002) Suppressor of Fused represses Gli-mediated transcription by recruiting the SAP18-mSin3 corepressor complex. Proc Natl Acad Sci U S A 99: 5442-5447 Cheung H. O., Zhang X., Ribeiro A., Mo R., Makino S., Puviindran V., Law K. K., Briscoe J., Hui C. C. (2009) The kinesin protein Kif7 is a critical regulator of Gli transcription factors in mammalian hedgehog signaling. Sci Signal 2: ra29 Chiang C, Litingtung Y, Lee E, Young KE, Corden JL, Westphal H, Beachy PA (1996) Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383: 407-413 Chiu J., Dawes I. W. (2012) Redox control of cell proliferation. Trends Cell Biol 22: 592-601 Choi A. M., Ryter S. W., Levine B. (2013) Autophagy in human health and disease. N Engl J Med 368: 651-662 Chou T. C. (2006) Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 58: 621-681 Chou T. C., Talalay P. (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22: 27-55 200

Chuang P. T., McMahon A. P. (1999) Vertebrate Hedgehog signalling modulated by induction of a Hedgehog-binding protein. Nature 397: 617-621 Chung U. I., Schipani E., McMahon A. P., Kronenberg H. M. (2001) Indian hedgehog couples chondrogenesis to osteogenesis in endochondral bone development. J Clin Invest 107: 295-304 Cicala C., Pompetti F., Carbone M. (1993) SV40 induces mesotheliomas in hamsters. Am J Pathol 142: 1524-1533 Clark V. E., Erson-Omay E. Z., Serin A., Yin J., Cotney J., Ozduman K., Avsar T., Li J., Murray P. B., Henegariu O., Yilmaz S., Gunel J. M., Carrion-Grant G., Yilmaz B., Grady C., Tanrikulu B., Bakircioglu M., Kaymakcalan H., Caglayan A. O., Sencar L., Ceyhun E., Atik A. F., Bayri Y., Bai H., Kolb L. E., Hebert R. M., Omay S. B., Mishra-Gorur K., Choi M., Overton J. D., Holland E. C., Mane S., State M. W., Bilguvar K., Baehring J. M., Gutin P. H., Piepmeier J. M., Vortmeyer A., Brennan C. W., Pamir M. N., Kilic T., Lifton R. P., Noonan J. P., Yasuno K., Gunel M. (2013) Genomic analysis of non-nf2 meningiomas reveals mutations in TRAF7, KLF4, AKT1, and SMO. Science 339: 1077-1080 Clarke P. G. (1990) Developmental cell death: morphological diversity and multiple mechanisms. Anat Embryol (Berl) 181: 195-213 Clement V., Sanchez P., de Tribolet N., Radovanovic I., Ruiz i Altaba A. (2007) HEDGEHOG-GLI1 signaling regulates human glioma growth, cancer stem cell selfrenewal, and tumorigenicity. Curr Biol 17: 165-172 Cohen M. M., Jr. (2003) The hedgehog signaling network. Am J Med Genet A 123A: 5-28 Comporti M. (1989) Three models of free radical-induced cell injury. Chem Biol Interact 72: 1-56 Cooper M. K., Porter J. A., Young K. E., Beachy P. A. (1998) Teratogen-mediated inhibition of target tissue response to Shh signaling. Science 280: 1603-1607 Coriat R., Nicco C., Chereau C., Mir O., Alexandre J., Ropert S., Weill B., Chaussade S., Goldwasser F., Batteux F. (2012) Sorafenib-induced hepatocellular carcinoma cell death depends on reactive oxygen species production in vitro and in vivo. Mol Cancer Ther 11: 2284-2293 Couve-Privat S., Bouadjar B., Avril M. F., Sarasin A., Daya-Grosjean L. (2002) Significantly high levels of ultraviolet-specific mutations in the smoothened gene in basal cell carcinomas from DNA repair-deficient xeroderma pigmentosum patients. Cancer Res 62: 7186-7189 Cregan I. L., Dharmarajan A. M., Fox S. A. (2013) Mechanisms of cisplatin-induced cell death in malignant mesothelioma cells: role of inhibitor of apoptosis proteins (IAPs) and caspases. Int J Oncol 42: 444-452 Crompton M. (1999) The mitochondrial permeability transition pore and its role in cell death. Biochem J 341 ( Pt 2): 233-249 Crompton M., Ellinger H., Costi A. (1988) Inhibition by cyclosporin A of a Ca2+dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. Biochem J 255: 357-360 Crompton T., Outram S. V., Hager-Theodorides A. L. (2007) Sonic hedgehog signalling in T-cell development and activation. Nat Rev Immunol 7: 726-735 201

Curran D., Sahmoud T., Therasse P., van Meerbeeck J., Postmus P. E., Giaccone G. (1998) Prognostic factors in patients with pleural mesothelioma: the European Organization for Research and Treatment of Cancer experience. J Clin Oncol 16: 145-152 Dai P., Akimaru H., Tanaka Y., Maekawa T., Nakafuku M., Ishii S. (1999) Sonic Hedgehog-induced activation of the Gli1 promoter is mediated by GLI3. J Biol Chem 274: 8143-8152 Das S., Harris L. G., Metge B. J., Liu S., Riker A. I., Samant R. S., Shevde L. A. (2009) The hedgehog pathway transcription factor GLI1 promotes malignant behavior of cancer cells by up-regulating osteopontin. J Biol Chem 284: 22888-22897 Das S., Samant R. S., Shevde L. A. (2013) Nonclassical activation of Hedgehog signaling enhances multidrug resistance and makes cancer cells refractory to Smoothened-targeting Hedgehog inhibition. J Biol Chem 288: 11824-11833 De Bruin M. L., Burgers J. A., Baas P., van 't Veer M. B., Noordijk E. M., Louwman M. W., Zijlstra J. M., van den Berg H., Aleman B. M., van Leeuwen F. E. (2009) Malignant mesothelioma after radiation treatment for Hodgkin lymphoma. Blood 113: 3679-3681 Deininger M. W., Goldman J. M., Lydon N., Melo J. V. (1997) The tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of BCR-ABL-positive cells. Blood 90: 3691-3698 Denef N, Neubuser D, Perez L, Cohen SM (2000) Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened. Cell 102: 521-531 Desch P., Asslaber D., Kern D., Schnidar H., Mangelberger D., Alinger B., Stoecher M., Hofbauer S. W., Neureiter D., Tinhofer I., Aberger F., Hartmann T. N., Greil R. (2010) Inhibition of GLI, but not Smoothened, induces apoptosis in chronic lymphocytic leukemia cells. Oncogene 29: 4885-4895 Dey A., Seshasayee D., Noubade R., French D. M., Liu J., Chaurushiya M. S., Kirkpatrick D. S., Pham V. C., Lill J. R., Bakalarski C. E., Wu J., Phu L., Katavolos P., LaFave L. M., Abdel-Wahab O., Modrusan Z., Seshagiri S., Dong K., Lin Z., Balazs M., Suriben R., Newton K., Hymowitz S., Garcia-Manero G., Martin F., Levine R. L., Dixit V. M. (2012) Loss of the tumor suppressor BAP1 causes myeloid transformation. Science 337: 1541-1546 Ding Q., Fukami S., Meng X., Nishizaki Y., Zhang X., Sasaki H., Dlugosz A., Nakafuku M., Hui C. (1999) Mouse suppressor of fused is a negative regulator of sonic hedgehog signaling and alters the subcellular distribution of Gli1. Curr Biol 9: 1119-1122 Dogan A. U., Baris Y. I., Dogan M., Emri S., Steele I., Elmishad A. G., Carbone M. (2006) Genetic predisposition to fiber carcinogenesis causes a mesothelioma epidemic in Turkey. Cancer Res 66: 5063-5068 Druker B. J., Tamura S., Buchdunger E., Ohno S., Segal G. M., Fanning S., Zimmermann J., Lydon N. B. (1996) Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 2: 561-566 Dunaeva M., Michelson P., Kogerman P., Toftgard R. (2003) Characterization of the physical interaction of Gli proteins with SUFU proteins. J Biol Chem 278: 5116-5122 Dyer M. A., Farrington S. M., Mohn D., Munday J. R., Baron M. H. (2001) Indian hedgehog activates hematopoiesis and vasculogenesis and can respecify prospective neurectodermal cell fate in the mouse embryo. Development 128: 1717-1730 Eastman A., Schulte N. (1988) Enhanced DNA repair as a mechanism of resistance to cisdiamminedichloroplatinum(ii). Biochemistry 27: 4730-4734 202

Echelard Y, Epstein DJ, St-Jacques B, Shen L, Mohler J, McMahon JA, McMahon AP (1993) Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75: 1417-1430 Edwards J. G., Abrams K. R., Leverment J. N., Spyt T. J., Waller D. A., O'Byrne K. J. (2000) Prognostic factors for malignant mesothelioma in 142 patients: validation of CALGB and EORTC prognostic scoring systems. Thorax 55: 731-735 Ellis P., Davies A. M., Evans W. K., Haynes A. E., Lloyd N. S., Lung Cancer Disease Site Group of Cancer Care Ontario's Program in Evidence-based Care (2006) The use of chemotherapy in patients with advanced malignant pleural mesothelioma: a systematic review and practice guideline. J Thorac Oncol 1: 591-601 Evans T., Boonchai W., Shanley S., Smyth I., Gillies S., Georgas K., Wainwright B., Chenevix-Trench G., Wicking C. (2000) The spectrum of patched mutations in a collection of Australian basal cell carcinomas. Hum Mutat 16: 43-48 Fan L., Pepicelli C. V., Dibble C. C., Catbagan W., Zarycki J. L., Laciak R., Gipp J., Shaw A., Lamm M. L., Munoz A., Lipinski R., Thrasher J. B., Bushman W. (2004) Hedgehog signaling promotes prostate xenograft tumor growth. Endocrinology 145: 3961-3970 Fang J., Seki T., Maeda H. (2009) Therapeutic strategies by modulating oxygen stress in cancer and inflammation. Adv Drug Deliv Rev 61: 290-302 Favaloro B., Allocati N., Graziano V., Di Ilio C., De Laurenzi V. (2012) Role of apoptosis in disease. Aging (Albany NY) 4: 330-349 Favoni R. E., Florio T. (2011) Combined chemotherapy with cytotoxic and targeted compounds for the management of human malignant pleural mesothelioma. Trends Pharmacol Sci 32: 463-479 FDA. (2012) U.S. Food and Drug Administration. FDA approves new treatment for most common type of skin cancer. Silver Spring (MD). Fedarko N. S., Jain A., Karadag A., Van Eman M. R., Fisher L. W. (2001) Elevated serum bone sialoprotein and osteopontin in colon, breast, prostate, and lung cancer. Clin Cancer Res 7: 4060-4066 Franken N. A., Rodermond H. M., Stap J., Haveman J., van Bree C. (2006) Clonogenic assay of cells in vitro. Nat Protoc 1: 2315-2319 Fransen M., Nordgren M., Wang B., Apanasets O. (2012) Role of peroxisomes in ROS/RNS-metabolism: implications for human disease. Biochim Biophys Acta 1822: 1363-1373 Fu J., Rodova M., Roy S. K., Sharma J., Singh K. P., Srivastava R. K., Shankar S. (2013) GANT-61 inhibits pancreatic cancer stem cell growth in vitro and in NOD/SCID/IL2R gamma null mice xenograft. Cancer Lett 330: 22-32 Gailani M. R., Stahle-Backdahl M., Leffell D. J., Glynn M., Zaphiropoulos P. G., Pressman C., Unden A. B., Dean M., Brash D. E., Bale A. E., Toftgard R. (1996) The role of the human homologue of Drosophila patched in sporadic basal cell carcinomas. Nat Genet 14: 78-81 Galluzzi L., Vitale I., Abrams J. M., Alnemri E. S., Baehrecke E. H., Blagosklonny M. V., Dawson T. M., Dawson V. L., El-Deiry W. S., Fulda S., Gottlieb E., Green D. R., Hengartner M. O., Kepp O., Knight R. A., Kumar S., Lipton S. A., Lu X., Madeo F., Malorni W., Mehlen P., Nunez G., Peter M. E., Piacentini M., Rubinsztein D. C., Shi Y., 203

Simon H. U., Vandenabeele P., White E., Yuan J., Zhivotovsky B., Melino G., Kroemer G. (2012) Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ 19: 107-120 Gambacorti-Passerini C., le Coutre P., Mologni L., Fanelli M., Bertazzoli C., Marchesi E., Di Nicola M., Biondi A., Corneo G. M., Belotti D., Pogliani E., Lydon N. B. (1997) Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL+ leukemic cells and induces apoptosis. Blood Cells Mol Dis 23: 380-394 Goetz S. C., Anderson K. V. (2010) The primary cilium: a signalling centre during vertebrate development. Nat Rev Genet 11: 331-344 Goldstein A. M. (2011) Germline BAP1 mutations and tumor susceptibility. Nat Genet 43: 925-926 Goldstein L. J., Galski H., Fojo A., Willingham M., Lai S. L., Gazdar A., Pirker R., Green A., Crist W., Brodeur G. M., et al. (1989) Expression of a multidrug resistance gene in human cancers. J Natl Cancer Inst 81: 116-124 Gorlick R., Goker E., Trippett T., Steinherz P., Elisseyeff Y., Mazumdar M., Flintoff W. F., Bertino J. R. (1997) Defective transport is a common mechanism of acquired methotrexate resistance in acute lymphocytic leukemia and is associated with decreased reduced folate carrier expression. Blood 89: 1013-1018 Gorlin R. J. (2004) Nevoid basal cell carcinoma (Gorlin) syndrome. Genet Med 6: 530-539 Gorrini C., Harris I. S., Mak T. W. (2013) Modulation of oxidative stress as an anticancer strategy. Nat Rev Drug Discov 12: 931-947 Graham D. M., Mesrobian R. B. (1963) Oxidation of N,N-Dimethylaniline.2. Reaction with Oxygen Catalyzed by Benzoyl Peroxide. Can J Chem 41: 2945-& Grigoriu B. D., Scherpereel A., Devos P., Chahine B., Letourneux M., Lebailly P., Gregoire M., Porte H., Copin M. C., Lassalle P. (2007) Utility of osteopontin and serum mesothelin in malignant pleural mesothelioma diagnosis and prognosis assessment. Clin Cancer Res 13: 2928-2935 Grover V. K., Valadez J. G., Bowman A. B., Cooper M. K. (2011) Lipid modifications of Sonic hedgehog ligand dictate cellular reception and signal response. PLoS One 6: e21353 Guleng B., Tateishi K., Ohta M., Asaoka Y., Jazag A., Lin L. J., Tanaka Y., Tada M., Seto M., Kanai F., Kawabe T., Omata M. (2006) Smoothened gene mutations found in digestive cancer have no aberrant Hedgehog signaling activity. J Gastroenterol 41: 1238-1239 Gupta S. C., Hevia D., Patchva S., Park B., Koh W., Aggarwal B. B. (2012) Upsides and downsides of reactive oxygen species for cancer: the roles of reactive oxygen species in tumorigenesis, prevention, and therapy. Antioxid Redox Signal 16: 1295-1322 Gupta S., Takebe N., Lorusso P. (2010) Targeting the Hedgehog pathway in cancer. Ther Adv Med Oncol 2: 237-250 Haber S. E., Haber J. M. (2011) Malignant Mesothelioma: A Clinical Study of 238 Cases. Ind Health 49: 166-172 Hafner C., Schmiemann V., Ruetten A., Coras B., Landthaler M., Reifenberger J., Vogt T. (2007) PTCH mutations are not mainly involved in the pathogenesis of sporadic trichoblastomas. Hum Pathol 38: 1496-1500 204

