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The Role of the Rho GEF Arhgef2 in RAS Tumorigenesis

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The Role of the Rho GEF Arhgef2 in RAS Tumorigenesis The Role of the Rho GEF Arhgef2 in RAS Tumorigenesis by Jane Cullis A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Medical Biophysics University of Toronto © by Jane Cullis 2013 The Role of Rho GEF Arhgef2 in RAS Tumorigenesis Jane Cullis Degree of Doctor of Philosophy, 2013 Graduate Department of Medical Biophysics, University of Toronto Abstract Tumorigenesis is driven by the sequential accumulation of genetic lesions within a cell, each which confer the cell with traits that enable its abnormal growth. The result is a mass of dysregulated cells, or tumor, which, upon further mutation, may spread, or metastasize, to other organs of the body. The dissemination of tumor cells makes treatment difficult, and thus confers cancer with its associated lethality. Over the past 30 years, the RAS genes have been critical in teaching us the mechanisms underlying the molecular progression of cancer. RAS is mutated in 33% of all cancers and is often an early event in its stepwise progression1. As a result, the RAS genes are widely accepted as ‘drivers’ or ‘initiators’ of human tumorigenesis. Unfortunately, efforts directed at targeting RAS in the clinic have as of yet been unsuccessful. This has triggered a need to identify genes that are required for RAS tumorigenesis that are therapeutically tractable. My research has focused on deciphering the potential role of the Rho GEF Arhgef2 in RAS- mediated tumorigenesis. I have found that Arhgef2 is a bona fide transcriptional target of RAS and is upregulated in human tumors harboring RAS mutations. Importantly, depletion of Arhgef2 in RAS-mutated cells inhibits their survival, proliferation, and tumor growth in murine models. In search of the mechanism underlying the requirement of Arhgef2 in RAS tumorigenesis, I have uncovered a novel function for Arhgef2 as a positive regulator of a central RAS pathway, the ii mitogen-activated protein kinase (MAPK) pathway. Thus, Arhgef2 is part of a positive feedback loop in which RAS-dependent increases in Arhgef2 expression results in the amplification of RAS signaling. Moreover, Arhgef2 confers tumor cells with properties favoring their malignant conversion, thereby implicating Arhgef2 in the formation of metastases. Together, these studies suggest that Arhgef2 plays an important role at multiple stages of tumorigenic progression and may therefore be a promising therapeutic target in RAS-mutated tumors. 1Karnoub et al., 2008 iii Acknowledgments First and foremost, I would like to thank my amazing family for their unwavering support and encouragement throughout my degree. My father, who not only inspires me with his science, but whose compassion and sense of fun I admire and strive to emulate. My mother, one of the strongest, most brilliant women I know: thank you for being not just a mother to me in the last 6 years but also one of my best friends. My brother Jepray, you have been a tremendous source of comfort, positivity and wisdom and I thank you for always being there for me. To my supervisor, Rob. You made it about more than science. You are the one who taught me how to run marathons. You taught me that the work you put in is the work you get out; that strength and endurance takes time and patience and cannot be forced; that the lows are worth the highs; that there are no shortcuts; that it’s not how fast you can sprint but how well you can push to the very end. Most importantly, you taught me never to give up until you’re there. Not many people can teach such hard lessons while expressing so much love, compassion and understanding, but you did. Thank you. Thank you to my committee members, Jane and Dr. Medin. I appreciate the time you took to oversee my project and improve my research with your excellent advice and encouragement. Thank you to my dear collaborators, Nikolina Radulovich and Dr. Ming Tsao, for your help and guidance with my animal and immunohistochemical studies. Dedi. You gave meaning to the word ‘Dedidit’ and together, we did it . You are a remarkable scientist but an even more amazing person and friend. Thank you for always putting things in perspective and for making science fun. Mauricio and Tim, my BFFs OMG. Mauricio, it is largely because of you that I was able to run Rob’s marathons. Thank you for being a constant source of support and fun during the last six years. You have made the difficult times bearable and the good times unbelievable. To all the members of the Rottapel lab for putting up with me and all my Western blots. Thank you for your encouragement and sense of humor – it is you guys that made coming to lab every day worth it, successful experiment or not. Also, thank you to the ladies from the Kislinger lab, iv especially Lusia, for either keeping me sane in the office or making the choice to go insane with me. To my training partners and running friends, the Angels. Thank you for constantly reminding me that there’s more to life than the lab (and for inspiring me to run real marathons). Nic, DocZ, MamaK and Jebs, thank you for your patience, wisdom and guidance in all aspects of life. To the others along the way that have inspired and encouraged me – Delilah (a.k.a. Topicoolis) and my beautiful cousin Sarika – you are two of the most important people in my life and it’s been so comforting knowing you were always there for me. You have each helped me in such different but crucial ways and I can’t thank you enough. Finally, I absolutely have to thank Goose for keeping me going during the last six years. You taught me to relax, gave me the energy to keep going and were always there when I needed you. Cheers. v Table of Contents Abstract ........................................................................................................................................... ii Acknowledgments.......................................................................................................................... iv Table of Contents ........................................................................................................................... vi List of Tables ................................................................................................................................. ix List of Figures ................................................................................................................................. x List of Appendices ........................................................................................................................ xii Appendix 1: Microarray Analysis of PANC-1 and H-RASV12-Transformed Fibroblast Cells Harboring Stable Arhgef2 Knockdown..................................................................................... xii List of Abbreviations and Symbols.............................................................................................. xiv Chapter 1 ....................................................................................................................................... 1 Introduction ..................................................................................................................................... 1 1.1 The RAS superfamily of small GTPases .............................................................................. 1 1.2 The RAS subfamily ............................................................................................................... 4 1.3 The Rho subfamily ................................................................................................................ 9 1.4 Guanine nucleotide exchange factors .................................................................................. 14 1.5 Arhgef2................................................................................................................................ 19 Chapter 2 ..................................................................................................................................... 25 Arhgef2 Provides a Positive Feedback Loop Required for Signaling Through the Oncogenic RAS Pathway ................................................................................................................................ 25 2.1 Abstract ............................................................................................................................... 25 2.2 Introduction ......................................................................................................................... 26 2.3 Experimental Procedures..................................................................................................... 29 2.4 Results ................................................................................................................................. 36 vi 2.4.1 Arhgef2 protein expression is acutely induced by the RAS/MAPK pathway .............. 36 2.4.2 ARHGEF2 is a transcriptional target of the RAS/MAPK pathway ............................. 38 2.4.3 Arhgef2 is required for cell survival downstream of oncogenic RAS ......................... 40 2.4.4 Arhgef2 contributes to RASV12-mediated cellular transformation in vitro and in vivo 43 2.4.5 Arhgef2 contributes to the increased proliferative capacity of RASV12-transformed fibroblasts in a GEF-independent manner ............................................................................. 45 2.4.6 Arhgef2 is required for MAPK pathway activation in response to oncogenic RAS .... 46 2.4.7 Arhgef2 is a component of the KSR-1 complex and is required for the dephosphorylation
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