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Cell Proliferation Control: from Intrinsic Transcriptional Programs to Extrinsic Stromal Networks

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Cell Proliferation Control: from Intrinsic Transcriptional Programs to Extrinsic Stromal Networks Cell Proliferation Control: from Intrinsic Transcriptional Programs to Extrinsic Stromal Networks DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Huayang Liu Graduate Program in Molecular Genetics The Ohio State University 2015 Dissertation Committee: Professor Gustavo Leone, Advisor Professor Helen Chamberlin Professor Harold Fisk Professor Tsonwin Hai Copyright by Huayang Liu 2015 Abstract Robust mechanisms to control cell proliferation have evolved to maintain the integrity of organ architecture and tissue homeostasis. Disruption of these mechanisms, either intracellular circuits or extracellular inputs for cell proliferation control, inevitably occur under oncogenic conditions, rendering cells with unlimited proliferative capacity and subsequent malignant transformation. Here, we investigated how two critical intracellular proliferative pathways, Myc and E2f, are integrated to control cell cycle transitions in normal and Rb deficient cells using a murine intestinal model. We show that in contrast to the current paradigm, Myc and E2f1-3 have little impact on normal G1- S transitions. Instead Myc and E2f1-3 synergistically control an S-G2 transcriptional program in intestinal crypts required for completing normal cell divisions and maintaining crypt-villi integrity. Surprisingly, Rb deficiency results in the Myc- dependent accumulation of E2f3a protein and the genome-wide repositioning of Myc and E2f3 on chromatin associated with ‘super activation’ of a G1-S transcriptional program, ectopic S phase entry and rampant cell proliferation. These findings reveal that Rb deficient cells hijack and redeploy Myc and E2f3 from an S-G2 program essential for normal cell cycles to a G1-S program that re-engages ectopic cell cycles, exposing an unanticipated addiction of Rb-null ‘cancer-like cells’ on Myc. ii We have also studied how cell non-autonomous inputs derived from stromal compartment in the tumor microenvironment impacts the proliferation of tumor compartment. We performed a genome-wide stroma-specific RNAi screen in ‘cancer sensitized’ C. elegans and discovered 39 stromal factors that suppress the proliferation of adjacent ‘Ras pathway-sensitized’ epithelial cells, with minimal impact on the proliferation of normal or ‘Wnt pathway-sensitized’ epithelial cells. These candidate genes encode histone variants and components of protein complexes known to converge on the control of chromatin dynamics, cytoplasmic polyadenylation and translation. An expression signature of the corresponding 33 human orthologs was selectively enriched in laser capture microdissected tumor stroma of breast cancer patients. Furthermore, depletion of stromal factors from human breast fibroblasts increased the proliferation of co-cultured breast cancer cells, but had no effect on normal mammary epithelial cells. This cross-species approach identified unanticipated stromal regulatory hubs with cell non-autonomous tumor suppressive function, exposing the conserved and selective nature of stroma-tumor communication. iii Dedication This document is dedicated to my family. iv Acknowledgments The time in the graduate school has been a memorable experience - it all seems like yesterday. I want to thank everyone who has been there to help me reach this point - thank you! I would like to thank my advisor, Dr. Gustavo Leone, for his passionate and unconditioned support during the study. Rather than giving me a fish for the day, he teaches me how to fish that I can feed myself on the scientific journey for a lifetime. I would also like to thank my co-advisor, Dr. Helen Chamberlin, who has always been there to offer help on the projects and more generally, being an supportive mentor all along. I would also like to thank my advisor committee, Dr. Harold Fisk and Dr. Tsonwin Hai, who have been critical and suggestive on my endeavors. A few hours on the committee meeting have always driven me to push the science to the next level. I am no doubt in debt to lab members, past or present, directly involved in the projects or just chatting over daily life. I am grateful when I reach out, you guys are always there to lend me a hand. v I am grateful to work with the undergraduate students, Amneet Bajwa, Karl Eiring, Nicholas Fackler, Benjamin Hemmelgarn, Michael Lause, Stephan Reyes and Kyle Toth. It has been a joyful experience, working with them as a mentor as well as a friend. I would always treasure