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Exploring the Roles of the Histone Deacetylase and Longevity Factor Sirt6 in Cancer

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Exploring the Roles of the Histone Deacetylase and Longevity Factor Sirt6 in Cancer EXPLORING THE ROLES OF THE HISTONE DEACETYLASE AND LONGEVITY FACTOR SIRT6 IN CANCER by Bernadette Margaretha Maria Zwaans A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular and Cellular Pathology) in the University of Michigan 2014 Doctoral Committee: Assistant Professor David B. Lombard, Chair Professor Kathleen R. Cho Associate Professor Yali Dou Professor Eric R. Fearon Associate Professor Patrick J. Hu To my parents, I hope I have made you proud. ii Acknowledgements Graduate school has been an immense learning experience for me, both at a professional and personal level. Many friends, family and colleagues, each in their own way, have contributed to my growth and completion of my PhD training at the University of Michigan and I’m grateful for having had such a great support system to fall back on. First I would like to thank my former mentor Dr. Diane Bielenberg. Prior to joining PIBS at the University of Michigan, I worked in Dr. Bielenberg’s laboratory as a research assistant. All the skills and the confidence that I obtained while working with Diane formed the foundation on which I further built my scientific career at the University of Michigan. I’m enormously thankful for having been able to work under her tutelage and hope that in the future our scientific paths will cross again. Throughout my PhD training I’ve had the pleasure of working with a number of extraordinary scientists. First and foremost I would like to show my gratitude to my mentor Dr. David Lombard, who has guided me through the daily twist and turns of scientific discovery. I admire his broad knowledge, enthusiasm for science, and positive attitude, and am thankful for his patience and flexibility. Secondly, I would like to thank my thesis committee members Dr. Fearon, Dr. Cho, Dr. Hu and Dr. Dou, for iii their support, valuable input, and for sharing reagents and protocols. Additionally, I’m very grateful for the guidance that I have gotten from the PIBS program director Dr. Isom, the pathology program directors Dr. Lukacs and Dr. Nikolovska-Coleska, and the support from our program coordinators Laura Hessler and Laura Labut. Furthermore, I would like to thank the Rackham graduate school for awarding me the Rackham predoctoral fellowship. I would also like to thank the current and former members of the Lombard lab: Mary Skinner, Billy Giblin, Jeongsoon Park, Surinder Kumar, Kylie Smith, Emily Sluiter, Ray Joe, Daniel Tishkoff, Shawn Loder, Bo Yang, Jiangjun Bao, and Mark Eckersdorf. We have spent countless hours working side-by-side, discussing follow-up experiments, hunting down free food and laughing about silly things. You have all contributed to the person I am today, and I thank you for that. My thesis work wouldn’t have been possible without our numerous collaborators. Thank you to Dr. Elenitoba-Johnson and Dr. Basrur for your help in identifying novel SIRT6 interactors, Dr. Rho and Dr. Giordano for providing human tissue samples for our IHC studies, Dr. Greenson for his pathological expertise, Dr. van Deursen and Janine Van Ree for help with our aneuploidy study, Dr. Cavey for performing the mass spectrometry to identify novel SIRT6 targets. Dr. Cho, Dr. Wu and Dr. Gruber for sharing Affymetrix data, and Dr. Lukacs and Dr. Mukherjee for our collaboration on the T-cell project, which is not discussed in my thesis work. Finally, I would like to thank Dr. iv Mostoslavsky and Dr. Sebastian for our productive collaboration on the identification of SIRT6’s tumor suppressor function (discussed in Chapter 3). Last, but definitely not least, I would like to thank my friends and family members for their love and support. In particular my friends Davina, Scott, the Liz’s, Molly, Seyth and Dave for being there for me through thick and thin, my husband Jake for putting a smile on my face every day and coping with my daily ups and downs, and my siblings for being the very best friends I could wish for. A special thank you goes out to my parents, without whose love and support my life wouldn’t be what it is today. They’ve worked hard their whole lives to be able to give their children every opportunity they’d like to pursue, even if that meant having them move far away from home. I thank them for their unconditional love, emotional as well as financial support, and the family vacations I was always able to look forward to. But most importantly, I thank them for creating this wonderful family, which, up to this day, I consider my strongest asset. My thesis work is the result of so many helping hands along the way and I hope I have made everybody proud of this joint accomplishment. v Table of contents