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Effects of Microrna Modulation of PLK1 in Breast Cancer in Combination with PLK1 Inhibitor NMS-P937

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Effects of Microrna Modulation of PLK1 in Breast Cancer in Combination with PLK1 Inhibitor NMS-P937 Effects of MicroRNA modulation of PLK1 in Breast Cancer in combination with PLK1 inhibitor NMS-P937 Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Nicole Zalles Graduate Program in Biomedical Sciences The Ohio State University 2019 Dissertation Committee Carlo M. Croce, M.D., Advisor Kay Huebner, Ph.D. William Carson, M.D. Larry Schlesinger, M.D. 1 Copyrighted by Nicole Zalles 2019 Abstract The elucidation of more universal therapeutic targets is key to effectively combat the heterogeneity of breast cancer (BrCa). MicroRNAs (miRNAs) are small noncoding RNAs that are an abundant class of endogenous regulatory molecules, which act by targeting mRNAs for cleavage and/or translational repression. Continuing studies into BrCa genetics show that the various subtypes of BrCa are also affected by miRNA activity, which can even influence drug response. This prompted our investigation into the nuances of how miRNAs may influence BrCa behavior and response to treatment with targeted therapies, focusing on the proto-oncogene Polo-like Kinase 1 (PLK1). PLK1 plays an important role in the cell cycle, and is considered to be a proto-oncogene. Inhibiting PLK1 in has shown promise in reducing tumor volume and promoting tumor cell death in various cancers. A member of the miRNA-183 cluster, miRNA-183-5p, was found to bind to the 3’ UTR of PLK1, with transiently-induced overexpression resulting in reduced expression levels of the active PLK1 protein. We also discovered that the miRNA-regulated reduction of PLK1 influenced the expression of other proteins in the PLK1 pathway, and affected cancer cell response to a PLK1-specific inhibitor, NMS-P937. The activity of miRNA-183-5p on PLK1 demonstrated an effect on cancer cell apoptosis following treatment with NMS- P937, suggesting a link with miRNA-183-5p expression and the efficacy of PLK1-specific inhibition in breast cancer cell lines. MiRNA-mediated regulation plays an important role ii in the initiation, progression and drug response of tumors. Understanding how miRNAs regulate PLK1 in breast cancer will improve our understanding of the PLK1 pathway, and whether this miRNA-directed regulation affects anti-PLK1 therapy. The work outlined here sets the stage for further inquiry into miRNA-governed pathways and their effects on drug response, something that should be considered carefully as putative targets are identified for breast cancer therapy. iii Dedication Dedicated to my family, my friends, and to the patients and families who bravely face the complications of this disease every day iv Acknowledgments I am very grateful to my advisor, Dr. Carlo M. Croce, for the opportunity to pursue this and other projects in his laboratory. Without his support, critique and guidance, none of this would be possible. The lessons and skills I have learned here over the past few years cannot be understated in terms of their value and impact. I should also like to thank both my advisory committee members, Dr. Kay Huebner, Dr. William Carson, and Dr. Larry Schlesinger for their help in every step of the way, from the candidacy exam all the way to this point. Your comments, critiques, and suggestions during committee meetings were positively invaluable as I worked to accomplish what I had set out to do. I also extend a special thank you to Dr. David Rimm for his support and mentorship so early on in my scientific career; without his help, direction and willingness to take a high school graduate into his laboratory, I would never have been exposed to research, nor would I have known about the prospect of becoming a physician-scientist, completely changing my life and career trajectory in the most profound way. I am also incredibly grateful to Dr. Charles Perou, who gave me the opportunity to further advance my scientific skills and training during my undergraduate studies, under the careful and patient tutelage of Dr. Charles Harrell. Your profound support and career advice cemented my research interests and goals. v I would like to thank the Biomedical Sciences Graduate Program (BSGP) for providing the resources and means to pursue my research interests. I would especially like to thank Mrs. Amy Lahmers for her constant patience and guidance with all questions and concerns I had throughout the PhD process. I am also inherently grateful to Dr. Pierluigi