the support from my family and my wife, Yian Ruan. It's their love that has been accompanying me all along this journey that is full of hope and surprises. It's their love that enables me for all the achievements. It's their love that will lead me to the next station for the exploration in the future. Thanks to everyone and best wishes! vi Vita 2004 to 2008 ..................................................B.S. Chemistry and Biology, Tsinghua University 2008 to 2009 ..................................................Graduate Teaching Associate, Department of Molecular Genetics, The Ohio State University 2009 to present ..............................................Graduate Research Associate, Department of Molecular Genetics, The Ohio State University Publications 1. Liu, H.†, Sullivan, N.J.†, Bertos, N., Dowdle, J.A., Rambani, K., Mair, M., Daniel, P., Toth, K., Lause, M., Harrigan, M.E., Eiring, K., Sullivan, C., Chang, S.W., Kladney, R., Tang, X., McElroy, J., Lu, Y., Tofigh, A., Fernandez, S.A., Parvin, J.D., Macrae, E., Majumder, S., Shapiro, C.L., Yee, L.D., Hallett, M., Ostrowski, M.C., Park, M., Chamberlin, H.M.*, Leone, G*. Discovery of stromal regulatory networks that suppress cancer cell proliferation. Manuscript under review. (†: co-first authors; *: co- correspondence authors) vii 2. Liu, H., Tang, X., Srivastava, A., Pécot, T., Daniel, P., Hemmelgarn, B., Reyes, S., Fackler, N., Bajwa, A., Kladney, R., Koivisto, C., Chen, Z., Wang, Q., Huang, K., Machiraju, R., Sáenz-Robles, M.T., Cantalupo, P., Pipas, J.M., Leone, G. Redeployment of Myc and E2f1-3 drives cancer-like cell cycles. Manuscript in revision. 3. Liu, H., Rakijas J., de Bruin, A., Leone, G. Emerging roles of E2Fs in cancer. Commissioned review in preparation. 4. Sáenz-Robles, M.T., Chong, J.L., Koivisto, C., Trimboli, A., Liu, H., Leone, G., Pipas, J.M. Viral oncogene expression in the stem/progenitor cell compartment of the mouse intestine induces adenomatous polyps. Mol Cancer Res. (2014); 12:1355-1364. 5. Coffman, V.C., Nile, A.H., Lee, I.J., Liu, H., Wu, J.Q. Roles of formin nodes and myosin motor activity in Mid1p-dependent contractile ring assembly during fission yeast cytokinesis. Mol. Biol. Cell (2009); 20: 5195-5210. Fields of Study Major Field: Molecular Genetics viii Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita .................................................................................................................................... vii List of Figures .................................................................................................................. xiii List of Tables .................................................................................................................... xv Chapter 1 Cell Proliferation Control ............................................................................. 1 1.1 Cell proliferation, cell cycle control and cyclin-dependent kinases.......................... 2 1.2 Retinoblastoma (Rb) family proteins ........................................................................ 3 1.3 E2f family transcription factors................................................................................. 3 1.4 Cdk-Rb-E2f axis in cancer ........................................................................................ 5 1.5 Myc transcription factor ............................................................................................ 6 1.6 Intracellular circuits and extracellular inputs for proliferation control ..................... 8 1.7 Proliferation signaling autonomy and inputs from tumor microenvironment in cancer .............................................................................................................................. 9 Chapter 2 Redeployment of Myc and E2f1-3 drives cancer-like cell cycle ................ 12 ix 2.1 Introduction ............................................................................................................. 12 2.2 Results ..................................................................................................................... 15 2.2.1 Combined ablation of Myc and E2f1-3 results in disruption of crypt-villus integrity ...................................................................................................................... 15 2.2.2 Combined Myc and E2f1-3 deficiency leads to S-G2 cell cycle arrest ............ 16 2.2.3 Synergistic regulation of an S-G2 transcriptional program by Myc and E2f1-3 ................................................................................................................................... 18 2.2.4 Myc and E2f3 bind to both G1-S and
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