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Modulating Hallmarks of Cholangiocarcinoma University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Fall 12-14-2018 Modulating Hallmarks of Cholangiocarcinoma Cody Wehrkamp University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Molecular Biology Commons Recommended Citation Wehrkamp, Cody, "Modulating Hallmarks of Cholangiocarcinoma" (2018). Theses & Dissertations. 337. https://digitalcommons.unmc.edu/etd/337 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]. MODULATING HALLMARKS OF CHOLANGIOCARCINOMA by Cody J. Wehrkamp A DISSERTATION Presented to the Faculty of the University of Nebraska Graduate College in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Biochemistry and Molecular Biology Graduate Program Under the Supervision of Professor Justin L. Mott University of Nebraska Medical Center Omaha, Nebraska November 2018 Supervisory Committee: Kaustubh Datta, Ph.D. Melissa Teoh‐Fitzgerald, Ph.D. Richard G. MacDonald, Ph.D. Acknowledgements This endeavor has led to scientific as well as personal growth for me. I am indebted to many for their knowledge, influence, and support along the way. To my mentor, Dr. Justin L. Mott, you have been an incomparable teacher and invaluable guide. You upheld for me the concept that science is intrepid, even when the experience is trying. Through my training, and now here at the end, I can say that it has been an honor to be your protégé. When you have shaped your future graduates to be and do great, I will be privileged to say that I was your first one. For your unwavering confidence in me and my abilities, even if mine faltered. I will always be grateful the time I was able to do science with you, and for the impact it has made on me, personally and professionally. For all you have done for me, I cannot adequately express my thanks; I only hope to deserve it. Thank you Justin. I thank the members of my supervisory committee, Dr. Richard MacDonald, Dr. Kaustubh Datta, and Dr. Melissa Teoh‐Fitzgerald, for their insight and guidance during our meetings, and for helping me to ask the right questions to properly test my hypotheses. I am additionally grateful to the entire BMB faculty for their varied expertise but moreover for their enthusiastic desire to impart it on us students. All of the BMB staff and especially Karen their sincere care and efforts to make my graduate career successful. My fellow students, who never ceased to impress me with the breadth of talents and intelligence they possess, I thank them for the comradery and standards they set. Thanks to the model organisms from which we learn about and improve our own malignancies. I love animals, and a career path in cancer research makes interaction specific and minimal, but more than none, so I am thankful for that. Thanks to the Mott Lab members for making true my oft‐repeated phrase, “teamwork makes the dream work.” To Sathish, your infectious work ethic, unyielding kindness, and lasting excitement for what we do is a motivation. To Ashley, thank you for listening to all and, having answers to most, of my questions, on the entire spectrum of quality, without judgement. Also for showing me what a Ph.D. that can do anything looks like. To Andrew and Matt, for challenging me to know the answer, or to at least be able to get there together. For allowing me to see the value and reward that comes with being in a mentor role, and making me want to be better at it. Thank you both for your help, and for letting me help. To Mary Anne, you add the laughter to the lab, and put a smile in the science. You remind us why we study these things, and for whom, and your continued creation of outreach is an inspiration. For making me a better person by knowing you. For being there. For everything. I am glad you forced me out of my shell those years ago. You made it all special. Thanks to my friends and family To Trevor, thanks for your true friendship, you are an always friend, family. Mom, thanks for being a good mom, and for always being there for Adam and I. Of the many things that are your strengths, perseverance is one I am thankful you ingrained in me. To Adam, you make me proud to be your brother. I value our bond, and our conversations about all things. I am inspired by your skill of living fully and by your effort to make change for the betterment of your fellow man. And to my father. While not a man of science by trade, my father always had a scientific curiosity and an interest in the natural world that I think was instilled in me. Since I was a boy I wanted to be a scientist, and here I am. I thank him for this, and for the example of what makes a good man. Thanks dad, I hope you would be proud. MODULATING HALLMARKS OF CHOLANGIOCARCINOMA Cody J. Wehrkamp, Ph.D. University of Nebraska Medical Center, 2018 Supervisor: Justin L. Mott, M.D., Ph.D. Abstract How are cholangiocarcinoma cells different from non‐malignant cholangiocytes? All of the hallmarks of cancer apply‐ those reported in this dissertation include cell proliferation, migration, apoptosis, and evasion of growth suppression. My studies began with testing how apoptosis might be regulated through embelin, a small molecule reported to sensitize cells to apoptosis by blocking XIAP. My data however revealed that embelin reduced the proliferative capacity in cholangiocarcinoma cells, but did not increase cell death. Malignant cells exhibit dysregulation of microRNA processing and expression. Hence, my studies seeking ways that cholangiocarcinoma eludes apoptosis transitioned to the oncomiR miR‐106b, which is overexpressed in cholangiocarcinoma. I observed that miR‐106b protected cholangiocarcinoma cells from apoptosis. Genome‐ wide screening identified the landscape of miR‐106b‐targeted genes in a cholangiocarcinoma cell line, some with roles in tumor biology. MiR‐106b targets included members of the Krüppel‐like factor (KLF) family of transcription factors. The function of KLF2 in biliary epithelia or cholangiocarcinoma is unknown. I describe in part how the cholangiocyte senses the environment through the primary cilium and translates this to regulation of KLF2, a flow‐responsive regulatory protein and tumor suppressor in several cancers. I observed lower expression of KLF2 in malignant cholangiocarcinoma cells and tumors compared to normal, and its enforced expression inhibited proliferation and migration while reducing sensitivity to apoptosis. In the normal bile duct epithelium, environmental cues are detected by the cholangiocyte primary cilium, a sensory organelle that functions as a signaling nexus for the cell. Cholangiocarcinoma cells are highly proliferative despite extracellular signals to remain quiescent. Cholangiocarcinoma cells often lose their cilia, resulting in altered communication and unchecked cell growth. I identified a cholangiocyte signaling axis in which the primary cilium maintains quiescence through enhanced KLF2 expression. I present the first finding of a ciliary‐dependent KLF2 flow response in cholangiocytes. Overall, this dissertation sought deeper understanding of biochemical and molecular features of cholangiocarcinoma and added to our understanding of microRNAs and mechanosensory pathways. i Table of Contents Acknowledgements ................................................................................................................... i Abstract .....................................................................................................................................iii List of Figures ........................................................................................................................... vi List of Tables .......................................................................................................................... viii List of Abbreviations ............................................................................................................... ix Chapter 1 ‐ Introduction .............................................................................................................. 1 Cholangiocyte biology .............................................................................................................. 1 Cholangiocarcinoma ................................................................................................................. 3 MicroRNAs ................................................................................................................................ 7 MicroRNAs in cancer ............................................................................................................. 10 miR‐106b ................................................................................................................................... 12 KLF2 review: “KLF2 regulation and function” .................................................................. 19 Development and cell differentiation .............................................................................. 19 Structure ............................................................................................................................... 20 Degradation ......................................................................................................................... 22 Regulation
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