IDENTIFICATION OF THE POLYCOMB PROTEIN CBX2 AS A POTENTIAL DRUG TARGET IN ADVANCED PROSTATE CANCER AND BEYOND by Pier-Luc Clermont B.Sc. (Honours), McGill University, 2012 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Interdisciplinary Oncology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2015 © Pier-Luc Clermont, 2015 Abstract Globally, prostate cancer (PCa) represents the most commonly diagnosed cancer in men. While localized PCa can often be cured, all patients with metastatic disease inevitably develop castration-resistant prostate cancer (CRPC) or neuroendocrine prostate cancer (NEPC). Increasing evidence suggests that epigenetic alterations involving the Polycomb Group (PcG) family drive PCa progression. Although the PcG protein CBX2 is required for prostate development, its implication in human cancer remains unexplored. I therefore hypothesized that CBX2 may become deregulated during PCa progression and induce transcriptional programs promoting PCa aggressiveness. Using patient-derived xenografts and clinical datasets, I have explored the epigenetic landscape of advanced PCa and identified the Polycomb Group protein and epigenetic reader CBX2 as a potential drug target. First, CBX2 was significantly up-regulated in metastatic and castration- resistant PCa tissues. Furthermore, CBX2 overexpression predicted lower overall survival and correlated with numerous adverse prognostic factors. In addition, CBX2 depletion induced proliferation arrest and apoptosis in metastatic PCa cell lines, implying that CBX2 is required for PCa cell survival. Microarray analysis conducted after CBX2 silencing revealed that CBX2 regulates many genes controlling cellular proliferation and differentiation. Given the rising incidence of NEPC in advanced PCa, I analyzed whether CBX2 was also involved in NEPC pathogenesis. Strikingly, CBX2 was consistently the most highly up-regulated epigenetic regulator across multiple clinical and xenograft tumor tissues. Furthermore, I derived a list of 185 genes down-regulated in NEPC that was preferentially enriched in PcG target genes and predicted poor clinical outcome, in line with a critical function for CBX2 in late-stage PCa. Since CBX2 has never been linked to human cancers, I conducted a comprehensive meta-analysis of CBX2 across many tumor types using previously published clinical data. Strikingly, these studies demonstrated that the CBX2 locus is rarely inactivated or down-regulated. In contrast, CBX2 was frequently amplified and over-expressed in many common tumors, where it correlated with metastatic dissemination and poor clinical outcome. Overall, this work identifies CBX2 as novel epigenetic driver of cancer progression and investigates the therapeutic potential of CBX2 in advanced solid malignancies. ii Preface Tumor tissues were obtained from patients through a protocol approved by the Clinical Research Ethics Board of the University of British Columbia (UBC) and the BC Cancer Agency (BCCA). All patients signed a consent form approved by the Ethics Board (UBC Ethics Board #: H09- 01628 and H04-60131; VCHRI #: V09-0320 and V07-0058). Animal care and experimental procedures were carried out in accordance with the guidelines of the Canadian Council of Animal Care (CCAC) under the approval of the Animal Care Committee of University of British Columbia (permit #: A10-0100). Portions of Chapter 1 have been adapted from [Clermont PL], Crea F, Helgason CD. Chapter 22: Trithorax genes in prostate cancer. In "Advances in Prostate Cancer" (ISBN 978-953-51-0932-7; edited by Gerhard Hamilton), InTech, 2013. I am the original writer of all portions included. Crea F and Helgason CD critically revised the manuscript. A version of Chapter 2 is being prepared for publication. [Clermont PL], Crea F, Chiang YT, Lin D, Zhang A, Wang JZL, Parolia A, Wu R, Xue H, Wang Y, Ding J, Thu KL, Lam WL, Shah SP, Collins CC, Wang Y, Helgason CD. (2015, in preparation) Identification of the epigenetic reader CBX2 as a potential drug target in advanced prostate cancer. I am first author of this manuscript. I designed experiments, collected and analyzed data, and wrote the manuscript. Lin D, Wu R, Xue H, Wang Y, and Wang Y were involved in the generation and characterization of the patient- derived xenograft models. Crea F, Chiang YT, Zhang A, Wang JZL, Parolia A, Ding J, and Thu KL provided assistance with experimental procedures and analysis. Lam WL, Shah SP, Collins CC, Wang Y, and Helgason CD critically revised the manuscript. A version of Chapters 1 and 3 has been accepted for publication. [Clermont PL], Lin D, Crea F, Wu R, Xue H, Wang Y, Thu KL, Lam WL, Collins CC, Wang Y, Helgason CD. (2015) Polycomb-mediated silencing in neuroendocrine prostate cancer. Clinical Epigenetics. I am first author of this manuscript. I designed experiments, collected and analyzed data, and wrote the manuscript. Lin D, Wu R, Xue H, Wang Y, and Wang Y were involved in the generation and characterization of the patient-derived xenograft models. Crea F and Thu KL provided assistance iii with experimental procedures and analysis. Lam WL, Collins CC, Wang Y, and Helgason CD critically revised the manuscript. A version of Chapters 1 and 4 has been published. [Clermont PL], Sun L, Crea F, Zhang A, Parolia A, Thu KL, Lam WL, Helgason CD. Genotranscriptomic meta-analysis of the polycomb gene CBX2 in human cancers: Initial evidence of an oncogenic role. British Journal of Cancer. 2014 Oct 14;111(8):1663-72. I am first author of this manuscript. I designed experiments, collected and analyzed data, and wrote the manuscript. Sun L, Crea F, Zhang A, Parolia A and Thu KL provided assistance with experimental procedures and analysis. Lam WL and Helgason CD critically revised the manuscript. iv Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iii Table of Contents .......................................................................................................................... v List of Tables ................................................................................................................................. ix List of Figures ............................................................................................................................... xi List of Abbreviations .................................................................................................................. xiii Acknowledgements ...................................................................................................................... xv Dedication .................................................................................................................................. xvii 1. Introduction .............................................................................................................................. 1 1.1. Prostate cancer ................................................................................................................... 1 1.1.1. Overview .................................................................................................................... 1 1.1.2. Prostate development and physiology ........................................................................ 3 1.1.3. Prostate tumorigenesis and metastasis ....................................................................... 4 1.1.4. Clinical management .................................................................................................. 5 1.1.5. Castration-resistant prostate cancer ............................................................................ 6 1.1.6. Neuroendocrine prostate cancer ................................................................................. 8 1.1.7. Experimental models .................................................................................................. 9 1.2. Polycomb group complexes ............................................................................................ 11 1.2.1. Epigenetic control of transcription ........................................................................... 11 1.2.2. Composition and molecular mechanisms ................................................................. 13 1.2.3. Functions in development and cancer ...................................................................... 15 1.2.4. Polycomb-mediated silencing in prostate cancer ..................................................... 16 v 1.2.5. CBX gene family ...................................................................................................... 18 1.3. CBX2 ............................................................................................................................... 19 1.3.1. Structure and function .............................................................................................. 19 1.3.2. Roles in development ............................................................................................... 21 1.4. Thesis theme and rationale .............................................................................................. 23 1.5. Hypotheses and specific aims ........................................................................................