Hahn H., Wicking C., Zaphiropoulous P. G., Gailani M. R., Shanley S., Chidambaram A., Vorechovsky I., Holmberg E., Unden A. B., Gillies S., Negus K., Smyth I., Pressman C., Leffell D. J., Gerrard B., Goldstein A. M., Dean M., Toftgard R., Chenevix-Trench G., Wainwright B., Bale A. E. (1996) Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 85: 841-851 Hahn W. C., Dessain S. K., Brooks M. W., King J. E., Elenbaas B., Sabatini D. M., DeCaprio J. A., Weinberg R. A. (2002) Enumeration of the simian virus 40 early region elements necessary for human cell transformation. Mol Cell Biol 22: 2111-2123 Han M. E., Lee Y. S., Baek S. Y., Kim B. S., Kim J. B., Oh S. O. (2009) Hedgehog signaling regulates the survival of gastric cancer cells by regulating the expression of Bcl- 2. Int J Mol Sci 10: 3033-3043 Hanahan D., Weinberg R. A. (2011) Hallmarks of cancer: the next generation. Cell 144: 646-674 Hanauske A. R., Chen V., Paoletti P., Niyikiza C. (2001) Pemetrexed disodium: a novel antifolate clinically active against multiple solid tumors. Oncologist 6: 363-373 He C., Klionsky D. J. (2009) Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 43: 67-93 Heisterkamp N., Stephenson J. R., Groffen J., Hansen P. F., de Klein A., Bartram C. R., Grosveld G. (1983) Localization of the c-ab1 oncogene adjacent to a translocation break point in chronic myelocytic leukaemia. Nature 306: 239-242 Helbock H. J., Beckman K. B., Ames B. N. (1999) 8-Hydroxydeoxyguanosine and 8- hydroxyguanine as biomarkers of oxidative DNA damage. Methods Enzymol 300: 156-166 Heretsch P., Buttner A., Tzagkaroulaki L., Zahn S., Kirchner B., Giannis A. (2011) Exocyclopamine--a stable and potent inhibitor of hedgehog-signaling. Chem Commun 47: 7362-7364 Hesson L. B., Cooper W. N., Latif F. (2007) Evaluation of the 3p21.3 tumour-suppressor gene cluster. Oncogene 26: 7283-7301 Hicks J. (2006) Biologic, cytogenetic, and molecular factors in mesothelial proliferations. Ultrastructural Pathology 30: 19-30 High A., Zedan W. (2005) Basal cell nevus syndrome. Curr Opin Oncol 17: 160-166 Hildeman D. A., Mitchell T., Aronow B., Wojciechowski S., Kappler J., Marrack P. (2003) Control of Bcl-2 expression by reactive oxygen species. Proc Natl Acad Sci U S A 100: 15035-15040 Holloway A. J., Diyagama D. S., Opeskin K., Creaney J., Robinson B. W., Lake R. A., Bowtell D. D. (2006) A molecular diagnostic test for distinguishing lung adenocarcinoma from malignant mesothelioma using cells collected from pleural effusions. Clin Cancer Res 12: 5129-5135 Hopkins-Donaldson S., Cathomas R., Simoes-Wust A. P., Kurtz S., Belyanskaya L., Stahel R. A., Zangemeister-Wittke U. (2003) Induction of apoptosis and chemosensitization of mesothelioma cells by Bcl-2 and Bcl-xL antisense treatment. Int J Cancer 106: 160-166 Huangfu D., Anderson K. V. (2005) Cilia and Hedgehog responsiveness in the mouse. Proc Natl Acad Sci U S A 102: 11325-11330 205

Huangfu D., Liu A., Rakeman A. S., Murcia N. S., Niswander L., Anderson K. V. (2003) Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature 426: 83-87 Huntzicker E. G., Estay I. S., Zhen H., Lokteva L. A., Jackson P. K., Oro A. E. (2006) Dual degradation signals control Gli protein stability and tumor formation. Genes Dev 20: 276-281 Hyman J. M., Firestone A. J., Heine V. M., Zhao Y., Ocasio C. A., Han K., Sun M., Rack P. G., Sinha S., Wu J. J., Solow-Cordero D. E., Jiang J., Rowitch D. H., Chen J. K. (2009) Small-molecule inhibitors reveal multiple strategies for Hedgehog pathway blockade. Proc Natl Acad Sci U S A 106: 14132-14137 IARC (2012) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: Arsenic, metals, fibres, and dusts. IARC Monogr Eval Carcinog Risks Hum 100: 311-316 Illei P. B., Rusch V. W., Zakowski M. F., Ladanyi M. (2003) Homozygous deletion of CDKN2A and codeletion of the methylthioadenosine phosphorylase gene in the majority of pleural mesotheliomas. Clin Cancer Res 9: 2108-2113 Imazu T., Shimizu S., Tagami S., Matsushima M., Nakamura Y., Miki T., Okuyama A., Tsujimoto Y. (1999) Bcl-2/E1B 19 kda-interacting protein 3-like protein (Bnip3L) interacts with bcl-2/bcl-xl and induces apoptosis by altering mitochondrial membrane permeability. Oncogene 18: 4523-4529 Inai K. () Pathology of mesothelioma. Environ Health Prev Med 13: 60-64 Incardona J. P., Gaffield W., Kapur R. P., Roelink H. (1998) The teratogenic Veratrum alkaloid cyclopamine inhibits sonic hedgehog signal transduction. Development 125: 3553-3562 Incardona J. P., Gruenberg J., Roelink H. (2002) Sonic hedgehog induces the segregation of patched and smoothened in endosomes. Curr Biol 12: 983-995 Ingham PW, McMahon AP (2001) Hedgehog signaling in animal development: paradigms and principles. Genes Dev 15: 3059-3087 Ingham PW, Nystedt S, Nakano Y, Brown W, Stark D, van den Heuvel M, Taylor AM (2000) Patched represses the Hedgehog signalling pathway by promoting modification of the Smoothened protein. Curr Biol 10: 1315-1318 Ishikawa H., Marshall W. F. (2011) Ciliogenesis: building the cell's antenna. Nat Rev Mol Cell Biol 12: 222-234 Ivanov S. V., Ivanova A. V., Goparaju C. M., Chen Y., Beck A., Pass H. I. (2009) Tumorigenic properties of alternative osteopontin isoforms in mesothelioma. Biochem Biophys Res Commun 382: 514-518 Iwai-Kanai E., Yuan H., Huang C., Sayen M. R., Perry-Garza C. N., Kim L., Gottlieb R. A. () A method to measure cardiac autophagic flux in vivo. Autophagy 4: 322-329 Janssen-Heininger Y. M., Mossman B. T., Heintz N. H., Forman H. J., Kalyanaraman B., Finkel T., Stamler J. S., Rhee S. G., van der Vliet A. () Redox-based regulation of signal transduction: principles, pitfalls, and promises. Free Radic Biol Med 45: 1-17 Jassem J, Ramlau R, Santoro A, Schuette W, Chemaissani A, Hong S, Blatter J, Adachi S, Hanauske A, Manegold C () Phase III trial of pemetrexed plus best supportive care compared with best supportive care in previously treated patients with advanced malignant pleural mesothelioma. J Clin Oncol 26: 1698-1704 206

Jenkins P, Milliner R, Salmon C (2011) Re-evaluating the role of palliative radiotherapy in malignant pleural mesothelioma. Eur J Cancer Jensen D. E., Proctor M., Marquis S. T., Gardner H. P., Ha S. I., Chodosh L. A., Ishov A. M., Tommerup N., Vissing H., Sekido Y., Minna J., Borodovsky A., Schultz D. C., Wilkinson K. D., Maul G. G., Barlev N., Berger S. L., Prendergast G. C., Rauscher F. J., 3rd (1998) BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression. Oncogene 16: 1097-1112 Jia J., Tong C., Wang B., Luo L., Jiang J. (2004) Hedgehog signalling activity of Smoothened requires phosphorylation by protein kinase A and casein kinase I. Nature 432: 1045-1050 Jiang F., Zhang Y., Dusting G. J. (2011) NADPH oxidase-mediated redox signaling: roles in cellular stress response, stress tolerance, and tissue repair. Pharmacol Rev 63: 218-242 Jiang J, Hui CC () Hedgehog signaling in development and cancer. Dev Cell 15: 801-812 Jiao X., Wood L. D., Lindman M., Jones S., Buckhaults P., Polyak K., Sukumar S., Carter H., Kim D., Karchin R., Sjoblom T. (2012) Somatic mutations in the Notch, NF-KB, PIK3CA, and Hedgehog pathways in human breast cancers. Genes Chromosomes Cancer 51: 480-489 Jimenez-Sanchez M., Menzies F. M., Chang Y. Y., Simecek N., Neufeld T. P., Rubinsztein D. C. (2012) The Hedgehog signalling pathway regulates autophagy. Nat Commun 3: 1200 Johnson R. L., Milenkovic L., Scott M. P. (2000) In vivo functions of the patched protein: requirement of the C terminus for target gene inactivation but not Hedgehog sequestration. Mol Cell 6: 467-478 Johnson R. L., Rothman A. L., Xie J., Goodrich L. V., Bare J. W., Bonifas J. M., Quinn A. G., Myers R. M., Cox D. R., Epstein E. H., Jr., Scott M. P. (1996) Human homolog of patched, a candidate gene for the basal cell nevus syndrome. Science 272: 1668-1671 Jones S, Zhang X, Parsons DW, Lin JC, Leary RJ, Angenendt P, Mankoo P, Carter H, Kamiyama H, Jimeno A, Hong SM, Fu B, Lin MT, Calhoun ES, Kamiyama M, Walter K, Nikolskaya T, Nikolsky Y, Hartigan J, Smith DR, Hidalgo M, Leach SD, Klein AP, Jaffee EM, Goggins M, Maitra A, Iacobuzio-Donahue C, Eshleman JR, Kern SE, Hruban RH, Karchin R, Papadopoulos N, Parmigiani G, Vogelstein B, Velculescu VE, Kinzler KW () Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 321: 1801-1806 Joshi T. K., Gupta R. K. (2004) Asbestos in developing countries: magnitude of risk and its practical implications. Int J Occup Med Environ Health 17: 179-185 Kagawa H., Shino Y., Kobayashi D., Demizu S., Shimada M., Ariga H., Kawahara H. (2011) A novel signaling pathway mediated by the nuclear targeting of C-terminal fragments of mammalian Patched 1. PLoS One 6: e18638 Kalmadi S. R., Rankin C., Kraut M. J., Jacobs A. D., Petrylak D. P., Adelstein D. J., Keohan M. L., Taub R. N., Borden E. C. () Gemcitabine and cisplatin in unresectable malignant mesothelioma of the pleura: a phase II study of the Southwest Oncology Group (SWOG 9810). Lung Cancer 60: 259-263 Kamada S., Shimono A., Shinto Y., Tsujimura T., Takahashi T., Noda T., Kitamura Y., Kondoh H., Tsujimoto Y. (1995) bcl-2 deficiency in mice leads to pleiotropic 207

abnormalities: accelerated lymphoid cell death in thymus and spleen, polycystic kidney, hair hypopigmentation, and distorted small intestine. Cancer Res 55: 354-359 Kameda C., Nakamura M., Tanaka H., Yamasaki A., Kubo M., Tanaka M., Onishi H., Katano M. (2010) Oestrogen receptor-alpha contributes to the regulation of the hedgehog signalling pathway in ERalpha-positive gastric cancer. Br J Cancer 102: 738-747 Kan Z., Jaiswal B. S., Stinson J., Janakiraman V., Bhatt D., Stern H. M., Yue P., Haverty P. M., Bourgon R., Zheng J., Moorhead M., Chaudhuri S., Tomsho L. P., Peters B. A., Pujara K., Cordes S., Davis D. P., Carlton V. E., Yuan W., Li L., Wang W., Eigenbrot C., Kaminker J. S., Eberhard D. A., Waring P., Schuster S. C., Modrusan Z., Zhang Z., Stokoe D., de Sauvage F. J., Faham M., Seshagiri S. (2010) Diverse somatic mutation patterns and pathway alterations in human cancers. Nature 466: 869-873 Kao S. C., Lee K., Armstrong N. J., Clarke S., Vardy J., van Zandwijk N., Reid G., Burn J., McCaughan B. C., Henderson D. W., Klebe S. (2011) Validation of tissue microarray technology in malignant pleural mesothelioma. Pathology 43: 128-132 Karhadkar S. S., Bova G. S., Abdallah N., Dhara S., Gardner D., Maitra A., Isaacs J. T., Berman D. M., Beachy P. A. (2004) Hedgehog signalling in prostate regeneration, neoplasia and metastasis. Nature 431: 707-712 Kasperczyk H., Baumann B., Debatin K. M., Fulda S. (2009) Characterization of sonic hedgehog as a novel NF-kappaB target gene that promotes NF-kappaB-mediated apoptosis resistance and tumor growth in vivo. FASEB J 23: 21-33 Katoh Y., Katoh M. (2009) Hedgehog target genes: mechanisms of carcinogenesis induced by aberrant hedgehog signaling activation. Curr Mol Med 9: 873-886 Kaufman A. J., Flores R. M. (2011) Surgical treatment of malignant pleural mesothelioma. Curr Treat Options Oncol 12: 201-216 Kaur H., Halliwell B. (1994) Evidence for nitric oxide-mediated oxidative damage in chronic inflammation. Nitrotyrosine in serum and synovial fluid from rheumatoid patients. FEBS Lett 350: 9-12 Kawabata N., Ijiri K., Ishidou Y., Yamamoto T., Nagao H., Nagano S., Maeda S., Komiya S., Setoguchi T. (2011) Pharmacological inhibition of the Hedgehog pathway prevents human rhabdomyosarcoma cell growth. Int J Oncol 39: 899-906 Kaye S. B., Fehrenbacher L., Holloway R., Amit A., Karlan B., Slomovitz B., Sabbatini P., Fu L., Yauch R. L., Chang I., Reddy J. C. (2012) A phase II, randomized, placebocontrolled study of vismodegib as maintenance therapy in patients with ovarian cancer in second or third complete remission. Clin Cancer Res 18: 6509-6518 Kenney A. M., Rowitch D. H. (2000) Sonic hedgehog promotes G(1) cyclin expression and sustained cell cycle progression in mammalian neuronal precursors. Mol Cell Biol 20: 9055-9067 Kim C., Kim J. Y., Kim J. H. () Cytosolic phospholipase A(2), lipoxygenase metabolites, and reactive oxygen species. BMB Rep 41: 555-559 Kim J., Lee J. J., Gardner D., Beachy P. A. (2010a) Arsenic antagonizes the Hedgehog pathway by preventing ciliary accumulation and reducing stability of the Gli2 transcriptional effector. Proc Natl Acad Sci U S A 107: 13432-13437 Kim J., Lee J. J., Kim J., Gardner D., Beachy P. A. (2010b) Arsenic antagonizes the Hedgehog pathway by preventing ciliary accumulation and reducing stability of the Gli2 transcriptional effector. Proc Natl Acad Sci U S A 107: 13432-13437 208

Kindler H. L. () Systemic treatments for mesothelioma: standard and novel. Curr Treat Options Oncol 9: 171-179 King M. P., Attardi G. (1989) Human cells lacking mtdna: repopulation with exogenous mitochondria by complementation. Science 246: 500-503 Kinzler K. W., Bigner S. H., Bigner D. D., Trent J. M., Law M. L., O'Brien S. J., Wong A. J., Vogelstein B. (1987) Identification of an amplified, highly expressed gene in a human glioma. Science 236: 70-73 Kinzler K. W., Ruppert J. M., Bigner S. H., Vogelstein B. (1988) The GLI gene is a member of the Kruppel family of zinc finger proteins. Nature 332: 371-374 Kinzler K. W., Vogelstein B. (1990) The GLI gene encodes a nuclear protein which binds specific sequences in the human genome. Mol Cell Biol 10: 634-642 Klionsky D. J. (2007) Autophagy: from phenomenology to molecular understanding in less than a decade. Nat Rev Mol Cell Biol 8: 931-937 Klionsky D. J., Emr S. D. (2000) Autophagy as a regulated pathway of cellular degradation. Science 290: 1717-1721 Kogerman P., Grimm T., Kogerman L., Krause D., Unden A. B., Sandstedt B., Toftgard R., Zaphiropoulos P. G. (1999) Mammalian suppressor-of-fused modulates nuclearcytoplasmic shuttling of Gli-1. Nat Cell Biol 1: 312-319 Kong Q., Beel J. A., Lillehei K. O. (2000) A threshold concept for cancer therapy. Med Hypotheses 55: 29-35 Kong Q., Lillehei K. O. (1998) Antioxidant inhibitors for cancer therapy. Med Hypotheses 51: 405-409 Kovac V., Zwitter M., Rajer M., Marin A., Debeljak A., Smrdel U., Vrankar M. (2012) A phase II trial of low-dose gemcitabine in a prolonged infusion and cisplatin for malignant pleural mesothelioma. Anticancer Drugs 23: 230-238 Kroemer G., Levine B. () Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol 9: 1004-1010 Kroemer G., Reed J. C. (2000) Mitochondrial control of cell death. Nat Med 6: 513-519 Kudo K., Gavin E., Das S., Amable L., Shevde L. A., Reed E. (2012) Inhibition of Gli1 results in altered c-jun activation, inhibition of cisplatin-induced upregulation of ERCC1, XPD and XRCC1, and inhibition of platinum-dna adduct repair. Oncogene 31: 4718-4724 Kundu M., Lindsten T., Yang C. Y., Wu J., Zhao F., Zhang J., Selak M. A., Ney P. A., Thompson C. B. () Ulk1 plays a critical role in the autophagic clearance of mitochondria and ribosomes during reticulocyte maturation. Blood 112: 1493-1502 Lai G. M., Ozols R. F., Smyth J. F., Young R. C., Hamilton T. C. (1988) Enhanced DNA repair and resistance to cisplatin in human ovarian cancer. Biochem Pharmacol 37: 4597-4600 Lam C. W., Xie J., To K. F., Ng H. K., Lee K. C., Yuen N. W., Lim P. L., Chan L. Y., Tong S. F., McCormick F. (1999) A frequent activated smoothened mutation in sporadic basal cell carcinomas. Oncogene 18: 833-836 209

