Characterizing and Selectively Targeting RNF20 Defects Within Colorectal Cancer Cells by Brent John Guppy a Thesis Submitted To
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Characterizing and Selectively Targeting RNF20 Defects Within Colorectal Cancer Cells by Brent John Guppy A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba In partial fulfillment of the requirements of the degree of Doctor of Philosophy Department of Biochemistry and Medical Genetics University of Manitoba Winnipeg Copyright © 2016 by Brent John Guppy ABSTRACT By 2030, the global colorectal cancer burden is projected to approximately double. This highlights the immediate need to expand our understanding of the etiological origins of colorectal cancer, so that novel therapeutic strategies can be identified and validated. The putative tumor suppressor gene RNF20 encodes a histone H2B mono-ubiquitin ligase and has been found altered/mutated in colorectal and numerous other cancer types. Several studies suggest that RNF20, and by extension mono-ubiquitinated histone H2B (H2Bub1), play important roles in maintaining genome stability in human cells. Indeed, hypomorphic RNF20 expression and/or function have been shown to underlie several phenotypes consistent with genome instability, making aberrant RNF20 biology a potential driver in oncogenesis. Through an evolutionarily conserved trans-histone pathway, RNF20 and H2Bub1 have been shown to modulate downstream di-methylation events at lysines 4 (H3K4me2) and 79 (H3K79me2) of histone H3. Accordingly, understanding the biology associated with RNF20, H2Bub1, H3K4me2, and H3K79me2 is an essential preliminary step towards understanding the etiological origins of cancer-associated RNF20 alterations and identifying a novel therapeutic strategy to selectively kill RNF20-deficient cancers. In this thesis, I employ single-cell imaging, and multiple biochemical techniques to investigate the spatial and temporal patterning and characterize the biology of RNF20, H2Bub1, H3K4me2 and H3K79me2 throughout the cell cycle. In addition, I employ the CRISPR-Cas9 genome editing system to generate RNF20-deficient HCT116 cells. Finally, I employ synthetic lethal strategies to selectively kill RNF20-depleted cells. In conclusion, the research chapters contained within this thesis have characterized putative drivers in cancer (Chapter 3), generated a valuable research reagent for CRISPR-Cas9 ii genome editing experiments (Chapter 4), and identified a novel therapeutic strategy to selectively kill certain cancer cells (Chapter 5). This thesis has increased our understanding of the etiological origins of cancer and generated novel reagents and treatments strategies that after further validation and clinical studies, could be employed to reduce morbidity and mortality rates associated with cancer. iii ACKNOWLEDGEMENTS First and foremost I would like to thank my supervisor, Dr. Kirk McManus, for providing me with guidance, support, and knowledge throughout the years. I am truly fortunate to have been a team member within the McManus lab. Kirk you are truly a science-mentor rockstar. I have enjoyed watching the lab grow over the past 6 years and look forward to the future. I would also like to thank my research committee, Drs. Mai, Davie, and Kung for providing me with valuable input and direction during my studies at the University of Manitoba. I would also like to thank the additional members of my Candidacy Examination Committee, Drs. Wigle, Ogilvie- Werbowetski, Kung, Gietz, and Murphy. I would like to acknowledge additional individuals for technical assistance during my PhD program including Zelda Lichtensztejn, Babu Sajesh, Laura Thompson, Shannon Healy, Amy Cisyk, Aaron MacAulay, Jeff Schacter, Signe Penner-Goeke, Raghvendra Vishwakarma, Alexandra Kuzyk, Nicole Wilkinson, Laryssa Sawchuk and Nermin Moujani. I would like to acknowledge the various agencies that have provided me funding in the form of studentships and travel awards during my tenure as a PhD student; the Mindel and Tom Olenick Research Studentship in Medicine, the Sheu L. Lee Family Scholarship in Oncology, the Manitoba Health Research Council PhD Studentship, the Dieter R. Hettig Scholarship in Cancer Biology, the Rt. Hon. Don Mazankowski Award for Excellence in Oncology Research, the Faculty of Graduate Studies Travel Award, the Canadian Cancer Research Alliance Conference Travel Award, and the Canadian Society for Molecular Biosciences Travel Award. I would like to also thank those who I consider family and who have always supported my dreams; Mom, Dad, Ted, Aaron, Ryan, Christine, Craig, Camille, Mark, Jill, Sajesh, and Zelda. Thank you very much for all your support. iv TABLE OF CONTENTS ABSTRACT………………………………………………………………………………... ii ACKNOWLEDGEMENTS………………………………………………………………... iv TABLE OF CONTENTS…………………………………………………………………... v LIST OF ABBREVIATIONS……………………………………………………………… ix LIST OF TABLES…………………………………………………………………………. xii LIST OF FIGURES………………………………………………………………………... xiv USED WITH PERMISSION AND CONTRIBUTION OF AUTHOR……………………. xvi Chapter 1 - INTRODUCTION 1.0.0 The Future Cancer Burden in Developed Nations…………………………………… 1 1.0.1 The Future Cancer Burden in Canada……………………………………… 2 1.0.2 A Focus on Colorectal Cancer in Canada………………………………….. 