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A Genome Wide Screen in C. Elegans Identifies Cell Non-Autonomous Regulators of Oncogenic Ras Mediated Over-Proliferation DISSER

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A genome wide screen in C. elegans identifies cell non-autonomous regulators of oncogenic Ras mediated over-proliferation DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Komal Rambani Graduate Program in Biomedical Sciences The Ohio State University 2016 Dissertation Committee: Gustavo Leone, PhD “Advisor" Helen Chamberlin, PhD Gregory Lesinski, PhD Joanna Groden, PhD Jeffrey Parvin, PhD Thomas Ludwig, PhD Copyright by Komal Rambani 2016 ABSTRACT Coordinated proliferative signals from the mesenchymal cells play a crucial role in the regulation of proliferation of epithelial cells during normal development, wound healing and several other normal physiological conditions. However, when epithelial cells acquire a set of malignant mutations, they respond differently to these extrinsic proliferative signals elicited by the surrounding mesenchymal cells. This scenario leads to a pathological signaling microenvironment that enhances abnormal proliferation of mutant epithelial cells and hence tumor growth. Despite mounting evidence that mesenchymal (stromal) cells influence the growth of tumors and cancer progression, it is unclear which specific genes in the mesenchymal cells regulate the molecular signals that promote the over-proliferation of the adjacent mutant epithelial cells. We hypothesized that there are certain genes in the mesenchymal (stromal) cells that regulate proliferation of the adjacent mutant cells. The complexity of various stromal cell types and their interactions in vivo in cancer mouse models and human tumor samples limits our ability to identify mesenchymal genes important in this process. Thus, we took a cross-species approach to use C. elegans vulval development as a model to understand the impact of mesenchymal (mesodermal) cells on the proliferation of epithelial (epidermal) cells. This model includes well-described signaling interactions between mesenchymal cells (anchor cell, gonad and muscle cells) and epidermal cells (vulva precursor cells, VPCs). In this system, three of the six equipotent VPCs normally divide to produce vulval tissue. These ii divisions result from coordination of Egf and Wnt signals from mesenchymal cells, and lateral Notch signaling between the VPCs. The anchor cell in the gonad (the mesenchymal cells) produces an LIN-3/EGF signal, and the most proximal VPC in the underlying epidermal cell (P6.p) is induced to adopt a vulval fate (termed 1°), and to promote its neighbors (P5.p and P7.p) to adopt a distinct vulval fate (termed 2°) via LIN- 12/Notch. Following signaling, these three cells go through three rounds of invariant cell division to form a symmetric vulva cavity comprised of 22 daughter cells. Abnormal activation of the Egf pathway, such as through introducing gain-of-function (gf) mutations in let-60/Ras, leads to the promotion of vulval fates in the remaining three VPCs, which then divide inappropriately to develop multiple ectopic vulval protrusions (the multivulva, or Muv, phenotype). To identify genes that function in mesodermal cells to inhibit the proliferation of Ras-mutant epidermal cells, we genetically engineered C. elegans to develop a model that possess let-60(gf)/Ras mutation mediated over- proliferation (Muv phenotype) and RNA interference (RNAi) competence specific to mesenchymal tissues only. This strain was subjected to a genome-wide RNAi screen using standard methods. Our screen identified 47 genes that, upon RNAi mediated knock- down of a gene in mesodermal cells, suppress over-proliferation of let-60(gf)/Ras epithelial cells in an allele-specific manner. Notably, these genes encode histone proteins, ribosomal proteins, vesicle transport proteins and metabolic factors, rather than secreted molecules. Importantly, candidate genes emerged from this screen are significantly enriched for the conserved genes and signaling-pathways from C. elegans to iii humans. These results form the basis of a comprehensive understanding of molecular factors in mesenchymal cells that impact oncogenic cell proliferation. iv Dedication This thesis is dedicated to my son, Ritav Das, my husband, my family and God – with infinite love and gratitude. My son, you are infinite source of my inspiration and window to look at things through the lens of your curious and creative ideas and questions. May you find your own ways to discover the world and contribute to solving significant and meaningful problems with your superb intellect, creativity and curiosity! v Acknowledgments Research is a challenging and time-consuming endeavor; the written portion is only a small portion of the total work done. For me, the greatest benefit