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Altered Levels of SOX2, and Its Associated Protein Musashi2, Disrupt Critical Cell Functions in Cancer and Embryonic Stem Cells

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Altered Levels of SOX2, and Its Associated Protein Musashi2, Disrupt Critical Cell Functions in Cancer and Embryonic Stem Cells University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Fall 12-16-2016 Altered Levels of SOX2, and Its Associated Protein Musashi2, Disrupt Critical Cell Functions in Cancer and Embryonic Stem Cells Erin L. Wuebben University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Recommended Citation Wuebben, Erin L., "Altered Levels of SOX2, and Its Associated Protein Musashi2, Disrupt Critical Cell Functions in Cancer and Embryonic Stem Cells" (2016). Theses & Dissertations. 154. https://digitalcommons.unmc.edu/etd/154 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. i ALTERED LEVELS OF SOX2, AND ITS ASSOCIATED PROTEIN MUSASHI2, DISRUPT CRITICAL CELL FUNCTIONS IN CANCER AND EMBRYONIC STEM CELLS BY ERIN LYNN WUEBBEN A DISSERTATION PRESENTED TO THE FACULTY OF THE GRADUATE COLLEGE AT THE UNIVERSITY OF NEBRASKA MEDICAL CENTER IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY CANCER RESEARCH GRADUATE PROGRAM EPPLEY INSTITUTE FOR CANCER RESEARCH AND ALLIED DISEASES UNDER THE SUPERVISION OF PROFESSOR ANGIE RIZZINO UNIVERSITY OF NEBRASKA MEDICAL CENTER OMAHA, NEBRASKA OCTOBER 2016 ii ACKNOWLEDGEMENTS My life has been filled with wonder at the beauty and complexity of the world surrounding me. This wonder blossomed under the education and guidance of phenomenal teachers and professors like Mrs. Marsha Stewart at Norfolk Catholic High School and Dr. Lawrence Harshman at the University of Nebraska – Lincoln. However, this wonder grew into true scientific inquiry under the supervision of my Ph.D. advisor Dr. Angie Rizzino. Angie has encouraged my growth as a scientist through daily discussions of recent cancer breakthroughs, experimental design, and effective communication through writing and presenting. However, beyond my development as a scientist, I believe the mentorship I have received from Angie has made me a better person by always challenging me to explore things for myself, to enhance my critical thinking, and to always remember that people are the reason we study this disease; to help save or improve the lives of people living with cancer. For these things, I am extremely grateful. This philosophy has been further expanded by the people of Eppley Institute and the Cancer Research Graduate Program. Faculty members of my supervisory committee, Drs. Tony Hollingsworth, Xu Luo, and Michel Oullette, as well as Dr. Amar Natarajan’s service on my comprehensive committee, have been a great source of knowledge and suggestions for furthering my graduate studies. Furthermore, Dr. Joyce Solheim has been a constant source of advice and listening ear for me and others, always keeping her door open, literally and figuratively, for support during times of uncertainty. Among the many people of Eppley, I want to extend my thanks to all the graduate students of the Eppley Institute for fostering a community of collaboration and fount of memories I’ll treasure for years to come. I must also acknowledge several former and current students and laboratory members who have helped me throughout my studies. Initially, I was taken under the wings of Dr. Sunil iii Mallanna who meticulously started the path for my first publication as he prepared his own dissertation. Additionally, Michelle Desler and Dr. Phillip Wilder have been crucial to the steady success of the Rizzino laboratory through not only keeping the laboratory running smoothly, but also through their patience in trouble shooting any problems that arise. Most importantly, I have to thank the former, senior students in Angie’s laboratory, Dr. Jesse Cox and Dr. Briana Ormsbee Golden, who carefully taught me so many protocols while also offering their thoughtful support in overcoming experimental roadblocks. Their patience and perseverance instilled in me a fortitude I hope to continue throughout my career. The additional support of several students outside of the laboratory made the ups and downs of graduate study so much more bearable. Dr. Jenna Preusker, Dr. Jennifer Kopanic, Dr. Dana Tabor, Dr. Kristi Anderson, Rachel Utter, Maria Steele, and Corey Suelter often provided the much needed happy distraction to balance the frenzy that is graduate work. Their unwavering support while balancing their own projects was so selfless and kind, and will be forever appreciated. Finally, I must extend my deepest gratitude to my family, especially the two people who have supported me the longest, Allen and Terri Wuebben. As my