Microscopy-based High-content Analyses Identify Novel Chromosome Instability Genes including SKP1 by Laura L. Thompson 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, Manitoba, Canada Copyright © 2018 by Laura Louise Thompson i ABSTRACT Cancer is a devastating disease responsible for ~80,000 deaths in Canada each year. Chromosome instability (CIN) is an abnormal phenotype frequently observed in cancer, characterized by an increase in the rate at which chromosomes or chromosomal fragments are gained or lost. CIN underlies the development of aggressive, drug resistant cancers, disease recurrence, and poor prognoses. Despite this, the majority of CIN genes have yet to be elucidated, highlighting the need for studies aimed at identifying the defective genes that underlie CIN. This thesis describes the development and utilization of imaged-based assays designed to detect CIN phenotypes (i.e. nuclear size changes and micronucleus formation) following gene silencing. Mitotic chromosome spread analyses validated that changes in CIN phenotypes corresponded with numerical and structural chromosome defects by silencing the established CIN gene, SMC1A (Chapter 3). The assays were multiplexed in a high-content, siRNA-based screen of 164 candidate genes in hTERT and HT1080, which identified 148 putative CIN genes. Validation of 10 genes (e.g. SKP1) was performed in hTERT and HCT116, which confirmed gene silencing induced chromosome changes (Chapter 4). SKP1 is a component of the SKP1- CUL1-F-box protein (SCF) E3 ubiquitin ligase complex, which regulates degradation of numerous proteins within pathways that maintain chromosome stability. Accordingly, the aberrant biology underlying CIN in SKP1 silenced cells was investigated further using immunofluorescence, microscopy, and biochemical techniques (Chapter 5). A high-content screen of all 68 F-box genes was performed to determine which F-box proteins/SCF complexes regulate chromosome stability. EMI1, SKP2, and FBXL7 induced CIN phenotypes similar to SKP1, suggesting that the corresponding SCF complexes are required for chromosome stability. ii Increases in the levels of SCF substrates Survivin and CCNE1, were detected in SKP1 silenced cells. Replication stress, DNA damage, centromeric protein mislocalization, and centrosome defects were also observed, all of which are mechanisms known to underlie CIN. Significantly, a novel screen for CIN phenotypes was developed and employed in this study, which expedited human CIN gene identification and provided critical insights into the fundamental biological mechanisms that regulate chromosome stability. Importantly, characterizing the aberrant genes/mechanisms that underlie CIN and oncogenesis could ultimately reveal novel cancer therapeutic targets. iii ACKNOWLEDGEMENTS First, I would like to express my sincerest gratitude to my supervisor Dr. Kirk McManus for his time, mentorship, scientific advice, and continuous support throughout my graduate studies. I am extremely fortunate to have had the opportunity to work, learn, and grow in the McManus lab. I would also like to thank my advisory committee; Dr. Mark Nachtigal, Dr. Yvonne Myal, and Dr. Tamra Werbowetski-Ogilvie, for their critical assessments of my research project, challenging questions, valuable input, support, and career advice. I would like to acknowledge all members of the McManus lab, past and present; Zelda Lichtensztejn, Sajesh Babu, Brent Guppy, Amy Cisyk, Erin McAndrew, Yasamin Asbaghi, Lucile Jeusset, Nicole Wilson, Chloe Lepage, Manisha Bungsy, Claire Morden, and all summer and co-op students that I have had the pleasure of working with. Thank you for your friendship, support, and for making the McManus lab a wonderful place to work. I would especially like to thank Zelda, Sajesh and Brent for training me in the laboratory and providing extensive technical assistance and trouble-shooting advice. I am extremely grateful to Allison Baergen, my summer student of three years, for her hard work and significant contributions to this research project. This research would not have been possible without the financial support of NSERC, Research Manitoba, University of Manitoba, CancerCare Manitoba, Terry Fox Institute and the Research Institute in Oncology and Hematology. I would also like to thank the donors that provided funding for the Caroline A. Cope Award for Excellence in Oncology Research, Mindel Rady Olenick Fellowship in Human Genetics, Emil & Lynette Hain Oncology Award, Human Genetics Endowment Fund Award, Mindel & Tom Olenick Research Studentship in Medicine, Sheu L. Lee Family Scholarship in Oncology Research, Nancie J. Mauro Scholarship in Oncology Research, and Phyllis J. McAlpine Graduate Fellowship. I am truly grateful to my family; Dad, Mom, Steve, Debbie, Travis, Janene, Troy, Brianna, Rob, Lucille, Robbie, and Sarah for their encouragement and support in all aspects of my life and studies. Lastly, I would like to extend a special thank you to Mike for his unwavering love, patience, and support. Thank you for always being there for me. iv TABLE OF CONTENTS ABSTRACT.............................................................................................................................. ii ACKNOWLEDGEMENTS .................................................................................................... iv TABLE OF CONTENTS ..........................................................................................................v LIST OF ABBREVIATIONS ................................................................................................. ix LIST OF TABLES .................................................................................................................xiv LIST OF FIGURES ...............................................................................................................xvi USED WITH PERMISSION and CONTRIBUTION OF AUTHOR ............................... xviii RESEARCH RATIONALE AND HYPOTHESES..................................................................1 CHAPTER 1: INTRODUCTION .............................................................................................3 1.0.0. Cancer Burden ...................................................................................................................3 1.0.1. The Global and National Impact of Cancer ....................................................................3 1.0.2. Colorectal Cancer Burden ..............................................................................................4 1.1.0. Overview of Colorectal Cancer ..........................................................................................5 1.1.1. Clinical Features ............................................................................................................5 1.1.2. Risk Factors and Screening ............................................................................................6 1.1.3. Diagnosis and Staging ....................................................................................................7 1.1.4. Current Standard of Care ...............................................................................................9 1.2.0. Molecular Pathogenesis of Colorectal Cancer ....................................................................9 1.2.1. Genome Instability in Colorectal Cancer ...................................................................... 10 1.2.2. Chromosome Instability ............................................................................................... 12 1.3.0. Chromosome Instability in Yeast and Cross-Species Approaches .................................... 14 1.4.0. Biological Pathways that Maintain Chromosome Stability ............................................... 15 1.4.1. Sister Chromatid Cohesion........................................................................................... 16 1.4.2. Centromere and Kinetochore Protein Recruitment and Regulation ............................... 18 1.4.3. Centrosome Regulation and Microtubule Dynamics ..................................................... 21 1.4.4. Spindle Assembly Checkpoint ..................................................................................... 25 1.4.5. DNA Replication ......................................................................................................... 27 1.4.6. DNA Damage Response .............................................................................................. 30 1.4.7. Ubiquitin-dependent Proteasomal Degradation ............................................................ 32 1.5.0. The SCF Complex ........................................................................................................... 35 1.5.1. S-Phase Kinase Associated Protein 1 ........................................................................... 37 1.5.2. SKP1 Alterations in Cancer .......................................................................................... 39 1.6.0. CIN Phenotypes............................................................................................................... 40 1.6.1. Nuclear Size Changes as an Emerging Indicator for Large-scale DNA Content Changes in Cancer ..................................................................................................................... 40 1.6.2. Micronucleus