3 1.0.3 Colorectal Cancer Diagnosis, Clinical Pathogenesis, and Treatment……… 4 1.1.0 The Chromosome Instability Phenotype……………………………………………... 5 1.1.1 Numerical and Structural Chromosome Instability………………………… 7 1.1.2 Mechanisms of CIN………………………………………………………… 7 1.1.3 The Biological Impact of Chromosome Instability………………………… 9 1.1.4 Therapeutic Interventions Targeting Chromosome Instability…………….. 10 1.2.0 Histone Post-Translational Modifications - Structure and Function………………… 11 1.2.1 The Regulation and Abundance of Histone PTMs Impact Genome Stability…………………………………………………………… 13 1.2.2 Regulation of Chromatin Structure via Histone PTMs and the Link to Oncogenesis………………………………………………………... 15 1.3.0 Histone H2B Ubiquitination…………………………………………………………. 16 1.3.1 H2Bub1, RNF20, RNF40 and their Roles in Oncogenesis………………… 17 1.3.2 H2Bub1 Promotes Tumor Suppressing Transcriptional Profiles…………... 20 1.3.3 H2Bub1 Plays a Role in DNA Damage Repair…………………………….. 22 1.3.4 Rnf20/Rnf40 are Suppressors of CIN……………………………………… 23 1.3.5 The H2Bub1 Trans-Histone Pathway………………………………………. 24 1.4.0 Histone H3 Methylation……………………………………………………………… 25 1.4.1 H3K79 Methylation………………………………………………………… 27 1.4.2 H3K79 Methylation, DOT1L (KMT4) and their Roles in Oncogenesis…… 29 1.5.0 Entering the Era of Highly-Efficient Human Genome Editing………………………. 31 1.5.1 Exploiting HRR and NHEJ for CRISPR-Cas9 Genome Editing…………... 32 1.5.2 CRISPR-Cas9 - A Historical Prospective………………………………….. 34 1.5.3 Employing CRISPR-Cas9 to Target Mammalian Cells……………………. 35 1.6.0 Synthetic Genetic Targeting in Cancer………………………………………………. 37 1.6.1 Synthetic Lethality…………………………………………………………. 38 1.6.2 Therapeutically Exploiting Genome Instability……………………………. 42 1.6.3 Knowledge-Based Direct Tests to Identify Synthetic Lethal Interactors…... 43 1.6.4 The Prototypic Human Synthetic Lethal Interaction Between BRCA1/2 and PARP1……………................................................................. 44 1.6.5 Employing Knowledge-Based Direct Tests to Identify Novel Synthetic Lethal Interactors……………........................................................ 46 v 1.7.0 Rationale, Hypotheses, and Aims……………………………………………………. 47 1.7.1 Chapter 3 - Mitotic Accumulation of Dimethylated Lysine 79 of Histone H3 is Important for Maintaining Genome Integrity During Mitosis in Human Cells………….................................................................. 47 1.7.2 Chapter 4 - Generation and Phenotypic Characterization of an HCT116-Cas9 Expressing Cell Line…………….......................................... 48 1.7.3 Chapter 5 - RNF20 and PARP1 are Synthetic Lethal Interactors in Human Cells……........................................................................................... 49 Chapter 2 - MATERIALS AND METHODS…………………………………………… 50 2.1 Reagents ……………………………………………………………………………… 50 2.2 Cell Lines and Tissue Culture…………………………………………………………. 50 2.3 Cell Synchronization and Mitotic Enrichment………………………………………… 52 2.4 Flow Cytometry………………………………………………………………………... 52 2.4.1 Ethanol Fixation……………………………………………………………. 52 2.4.2 Propidium Iodide Staining………………………………………………….. 53 2.4.3 Flow Cytometric Analysis…………………………………………………. 53 2.5 Immunoblotting………………………………………………………………………… 54 2.5.1 Whole Cell Lysate Preparation…………………………………………….. 56 2.5.2 Acid-Extraction of Nuclear Proteins……………………………………….. 56 2.5.3 Protein Quantification via Bicinchoninic Acid Assay……………………... 57 2.5.4 Protein Resolution via Electrophoresis and Immunoblotting………………. 57 2.5.5 Semi-Quantitative Immunoblot Analysis…………………………………... 59 2.6 siRNA-Based Silencing………………………………………………………………... 59 2.7 DO1L Inhibition………………………………………………………………………... 60 2.8 Mitotic Chromosome Spreads………………………………………………………….. 60 2.9 Mitotic Defect Analysis Based on Chromatin Morphology…………………………… 61 2.10 Lentiviral and Stable Cell Line Production…………………………………………… 62 2.10.1 pGIPZ Lentiviral shRNA Plasmid Preparations…………………………... 62 2.10.2 Lentiviral Production……………………………………………………… 62 2.10.3 Lentiviral Transduction……………………………………………………. 63 2.11 Immunofluorescent Labeling…………………………………………………………. 64 2.11.1 Indirect Immunofluorescent Labeling…………………………………….. 64 2.12 Antibody Validation - Peptide Competition and Dot Blot Assays…………………… 65 2.13 Image Acquisition……………………………………………………………………. 66 2.14 Image Analyses……………………………………………………………………….. 66 2.14.1 Semi-Quantitative Signal Intensity Normalization and Analyses………… 67 2.14.2 Line Scans………………………………………………………………….