Lauth M., Bergstrom A., Shimokawa T., Toftgard R. (2007) Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists. Proc Natl Acad Sci U S A 104: 8455-8460 Lauth M., Toftgard R. (2007) Non-canonical activation of GLI transcription factors: implications for targeted anti-cancer therapy. Cell Cycle 6: 2458-2463 Lavie Y., Harel-Orbital T., Gaffield W., Liscovitch M. (2001) Inhibitory effect of steroidal alkaloids on drug transport and multidrug resistance in human cancer cells. Anticancer Res 21: 1189-1194 Le G. V., Takahashi K., Park E. K., Delgermaa V., Oak C., Qureshi A. M., Aljunid S. M. (2011) Asbestos use and asbestos-related diseases in Asia: past, present and future. Respirology 16: 767-775 Lee JJ, Ekker SC, von Kessler DP, Porter JA, Sun BI, Beachy PA (1994) Autoproteolysis in hedgehog protein biogenesis. Science 266: 1528-1537 Lee Y., Miller H. L., Jensen P., Hernan R., Connelly M., Wetmore C., Zindy F., Roussel M. F., Curran T., Gilbertson R. J., McKinnon P. J. (2003) A molecular fingerprint for medulloblastoma. Cancer Res 63: 5428-5437 Lee Y., Miller H. L., Russell H. R., Boyd K., Curran T., McKinnon P. J. (2006) Patched2 modulates tumorigenesis in patched1 heterozygous mice. Cancer Res 66: 6964-6971 Lemasters J. J., Nieminen A. L., Qian T., Trost L. C., Elmore S. P., Nishimura Y., Crowe R. A., Cascio W. E., Bradham C. A., Brenner D. A., Herman B. (1998) The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta 1366: 177-196 Levine B., Yuan J. (2005) Autophagy in cell death: an innocent convict? J Clin Invest 115: 2679-2688 Li D., Duell E. J., Yu K., Risch H. A., Olson S. H., Kooperberg C., Wolpin B. M., Jiao L., Dong X., Wheeler B., Arslan A. A., Bueno-de-Mesquita H. B., Fuchs C. S., Gallinger S., Gross M., Hartge P., Hoover R. N., Holly E. A., Jacobs E. J., Klein A. P., LaCroix A., Mandelson M. T., Petersen G., Zheng W., Agalliu I., Albanes D., Boutron-Ruault M. C., Bracci P. M., Buring J. E., Canzian F., Chang K., Chanock S. J., Cotterchio M., Gaziano J. M., Giovannucci E. L., Goggins M., Hallmans G., Hankinson S. E., Hoffman Bolton J. A., Hunter D. J., Hutchinson A., Jacobs K. B., Jenab M., Khaw K. T., Kraft P., Krogh V., Kurtz R. C., McWilliams R. R., Mendelsohn J. B., Patel A. V., Rabe K. G., Riboli E., Shu X. O., Tjonneland A., Tobias G. S., Trichopoulos D., Virtamo J., Visvanathan K., Watters J., Yu H., Zeleniuch-Jacquotte A., Amundadottir L., Stolzenberg-Solomon R. Z. (2012a) Pathway analysis of genome-wide association study data highlights pancreatic development genes as susceptibility factors for pancreatic cancer. Carcinogenesis 33: 1384-1390 Li H., Li J., Li Y., Singh P., Cao L., Xu L. J., Li D., Wang Y., Xie Z., Gui Y., Zheng X. L. (2012b) Sonic hedgehog promotes autophagy of vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 303: H1319-1331 Li H., Lui N., Cheng T., Tseng H. H., Yue D., Giroux-Leprieur E., Do H. T., Sheng Q., Jin J. Q., Luh T. W., Jablons D. M., He B. (2013) Gli as a novel therapeutic target in malignant pleural mesothelioma. PLoS One 8: e57346 Liboutet M., Portela M., Delestaing G., Vilmer C., Dupin N., Gorin I., Saiag P., Lebbe C., Kerob D., Dubertret L., Grandchamp B., Basset-Seguin N., Soufir N. (2006) MC1R and PTCH gene polymorphism in French patients with basal cell carcinomas. J Invest Dermatol 126: 1510-1517 210

Lim C. B., Fu P. Y., Ky N., Zhu H. S., Feng X., Li J., Srinivasan K. G., Hamza M. S., Zhao Y. (2012) NF-kappaB p65 repression by the sesquiterpene lactone, Helenalin, contributes to the induction of autophagy cell death. BMC Complement Altern Med 12: 93 Lin T. L., Matsui W. (2012) Hedgehog pathway as a drug target: Smoothened inhibitors in development. Onco Targets Ther 5: 47-58 Lindstrom E., Shimokawa T., Toftgard R., Zaphiropoulos P. G. (2006) PTCH mutations: distribution and analyses. Hum Mutat 27: 215-219 Ling Y. H., Liebes L., Zou Y., Perez-Soler R. (2003) Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic response to Bortezomib, a novel proteasome inhibitor, in human H460 non-small cell lung cancer cells. J Biol Chem 278: 33714-33723 Lipinski R. J., Gipp J. J., Zhang J., Doles J. D., Bushman W. (2006) Unique and complimentary activities of the Gli transcription factors in Hedgehog signaling. Exp Cell Res 312: 1925-1938 Lipinski R. J., Hutson P. R., Hannam P. W., Nydza R. J., Washington I. M., Moore R. W., Girdaukas G. G., Peterson R. E., Bushman W. () Dose- and route-dependent teratogenicity, toxicity, and pharmacokinetic profiles of the hedgehog signaling antagonist cyclopamine in the mouse. Toxicol Sci 104: 189-197 Livak K. J., Schmittgen T. D. (2001) Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402-408 Lo Muzio L. () Nevoid basal cell carcinoma syndrome (Gorlin syndrome). Orphanet J Rare Dis 3: 32 LoRusso P. M., Rudin C. M., Reddy J. C., Tibes R., Weiss G. J., Borad M. J., Hann C. L., Brahmer J. R., Chang I., Darbonne W. C., Graham R. A., Zerivitz K. L., Low J. A., Von Hoff D. D. (2011) Phase I trial of hedgehog pathway inhibitor vismodegib (GDC-0449) in patients with refractory, locally advanced or metastatic solid tumors. Clin Cancer Res 17: 2502-2511 Lu X., Liu S., Kornberg T. B. (2006) The C-terminal tail of the Hedgehog receptor Patched regulates both localization and turnover. Genes Dev 20: 2539-2551 Lum J. J., DeBerardinis R. J., Thompson C. B. (2005) Autophagy in metazoans: cell survival in the land of plenty. Nat Rev Mol Cell Biol 6: 439-448 Maesawa C., Tamura G., Iwaya T., Ogasawara S., Ishida K., Sato N., Nishizuka S., Suzuki Y., Ikeda K., Aoki K., Saito K., Satodate R. (1998) Mutations in the human homologue of the Drosophila patched gene in esophageal squamous cell carcinoma. Genes Chromosomes Cancer 21: 276-279 Mahindroo N., Punchihewa C., Fujii N. (2009) Hedgehog-Gli signaling pathway inhibitors as anticancer agents. J Med Chem 52: 3829-3845 Majander A., Finel M., Wikstrom M. (1994) Diphenyleneiodonium inhibits reduction of iron-sulfur clusters in the mitochondrial NADH-ubiquinone oxidoreductase (Complex I). J Biol Chem 269: 21037-21042 Manda Gina, Nechifor Marina T, Neagu Teodora-Monica (2009) Reactive oxygen species, cancer and anti-cancer therapies. Curr Chem Biol 3: 22-46 Manning L. S., Whitaker D., Murch A. R., Garlepp M. J., Davis M. R., Musk A. W., Robinson B. W. (1991) Establishment and characterization of five human malignant mesothelioma cell lines derived from pleural effusions. Int J Cancer 47: 285-290 211

Marigo V., Davey R. A., Zuo Y., Cunningham J. M., Tabin C. J. (1996) Biochemical evidence that patched is the Hedgehog receptor. Nature 384: 176-179 Martin S. T., Sato N., Dhara S., Chang R., Hustinx S. R., Abe T., Maitra A., Goggins M. (2005) Aberrant methylation of the Human Hedgehog interacting protein (HHIP) gene in pancreatic neoplasms. Cancer Biol Ther 4: 728-733 Matherly L. H., Hou Z., Deng Y. (2007) Human reduced folate carrier: translation of basic biology to cancer etiology and therapy. Cancer Metastasis Rev 26: 111-128 Mazumdar T., Devecchio J., Agyeman A., Shi T., Houghton J. A. (2011a) Blocking Hedgehog survival signaling at the level of the GLI genes induces DNA damage and extensive cell death in human colon carcinoma cells. Cancer Res 71: 5904 5914 Mazumdar T., DeVecchio J., Shi T., Jones J., Agyeman A., Houghton J. A. (2011b) Hedgehog signaling drives cellular survival in human colon carcinoma cells. Cancer Res 71: 1092-1102 McAleer M. F., Tsao A. S., Liao Z. (2009) Radiotherapy in malignant pleural mesothelioma. Int J Radiat Oncol Biol Phys 75: 326-337 McGarvey T. W., Maruta Y., Tomaszewski J. E., Linnenbach A. J., Malkowicz S. B. (1998) PTCH gene mutations in invasive transitional cell carcinoma of the bladder. Oncogene 17: 1167-1172 McLendon R, Friedman A, Bigner D, Van Meir EG, Brat DJ, Mastrogianakis GM, Olson JJ Mikkelsen T, Lehman N, Aldape K, Yung WK, Bogler O, Weinstein JN, VandenBerg S Berger M, Prados M, Muzny D, Morgan M, Scherer S, Sabo A, Nazareth L, Lewis L, Hall O, Zhu Y, Ren Y, Alvi O, Yao J, Hawes A, Jhangiani S, Fowler G, San Lucas A, Kovar C, Cree A, Dinh H, Santibanez J, Joshi V, Gonzalez-Garay ML, Miller CA, Milosavljevic A, Donehower L, Wheeler DA, Gibbs RA, Cibulskis K, Sougnez C, Fennell T, Mahan S, Wilkinson J, Ziaugra L, Onofrio R, Bloom T, Nicol R, Ardlie K, Baldwin J, Gabriel S, Lander ES, Ding L, Fulton RS, McLellan MD, Wallis J, Larson DE, Shi X, Abbott R, Fulton L, Chen K, Koboldt DC, Wendl MC, Meyer R, Tang Y, Lin L, Osborne JR, Dunford-Shore BH, Miner TL, Delehaunty K, Markovic C, Swift G, Courtney W, Pohl C, Abbott S, Hawkins A, Leong S, Haipek C, Schmidt H, Wiechert M, Vickery T, Scott S, Dooling DJ, Chinwalla A, Weinstock GM, Mardis ER, Wilson RK, Getz G, Winckler W, Verhaak RG, Lawrence MS, O'Kelly M, Robinson J, Alexe G, Beroukhim R, Carter S, Chiang D, Gould J, Gupta S, Korn J, Mermel C, Mesirov J, Monti S, Nguyen H, Parkin M, Reich M, Stransky N, Weir BA, Garraway L, Golub T, Meyerson M, Chin L, Protopopov A, Zhang J, Perna I, Aronson S, Sathiamoorthy N, Ren G, Yao J, Wiedemeyer WR, Kim H, Kong SW, Xiao Y, Kohane IS, Seidman J, Park PJ, Kucherlapati R, Laird PW, Cope L, Herman JG, Weisenberger DJ, Pan F, Van den Berg D, Van Neste L, Yi JM, Schuebel KE, Baylin SB, Absher DM, Li JZ, Southwick A, Brady S, Aggarwal A, Chung T, Sherlock G, Brooks JD, Myers RM, Spellman PT, Purdom E, Jakkula LR, Lapuk AV, Marr H, Dorton S, Choi YG, Han J, Ray A, Wang V, Durinck S, Robinson M, Wang NJ, Vranizan K, Peng V, Van Name E, Fontenay GV, Ngai J, Conboy JG, Parvin B, Feiler HS, Speed TP, Gray JW, Brennan C, Socci ND, Olshen A, Taylor BS, Lash A, Schultz N, Reva B, Antipin Y, Stukalov A, Gross B, Cerami E, Wang WQ, Qin LX, Seshan VE, Villafania L, Cavatore M, Borsu L, Viale A, Gerald W, Sander C, Ladanyi M, Perou CM, Hayes DN, Topal MD, Hoadley KA, Qi Y, Balu S, Shi Y, Wu J, Penny R, Bittner M, Shelton T, Lenkiewicz E, Morris S, Beasley D, Sanders S, Kahn A, Sfeir R, Chen J, Nassau D, Feng L, Hickey E, Barker A, Gerhard DS, Vockley J, Compton C, Vaught J, Fielding P, Ferguson ML, Schaefer C, Zhang J, Madhavan S, Buetow KH, Collins F, Good P, Guyer M, Ozenberger B, Peterson J, Thomson E. () Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455: 1061-1068 212

Meloni A. R., Fralish G. B., Kelly P., Salahpour A., Chen J. K., Wechsler-Reya R. J., Lefkowitz R. J., Caron M. G. (2006) Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2. Mol Cell Biol 26: 7550-7560 Metintas M., Metintas S., Ucgun I., Gibbs A. R., Harmanci E., Alatas F., Erginel S., Tel N., Pasaoglu O. (2001) Prognostic factors in diffuse malignant pleural mesothelioma: effects of pretreatment clinical and laboratory characteristics. Respir Med 95: 829-835 Meyers-Needham M., Lewis J. A., Gencer S., Sentelle R. D., Saddoughi S. A., Clarke C. J., Hannun Y. A., Norell H., da Palma T. M., Nishimura M., Kraveka J. M., Khavandgar Z., Murshed M., Cevik M. O., Ogretmen B. (2012) Off-target function of the Sonic hedgehog inhibitor cyclopamine in mediating apoptosis via nitric oxide-dependent neutral sphingomyelinase 2/ceramide induction. Mol Cancer Ther 11: 1092-1102 Michimukai E., Kitamura N., Zhang Y., Wang H., Hiraishi Y., Sumi K., Hayashido Y., Toratani S., Okamoto T. (2001) Mutations in the human homologue of the Drosophila segment polarity gene patched in oral squamous cell carcinoma cell lines. In Vitro Cell Dev Biol - Animal 37: 459-464 Minami T., Adachi M., Kawamura R., Zhang Y., Shinomura Y., Imai K. (2005) Sulindac enhances the proteasome inhibitor bortezomib-mediated oxidative stress and anticancer activity. Clin Cancer Res 11: 5248-5256 Misaghi S., Ottosen S., Izrael-Tomasevic A., Arnott D., Lamkanfi M., Lee J., Liu J., O'Rourke K., Dixit V. M., Wilson A. C. (2009) Association of C-terminal ubiquitin hydrolase BRCA1-associated protein 1 with cell cycle regulator host cell factor 1. Mol Cell Biol 29: 2181-2192 Mizushima N. (2007) Autophagy: process and function. Genes Dev 21: 2861-2873 Mizushima N., Levine B. (2010) Autophagy in mammalian development and differentiation. Nat Cell Biol 12: 823-830 Montezano A. C., Touyz R. M. (2012) Reactive oxygen species and endothelial function-- role of nitric oxide synthase uncoupling and Nox family nicotinamide adenine dinucleotide phosphate oxidases. Basic Clin Pharmacol Toxicol 110: 87-94 Moolgavkar S. H., Meza R., Turim J. (2009) Pleural and peritoneal mesotheliomas in SEER: age effects and temporal trends, 1973-2005. Cancer Causes Control 20: 935-944 Mossman B., Light W., Wei E. (1983) Asbestos: mechanisms of toxicity and carcinogenicity in the respiratory tract. Annu Rev Pharmacol Toxicol 23: 595-615 Motoyama J., Milenkovic L., Iwama M., Shikata Y., Scott M. P., Hui C. C. (2003) Differential requirement for Gli2 and Gli3 in ventral neural cell fate specification. Dev Biol 259: 150-161 Mujoomdar A. A., Tilleman T. R., Richards W. G., Bueno R., Sugarbaker D. J. (2010) Prevalence of in vitro chemotherapeutic drug resistance in primary malignant pleural mesothelioma: result in a cohort of 203 resection specimens. J Thorac Cardiovasc Surg 140: 352-355 Murone M, Rosenthal A, de Sauvage FJ (1999) Sonic hedgehog signaling by the patchedsmoothened receptor complex. Curr Biol 9: 76-84 Murone M., Luoh S. M., Stone D., Li W., Gurney A., Armanini M., Grey C., Rosenthal A., de Sauvage F. J. (2000) Gli regulation by the opposing activities of fused and suppressor of fused. Nat Cell Biol 2: 310-312 213