parents, they have always reminded me that I’m capable of anything I set my mind to and have always been there to pick me up when I stumble. Their faith in me has given me the confidence to be independent and pursue my dreams. I have been extremely blessed to have them as my parents and as friends. iv Altered levels of SOX2, and its associated protein Musashi2, disrupt critical cell functions in cancer and embryonic stem cells Erin Lynn Wuebben, Ph.D. University of Nebraska, 2016 Advisor: Angie Rizzino, Ph.D. The transcription factor SOX2 is widely recognized for its critical roles during mammalian embryogenesis. SOX2 has also been examined in cancer; and it has been implicated in the growth, tumorigenicity, drug resistance, or metastasis of over 20 different cancers, including cancers of the brain and pancreas. Thus, we hypothesized that SOX2 is a major player in cancer and may be a potential therapeutic target; however, the effects of SOX2 on the many facets of human cancer have only begun to be explored. Recently, efforts to understand the mechanisms by which SOX2 mediates its effects have explored SOX2 protein-protein interaction landscapes in a number of cellular systems. Previous studies in our laboratory identified proteins, like the RNA-binding protein Musashi2 (Msi2), which interact with SOX2 in multiple cell types, including embryonic stem cells and brain tumor cells. We hypothesized that proteins that interact with SOX2 in multiple cell types are likely to be necessary for the continued growth and function of these cells. The studies presented in this dissertation demonstrate that ESC require Msi2 to maintain self-renewal and pluripotency; and that MSI2 is also required to support the growth and survival of DAOY, U87, and U118 brain tumor cell lines. This dissertation also examined the roles of SOX2 in pancreatic adenocarcinoma (PDAC). Multiple PDAC cell lines were engineered for either inducible overexpression of SOX2 or inducible knockdown of SOX2. Through in vitro growth and tumorigenicity studies with these inducible PDAC cell lines, we determined that SOX2 functions as a biphasic molecular rheostat in PDAC. Furthermore, we determined that inducible elevation of SOX2 in PDAC cells reduces the growth inhibitory effects of MEK and v AKT inhibitors, while the inducible knockdown of SOX2 enhanced growth inhibition in the presence of these inhibitors. Altogether, the work presented in this dissertation extends and strengthens our knowledge of SOX2 and its function as a master regulator in multiple cell types, and provides useful platforms for the continued study of these highly deadly malignancies. vi TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................................. ii ABSTRACT .................................................................................................................................... iv TABLE OF CONTENTS ................................................................................................................ vi LIST OF FIGURES AND TABLES ............................................................................................... ix ABBREVIATIONS ....................................................................................................................... xii CHAPTER 1: Introduction ....................................................................................................... 1 1.1 Introduction ............................................................................................................... 2 1.2 Sox2 Gene Structure and Regulation ........................................................................ 3 1.2.1 Transcriptional Regulation of Sox2 .......................................................... 3 1.2.2 MicroRNAs and SOX2 Expression .......................................................... 6 1.2.3 Long Noncoding RNAs and SOX2 Expression ...................................... 10 1.2.4 Post-translational Modifications of SOX2 .............................................. 13 1.3 SOX2 is Essential during Mammalian Embryogenesis .......................................... 18 1.4 Sox2 levels are optimized to support both self-renewal and pluripotency of ESC . 20 1.5 SOX2-Protein Interactomes Identify Critical Understudied Proteins, like Musashi2, in Multiple Cell Types ...................................................................................................... 22 1.6 SOX2 Expression in Cancer: Amplification, Prognosis, and Survival ................... 23 1.7 SOX2 and Tumor-Initiating Cells/Cancer Stem Cells ............................................ 27 1.8 SOX2 and Drug Resistance ...................................................................................
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