University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2017 The Role of the ING3 Epigenetic Regulator in Prostate Cancer Nabbi, Arash Nabbi, A. (2017). The Role of the ING3 Epigenetic Regulator in Prostate Cancer (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/28360 http://hdl.handle.net/11023/3584 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY The Role of the ING3 Epigenetic Regulator in Prostate Cancer by Arash Nabbi A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN MEDICAL SCIENCE CALGARY, ALBERTA JANUARY, 2017 © Arash Nabbi 2017 Abstract INhibitor of growth (ING) proteins are epigenetic regulators and stoichiometric members of histone acetyltransferase (KAT) or histone deacetylase (KDAC) complexes. By reading the histone mark H3K4me3, they direct their complexes to chromatin to alter gene expression. This thesis focuses on the role of ING3 in prostate cancer biology. Since rigorous characterization of antibodies is a prerequisite to acquire reliable results, we began by characterizing a new mouse monoclonal antibody against ING3. We profiled the expression of ING3 protein in normal human tissues and found that it is highly expressed in bone marrow, suggesting high expression in hematopoietic cell precursors. We also reported that ING3 protein levels are highest in proliferating tissues of the small intestine and epidermis. These data suggest a role for ING3 in promoting cell growth and renewal. In the second part of this study, we investigated the effects of ING3 on the androgen receptor (AR) pathway in prostate cancer (PC). We hypothesized that ING3 by virtue of being an essential member of TIP60 KAT complex, plays a role in post-translational modifications of AR protein and thereby contributes to PC progression. We found that the levels of ING3 and AR are positively correlated in patient samples and cell lines. ING3 potentiates androgen effects, activating expression of androgen responsive genes and AR-regulated reporters. We showed that ING3 interacts with the binding domain of AR and this interaction happens in the cytoplasm in the absence of androgens. ING3 increases AR-TIP60 interaction, promoting AR acetylation and nuclear translocation. The activating role of ING3 is independent of its ability to target the TIP60 complex to H3K4me3, identifying a previously unknown function for ING3. Knockdown of ING3 inhibits PC cell proliferation and migration, establishing ING3 as a positive regulator of growth in PC. ii Lastly, we asked whether ING3 could serve as a biomarker to distinguish latent versus aggressive PC. ING3 levels are higher in aggressive PC, with high levels of ING3 predicting shorter overall survival. Analysis with other predictive factors shows that including ING3 levels provides more accurate prognosis in PC. iii Acknowledgements I would like to express my gratitude to my doctoral supervisor Dr Karl Riabowol for the opportunity to pursue my graduate studies and to be part of his research group. His patience, support and supervision have been undoubtedly crucial to develop this project from start to completion and to help me think independently and test my own ideas and hypotheses. I would like to thank my supervisory committee members, Dr Randal Johnston, Dr Frank Jirik and Dr Tarek Bismar, who have been tremendously helpful with their advice and guidance during my PhD program. I would also like to thank Dr John Lewis and Dr Donald Morris for taking time out of their schedule to attend my PhD defense as external examiners. I would like to thank all past and present members of Riabowol laboratory - Keiko, Uma, Alex, Laura, Yang, Fangwu, Satbir and Tae-sun for their help, friendship and scientific suggestions throughout my work. My special thanks goes to Subhash, who has been always kind and patient to teach me the nuts and bolts of techniques and to provide me with his invaluable comments during my journey in science. It would not have been possible nor enjoyable without his friendliness and encouragement. This work would not have been possible without the inter- and intra-departmental support. Thanks to Ms Donna Boland from SACRI antibody facility, who generated and screened numerous hybridoma antibodies and provided me with unending supply of antibodies to perform my experiments. I would like to express my appreciation to members of translational laboratories, especially Dr Emeka Enwere and Ms Michelle Dean for their contributions to my study on tissue samples. I would like to thank Dr Shirin Bonni and her laboratory for helping me optimize my luciferase system and accommodating me to use their equipment in a set of my experiments. I would like to appreciate Dr Olivier Binda and Dr Ula McClurg from Newcastle iv University and Dr Dieter Fink from Vienna University for ongoing collaborations to take this project to next step. And finally, special thanks to my parents for their constant care and encouragement during difficult times and my siblings, Christoph, Negi and Ali who have never left me without support, laugh and happiness in face of disappointment and frustration. v Table of Contents Abstract ............................................................................................................................... ii! Acknowledgements ............................................................................................................ iv! Table of Contents ............................................................................................................... vi! List of Tables ................................................................................................................... viii! List of Figures and Illustrations ......................................................................................... ix! List of Symbols, Abbreviations and Nomenclature ........................................................... xi! CHAPTER ONE: INTRODUCTION ..................................................................................1! 1.1. Prostate cancer epidemiology ...................................................................................2! 1.2. Diagnosis and grades of prostate cancer and current challenges ..............................5! 1.3. Management of prostate cancer ................................................................................9! 1.4. Molecular mechanisms of prostate cancer progression ..........................................16! 1.4.1. Androgen receptor (AR) pathway ..................................................................16! 1.4.2. Mechanisms of Castrate Resistant Prostate Cancer (CRPC) progression ......21! 1.5. The INhibitor of growth (ING) family of epigenetic regulators .............................28! 1.5.1. ING3 is a member of the TIP60 complex ......................................................36! 1.6. Hypothesis and specific aims ..................................................................................37! CHAPTER TWO: MATERIALS AND METHODS ........................................................39! 2.1.Cell culture and transfections ..................................................................................40! 2.2. Generation of ING3 mouse monoclonal antibody ..................................................40! 2.3. Cloning and viral preparations ................................................................................41! 2.4. SDS-PAGE and western blotting ............................................................................42! 2.5. Immunoprecipitation (IP) .......................................................................................42! 2.6. In vitro acetylation assay ........................................................................................42! 2.7. Luciferase assay ......................................................................................................43! 2.8. Chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) ................44! 2.9. Immunofluorescence ...............................................................................................47! 2.10. Alamar Blue metabolic survival assay ..................................................................47! 2.11. Colony forming assay ...........................................................................................47! 2.12. Patient cohort ........................................................................................................47! 2.13. Immunohistochemistry and automated immunofluorescence ..............................50! 2.14.Transwell migration assay .....................................................................................51! 2.15. Wound healing assay ............................................................................................51! 2.16. Statistical analysis .................................................................................................51! CHAPTER THREE: RESULTS ........................................................................................53!