Murrow L., Debnath J. (2013) Autophagy as a stress-response and quality-control mechanism: implications for cell injury and human disease. Annu Rev Pathol 8: 105-137 Murthy S. S., Testa J. R. (1999) Asbestos, chromosomal deletions, and tumor suppressor gene alterations in human malignant mesothelioma. J Cell Physiol 180: 150-157 Nagao H., Ijiri K., Hirotsu M., Ishidou Y., Yamamoto T., Nagano S., Takizawa T., Nakashima K., Komiya S., Setoguchi T. (2011) Role of GLI2 in the growth of human osteosarcoma. J Pathol 224: 169-179 Neumann V., Rutten A., Scharmach M., Muller K. M., Fischer M. (2004) Factors influencing long-term survival in mesothelioma patients - results of the German mesothelioma register. Int Arch Occup Environ Health 77: 191-199 Ng P. C., Henikoff S. (2003) SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res 31: 3812-3814 Nilsonne G., Sun X., Nystrom C., Rundlof A. K., Potamitou Fernandes A., Bjornstedt M., Dobra K. (2006) Selenite induces apoptosis in sarcomatoid malignant mesothelioma cells through oxidative stress. Free Radic Biol Med 41: 874-885 Njauw C. N., Kim I., Piris A., Gabree M., Taylor M., Lane A. M., DeAngelis M. M., Gragoudas E., Duncan L. M., Tsao H. (2012) Germline BAP1 inactivation is preferentially associated with metastatic ocular melanoma and cutaneous-ocular melanoma families. PLoS One 7: e35295 Nojiri S., Gemba K., Aoe K., Kato K., Yamaguchi T., Sato T., Kubota K., Kishimoto T. (2011) Survival and Prognostic Factors in Malignant Pleural Mesothelioma: A Retrospective Study of 314 patients in the West Part of Japan. Jpn J Clin Oncol 41: 32-39 Nowak A. K. (2012) Chemotherapy for malignant pleural mesothelioma: a review of current management and a look to the future. Ann Cardiothorac Surg 1: 508-515 Nowak AK, Byrne MJ, Williamson R, Ryan G, Segal A, Fielding D, Mitchell P, Musk AW, Robinson BW (2002) A multicentre phase II study of cisplatin and gemcitabine for malignant mesothelioma. Br J Cancer 87: 491-496 Nurse P. (2000) A long twentieth century of the cell cycle and beyond. Cell 100: 71-78 O'Brien S. G., Guilhot F., Larson R. A., Gathmann I., Baccarani M., Cervantes F., Cornelissen J. J., Fischer T., Hochhaus A., Hughes T., Lechner K., Nielsen J. L., Rousselot P., Reiffers J., Saglio G., Shepherd J., Simonsson B., Gratwohl A., Goldman J. M., Kantarjian H., Taylor K., Verhoef G., Bolton A. E., Capdeville R., Druker B. J. (2003) Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 348: 994-1004 O'Kane S. L., Pound R. J., Campbell A., Chaudhuri N., Lind M. J., Cawkwell L. (2006) Expression of bcl-2 family members in malignant pleural mesothelioma. Acta Oncol 45: 449-453 Oeste C. L., Seco E., Patton W. F., Boya P., Perez-Sala D. (2013) Interactions between autophagic and endo-lysosomal markers in endothelial cells. Histochem Cell Biol 139: 659-670 Ohashi R., Tajima K., Takahashi F., Cui R., Gu T., Shimizu K., Nishio K., Fukuoka K., Nakano T., Takahashi K. (2009) Osteopontin modulates malignant pleural mesothelioma cell functions in vitro. Anticancer Res 29: 2205-2214 214

Ohki K., Kumamoto H., Ichinohasama R., Sato T., Takahashi N., Ooya K. (2004) PTC gene mutations and expression of SHH, PTC, SMO and GLI-1 in odontogenic keratocysts. Int J Oral Maxillofac Surg 33: 584-592 Oldberg A., Franzen A., Heinegard D. (1986) Cloning and sequence analysis of rat bone sialoprotein (osteopontin) cdna reveals an Arg-Gly-Asp cell-binding sequence. Proc Natl Acad Sci U S A 83: 8819-8823 Oliver M. H., Harrison N. K., Bishop J. E., Cole P. J., Laurent G. J. (1989) A rapid and convenient assay for counting cells cultured in microwell plates: application for assessment of growth factors. J Cell Sci 92 ( Pt 3): 513-518 Opferman J. T., Iwasaki H., Ong C. C., Suh H., Mizuno S., Akashi K., Korsmeyer S. J. (2005) Obligate role of anti-apoptotic MCL-1 in the survival of hematopoietic stem cells. Science 307: 1101-1104 Opferman J. T., Letai A., Beard C., Sorcinelli M. D., Ong C. C., Korsmeyer S. J. (2003) Development and maintenance of B and T lymphocytes requires antiapoptotic MCL-1. Nature 426: 671-676 Oro A. E. (2007) The primary cilia, a 'Rab-id' transit system for hedgehog signaling. Curr Opin Cell Biol 19: 691-696 Oro A. E., Higgins K. M., Hu Z., Bonifas J. M., Epstein E. H., Jr., Scott M. P. (1997) Basal cell carcinomas in mice overexpressing sonic hedgehog. Science 276: 817-821 Orrenius S., Gogvadze V., Zhivotovsky B. (2007) Mitochondrial oxidative stress: implications for cell death. Annu Rev Pharmacol Toxicol 47: 143-183 Ozvaran M. K., Cao X. X., Miller S. D., Monia B. A., Hong W. K., Smythe W. R. (2004) Antisense oligonucleotides directed at the bcl-xl gene product augment chemotherapy response in mesothelioma. Mol Cancer Ther 3: 545-550 Paglin S., Hollister T., Delohery T., Hackett N., McMahill M., Sphicas E., Domingo D., Yahalom J. (2001) A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res 61: 439-444 Palma V., Lim D. A., Dahmane N., Sanchez P., Brionne T. C., Herzberg C. D., Gitton Y., Carleton A., Alvarez-Buylla A., Ruiz i Altaba A. (2005) Sonic hedgehog controls stem cell behavior in the postnatal and adult brain. Development 132: 335-344 Pan D., Li Y., Li Z., Wang Y., Wang P., Liang Y. (2012) Gli inhibitor GANT61 causes apoptosis in myeloid leukemia cells and acts in synergy with rapamycin. Leuk Res 36: 742-748 Pan Y., Bai C. B., Joyner A. L., Wang B. (2006) Sonic hedgehog signaling regulates Gli2 transcriptional activity by suppressing its processing and degradation. Mol Cell Biol 26: 3365-3377 Parker R. J., Eastman A., Bostick-Bruton F., Reed E. (1991) Acquired cisplatin resistance in human ovarian cancer cells is associated with enhanced repair of cisplatin-dna lesions and reduced drug accumulation. J Clin Invest 87: 772-777 Parmantier E., Lynn B., Lawson D., Turmaine M., Namini S. S., Chakrabarti L., McMahon A. P., Jessen K. R., Mirsky R. (1999) Schwann cell-derived Desert hedgehog controls the development of peripheral nerve sheaths. Neuron 23: 713-724 Pass H. I., Lott D., Lonardo F., Harbut M., Liu Z., Tang N., Carbone M., Webb C., Wali A. (2005) Asbestos exposure, pleural mesothelioma, and serum osteopontin levels. N Engl J Med 353: 1564-1573 215

Pathi S, Pagan-Westphal S, Baker DP, Garber EA, Rayhorn P, Bumcrot D, Tabin CJ, Blake Pepinsky R, Williams KP (2001) Comparative biological responses to human Sonic, Indian, and Desert hedgehog. Mech Dev 106: 107-117 Pavletich N. P., Pabo C. O. (1993) Crystal structure of a five-finger GLI-DNA complex: new perspectives on zinc fingers. Science 261: 1701-1707 Pazour G. J., Witman G. B. (2003) The vertebrate primary cilium is a sensory organelle. Curr Opin Cell Biol 15: 105-110 Pei X. Y., Dai Y., Grant S. (2004) Synergistic induction of oxidative injury and apoptosis in human multiple myeloma cells by the proteasome inhibitor bortezomib and histone deacetylase inhibitors. Clin Cancer Res 10: 3839-3852 Pepinsky RB, Zeng C, Wen D, Rayhorn P, Baker DP, Williams KP, Bixler SA, Ambrose CM, Garber EA, Miatkowski K, Taylor FR, Wang EA, Galdes A (1998) Identification of a palmitic acid-modified form of human Sonic hedgehog. J Biol Chem 273: 14037-14045 Perez-Galan P., Roue G., Villamor N., Montserrat E., Campo E., Colomer D. (2006) The proteasome inhibitor bortezomib induces apoptosis in mantle-cell lymphoma through generation of ROS and Noxa activation independent of p53 status. Blood 107: 257-264 Petiot A., Ogier-Denis E., Blommaart E. F., Meijer A. J., Codogno P. (2000) Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem 275: 992-998 Peto J., Decarli A., La Vecchia C., Levi F., Negri E. (1999) The European mesothelioma epidemic. Br J Cancer 79: 666-672 Petralia R. S., Schwartz C. M., Wang Y. X., Kawamoto E. M., Mattson M. P., Yao P. J. (2013) Sonic hedgehog promotes autophagy in hippocampal neurons. Biol Open 2: 499-504 Pilarsky C., Wenzig M., Specht T., Saeger H. D., Grutzmann R. (2004) Identification and validation of commonly overexpressed genes in solid tumors by comparison of microarray data. Neoplasia 6: 744-750 Ping X. L., Ratner D., Zhang H., Wu X. L., Zhang M. J., Chen F. F., Silvers D. N., Peacocke M., Tsou H. C. (2001) PTCH mutations in squamous cell carcinoma of the skin. J Invest Dermatol 116: 614-616 Popova T., Hebert L., Jacquemin V., Gad S., Caux-Moncoutier V., Dubois-d'Enghien C., Richaudeau B., Renaudin X., Sellers J., Nicolas A., Sastre-Garau X., Desjardins L., Gyapay G., Raynal V., Sinilnikova O. M., Andrieu N., Manie E., de Pauw A., Gesta P., Bonadona V., Maugard C. M., Penet C., Avril M. F., Barillot E., Cabaret O., Delattre O., Richard S., Caron O., Benfodda M., Hu H. H., Soufir N., Bressac-de Paillerets B., Stoppa-Lyonnet D., Stern M. H. (2013) Germline BAP1 mutations predispose to renal cell carcinomas. Am J Hum Genet 92: 974-980 Porter JA, Young KE, Beachy PA (1996) Cholesterol modification of hedgehog signaling proteins in animal development. Science 274: 255-259 Prestayko A. W., D'Aoust J. C., Issell B. F., Crooke S. T. (1979) Cisplatin (cisdiamminedichloroplatinum II). Cancer Treat Rev 6: 17-39 Putoux A., Thomas S., Coene K. L., Davis E. E., Alanay Y., Ogur G., Uz E., Buzas D., Gomes C., Patrier S., Bennett C. L., Elkhartoufi N., Frison M. H., Rigonnot L., Joye N., Pruvost S., Utine G. E., Boduroglu K., Nitschke P., Fertitta L., Thauvin-Robinet C., Munnich A., Cormier-Daire V., Hennekam R., Colin E., Akarsu N. A., Bole-Feysot C., 216

Cagnard N., Schmitt A., Goudin N., Lyonnet S., Encha-Razavi F., Siffroi J. P., Winey M., Katsanis N., Gonzales M., Vekemans M., Beales P. L., Attie-Bitach T. KIF7 mutations cause fetal hydrolethalus and acrocallosal syndromes. Nat Genet 43: 601-606 Queiroz K. C., Ruela-de-Sousa R. R., Fuhler G. M., Aberson H. L., Ferreira C. V., Peppelenbosch M. P., Spek C. A. (2010) Hedgehog signaling maintains chemoresistance in myeloid leukemic cells. Oncogene 29: 6314-6322 Ramael M., van den Bossche J., Buysse C., van Meerbeeck J., Segers K., Vermeire P., van Marck E. (1992) Immunoreactivity for P-170 glycoprotein in malignant mesothelioma and in non-neoplastic mesothelium of the pleura using the murine monoclonal antibody JSB-1. J Pathol 167: 5-8 Ramanathan B., Jan K. Y., Chen C. H., Hour T. C., Yu H. J., Pu Y. S. (2005) Resistance to paclitaxel is proportional to cellular total antioxidant capacity. Cancer Res 65: 8455-8460 Ranzato E., Biffo S., Burlando B. (2011) Selective ascorbate toxicity in malignant mesothelioma: a redox Trojan mechanism. Am J Respir Cell Mol Biol 44: 108-117 Rasband W.S. (1997-2012) ImageJ. National Institutes of Health, Bethesda, Maryland, USA. Reed J. C. (1999) Dysregulation of apoptosis in cancer. J Clin Oncol 17: 2941-2953 Regl G., Kasper M., Schnidar H., Eichberger T., Neill G. W., Ikram M. S., Quinn A. G., Philpott M. P., Frischauf A. M., Aberger F. (2004a) The zinc-finger transcription factor GLI2 antagonizes contact inhibition and differentiation of human epidermal cells. Oncogene 23: 1263-1274 Regl G., Kasper M., Schnidar H., Eichberger T., Neill G. W., Philpott M. P., Esterbauer H., Hauser-Kronberger C., Frischauf A. M., Aberger F. (2004b) Activation of the BCL2 promoter in response to Hedgehog/GLI signal transduction is predominantly mediated by GLI2. Cancer Res 64: 7724-7731 Reifenberger J., Wolter M., Knobbe C. B., Kohler B., Schonicke A., Scharwachter C., Kumar K., Blaschke B., Ruzicka T., Reifenberger G. (2005) Somatic mutations in the PTCH, SMOH, SUFUH and TP53 genes in sporadic basal cell carcinomas. Br J Dermatol 152: 43-51 Reifenberger J., Wolter M., Weber R. G., Megahed M., Ruzicka T., Lichter P., Reifenberger G. (1998) Missense mutations in SMOH in sporadic basal cell carcinomas of the skin and primitive neuroectodermal tumors of the central nervous system. Cancer Res 58: 1798-1803 Richardson P. G., Hideshima T., Mitsiades C., Anderson K. C. (2007) The emerging role of novel therapies for the treatment of relapsed myeloma. J Natl Compr Canc Netw 5: 149-162 Richter C., Park J. W., Ames B. N. (1988) Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc Natl Acad Sci U S A 85: 6465-6467 Riobo N. A., Lu K., Ai X., Haines G. M., Emerson C. P., Jr. (2006) Phosphoinositide 3- kinase and Akt are essential for Sonic Hedgehog signaling. Proc Natl Acad Sci U S A 103: 4505-4510 Robarge K. D., Brunton S. A., Castanedo G. M., Cui Y., Dina M. S., Goldsmith R., Gould S. E., Guichert O., Gunzner J. L., Halladay J., Jia W., Khojasteh C., Koehler M. F., Kotkow K., La H., Lalonde R. L., Lau K., Lee L., Marshall D., Marsters J. C., Jr., Murray L. J., Qian C., Rubin L. L., Salphati L., Stanley M. S., Stibbard J. H., Sutherlin D. P., 217