Gasparini for his time, patience and tutelage. He always motivated me to work diligently and think scientifically, and has always been there to provide guidance and focus. Thanks to Gianni, Dario and Rosario for their tremendous assistance in the analysis of the patient expression data for this project, and thank you to Nicola for his invaluable help in all thing mouse-related. Thanks also to Dr. Bruce Kimler, whose guidance and perspective has always been a welcome source of wisdom as I’ve developed and solidified my career goals. I would like to thank Dr. Dorothee Wernicke, Mrs. Sharon Palko and Mrs. Marika Arledge for their everyday support and assistance. I would like to give many thanks to all previous and current members of the laboratory and our neighbors, especially Dr. Francesca Lovat, Dr. Federica Calore, Dr. Marina Capece, Dr. Pooja Joshi, Dr. Douglas Cheung, Dr. Alessandra Drusco, Dr. Dario Veneziano, Dr. Anna Tesari, and Dr. Veronica Baletti. I would also like to thank all the staff at the University Laboratory Animal Resources (ULAR) for providing all the support on mouse experiments. I would like to thank the Genomics Shared Resource (GSR) and Dr. Paolo Fadda for the technical advice and support. I would like to give special thanks to the Medical Scientist Training Program (MSTP), who gave me the opportunity to pursue my dream of combining the medical profession with scientific inquiry. In particular, I am grateful to Ashley Bertran and Aaron vi Thomas for their administrative and personal support. I of course would be remiss to mention a few key classmates who have been with me since the first day of pursuing this long but rewarding training process- Eileen, Ellen, Lucy, Giancarlo and Kavin, who have all helped me get through some of the tougher times throughout medical and graduate school. I am also incredibly grateful to my other friends Alisha, Tiffany, Abbie, Gabrielle, Tony, Joyce, and the Malleske family. I couldn’t have made it through the day-to-day without all of you. Last, but most importantly, I would like to give special recognition to my family, first and foremost to Dr. Maria Carola Zalles, my mother and guiding light for all aspects of my life. She instilled in me the importance of hard work, and inspired me to pursue a career in service to others, shaping me into the person I am today. She sparked my love for the sciences and medicine as a whole, and is my constant source of love and support. I want to thank my brother Daniel, for his grounded and practical perspective in all things, and in sharing my excitement for the simple things that make life worthwhile. I would also like to dedicate this work to the memory of my uncle Alfredo Bouchon Jr., and my grandfather Alfredo Bouchon Sr. They always made me feel as if I could do anything from the time I was very young. My grandfather showed through example how excellence is not simply given; it is earned. I miss them both dearly, and hope I have been able to make them proud. vii Vita 2009 to 2013 .................................................. B.A. Biology (Cum Laude), B.A. Anthropology University of North Carolina at Chapel Hill 2013 to 2015 ................................................. Medical Student, The Ohio State University, Columbus, Ohio 2015 to present ..............................................Graduate Research Associate, The Ohio State University, Columbus, Ohio Publications 1. Zalles, C., Zalles, N., Mahooti, S., Zahid, F., Khan, S., and Rimm, D. (2009). Objective spectral-spatial analysis of Random Pariareolar Fine Needle Aspiration of Women at High Risk for Breast Cancer. Poster presented at San Antonio Breast Cancer Symposium 2. Harrell, JC., Pfefferle, AD., Zalles, N., Prat, A., Fan, C., Khramtsov, A., Olopade, OI., Troester, MA., Dudley, AC., and Perou, CM. (2013). Endothelial-like properties of claudin-low breast cancer cells promote tumor vascular permeability. Clin Exp Metastasis. 31(1): 33-45. 3. Zalles, N., Gasparini, P., and Croce, C.M (2018). The Effects of microRNA modulation of Polo-like Kinase 1 in Breast Cancer cell lines. Poster presented at San Antonio Breast Cancer symposium viii Fields of Study Major Field: Biomedical Sciences ix Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita ................................................................................................................................... viii List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xiii Chapter 1. Introduction ......................................................................................................
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