Dedication……………………………………………………………………………………….ii Acknowledgements…………………………………………………………………………….iii List of Figures…………………………………………………………………………………viii List of Tables…………………………………………………………………………………...xi List of Abbreviations…………………………………………………………………………..xii Abstract………………………………………………………………………………………...xv Chapter 1: Introduction: The Diverse Roles of SIRT6 in Mammalian Healthspan Introduction to SIRT6…………………………………………..................................1 SIRT6 is a master metabolic regulator……………………………………………....6 Regulation of SIRT6………………………………………………………………….13 SIRT6 regulates inflammation……………………………………………………….16 SIRT6 promotes genomic stability via diverse mechanisms……………………..19 SIRT6 suppresses cardiac hypertrophy and promotes cardiac stress resistance……………………………………………………………………………...22 SIRT6 prolongs mammalian lifespan……………………………………………….25 Rationale……………………………………………………………………………….28 Chapter 2: Identification of Novel SIRT6 Targets Abstract………………………………………………………………………………...31 Introduction…………………………………………………………………………….32 Materials and Methods……………………………………………………………….35 Results…………………………………………………………………………………40 Discussion……………………………………………………………………………..47 Future Directions……………………………………………………………………...50 Chapter 3: SIRT6 Acts as a Tumor Suppressor Through Inhibition of Glycolysis Abstract………………………………………………………………………………...52 vi Introduction…………………………………………………………………………….53 Materials and Methods……………………………………………………………….63 Results…………………………………………………………………………………71 Discussion……………………………………………………………………………..87 Future Directions……………………………………………………………………...91 Chapter 4: SIRT6 Protects Against Aneuploidy Abstract………………………………………………………………………………...94 Introduction…………………………………………………………………………….95 Materials and Methods……………………………………………………………….97 Results………………………………………………………………………………..105 Discussion……………………………………………………………………………121 Future Directions…………………………………………………………………….123 Chapter 5: A Study of SIRT6 Localization in Cancer Abstract……………………………………………………………………………….125 Introduction……………………………………………………………………...…...126 Materials and Methods……………………………………………………………...126 Results………………………………………………………………………………..133 Discussion……………………………………………………………………………148 Future Directions…………………………………………………………………….153 Chapter 6: Concluding Remarks…………………………………………………………..154 References…………………………………………………………………………………...156 vii List of Figures Figure 1.1: Mammalian Sirtuin family………………………………………………………...3 Figure 1.2: SIRT6 biochemical functions…………………………………………..………..4 Figure 1.3: SIRT6 as a master regulator of glucose metabolism…………………………7 Figure 1.4: SIRT6 regulates glycolysis………………………………………………………9 Figure 1.5: Mechanisms of SIRT6 regulation…………………………………….………..14 Figure 1.6: SIRT6’s regulation of inflammation……………………………………………17 Figure 1.7: Schematic overview of SIRT6 functions………………………………………24 Figure 1.8: Implications of SIRT6 in diseases……………………………………………..27 Figure 1.9: The revised hallmarks of cancer……………………………………………….29 Figure 2.1: Illustration of in vitro deacetylation assay……………………………............39 Figure 2.2: SIRT6 deacetylates Histone H3 Lysine 56…………………………………...41 Figure 2.3: In vitro deacetylation of Histone H3 residues by SIRT6…………………….43 Figure 2.4: Immunocytochemistry of histone marks in Sirt6 KO MEFs………………...44 Figure 2.5: SIRT6 deacetylates H3K18ac in the brain……………………………………46 Figure 2.6: SIRT6 deacetylates histone H3 in vivo………………………………............46 Figure 2.7: Histone H3 acetylation changes at SIRT6 target promoter regions……………………………………………………………………………...51 Figure 3.1: Illustration of the Warburg Effect………………………………………………54 viii Figure 3.2: HIF-1 transcriptional activity positively regulates glycolysis and lactate production……………………………………………………………………............57 Figure 3.3: Adenoma-prone mouse model breeding scheme…………………………...70 Figure 3.4: SIRT6-deficient cells are tumorigenic………………………………………...73 Figure 3.5: SIRT6-deficient cells and tumors show elevated aerobic glycolysis………………………………………………………………………………………75 Figure 3.6: Inhibition of glycolysis suppresses tumorigenesis in Sirt6 KO cells…………………………………………………………………………………………….77 Figure 3.7: SIRT6 inhibits ribosomal gene expression by co-repressing MYC transcriptional activity………………………………………………………………….78 Figure 3.8: MYC regulates growth of SIRT6 deficient cells………………………………81 Figure 3.9: Increased tumorigenesis in SIRT6-deficient intestinal mouse model…………………………………………………………………………………………..83 Figure 3.10: Enhanced glycolytic and ribosomal gene expression in mouse adenomas……………………………………………………………………………………...84 Figure 3.11: Inhibition of glycolysis decreases tumorigenesis of SIRT6-deficient adenomas……………………………………………………………………………………...85 Figure 3.12: SIRT6 expression is decreased in human adenomas……………………..86 Figure 3.13: Schematic overview of the role of SIRT6 as a tumor suppressor……………………………………………………………………………............88 Figure 3.14: Polyp number in Sirt6 overexpressing mice……………………….............92
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