Ubhayaker S., Wang S., Wong S., Xie M. (2009) GDC-0449-a potent inhibitor of the hedgehog pathway. Bioorg Med Chem Lett 19: 5576-5581 Robinson Bruce W. S., Chahinian A. Philippe (2002) Mesothelioma, London: M. Dunitz. Robinson BW, Lake RA (2005) Advances in malignant mesothelioma. N Engl J Med 353: 1591-1603 Robinson C., Walsh A., Larma I., O'Halloran S., Nowak A. K., Lake R. A. (2011) MexTAg mice exposed to asbestos develop cancer that faithfully replicates key features of the pathogenesis of human mesothelioma. Eur J Cancer 47: 151-161 Rohatgi R., Milenkovic L., Corcoran R. B., Scott M. P. (2009) Hedgehog signal transduction by Smoothened: pharmacologic evidence for a 2-step activation process. Proc Natl Acad Sci U S A 106: 3196-3201 Rohatgi R., Milenkovic L., Scott M. P. (2007) Patched1 regulates hedgehog signaling at the primary cilium. Science 317: 372-376 Romagnoli S., Fasoli E., Vaira V., Falleni M., Pellegrini C., Catania A., Roncalli M., Marchetti A., Santambrogio L., Coggi G., Bosari S. (2009) Identification of potential therapeutic targets in malignant mesothelioma using cell-cycle gene expression analysis. Am J Pathol 174: 762-770 Romer J. T., Kimura H., Magdaleno S., Sasai K., Fuller C., Baines H., Connelly M., Stewart C. F., Gould S., Rubin L. L., Curran T. (2004) Suppression of the Shh pathway using a small molecule inhibitor eliminates medulloblastoma in Ptc1(+/-)p53(-/-) mice. Cancer Cell 6: 229-240 Ronen A., Glickman B. W. (2001) Human DNA repair genes. Environ Mol Mutagen 37: 241-283 Rosato R. R., Almenara J. A., Grant S. (2003) The histone deacetylase inhibitor MS-275 promotes differentiation or apoptosis in human leukemia cells through a process regulated by generation of reactive oxygen species and induction of p21cip1/waf1 1. Cancer Res 63: 3637-3645 Roushdy-Hammady I., Siegel J., Emri S., Testa J. R., Carbone M. (2001) Geneticsusceptibility factor and malignant mesothelioma in the Cappadocian region of Turkey. Lancet 357: 444-445 Roy S. (2012) Cilia and Hedgehog: when and how was their marriage solemnized? Differentiation 83: S43-48 Rubbo H., Radi R., Trujillo M., Telleri R., Kalyanaraman B., Barnes S., Kirk M., Freeman B. A. (1994) Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives. J Biol Chem 269: 26066-26075 Rubin L. L., de Sauvage F. J. (2006) Targeting the Hedgehog pathway in cancer. Nat Rev Drug Discov 5: 1026-1033 Rudin C. M., Hann C. L., Laterra J., Yauch R. L., Callahan C. A., Fu L., Holcomb T., Stinson J., Gould S. E., Coleman B., LoRusso P. M., Von Hoff D. D., de Sauvage F. J., Low J. A. (2009) Treatment of medulloblastoma with hedgehog pathway inhibitor GDC- 0449. N Engl J Med 361: 1173-1178 Ruefli A. A., Ausserlechner M. J., Bernhard D., Sutton V. R., Tainton K. M., Kofler R., Smyth M. J., Johnstone R. W. (2001) The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death 218

pathway characterized by cleavage of Bid and production of reactive oxygen species. Proc Natl Acad Sci U S A 98: 10833-10838 Ruiz i Altaba A, Mas C, Stecca B (2007) The Gli code: an information nexus regulating cell fate, stemness and cancer. Trends Cell Biol 17: 438-447 Ruiz i Altaba A. (1999) Gli proteins encode context-dependent positive and negative functions: implications for development and disease. Development 126: 3205-3216 Ruppert J. M., Kinzler K. W., Wong A. J., Bigner S. H., Kao F. T., Law M. L., Seuanez H. N., O'Brien S. J., Vogelstein B. (1988) The GLI-Kruppel family of human genes. Mol Cell Biol 8: 3104-3113 Sanchez P., Clement V., Ruiz i Altaba A. (2005) Therapeutic targeting of the Hedgehog- GLI pathway in prostate cancer. Cancer Res 65: 2990-2992 Sanchez P., Hernandez A. M., Stecca B., Kahler A. J., DeGueme A. M., Barrett A., Beyna M., Datta M. W., Datta S., Ruiz i Altaba A. (2004) Inhibition of prostate cancer proliferation by interference with SONIC HEDGEHOG-GLI1 signaling. Proc Natl Acad Sci U S A 101: 12561-12566 Sandberg A. A., Bridge J. A. (2001) Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors. Mesothelioma. Cancer Genetics and Cytogenetics 127: 93-110 Santos C. X., Tanaka L. Y., Wosniak J., Laurindo F. R. (2009) Mechanisms and implications of reactive oxygen species generation during the unfolded protein response: roles of endoplasmic reticulum oxidoreductases, mitochondrial electron transport, and NADPH oxidase. Antioxid Redox Signal 11: 2409-2427 Satoh M., Takemura Y., Hamada H., Sekido Y., Kubota S. (2013) EGCG induces human mesothelioma cell death by inducing reactive oxygen species and autophagy. Cancer Cell Int 13: 19 Sauna Z. E., Kimchi-Sarfaty C. (2011) Understanding the contribution of synonymous mutations to human disease. Nat Rev Genet 12: 683-691 Scales S. J., de Sauvage F. J. (2009) Mechanisms of Hedgehog pathway activation in cancer and implications for therapy. Trends Pharmacol Sci 30: 303-312 Segers K., Ramael M., Singh S. K., Weyler J., Van Meerbeeck J., Vermeire P., Van Marck E. (1994) Immunoreactivity for bcl-2 protein in malignant mesothelioma and nonneoplastic mesothelium. Virchows Archiv 424: 631-634 Seglen P. O., Gordon P. B. (1982) 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci U S A 79: 1889-1892 Sekido Y., Pass H. I., Bader S., Mew D. J. Y., Christman M. F., Gazdar A. F., Minna J. D. (1995) Neurofibromatosis Type-2 (Nf2) Gene Is Somatically Mutated in Mesothelioma but Not in Lung-Cancer. Cancer Res 55: 1227-1231 Sekulic A., Migden M. R., Oro A. E., Dirix L., Lewis K. D., Hainsworth J. D., Solomon J. A., Yoo S., Arron S. T., Friedlander P. A., Marmur E., Rudin C. M., Chang A. L., Low J. A., Mackey H. M., Yauch R. L., Graham R. A., Reddy J. C., Hauschild A. (2012) Efficacy and safety of vismodegib in advanced basal-cell carcinoma. N Engl J Med 366: 2171-2179 Selikoff I. J., Hammond E. C., Seidman H. (1980) Latency of Asbestos Disease among Insulation Workers in the United-States and Canada. Cancer 46: 2736-2740 219

Sena L. A., Chandel N. S. (2012) Physiological roles of mitochondrial reactive oxygen species. Mol Cell 48: 158-167 Shafaee Z., Schmidt H., Du W., Posner M., Weichselbaum R. (2006) Cyclopamine increases the cytotoxic effects of paclitaxel and radiation but not cisplatin and gemcitabine in Hedgehog expressing pancreatic cancer cells. Cancer Chemother Pharmacol 58: 765-770 Shahi M. H., Afzal M., Sinha S., Eberhart C. G., Rey J. A., Fan X., Castresana J. S. (2011) Human hedgehog interacting protein expression and promoter methylation in medulloblastoma cell lines and primary tumor samples. J Neurooncol 103: 287-296 Sharif S, Zahid I, Routledge T, Scarci M (2011) Extrapleural pneumonectomy or supportive care: treatment of malignant pleural mesothelioma? Interact Cardiovasc Thorac Surg 12: 1040-1045 Shi T., Mazumdar T., Devecchio J., Duan Z. H., Agyeman A., Aziz M., Houghton J. A. (2010) cdna microarray gene expression profiling of hedgehog signaling pathway inhibition in human colon cancer cells. PLoS One 5 Shi Y., Moura U., Opitz I., Soltermann A., Rehrauer H., Thies S., Weder W., Stahel R. A., Felley-Bosco E. (2012) Role of hedgehog signaling in malignant pleural mesothelioma. Clin Cancer Res 18: 4646-4656 Shin S. H., Kogerman P., Lindstrom E., Toftgard R., Biesecker L. G. (1999) GLI3 mutations in human disorders mimic Drosophila cubitus interruptus protein functions and localization. Proc Natl Acad Sci U S A 96: 2880-2884 Shintani T., Klionsky D. J. (2004) Autophagy in health and disease: a double-edged sword. Science 306: 990-995 Sims-Mourtada J., Izzo J. G., Ajani J., Chao K. S. (2007) Sonic Hedgehog promotes multiple drug resistance by regulation of drug transport. Oncogene 26: 5674-5679 Singh R. R., Kim J. E., Davuluri Y., Drakos E., Cho-Vega J. H., Amin H. M., Vega F. (2010) Hedgehog signaling pathway is activated in diffuse large B-cell lymphoma and contributes to tumor cell survival and proliferation. Leukemia 24: 1025-1036 Sjoblom T., Jones S., Wood L. D., Parsons D. W., Lin J., Barber T. D., Mandelker D., Leary R. J., Ptak J., Silliman N., Szabo S., Buckhaults P., Farrell C., Meeh P., Markowitz S. D., Willis J., Dawson D., Willson J. K., Gazdar A. F., Hartigan J., Wu L., Liu C., Parmigiani G., Park B. H., Bachman K. E., Papadopoulos N., Vogelstein B., Kinzler K. W., Velculescu V. E. (2006) The consensus coding sequences of human breast and colorectal cancers. Science 314: 268-274 Slade I., Murray A., Hanks S., Kumar A., Walker L., Hargrave D., Douglas J., Stiller C., Izatt L., Rahman N. (2011) Heterogeneity of familial medulloblastoma and contribution of germline PTCH1 and SUFU mutations to sporadic medulloblastoma. Fam Cancer 10: 337-342 Smyth I., Narang M. A., Evans T., Heimann C., Nakamura Y., Chenevix-Trench G., Pietsch T., Wicking C., Wainwright B. J. (1999) Isolation and characterization of human patched 2 (PTCH2), a putative tumour suppressor gene inbasal cell carcinoma and medulloblastoma on chromosome 1p32. Hum Mol Genet 8: 291-297 Society American Cancer. (2013) Cancer Facts and Figures 2013. American Cancer Society, Atlanta. 220

Soini Y., Kinnula V., Kaarteenaho-Wiik R., Kurttila E., Linnainmaa K., Paakko P. (1999) Apoptosis and expression of apoptosis regulating proteins bcl-2, mcl-1, bcl-x, and bax in malignant mesothelioma. Clin Cancer Res 5: 3508-3515 Song B., Liu X. S., Rice S. J., Kuang S., Elzey B. D., Konieczny S. F., Ratliff T. L., Hazbun T., Chiorean E. G., Liu X. (2013a) Plk1 phosphorylation of orc2 and hbo1 contributes to gemcitabine resistance in pancreatic cancer. Mol Cancer Ther 12: 58-68 Song Y. L., Zhang W. F., Peng B., Wang C. N., Wang Q., Bian Z. (2006) Germline mutations of the PTCH gene in families with odontogenic keratocysts and nevoid basal cell carcinoma syndrome. Tumour Biol 27: 175-180 Song Y., Tian Y., Zuo Y., Tu J. C., Feng Y. F., Qu C. J. (2013b) Altered expression of PTCH and HHIP in gastric cancer through their gene promoter methylation: novel targets for gastric cancer. Mol Med Rep 7: 1159-1168 St-Jacques B., Hammerschmidt M., McMahon A. P. (1999) Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev 13: 2072-2086 Stadtman E. R., Levine R. L. (2000) Protein oxidation. Ann N Y Acad Sci 899: 191-208 Stanton B. Z., Peng L. F., Maloof N., Nakai K., Wang X., Duffner J. L., Taveras K. M., Hyman J. M., Lee S. W., Koehler A. N., Chen J. K., Fox J. L., Mandinova A., Schreiber S. L. (2009) A small molecule that binds Hedgehog and blocks its signaling in human cells. Nat Chem Biol 5: 154-156 Stapelberg M., Gellert N., Swettenham E., Tomasetti M., Witting P. K., Procopio A., Neuzil J. (2005) Alpha-tocopheryl succinate inhibits malignant mesothelioma by disrupting the fibroblast growth factor autocrine loop: mechanism and the role of oxidative stress. J Biol Chem 280: 25369-25376 Stecca B., Mas C., Clement V., Zbinden M., Correa R., Piguet V., Beermann F., Ruiz I. Altaba A. (2007) Melanomas require HEDGEHOG-GLI signaling regulated by interactions between GLI1 and the RAS-MEK/AKT pathways. Proc Natl Acad Sci U S A 104: 5895-5900 Steele J. P., Klabatsa A. (2005) Chemotherapy options and new advances in malignant pleural mesothelioma. Ann Oncol 16: 345-351 Stegmeier F., Warmuth M., Sellers W. R., Dorsch M. (2010) Targeted cancer therapies in the twenty-first century: lessons from imatinib. Clin Pharmacol Ther 87: 543-552 Sterman D. H., Kaiser L. R., Albelda S. M. (1999) Advances in the treatment of malignant pleural mesothelioma. Chest 116: 504-520 Stone D. M., Murone M., Luoh S., Ye W., Armanini M. P., Gurney A., Phillips H., Brush J., Goddard A., de Sauvage F. J., Rosenthal A. (1999) Characterization of the human suppressor of fused, a negative regulator of the zinc-finger transcription factor Gli. J Cell Sci 112 ( Pt 23): 4437-4448 Stone DM, Hynes M, Armanini M, Swanson TA, Gu Q, Johnson RL, Scott MP, Pennica D, Goddard A, Phillips H, Noll M, Hooper JE, de Sauvage F, Rosenthal A (1996) The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature 384: 129-134 Sugarbaker D. J., Strauss G. M., Lynch T. J., Richards W., Mentzer S. J., Lee T. H., Corson J. M., Antman K. H. (1993) Node Status Has Prognostic-Significance in the Multimodality Therapy of Diffuse, Malignant Mesothelioma. J Clin Oncol 11: 1172-1178 221

Suh K. S., Goy A. () Bortezomib in mantle cell lymphoma. Future Oncol 4: 149-168 Sun L. S., Li X. F., Li T. J. () PTCH1 and SMO gene alterations in keratocystic odontogenic tumors. J Dent Res 87: 575-579 Sun Y., Guo W., Ren T., Liang W., Zhou W., Lu Q., Jiao G., Yan T. (2014) Gli1 inhibition suppressed cell growth and cell cycle progression and induced apoptosis as well as autophagy depending on ERK1/2 activity in human chondrosarcoma cells. Cell Death Dis 5: e979 Suzuki Y. (2001) Pathology of human malignant mesothelioma--preliminary analysis of 1,517 mesothelioma cases. Ind Health 39: 183-185 Svard J., Heby-Henricson K., Persson-Lek M., Rozell B., Lauth M., Bergstrom A., Ericson J., Toftgard R., Teglund S. (2006) Genetic elimination of Suppressor of fused reveals an essential repressor function in the mammalian Hedgehog signaling pathway. Dev Cell 10: 187-197 Szatrowski T. P., Nathan C. F. (1991) Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res 51: 794-798 Tada M., Kanai F., Tanaka Y., Tateishi K., Ohta M., Asaoka Y., Seto M., Muroyama R., Fukai K., Imazeki F., Kawabe T., Yokosuka O., Omata M. () Down-regulation of hedgehog-interacting protein through genetic and epigenetic alterations in human hepatocellular carcinoma. Clin Cancer Res 14: 3768-3776 Taipale J, Chen JK, Cooper MK, Wang B, Mann RK, Milenkovic L, Scott MP, Beachy PA (2000) Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature 406: 1005-1009 Taipale J., Cooper M. K., Maiti T., Beachy P. A. (2002) Patched acts catalytically to suppress the activity of Smoothened. Nature 418: 892-897 Tang Yang, Cai Song-Bai, Zhang Xu, Wang Song-Po (2012) Effect of abnormal Hedgehog signaling pathway on multidrug resistance of human colon adenocarcinoma cells. Tumor 32: 886-891 Taylor M. D., Liu L., Raffel C., Hui C. C., Mainprize T. G., Zhang X., Agatep R., Chiappa S., Gao L., Lowrance A., Hao A., Goldstein A. M., Stavrou T., Scherer S. W., Dura W. T., Wainwright B., Squire J. A., Rutka J. T., Hogg D. (2002) Mutations in SUFU predispose to medulloblastoma. Nat Genet 31: 306-310 Teglund S., Toftgard R. (2010) Hedgehog beyond medulloblastoma and basal cell carcinoma. Biochim Biophys Acta 1805: 181-208 Teh M. T., Wong S. T., Neill G. W., Ghali L. R., Philpott M. P., Quinn A. G. (2002) FOXM1 is a downstream target of Gli1 in basal cell carcinomas. Cancer Res 62: 4773-4780 Tempe D., Casas M., Karaz S., Blanchet-Tournier M. F., Concordet J. P. (2006) Multisite protein kinase A and glycogen synthase kinase 3beta phosphorylation leads to Gli3 ubiquitination by SCFbetaTrCP. Mol Cell Biol 26: 4316-4326 Tenzen T., Allen B. L., Cole F., Kang J. S., Krauss R. S., McMahon A. P. (2006) The cell surface membrane proteins Cdo and Boc are components and targets of the Hedgehog signaling pathway and feedback network in mice. Dev Cell 10: 647-656 Testa J. R., Cheung M., Pei J., Below J. E., Tan Y., Sementino E., Cox N. J., Dogan A. U., Pass H. I., Trusa S., Hesdorffer M., Nasu M., Powers A., Rivera Z., Comertpay S., 222

