Epigenetic based therapeutic strategies in breast cancer Niaz Mahmood Division of Experimental Medicine Department of Medicine McGill University, Montreal Quebec, Canada August 2020 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © Niaz Mahmood, 2020 1 TABLE OF CONTENTS ABSTRACT .......................................................................................................................10 RÉSUMÉ ...........................................................................................................................12 ACKNOWLEDGMENTS .................................................................................................15 PREFACE ..........................................................................................................................18 CONTRIBUTION OF AUTHORS....................................................................................22 CONTRIBUTION TO ORIGINAL KNOWLEDGE ........................................................25 LIST OF ABBREVIATIONS ............................................................................................28 Chapter One: Introduction and literature review .............................................................30 EPIGENETICS AND CANCER .......................................................................................31 DNA METHYLATION .....................................................................................................33 DNA methylation is evolutionarily primitive ................................................................33 DNA methylation writers, readers, and erasers: Role in gene regulation ......................34 BREAST CANCER HETEROGENEITY: A CHALLENGE FOR THERAPEUTIC INTERVENTIONS ...................................................................................................40 ABERRANT DNA METHYLATION IN CANCER ........................................................41 DNA HYPOMETHYLATION AND CANCER ...............................................................43 Factors contributing to DNA hypomethylation .............................................................44 Impact of DNA hypomethylation ..................................................................................47 DNA hypomethylation in diagnosis and prognosis of cancer .......................................50 HYPERMETHYLATION IN CANCER: ..........................................................................53 TARGETING ABNORMAL DNA METHYLATION AS AN ANTI-CANCER STRATEGY ..............................................................................................................55 Targeting hypomethylation ............................................................................................55 Targeting hypermethylation ...........................................................................................66 Targeting DNA methylation readers ..............................................................................77 Current state of knowledge on targeted Epi-therapies ...................................................82 RATIONALE OF THE PROJECT ....................................................................................86 OBJECTIVES OF THE PROJECT ...................................................................................87 Chapter Two: Methyl Donor S-adenosylmethionine (SAM) supplementation Attenuates Breast Cancer Growth, Invasion, and Metastasis in vivo; therapeutic and chemopreventive applications ..............................................................................88 PREFACE ..........................................................................................................................89 ABSTRACT .......................................................................................................................90 2 INTRODUCTION .............................................................................................................91 RESULTS ..........................................................................................................................93 SAM-treatment suppresses cell proliferation, migration, invasion, anchorage- independent growth and potentiates apoptosis in vitro ..........................................93 SAM-treatment reduces tumorigenesis and metastasis in MDA-MB-231 xenograft mice model .............................................................................................................95 SAM-treatment differentially regulates genes implicated in cancer progression and metastasis ...............................................................................................................97 SAM-treatment changes promoter methylation status and protein expression of prometastatic genes ................................................................................................99 SAM is bioavailable in the serum of experimental animal with no adverse behavioral and physiological changes .................................................................100 DISCUSSION ..................................................................................................................102 MATERIALS AND METHODS .....................................................................................108 Cell culture and treatments ..........................................................................................108 Cell proliferation, migration, invasion and anchorage-independent growth assay ......109 Apoptosis assay ............................................................................................................109 Study approval and in vivo xenograft model ...............................................................110 RNA extraction and quantitative real-time PCR (qPCR) ............................................111 Gene expression microarrays .......................................................................................111 Determination of SAM levels in the serum by ELISA ................................................112 DNA extraction, Bisulfite conversion, and Pyrosequencing .......................................112 Western blot and immunohistochemistry ....................................................................113 Behavior test ................................................................................................................113 Statistical analysis ........................................................................................................113 ACKNOWLEDGEMENT ...............................................................................................114 CONFLICT OF INTEREST ............................................................................................114 REFERENCE ...................................................................................................................114 SUPPLEMENTALS FOR CHAPTER 2 .........................................................................126 Chapter Three: S-adenosylmethionine in combination with Decitabine shows enhanced anti-cancer effects in repressing breast cancer growth and metastasis ...............139 PREFACE ........................................................................................................................140 ABSTRACT .....................................................................................................................141 INTRODUCTION ...........................................................................................................142 3 MATERIAL AND METHODS .......................................................................................144 Cell culture and treatments ..........................................................................................144 Cell proliferation and anchorage-independent growth assay .......................................145 Apoptosis assay ............................................................................................................146 In vivo xenograft model of breast cancer ....................................................................146 RNA extraction and qPCR ...........................................................................................147 RNA-Seq and analysis pipeline ...................................................................................147 DNA isolation and MethylationEpic 850 K BeadChip microarray .............................148 Western blot .................................................................................................................149 Immunohistochemistry ................................................................................................149 Statistical analysis ........................................................................................................149 RESULTS ........................................................................................................................150 Effect of SAM+5AzadC combination on TNBC cell lines in vitro .............................150 Effect of SAM+5AzadC combination on MDA-MB-231 xenograft model of breast cancer ...................................................................................................................151 Effect of SAM+5AzadC combination on the MDA-MB-231 transcriptome ..............154 Validation of differentially expressed cancer-related genes affected by the combination treatment in MDA-MB-231 ............................................................155 Effect