of doing PhD is to learn about a new field of research (cancer) and develop as an independent thinker and problem solver – which have prepared me for my future endeavors. Not only I learned about various thinking and experimental styles to solve scientific problems, I also learned a big deal to work with various personalities in team-oriented projects. I sincerely thank my dissertation committee members and my adivsor, for advice and valuable suggestions to my research and graduate work. Their guidance was instrumental in focusing efforts and completing work presented herein. I am tremendously thankful to BSGP directors, Dr. Groden and Dr. Parvin, and administrators, Amy Lahmers and Lauren Graham, for their incredible mentorship, guidance and support through these years. After Dr. Bloomston moved from OSU, Dr. Parvin and Dr. Groden generously accepted to mentor me as committee members in place of him. When I joined the lab, I had some career goals; I discussed and took steps to achieve them – some succeeded, some did not, irrespective my efforts. I want to pursue translational cancer research even though this project culminated with basic research part only. From this, I have learned several important skills that will benefit in future. All of the BSGP classes were instrumental in gaining broad and rich knowledge on various topics and honing critical thinking; some of them will stay in my memories for long. Several lectures of Professors Tsowin Hai, Larry Kirschner, Thomas Boyd, James Waldman, Deborah Parris, Harold Fisk, James Chen, Raghu Machiraju, and Kun Huang had special impact on me. They brought knowledge, excitement, challenge, thinking on- the-fly and an urge to students to draw rational conclusions along with take-home messages like “Think alternatives” and “correlations are not always causal relationships”. The grant writing class of BSGP is very valuable and was instrumental in success of my vi fellowship applications. While taking these classes and beyond, I am thankful for great company of my classmates and friends. I am also thankful to all the past and present members of the Leone lab. I thank Raleigh Kladney, Serena Chang, Markus Harrigan, James Dowdle, Elizabeth Brunner, Xing Tang for their contributions to work presented in this thesis. I am grateful to Susan Lutz for her support and help on lab related administrative tasks. I am tremendously thankful to a long list of my friends on BRT 5th floor and my friends across the campus for their company and good times spent with them! A special thanks to Jyotsana, Vijay, Kirana, Jaya, Sriram, Margi, Tarak, Lauren, Sarika, Neelam and Arunima for their unconditional friendship! Above all, I am grateful to my incredible son and husband for their unwavering support throughout these years – without which, I am certain, I would not be writing this thesis today. A special thanks goes to my family members: Ritav, Jayajit, Vikas, Vishal, Arijit, Suruchi, Jasleen, Aratrika, Aariv, Aniket, my mother, my parents-in-law and my extended family for their immense love and support in numerous ways. Without their loving support and understanding, I would not have made through this experience. I am lucky to have delightful and marvelous sister-in-laws, Jasleen, Suruchi and Aratika, who always stand by my side in all my high-n-low times and I cherish their loving company, whenever we talk or meet. I am fortunate to have brothers like Vikas, Vishal and Arijit, whose unconditional love, support and respect makes me proudest women. My darlings – Ritav, Aariv and Aniket – are the sweetest charm of my life whose child-wisdom added to my ability to manage my time efficiently at work. Through these years, I also endured pain of missing those family members whom I would never be able to meet again ever – Sita didi, Onkar Jijaji, baby Anushka and Humpy Dada, my next visit to home would certainly make me realize your absence more than ever! vii Vita 2007 ...............................................................M.S., Georgia Institute of Technology, Atlanta, GA 2010 - present ................................................Graduate Research Associate, The Ohio State University, Columbus, OH 2013-2015 …………………………………...Pelotonia Graduate Fellow, The Ohio State University, Columbus, OH Publications 1. Rambani K, Kladney RD, Chang SW, Harrigan ME, Dowdle JE, Tang X, Liu H, Brunner E, Chamberlin HM, and Leone G. A genome-wide screen in C. elegans identifies cell non-autonomous suppressors of oncogenic Ras mediated over- proliferation. in preparation. 2. Liu H*, Dowdle JA*, Khurshid S*, Sullivan NJ*, Bertos N, Rambani K, Mair M, Daniel P, Toth K, Lause M, Harrigan ME, Eiring K, Sullivan C, Chang SW, Kladney RD, Tang X, McElroy J, Lu Y, Tofigh A, Fernandez SA, Parvin JD, Macrae E, Majumder S, Shapiro CL, Yee LD, Hallett
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