Tanji M., Gaudino G., Yang H., Carbone M. (2011) Germline BAP1 mutations predispose to malignant mesothelioma. Nat Genet 43: 1022-1025 Thayer S. P., di Magliano M. P., Heiser P. W., Nielsen C. M., Roberts D. J., Lauwers G. Y., Qi Y. P., Gysin S., Fernandez-del Castillo C., Yajnik V., Antoniu B., McMahon M., Warshaw A. L., Hebrok M. (2003) Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature 425: 851-856 Thomas Z. I., Gibson W., Sexton J. Z., Aird K. M., Ingram S. M., Aldrich A., Lyerly H. K., Devi G. R., Williams K. P. (2011) Targeting GLI1 expression in human inflammatory breast cancer cells enhances apoptosis and attenuates migration. Br J Cancer 104: 1575-1586 Tooze S. A., Yoshimori T. (2010) The origin of the autophagosomal membrane. Nat Cell Biol 12: 831-835 Tostar U., Toftgard R., Zaphiropoulos P. G., Shimokawa T. (2010) Reduction of human embryonal rhabdomyosarcoma tumor growth by inhibition of the hedgehog signaling pathway. Genes Cancer 1: 941-951 Trachootham D., Lu W., Ogasawara M. A., Nilsa R. D., Huang P. () Redox regulation of cell survival. Antioxid Redox Signal 10: 1343-1374 Traganos F., Darzynkiewicz Z. (1994) Lysosomal proton pump activity: supravital cell staining with acridine orange differentiates leukocyte subpopulations. Methods Cell Biol 41: 185-194 Travis William D., World Health Organization., International Agency for Research on Cancer., International Association for the Study of Lung Cancer., International Academy of Pathology. (2004) Pathology and genetics of tumours of the lung, pleura, thymus, and heart, Lyon: IARC Press. Tsang W. P., Chau S. P., Kong S. K., Fung K. P., Kwok T. T. (2003) Reactive oxygen species mediate doxorubicin induced p53-independent apoptosis. Life Sci 73: 2047-2058 Tsao A. S., Wistuba I., Roth J. A., Kindler H. L. (2009) Malignant pleural mesothelioma. J Clin Oncol 27: 2081-2090 Tukachinsky H, Lopez LV, Salic A (2010) A mechanism for vertebrate Hedgehog signaling: recruitment to cilia and dissociation of SuFu-Gli protein complexes. J Cell Biol 191: 415-428 Turrens J. F. (2003) Mitochondrial formation of reactive oxygen species. J Physiol 552: 335-344 Twig G., Elorza A., Molina A. J., Mohamed H., Wikstrom J. D., Walzer G., Stiles L., Haigh S. E., Katz S., Las G., Alroy J., Wu M., Py B. F., Yuan J., Deeney J. T., Corkey B. E., Shirihai O. S. () Fission and selective fusion govern mitochondrial segregation and elimination by autophagy. EMBO J 27: 433-446 van den Brink G. R. (2007) Hedgehog signaling in development and homeostasis of the gastrointestinal tract. Physiol Rev 87: 1343-1375 van den Heuvel M., Ingham P. W. (1996) smoothened encodes a receptor-like serpentine protein required for hedgehog signalling. Nature 382: 547-551 van Gelder T., Hoogsteden H. C., Vandenbroucke J. P., van der Kwast T. H., Planteydt H. T. (1991) The influence of the diagnostic technique on the histopathological diagnosis in malignant mesothelioma. Virchows Arch A Pathol Anat Histopathol 418: 315-317 223

van Haarst J. M., Baas P., Manegold Ch, Schouwink J. H., Burgers J. A., de Bruin H. G., Mooi W. J., van Klaveren R. J., de Jonge M. J., van Meerbeeck J. P. (2002) Multicentre phase II study of gemcitabine and cisplatin in malignant pleural mesothelioma. Br J Cancer 86: 342-345 van Meerbeeck J. P., Scherpereel A., Surmont V. F., Baas P. (2011) Malignant pleural mesothelioma: the standard of care and challenges for future management. Crit Rev Oncol Hematol 78: 92-111 Varin E., Denoyelle C., Brotin E., Meryet-Figuiere M., Giffard F., Abeilard E., Goux D., Gauduchon P., Icard P., Poulain L. (2010) Downregulation of Bcl-xL and Mcl-1 is sufficient to induce cell death in mesothelioma cells highly refractory to conventional chemotherapy. Carcinogenesis 31: 984-993 Varjosalo M, Li SP, Taipale J (2006) Divergence of hedgehog signal transduction mechanism between Drosophila and mammals. Dev Cell 10: 177-186 Varjosalo M, Taipale J () Hedgehog: functions and mechanisms. Genes Dev 22: 2454-2472 Ventii K. H., Devi N. S., Friedrich K. L., Chernova T. A., Tighiouart M., Van Meir E. G., Wilkinson K. D. () BRCA1-associated protein-1 is a tumor suppressor that requires deubiquitinating activity and nuclear localization. Cancer Res 68: 6953-6962 Vercesi A. E., Kowaltowski A. J., Grijalba M. T., Meinicke A. R., Castilho R. F. (1997) The role of reactive oxygen species in mitochondrial permeability transition. Biosci Rep 17: 43-52 Vogelzang NJ, Rusthoven JJ, Symanowski J, Denham C, Kaukel E, Ruffie P, Gatzemeier U, Boyer M, Emri S, Manegold C, Niyikiza C, Paoletti P (2003) Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J Clin Oncol 21: 2636-2644 Von Hoff D. D., LoRusso P. M., Rudin C. M., Reddy J. C., Yauch R. L., Tibes R., Weiss G. J., Borad M. J., Hann C. L., Brahmer J. R., Mackey H. M., Lum B. L., Darbonne W. C., Marsters J. C., Jr., de Sauvage F. J., Low J. A. (2009) Inhibition of the hedgehog pathway in advanced basal-cell carcinoma. N Engl J Med 361: 1164-1172 Vorechovsky I., Unden A. B., Sandstedt B., Toftgard R., Stahle-Backdahl M. (1997) Trichoepitheliomas contain somatic mutations in the overexpressed PTCH gene: support for a gatekeeper mechanism in skin tumorigenesis. Cancer Res 57: 4677-4681 Wagner J. C., Skidmore J. W., Hill R. J., Griffiths D. M. (1985) Erionite exposure and mesotheliomas in rats. Br J Cancer 51: 727-730 Wagner K. U., Claudio E., Rucker E. B., 3rd, Riedlinger G., Broussard C., Schwartzberg P. L., Siebenlist U., Hennighausen L. (2000) Conditional deletion of the Bcl-x gene from erythroid cells results in hemolytic anemia and profound splenomegaly. Development 127: 4949-4958 Wang B., Fallon J. F., Beachy P. A. (2000) Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell 100: 423-434 Wang B., Li Y. (2006) Evidence for the direct involvement of {beta}trcp in Gli3 protein processing. Proc Natl Acad Sci U S A 103: 33-38 Wang K., Pan L., Che X., Cui D., Li C. (2010) Gli1 inhibition induces cell-cycle arrest and enhanced apoptosis in brain glioma cell lines. J Neurooncol 98: 319-327 224

Wang L. H., Choi Y. L., Hua X. Y., Shin Y. K., Song Y. J., Youn S. J., Yun H. Y., Park S. M., Kim W. J., Kim H. J., Choi J. S., Kim S. H. (2006) Increased expression of sonic hedgehog and altered methylation of its promoter region in gastric cancer and its related lesions. Mod Pathol 19: 675-683 Wang X. D., Inzunza H., Chang H., Qi Z., Hu B., Malone D., Cogswell J. (2013a) Mutations in the hedgehog pathway genes SMO and PTCH1 in human gastric tumors. PLoS One 8: e54415 Wang Y., Han C., Lu L., Magliato S., Wu T. (2013b) Hedgehog signaling pathway regulates autophagy in human hepatocellular carcinoma cells. Hepatology 58: 995-1010 Wang Y., McMahon A. P., Allen B. L. (2007) Shifting paradigms in Hedgehog signaling. Curr Opin Cell Biol 19: 159-165 Wang Y., Zhao R., Goldman I. D. (2003) Decreased expression of the reduced folate carrier and folypolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukemia cell line. Biochem Pharmacol 65: 1163-1170 Waris G., Ahsan H. (2006) Reactive oxygen species: role in the development of cancer and various chronic conditions. J Carcinog 5: 14 Watkins D. N., Berman D. M., Burkholder S. G., Wang B., Beachy P. A., Baylin S. B. (2003) Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer. Nature 422: 313-317 Wheatley D. N., Wang A. M., Strugnell G. E. (1996) Expression of primary cilia in mammalian cells. Cell Biol Int 20: 73-81 Wicking C., Shanley S., Smyth I., Gillies S., Negus K., Graham S., Suthers G., Haites N., Edwards M., Wainwright B., Chenevix-Trench G. (1997) Most germ-line mutations in the nevoid basal cell carcinoma syndrome lead to a premature termination of the PATCHED protein, and no genotype-phenotype correlations are evident. Am J Hum Genet 60: 21-26 Wickstrom M., Dyberg C., Shimokawa T., Milosevic J., Baryawno N., Fuskevag O. M., Larsson R., Kogner P., Zaphiropoulos P. G., Johnsen J. I. (2013) Targeting the hedgehog signal transduction pathway at the level of GLI inhibits neuroblastoma cell growth in vitro and in vivo. Int J Cancer 132: 1516-1524 Wierstra I., Alves J. (2007) FOXM1, a typical proliferation-associated transcription factor. Biol Chem 388: 1257-1274 Wiesner T., Obenauf A. C., Murali R., Fried I., Griewank K. G., Ulz P., Windpassinger C., Wackernagel W., Loy S., Wolf I., Viale A., Lash A. E., Pirun M., Socci N. D., Rutten A., Palmedo G., Abramson D., Offit K., Ott A., Becker J. C., Cerroni L., Kutzner H., Bastian B. C., Speicher M. R. (2011) Germline mutations in BAP1 predispose to melanocytic tumors. Nat Genet 43: 1018-1021 Wolter M., Reifenberger J., Sommer C., Ruzicka T., Reifenberger G. (1997) Mutations in the human homologue of the Drosophila segment polarity gene patched (PTCH) in sporadic basal cell carcinomas of the skin and primitive neuroectodermal tumors of the central nervous system. Cancer Res 57: 2581-2585 Wondrak G. T. (2009) Redox-directed cancer therapeutics: molecular mechanisms and opportunities. Antioxid Redox Signal 11: 3013-3069 Wong A. J., Bigner S. H., Bigner D. D., Kinzler K. W., Hamilton S. R., Vogelstein B. (1987) Increased expression of the epidermal growth factor receptor gene in malignant 225

gliomas is invariably associated with gene amplification. Proc Natl Acad Sci U S A 84: 6899-6903 Xie J., Johnson R. L., Zhang X., Bare J. W., Waldman F. M., Cogen P. H., Menon A. G., Warren R. S., Chen L. C., Scott M. P., Epstein E. H., Jr. (1997) Mutations of the PATCHED gene in several types of sporadic extracutaneous tumors. Cancer Res 57: 2369-2372 Xie J., Murone M., Luoh S. M., Ryan A., Gu Q., Zhang C., Bonifas J. M., Lam C. W., Hynes M., Goddard A., Rosenthal A., Epstein E. H., Jr., de Sauvage F. J. (1998) Activating Smoothened mutations in sporadic basal-cell carcinoma. Nature 391: 90-92 Xu X. F., Guo C. Y., Liu J., Yang W. J., Xia Y. J., Xu L., Yu Y. C., Wang X. P. (2009) Gli1 maintains cell survival by up-regulating IGFBP6 and Bcl-2 through promoter regions in parallel manner in pancreatic cancer cells. J Carcinog 8: 13 Xu Y., An Y., Wang X., Zha W., Li X. (2014) Inhibition of the Hedgehog pathway induces autophagy in pancreatic ductal adenocarcinoma cells. Oncol Rep 31: 707-712 Yamamoto A., Tagawa Y., Yoshimori T., Moriyama Y., Masaki R., Tashiro Y. (1998) Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct 23: 33-42 Yan M., Wang L., Zuo H., Zhang Z., Chen W., Mao L., Zhang P. (2011a) HH/GLI signalling as a new therapeutic target for patients with oral squamous cell carcinoma. Oral Oncol 47: 504-509 Yan R., Peng X., Yuan X., Huang D., Chen J., Lu Q., Lv N., Luo S. (2013) Suppression of growth and migration by blocking the Hedgehog signaling pathway in gastric cancer cells. Cell Oncol (Dordr) 36: 421-435 Yan T. D., Cao C. Q., Boyer M., Tin M. M., Kennedy C., McLean J., Bannon P. G., McCaughan B. C. (2011b) Improving survival results after surgical management of malignant pleural mesothelioma: an Australian institution experience. Ann Thorac Cardiovasc Surg 17: 243-249 Yang L., Xie G., Fan Q., Xie J. (2010) Activation of the hedgehog-signaling pathway in human cancer and the clinical implications. Oncogene 29: 469-481 Yang Z. J., Chee C. E., Huang S., Sinicrope F. (2011) Autophagy modulation for cancer therapy. Cancer Biol Ther 11: 169-176 Yauch R. L., Dijkgraaf G. J., Alicke B., Januario T., Ahn C. P., Holcomb T., Pujara K., Stinson J., Callahan C. A., Tang T., Bazan J. F., Kan Z., Seshagiri S., Hann C. L., Gould S. E., Low J. A., Rudin C. M., de Sauvage F. J. (2009) Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma. Science 326: 572-574 Yauch R. L., Gould S. E., Scales S. J., Tang T., Tian H., Ahn C. P., Marshall D., Fu L., Januario T., Kallop D., Nannini-Pepe M., Kotkow K., Marsters J. C., Rubin L. L., de Sauvage F. J. () A paracrine requirement for hedgehog signalling in cancer. Nature 455: 406-410 Yoon J. W., Kita Y., Frank D. J., Majewski R. R., Konicek B. A., Nobrega M. A., Jacob H., Walterhouse D., Iannaccone P. (2002) Gene expression profiling leads to identification of GLI1-binding elements in target genes and a role for multiple downstream pathways in GLI1-induced cell transformation. J Biol Chem 277: 5548-5555 226

Yoon J. W., Liu C. Z., Yang J. T., Swart R., Iannaccone P., Walterhouse D. (1998) GLI activates transcription through a herpes simplex viral protein 16-like activation domain. J Biol Chem 273: 3496-3501 You M., Varona-Santos J., Singh S., Robbins D. J., Savaraj N., Nguyen D. M. (2013) Targeting of the Hedgehog signal transduction pathway suppresses survival of malignant pleural mesothelioma cells in vitro. J Thorac Cardiovasc Surg 147: 508-516 Zahid I, Sharif S, Routledge T, Scarci M (2011) Is pleurectomy and decortication superior to palliative care in the treatment of malignant pleural mesothelioma? Interact Cardiovasc Thorac Surg 12: 812-817 Zamzami N., Marchetti P., Castedo M., Decaudin D., Macho A., Hirsch T., Susin S. A., Petit P. X., Mignotte B., Kroemer G. (1995) Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med 182: 367-377 Zangar R. C., Davydov D. R., Verma S. (2004) Mechanisms that regulate production of reactive oxygen species by cytochrome P450. Toxicol Appl Pharmacol 199: 316-331 Zaphiropoulos P. G., Unden A. B., Rahnama F., Hollingsworth R. E., Toftgard R. (1999) PTCH2, a novel human patched gene, undergoing alternative splicing and up-regulated in basal cell carcinomas. Cancer Res 59: 787-792 Zelenin A. V. (1966) Fluorescence microscopy of lysosomes and related structures in living cells. Nature 212: 425-426 Zhang Q., Zhang L., Wang B., Ou C. Y., Chien C. T., Jiang J. (2006) A hedgehoginduced BTB protein modulates hedgehog signaling by degrading Ci/Gli transcription factor. Dev Cell 10: 719-729 Zhang X., Harrington N., Moraes R. C., Wu M. F., Hilsenbeck S. G., Lewis M. T. (2009a) Cyclopamine inhibition of human breast cancer cell growth independent of Smoothened (Smo). Breast Cancer Res Treat 115: 505-521 Zhang X., Varin E., Allouche S., Lu Y., Poulain L., Icard P. (2009b) Effect of citrate on malignant pleural mesothelioma cells: a synergistic effect with cisplatin. Anticancer Res 29: 1249-1254 Zhang X., Varin E., Briand M., Allouche S., Heutte N., Schwartz L., Poulain L., Icard P. (2009c) Novel therapy for malignant pleural mesothelioma based on anti-energetic effect: an experimental study using 3-Bromopyruvate on nude mice. Anticancer Res 29: 1443-1448 Zhang Y., He J., Zhang F., Li H., Yue D., Wang C., Jablons D. M., He B., Lui N. (2013) SMO expression level correlates with overall survival in patients with malignant pleural mesothelioma. J Exp Clin Cancer Res 32: 7 Zhao C., Chen A., Jamieson C. H., Fereshteh M., Abrahamsson A., Blum J., Kwon H. Y., Kim J., Chute J. P., Rizzieri D., Munchhof M., VanArsdale T., Beachy P. A., Reya T. (2009) Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia. Nature 458: 776-779 Zorov D. B., Juhaszova M., Sollott S. J. (2006) Mitochondrial ROS-induced ROS release: an update and review. Biochim Biophys Acta 1757: 509-517 Zwelling L. A., Anderson T., Kohn K. W. (1979) DNA-protein and DNA interstrand cross-linking by cis- and trans-platinum(ii) diamminedichloride in L1210 mouse leukemia cells and relation to cytotoxicity. Cancer Res 39: 365-369 227

APPENDICES 228

APPENDIX A Intronic primer sequences for amplification of exons of Hedgehog pathway genes Gene Symbol Exon GLI1 1 GLI1 1 GLI1 2 GLI1 3 GLI1 4 GLI1 5 GLI1 6 GLI1 7 GLI1 8 GLI1 9 GLI1 10 GLI1 GLI1 GLI1 GLI1 GLI1 11a 11b 11c 11d 11e GLI2 1 GLI2 2 GLI2 3 GLI2 4 GLI2 5 GLI2 6 GLI2 7 GLI2 8 GLI2 9 GLI2 10 GLI2 11 GLI2 12 GLI2 GLI2 13a 13b Forward primer sequence CCAGGATTCTGTGC CTGAGAC CATATGGACCTGGA ATCTGGG GCATCAATAGGGCA AAGCAG ATCCTCCAAATAGC CCAATCC AGGAGGAAGTCCCT TCCAAAC CAGGAGCTTTACCT CTGGGAC GTGGAGGGAAGGT CTGTTCTG CTGTTGGAGATTGA GGGTTCC CCTCCTTACTTCCTT TGGTGC AGTCACTGGGACAC AGGCTTC CCAGAAACAGAAT AGGCATGG AGATATGCTTCAGC CAGAGGG AACCCCTATATGGA CTTCCCA AGCCACCCCCTGAT TATCTT ACTTGCTATGGGCC TCTCAA GTTTGGGCTCAGTG TTGGTG TTGTCTTTGTCAAG AGCGCAGT TTTCCAGGAGAGAC AAGGACTG CCAGGTGTGCATTT CTCTCTG TCCTTGCAGGCTCT TCCTATC CTGGGCAAGGTTCT CTCTGTC TCTGTGCGGAGAGA TCCTAGAG CACACACTTGCATC CACACC GGATGGTCTGCACA CAGGTC TTGTACCTTTCAGC CCTTTCC GCTTCAGGAGAACA GGGAGAG CAGGCCTAGAGGCA GGACC GATGACTGAGCACG GTCAAAG CTTCCACAGCACCC ACAAC Reverse primer sequence Amplicon size (bp) PCR protocol CCCAGATTCCAGGT CCATATG 336 1 GCTCTGACGCCTCA CATCTC 312 1 TAGTGGTTGAGGCA GTCCCA 466 10 ATCCCTTGTCCTTCC TTCACC 309 1 AATCTCAGGGTCAC TTGGGTG 301 1 CTGAGCTTCTGCCT ACCCAAC 353 1 TCTTTATGCCAACA CAGTCACAC 374 1 GTGCTGGGCTAAGG GATTTC 329 1 ATGGATGGGTTGGG AAGTAAC 440 1 CAAGGGTGACTTCC TCCTCTC 433 1 AAGACCACCTATCC GATCCAG 431 1 CAGAGTGGGAAGG GAACTCAC 589 1 CAGTAGCTCCTGTT GAGATTGAAC 573 1 ACATACCTGTCCTT CGGGAG 503 1 ACTATACATAGCCC CCAGCC 468 1 ACAGGTGACCTTCA TGGTGC 392 1 AGGCCATGATAGCA AAGGTGA 501 1 GACCAAGGCTGAG GAGTTGAC 400 1 ACAGCGAGTGACCT TCTCAGC 374 1 CAGGTCCTGTCTTT CTCCTCG 406 1 AGGCCCTCCACAAG TTCTTAG 400 1 TTCACCACCAAGGG TACAGC 302 1 TCTAGTAGAGGCCC TGGAAGG 350 1 AAGTCCATCAAGGT CTGGGAC 483 1 GGACTACAGATGCC TGGAAGC 422 1 GAGAGGAGGGATA TCGCTGTG 482 1 TGAGATAGCACTGG TGCTTGG 502 1 GGTCGTAGGAGTCA GCGG 348 1 TCAGAGAAGCCAGT GCTTTCC 327 1 Reference Current study Current study Current study Current study Current study Current study 229

GLI2 13c GLI2 13d GLI2 13e GLI2 13f GLI2 13g GLI2 13h GLI2 13i GLI2 13k GLI2 13L GLI2 13m GLI3 1 GLI3 2 GLI3 3 GLI3 4 GLI3 5 GLI3 6 GLI3 7 GLI3 8 GLI3 9 GLI3 10 GLI3 11 GLI3 12 GLI3 13a GLI3 13b GLI3 14a GLI3 14b GLI3 14c GLI3 14d GLI3 14e GLI3 14f GLI3 14g GLI3 14h GLI3 14i GLI3 14j TAGCATCAGCGAGA ACGTGG ACGTGGTGCAGTAC ATCAAGG GAGGATGCCAGTTA GGCTTTG GGCCCTCAACCAGT TCCC CACCTCACCCAGTC CAGAG TTTGGCCTAGTGCA GCCC ATGTACGAACAGGA TGGAGGC AGCGACGGGCCGA CCTATGG CTGCTCTCCCGCAG GCTCCATCCT AGCAGTACAGCCTG CGGGCCAAGTA TGCTCCATTTGCTTT CTTTAGC TGTGTACTTTGGAC CCTCATTC CTTCAGTGAACCCA CGAACAG TTATACACGTCCCG AGTGAGG GGTGGTTCCACTTT CTCCTCC TGCATCACTACAGG TGCAAAC GATTAACAGCTGAC GTGGTGG ATGCAGTGGACAAG GAACTGC TGACTCAGCTCAGG GTCAGAG GAGCTGGTGTCATC AGTTTGC ACAGAACACACTGC GCCTTC TGTCCCTCTATGCA CCCTACC TCAGAATATGAGTC ATGCACAGTC TTTCCTATGAGAGG AGAGACCG AAACTGAAATGGA AGACAGTTTCTC TCCTATTGATTTCC GTTGGTTG GGAGCTATGGGAA AGGTTCTG TCTTGATACCATTC ACCCTGC GCTGACTCATTTGG CGCTAC CTGTTGTGGAGCAT CAAGTGC GGGAATTTAAATAC TGCACCACG GTCAGGGACTCCAG GGTGAC GCATGAACTGGAGG CAGG GCGTCTTCAGGCTC ATCCTC AAAGCCTAACTGGC ATCCTCC 307 1 GGTAGTCATGCCAG GACACTG 524 1 GTAGCTCTGGACTG GGTGAGG 590 1 CTGCTGCACAGCAC GTACC 391 1 TCCTGTTCGTACAT GTGGATCT 430 1 GATGGCATCGAAGT CAATCTG 450 1 AACATCTGTCATCT GAAGCGG 551 1 CCACGTTCTCGCTG ATGCTA 286 1 GCAGGCTCATGCGC TCCAGGC 461 7 CTCCATCGCCACGT TCTCGCT 461 1 TTTGGAAAGTTGAT GGCTCTG 331 1 AACTTCATAAAGCG CGCACAC 550 1 CAATGTTGCTTTGT GAATCGG 347 1 AGAGACAGCCTCTG CCTGTG 350 1 TGATGTGGGTTGTG TAATGGAC 369 1 TTCTTCCACGTAGG CAAGTAGC 360 1 GATGCTTGGCAATA ATCCTACC 436 1 AGCCAAATAAGACC GCTTGTC 340 1 TTCTTGTCTTCCCTC CTGTTG 300 5 GGAAGCATGCATAC ACAGTTAGC 434 9 AAAGCTGCTGACCC TTGAAAC 363 9 CCTCAAAGCCTTGT GAAAGTG 491 1 CCTCTGACCGATGG AGGTAGT 475 1 GGTGGTCCATCCGT CATTC 507 1 CCCTGAGCCCAAGT ATCATTC 390 1 TGCAGGGTGAATGG TATCAAG 540 1 CATCTACCAAGGGC CAGAGAG 541 1 GAGCACTTGATGCT CCACAAC 589 1 CCAAGCTCAAGTGT GGGC 391 1 TCGTGCTTCAGAAT TACACGC 571 1 CTGCCTCCAGTTCA TGCC 400 1 CAACATGGAGAGG ATGAGCC 399 1 CTCCTCAGGGATCT CGCC 336 1 GGGTGGACGTCACT ATGCTG 363 1 Current study Current study Current study Current study 230

GLI3 14k GLI3 14L GLI3 14m IHH 1a IHH 1b IHH 2 IHH 3a IHH 3b IHH 3c IHH 3d PTCH1 1 PTCH1 2 PTCH1 3 PTCH1 4 PTCH1 5 PTCH1 6 PTCH1 7 PTCH1 8 PTCH1 9 PTCH1 10 PTCH1 11 PTCH1 12 PTCH1 13 PTCH1 14a PTCH1 14b PTCH1 15 PTCH1 16 PTCH1 17 PTCH1 18 PTCH1 19 PTCH1 20 PTCH1 21 PTCH1 22 PTCH1 23a TAGGAGTCGGCCAC GCTC CTATCAGACCCTCG GGGAGAA AAATGAGTCAGCTG GCAGCAT ATCTTGGGCAGGAG GCAG TCGTCCTTGAAGAT GATGTCTG TTCTCGGCACTACT CCTCCTG CAGCTCAGGTCCCT TCCAG AGTACAGCAGTTCC AGGAGGG ACATCCTCCACCAC CAGTGTC GTCAGCCGTAAAGA GCAGGTG GTGTTTGTGTGTGG CGGG GCGCTGGCGAATAT CTCTATC GCCTAAACCAGCAG CCTTC GAGGCCATGCGTTA GGTTAAG TGAATGAAATTTAA TGACGCCTAC AGGCTAATGGGAG GTGTATGG TACACTTGCCGATG TCAGGAG TCATCCCATCAAGT TCCCAG ACGCTCTCTCTGTC CTGGATG AGGACACACAGCA CACAGGAG TGACACATCATCTG ACATGGG AGCCTCAAACACAG GCATTTC GTTCTCCACACCAG CACAAAC ACTCCCATGGAAGA TGACCTC GTTGTGGCAGATTA CCTTGGC TCATAATCATGACA AAGGAACCTG CTTTCTACCAGCTC CCAGTGC TGAAGGCTGTTGCT GAGTTTG ACTTCCCGGCTGCA GAAAG GGTTCCCACTTGGA GACAAAC TCCTTGACCTTCTG ATCCACC AGTATCGAAGTGAA GAGCGGC CCATCTGCCTGTGT GATGTG AGCTTGGACACATC AGCCTTG GTAGTTGTGAGGCA GGCAATG 398 1 CTCTCTGGCCCTTG GTAGATG 450 1 TCGTGTCTTGCTGA CTGAAGC 464 1 CACTTCTGCCTGGT CCTGTT 434 11 CAGCCCACCAGGAG ACCT 328 1 TCTGTTCCTAAGGG AGAGGGAC 490 1 AGCTGTCTCTACAC ACGTGGC 375 1 CTGAGAGCCTTCCA GGTCATC 514 10 CTTCATGTCCCTTTC TCCCAC 474 1 GAAGTATGGGTTCA AGCCTGC 440 1 GCTGGTCTGTCAAC CGGAG 440 2 AGTCTCGAGGGCGA GTCC 411 1 GCTCACACATCAGC CAGTCTC 391 1 AAGCTTGCTGGGTC TCTACTTG 304 1 AGAAACAGGTTACA TGGATCAGG 430 1 GCGCAGCCGTGTTA CTTTAC 459 1 CTCTCTGAAACACA CAAGCCC 306 1 AGAATTGCAGCCAG TGAGTTG 383 1 CTGAGATCTGTGCT GTCGAGG 334 1 ATGGGTGGAGGGA AACATTAG 328 1 TGCTTCAGGAGCTG TTAGGTG 332 1 CTCTGTTTCCCTAAT GCCAGC 392 1 TGTCACGGTTTCAA ATGCTTC 371 1 GGTTGAACCTCAGG CCTACAC 577 1 GATGTTATCAACCA GGCGATG 585 1 GATAAATCAGTTTA AGTGTGGTGGTG 505 1 TTCAGTCAAAGTGG ATGTGGG 426 1 TGCTCTCAAGGCAG AAGTGTG 334 1 GAGGCTATGATCAG CATTGTTTG 497 1 CTGAACCGAGGACA CCTTAGC 315 1 TGAGCAGTTCTGAG AGCTTGTAAC 362 1 TGTGAACTGCGGTT GGATAAC 324 1 TACCGTGCTTTGAG CTTTGAG 441 1 GCATTCTGGCCCTA GCAATAG 526 1 Current study Current study 231

PTCH1 23b SHH 1 SHH 2 SHH 3a SHH 3a SHH 3b SHH 3c SHH 3d SHH 3e SMO 1 SMO 2 SMO 3 SMO 4 SMO 5 SMO 6 SMO 7 SMO 8 SMO 9 SMO 10 SMO 11 SMO 12 PTCH2 1a PTCH2 2 PTCH2 3 PTCH2 4 PTCH2 5 PTCH2 6 PTCH2 7 PTCH2 8 PTCH2 9 PTCH2 10 PTCH2 11 PTCH2 12 PTCH2 13 CTGCAGCTCAATGA CTTCCAC AACCCAAGGAGGG AAGTGTG 579 3 AGTCTGGAAGTGTT GAGGAAGGGAAAG CGGCTTC CGCAAG 480 4 GAAACGCAGTCATC CCTTGAGACTGGGC GCCC AGGG 493 1 GCAGAGTAGCCCTA ACCGCT 8 CTAAGCACAACAAC AAAACTTCC 437 8 AGCCAGGTGCCTAT GTGCACAGCGTGAC TTGGTAG CCTAAG 344 1 CAGCACCTGGAGCG ACAGCGACTTCCTC GTTAG ACTTTCC 558 1 TCCTCGATGACCGC TGCCCTGTCCTCTCT GTAG TCTTTC 583 1 GGAAAGTGAGGAA TGCCCTGTCCTCTCT GTCGCTGTAG TCTTTC 201 1 AAAGTTTGCGAAGT AGCCTGTTCTTTCC TGGGC AAGGGT 595 1 AAGAACTGTCCTGC CCACTGGACCCTGC CCAGATG CCTATAC 440 1 AATAATTTGCCAAG CTTCTGATCATGAC CCAGCC CCTTCCC 473 1 AGGGTCATGATCAG AGTATGCAGTAGGG AAGGGTC CAGAGCC 397 1 CTGACTTCTGGGAA GACAGAAGGTGGG CCTCCAG TTACTGGC 411 1 GTGGCGCAGGTATA GCCCTATAGGAGCT GTGACTG AGCTGGG 348 1 GACTCCAGAGCCTT TCCCTATGGCTAAC AGGACCC TTGTCCC 304 1 AAGCAGTTCTTGGA CCATCCATTGAATC CTGAGCC TGCTGTC 384 1 AGTTGGAAGCTGCA CAAGGCTGTGCTAG GTGGG AGGCAG 416 1 CTCTGGAAAGAATG TTCCAAATAATCTG GCATCG TGTGCCC 336 1 AATGGCACTGACTA CCACTCTTCAGATC TGGGAGG CTCTGGG 344 1 AACAGGTTAAGTGC CATGCTCGGTGAGG TCCCAGG AAGAAG 585 1 TTAACACCCACACC ATTCTCACTGGCCA CCACAGT GACGATG 427 1 TCCTAGATGCCCAG CTGGGCAGCAGATA GTGTAGG TACAGGG 429 1 AAATCCCAGGACCT ACCTCTCCCACTTG GCAATAG TCTGGG 414 1 CAACCTTTGTAGGA TATGGGAAGTGAGT TGCCCTC CTGGCTG 324 1 GAGGATGTGGAGA GACTCAGAGACCGA GAGCCTTG GCAAGC 383 1 GTGGGAGCAGGGT AATCGGCAGATAAG AATAATGG AGGAGGG 436 1 AACACCTGGTGAGG TCCTGTCTGCGGGA GATGTG TACTAGC 408 1 TGGAGAAACAGGG CATACTGTAATGCC TGGATAGG AGGCAGC 326 1 AGCATGGTCACACA GACACATGACATTG GGCATAG GCTGGAG 400 1 GATTCCCAGAGCCA GCTATCTGCTCATG AGAAGG GTGGGTC 368 1 AGAGGCACCGAGG CACCTTCAATGCTG TTCATTAG CCACTAC 303 1 GCTACAAAGGTGCA CCCAATAAATGCTA GCCAAC GCCACTG 382 1 ACAGGTCTGTGCCT GCACCAGTGTCGTA TGAAATG CTCACATC 386 1 Current study Current study 232

PTCH2 14 PTCH2 15 PTCH2 16a PTCH2 17 PTCH2 18 PTCH2 19 PTCH2 20 PTCH2 21 PTCH2 22 PTCH2 23 STK36 1 STK36 2 STK36 3 STK36 4 STK36 5 STK36 6 STK36 7 STK36 8 STK36 9 STK36 10 STK36 11 STK36 12 STK36 13 STK36 14 STK36 15 STK36 16 STK36 17 STK36 18 STK36 19 STK36 20 STK36 21 STK36 22 STK36 23 STK36 24 AGTGGACTAAGCCC TCTCTGC GGTTCTATTAGCTG GTGGCCC AGCGCTTCAGTTCC CTCAAG AAACTCCAAGGGCT GAGCTG CTGGCAGGAGGGAT GACAG CAGGAATGAGCTAC CACGTCC AGGGTGTGGTCACC TCTGG GGGAGGTACCTGTC TGTGGG CGTCTAACACCAGA CCCAGTG AGGGAGGCTGCATT TGGGTC GATGTTGTGGAACT GTCCCTG ATGGAAGCGGGAG ACTAACAG GTTCCAGGAATTTC CCAACC GAATGAATGAGCA AATGAAAGAGTG CCTAATCAAAGGTG CTCCCTC AGGATGGATGTGGG ATTCTTG CACAGAAGCCTGGA GACACTG TTTCTAGGGTTGGA GAAGGGC CCCACATCCCAGAA AGAAGTC CCATGGTTTCTACT GGTTGGG AGGAATGCAAGCAT GTGGTC TTGGCAGATTCTCT GTTACCC GACTGACCCATTCA CCACATC CCTTGAAGCATGAA TGAAAGC CCATGTCGGTGAGT ACTGGTG CTGGTGAATGTGGG AAGAGTG GGGTTCTCTCACAC TCCCAAG TTATGTTGATTCAG GGCAAGG GGAATTGGGATAGA GAGCGTG CATCTGTTTCTGGA GGCATTC CTCCATGGCACCAT CACTG TACTAGGTGGGAAA GCGGAAG AATAGGTGTAGCCC TGGGAGC CAAAGGAATTGGA GGAGGGAG CTTCCTCCCGTGAC CCAC 504 1 AAGGGCAGAGAGG GCTTAGTC 467 1 ACACGGTCAGCCCC ATGTAG 482 1 ATTTCAGCCAGGTT GGGAGAG 435 1 GGAGAACCTTCGCA GTGAGTC 499 1 GTGCTCTGCTGCTC CTCAAC 408 1 TGAAGACAGATACA GCTCGGG 412 1 GCTGGTTCCCACTT TGACTTC 308 1 CCCAACAGTCATGG TAATCCC 421 1 GCAGAGGGAATGG CGTATGA 308 1 TGCACCCAAATAAT CCAACTTC 330 1 CCAGATTCATGCAT TCTGAGG 366 1 TGAGACCCAGCTAG GAAGGAG 374 1 AAAAGATTCCGTGT GGAACT 368 1 TTCAGTAACTGCAA AGTCCCG 477 1 GTTCAAGCTGCTGG AGGAAAG 310 1 TCCAACCCTAGAAA GTCACCC 350 1 GGCTAGTGACAGGG ACCATTC 376 1 TTAGGGAGTTGGGA ATCACTG 340 1 AAGAAACCTGAGG GCTGCTC 407 1 TGGCAGACCTTATC TGCCTG 480 1 TTCCTAAATGGGAA GGGCAC 345 1 CCTAGTCCCTTGAA GGCAGG 354 1 ATGGCAATAGAAA GAGCGGAG 405 1 GGGAGCATCTGTTT CTGGAAG 340 1 CAGAAAGGAGTCTT CCAAGGG 373 1 GCCGAAAGACAAG AAGGGAG 372 1 CACGCTCTCTATCC CAATTCC 303 1 TGCAAATTGGATAG TGACTCCC 329 1 CAGTTTACTCCAGG CTTGCTG 329 1 AGTTCCAAGGCTAT CACAGGC 301 1 GTGCAGACACTGCT TCCTGTC 438 1 GCCACTACTTAGGT TTCCAGCC 375 1 CAGCTGTTCCATGC TTACAGG 343 1 Current study Current study Current study 233

STK36 26 STK36 25a STK36 25b STK36 25c DHH 1 DHH 2 DHH 3 DHH 3 DHH 3 DHH 3 SUFU 1 SUFU 2 SUFU 3 SUFU 4 SUFU 5 SUFU 6 SUFU 7 SUFU 8 SUFU 9 SUFU 10 SUFU 11 SUFU 12 HHIP 1 HHIP 2 HHIP 3 HHIP 4 HHIP 5 HHIP 6 HHIP 7 HHIP 8 HHIP 9 HHIP 10 HHIP 11 HHIP 12 TGGGTAAATGAGAG TCCTCCG GCCCTTGCAAAGTA AGGAATG GTGAACACAGTGTC TGCCTCC TATCCAAGAGCTTC TGGCTGG GTGAAGCTGGACTC ACCCAC ACTAAAGCCCGCTT GGTCTC GCTTCGAGGTTTCT ATGCCTG TCCTCTCAGTACGA GGTTGCC TCTCCACAGCCACA AATGAAG TAGCGCGTGCAGCA GTCT GGAGTCTCACCCAC CGAGTC GCCTTAGCTGTCCA TCCCTTAG GATTTGGATACTGA GGCCACC AGCCTGGGCTAGTG AGATCC CCTGGGTAGCTGAC CTTCTTG TCCCTGACCACGAA CTATTCC CTGCTCTCCAGCAT TTGTCC GATGCGTAGAGTGG GCTCAG CTGGAGACTCCCAT CTTGCTC AGCGTGTTTGGATA CAGTCCC TCATCTCTCCTCCAT GGTCAG CCTGTCCTATCCCT AGCTCCC CCTCCCTCTGTCTCT GGAGTG GCCCAGCTCCAGAG ACTATAC TGGTGGGATTACAG GACTGTTT CCTTGGGTTAGCAA GCACTTC AAGACAATTGTCTT GTTCTAACCC TCCAGCAATTTCTG TTAGCTCC CCTTGAACTCTTGG CATTAAAC ACAAAGGGCTCCTT CCTATGT CTAGCAAGAAACCC TTTGGTG AGCCAGGTCTATGG CAAAGC AAGAACAAGAGTG GCAACAGG TAAGAGGCATGCTT TGTAAGCC GAGTTGGCGGCTTG TGTAGTAG 549 1 GGAGCCTATAAGTG TGTGCCC 575 1 CCACATGCCATTTC TAGGAGC 600 1 CCCGATTCCAATTC CTTACAG 308 1 GCCTGTGGAGATGC CTAACTG 468 1 GAATCAACCCTCCT ATCCAGG 446 1 CTCCACGCAAACTG TTGCTC 358 1 GCTGCTGGTGAACG ATGTC 437 1 GGGCCAGTCAGATT AACGG 572 1 TGGTCTTGATTCAA TCCTCCC 327 1 ACAAATTACTTCGC CCTCGG 352 1 CCACGGAGAAGTAC CATGCTC 412 1 AAAGGCCACTCCAC AAACATC 345 1 AAACAGAAATCAG GACGAGGC 354 1 GGGACTGTCAGGGA GTGAGTC 311 1 GTGCTCTGCAGAAT TCAGGG 351 1 GCTGGAAGACTTCA CTTGCTG 443 4 CAAGGGCTATGTGC ATTGTTC 349 1 ATCAGGGAGACAG GGAGACG 367 1 CTGCCAAGGGAGA AGAGGAG 384 1 GCCACACAGATACA TGCAAGC 305 4 GCAGTCTTCCTCAC ACTTGTCC 317 2 TTGACCTTCTTACA ACCACCG 504 1 AAATTTGGAAATGT TAATTATGAGGT 528 3 CCACAAGATTTGTC AGTTGGG 374 1 AATGTCACTGTCCT AATTCAGGTG 509 1 CACCTTATGGTTAC TTATTTGCAAC 466 1 TGGTTCTTAATTCA CTGGTGCC 416 1 TTCTCAATCTGCTTC TCACAGTAAT 554 1 GAACCCTCAGGGCT TGTAAGA 403 1 CAAGCCTGTTGAGA GAGCATC 461 1 CTTCAGGGCAGAAC AAAGCC 318 1 GACAGCAAGGACG ACATGAAT 600 1 TGATCTAGTTGTGC TATTTGCATTT 528 1 Current study Current study 234

HHIP 13 KIF7 1 KIF7 2 KIF7 4a KIF7 4b KIF7 3 KIF7 5 KIF7 6 KIF7 7 KIF7 8+9 KIF7 10 KIF7 11 KIF7 12 KIF7 13 KIF7 14 KIF7 15 KIF7 16 KIF7 17 KIF7 18a KIF7 18b TTTGTTGGTCTCAT GCTCAGTC AGGAGCTGCAGGAT CTGG CACTGCCTTCTCCA TCCTAGAG ACCCTACCGCGACT CCAAGATC GAGACCCTCAACAC CCTCAAC GGTCAGGAAGGGC AGGTT CACCAGTGGGTCTG GAGTTC TTTGACACTCCTCA TTTTCAGC CAGAGGGGTCATTT GAGCTG TCTGCCAGACTCAC ATCCTG CAAAGGCTTGGTGA AACCC CCGGCCTCTGTTAT TATTATTGG CTAAGTGTTTGCGT CTATTTGCC CATGAGCAACAGCA GAAGATCC GCTGAGCAGAACAT TTGGAG GCTGACTTGGCCCT TGG GGGAAGACCCTGGT GATTTC ACAGCCCCTGCACC TACA TGGGGAAAGCTTAG AGACCA GACAGGCTCCTGGA AATGAA AGAGGATCTGCACA TTTCTGG 422 1 CACAGATCCCTGTC TAGGAAATC 591 1 GCTAAAGCAAAACC TCCCAG 458 1 TGGATGATGCGGGT CTCGGAG 460 6 CTCTTCCTTCCTCTC CACCAC 506 6 CCCAGCGAGTCTTT GAGGAT 717 1 GACTCTACACCCCT ACCCCG 376 1 ACAGCTGAAAGCA GCCTCTC 494 1 CATCCTAGGACCCA GAGGC 386 1 AGGCTGGGCTGAGT ATCAAA 721 1 CTATACCAGCCTCA CCCTGC 472 1 CAGGCTAAGTGACC TGCTTTC 467 1 CCACTTCTTCTGCTC CTCAAT 473 1 AAACTTACTTGGCA GGAGCTCA 530 6 AGTGAAAACTTGGG TCTGCC 474 1 CTGCAGATGAGTTG GTCCTG 473 1 GCCGGGGTTGTGAG CCAT 358 1 AAGCAACCTGTGAA TTGAGC 398 1 ACAAAGGCCCAAA GTTCCAG 445 1 TTGATCCCAGTGAG GGTACAG 473 1 Putoux et al., 2011 Putoux et al., 2011 Current study Current study Putoux et al., 2011 Putoux et al., 2011 Putoux et al., 2011 Putoux et al., 2011 Putoux et al., 2011 Putoux et al., 2011 Putoux et al., 2011 Current study Current study Putoux et al., 2011 Putoux et al., 2011 Putoux et al., 2011 Putoux et al., 2011 Putoux et al., 2011 Putoux et al., 2011 Refer to Appendix B for the respective PCR protocol. 235

APPENDIX B PCR protocols for amplification of Hedgehog pathway genes Protocol 1 Step Cycle Temp ( o C) Time 1 1 94 2 min 2 3 94 10 s 64 10 s 72 30 s 3 3 94 10 s 61 10 s 72 30 s 4 3 94 10 s 58 10 s 72 30 s 5 40 94 10 s 57 10 s 72 30 s 6 1 72 5 min 7 1 20 Protocol 2 Step Cycle Temp ( o C) Time 1 1 94 2 min 2 40 94 10 s 64 10 s 72 30 s 3 1 72 5 min 4 1 20 Protocol 3 Step Cycle Temp ( o C) Time 1 1 94 2 min 2 40 94 10 s 57 10 s 72 30 s 3 1 72 5 min 4 1 20 236

Protocol 4 Step Cycle Temp ( o C) Time 1 1 95 2 min 2 40 95 10 s 61 10 s 72 30 s 3 1 72 5 min 4 1 20 Protocol 5 Step Cycle Temp ( o C) Time 1 1 94 2 min 2 40 94 10 s 60 10 s 72 1 min 3 1 72 5 min 4 1 20 Protocol 6 Step Cycle Temp ( o C) Time 1 1 94 2 min 2 40 94 10 s 60 10 s 72 30 s 3 1 72 5 min 4 1 20 Protocol 7 Step Cycle Temp ( o C) Time 1 1 94 2 min 2 40 94 10 s 68 10 s 72 30 s 3 1 72 5 min 4 1 20 237

Protocol 8 Step Cycle Temp ( o C) Time 1 1 94 2 min 2 35 94 30 s 61.5 30 s 72 30 s 3 1 72 5 min 4 1 20 Protocol 9 Step Cycle Temp ( o C) Time 1 1 94 2 min 2 40 94 10 s 57 10 s 72 1 min 3 1 72 5 min 4 1 20 Protocol 10 Step Cycle Temp ( o C) Time 1 1 94 2 min 2 40 94 10 s 64 10 s 72 30 s 3 1 72 5 min 4 1 20 PCR product was loaded onto a 2% agarose gel and electrophoresed for 1.5 h at 100V. Under UV illumination, the DNA band of interest was stabbed with a sterile pipette tip that was then dipped into a new PCR reaction mix. PCR was carried out using the thermal cycling condition as above. Protocol 11 Step Cycle Temp ( o C) Time 1 1 95 2 min 2 40 95 10 s 61 10 s 72 30 s 3 1 72 5 min 4 1 20 238

PCR product was loaded onto a 2% agarose gel and electrophoresed for 1.5 h at 100V. Under UV illumination, the DNA band of interest was stabbed with a sterile pipette tip that was then dipped into a new PCR reaction mix. PCR was carried out using the thermal cycling condition as above. 239

APPENDIX C Exonic primer sequences for PCR assay for detection of PTCH1 exon deletions Gene Exon PTCH1 18 PTCH1 19 PTCH1 20 PTCH1 21 PTCH1 22 PTCH1 23 Forward primer sequence CATCGAGTATGCCC AGTTCCCTT GCTGATGACGGTCG AGCTGTTC CCTTTCTGACGGCCA TCGGCGACAA GTATTTCTTTGCTGT GCTGGCG CAGACGACAGTGTC AGGCCTCA CGTTCTCACAACCCT CGGAACC Reverse primer Amplicon sequence size (bp) GGTCCTTACTTTTTC AATTGCCTCC 143 GGTGAACTCCACTCC TATGCCA 109 TCAGATCCCGCCAG CATCAGCACTC 124 AAAAGCACGGGAAG CAAAACCA 75 TGGCTTCCACGATCA CTTGGTG 152 CAGTGGTGATGGGC TGGCAGTA 243 Reference Current study 240

APPENDIX D Buffers and solutions 10x Phosphate Buffered Saline (PBS) 1.49 M NaCl 160 mm Na 2 HPO 4 52 mm NaH 2 PO 4.H 2 O 1 L double distilled H 2 O (ddh 2 O) ph to 7.4 1x SDS-PAGE Running Buffer 250 mm Tris Base 190 mm Glycine 17 mm SDS 1x Transfer Buffer 25mM Tris (ph 8.3) 192 mm Glycine 20% Methanol 10x Tris Buffered Saline (TBS) 200 mm Tris (ph 7.4) 1.37 M NaCl 1 L ddh 2 O 1x TBS-0.1% Tween 20 0.1% Tween 20 in 1x TBS 5% Blocking Buffer 5g Skim Milk Powder 100 ml 1x TBS-0.1% Tween 20 50x 0.5 M Borate buffer 15.4 g Boric acid 6.75 ml 10 M NaOH Solution was made up to 500ml using ddh 2 O ph to 8.5. 2% Methylene Blue 2 g Methylene blue 100 ml 0.01M borate buffer (10 ml of 0.5 M borate buffer made up to 500 ml) Solution was filtered prior to use. 241

50x TAE Buffer 242 g Tris base 57.10 ml Glacial acetic acid 5 mm EDTA (ph 8.0) 1 L ddh 2 O Agarose Gel 1, 1.5, 2 or 4% Agarose powder 20 ml 50 x TAE buffer 80 ml ddh 2 O 242