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Regulation of Clathrin Mediated Endocytosis and Its Role in Cancer

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Regulation of Clathrin Mediated Endocytosis and Its Role in Cancer REGULATION OF CLATHRIN MEDIATED ENDOCYTOSIS AND ITS ROLE IN CANCER PROGRESSION APPROVED BY SUPERVISORY COMMITTEE Sandra Schmid, Ph.D., Advisor John Minna, M.D., Chairman Jerry Shay, Ph.D. Gaudenz Danuser, Ph.D DEDICATION I would like to thank my husband, Mac, my parents, and my family for their love and support, which has allowed me to reach this milestone in my life. I would also like to thank my mentor and the members of my Graduate Committee for thoughtful discussion and helping me reach my true potential. REGULATION OF CLATHRIN MEDIATED ENDOCYTOSIS AND ITS ROLE IN CANCER PROGRESSION by SARAH ELKIN CONNELLY DISSERTATION Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas, Texas August 2017 Copyright by Sarah Elkin Connelly, 2017 All Rights Reserved REGULATION OF CLATHRIN MEDIATED ENDOCYTOSIS AND ITS ROLE IN CANCER PRGRESSION Publication No. Sarah Elkin Connelly, Ph.D The University of Texas Southwestern Medical Center at Dallas, 2017 Supervising Professor: Sandra Schmid, Ph.D. Metastasis is a multistep process requiring cancer cell signaling, invasion, migration, survival, and proliferation. These processes require dynamic modulation of cell surface proteins by endocytosis. Given this functional connection, it has been suggested that endocytosis is dysregulated in cancer. To test this, we developed In-Cell ELISA assays to measure three different endocytic pathways: clathrin-mediated endocytosis, caveolae- mediated endocytosis, and clathrin-independent endocytosis and compared these activities in 29 independently isolated non–small cell lung cancer (NSCLC) cell lines to determine whether there were systematic changes in the three different endocytic pathways. However v we observed significant heterogeneity. Nonetheless, using hierarchical clustering based on their combined endocytic properties we identified two phenotypically distinct clusters of NSCLCs. One co-clustered with mutations in KRAS, a mesenchymal phenotype, increased invasion through collagen and decreased growth in soft agar, whereas the second was enriched in cells with an epithelial phenotype. We also used the In-Cell ELISA assay to characterize Ikarugamycin (IKA), a previously discovered antibiotic, which inhibits the uptake of oxidized low-density lipoproteins in macrophages, as well as clathrin-mediated endocytosis (CME) in plant cell lines. However, detailed characterization of IKA had yet been performed. Therefore, we performed biochemistry and microscopy experiments to further characterize the effects of IKA on CME. We showed that IKA acutely inhibits CME, but not other endocytic pathways with an IC50 of 2.7 µM. Although long-term incubation with IKA has cytotoxic effects, the short-term inhibitory effects on CME were reversible. Thus, IKA can be a useful tool for probing routes of endocytic trafficking. Finally, we investigated possible mechanisms that lead to altered endocytosis in cancer cells. We discovered that dynamin 1 (Dyn1), previously thought to be neuron specific is frequently upregulated and postranslationally regulated in cancer cells. Dyn1 expression alters the proliferation rates, growth in soft agar, and tumor growth of cancer cells. We hypothesize that these changes are due to alteration in cell surface protein expression and downstream signaling pathways and have developed protocols to test these hypothesizes. Taken together, our results suggest that endocytic alterations in cancer cells can significantly influence cancer-relevant phenotypes. vi TABLE OF CONTENTS ABSTRACT ............................................................................................................................ v TABLE OF CONTENTS ....................................................................................................... vii PRIOR PUBLICATIONS ..................................................................................................... ix LIST OF FIGURES ............................................................................................................... x LIST OF TABLES .............................................................................................................. xiii LIST OF ABBREVIATIONS ............................................................................................. xiv CHAPTER ONE: AN OVERVIEW OF ENDOCYTIC PATHWAYS AND THEIR POSSIBLE ROLE IN CANCER PROGRESSION .......................................................... 1 INTRODUCTION ........................................................................................................... 1 MULTIPLE MECHANISMS FOR UPTAKE INTO CELLS ......................................... 2 CLATHRIN MEDIATED ENDOCYTOSIS ............................................................. 2 CAVEOLAE MEDIATED ENDOCYTOSIS ........................................................... 4 CLATHRIN INDEPENDENT ENDOCYTOSIS ...................................................... 5 ENDOCYTOSIS IN HUMAN DISEASE ....................................................................... 7 ENDOCYTOSIS AND CANCER ................................................................................... 8 CHAPTER TWO: IKARUGAMYCIN: A NATURAL PRODUCT INHIBITOR OF CLATHRIN-MEDIATED ENDOCYTOSIS ................................................................... 14 ABSTRACT ................................................................................................................... 14 INTRODUCTION ......................................................................................................... 14 MATERIALS AND METHODS ................................................................................... 16 vii RESULTS ...................................................................................................................... 24 DISCUSSION ................................................................................................................ 38 SUPPLEMENTAL MATERIAL .................................................................................... 40 CHAPTER THREE: A SYSTEMATIC ANALYSIS REVEALS HETEROGENEOUS CHANGES IN THE ENDOCYTIC ACTIVITIES OF CANCER CELLS ................... 43 ABSTRACT ................................................................................................................... 43 INTRODUCTION ......................................................................................................... 44 MATERIALS AND METHODS ................................................................................... 46 RESULTS ...................................................................................................................... 52 DISCUSSION ................................................................................................................ 68 SUPPLEMENTAL MATERIAL .................................................................................... 74 CHAPTER FOUR: THE ROLE OF DYNAMIN 1 IN CANCER CELL AGGRESSION .................................................................................................................................... 84 ABSTRACT ................................................................................................................... 84 INTRODUCTION ......................................................................................................... 85 RESULTS ...................................................................................................................... 87 DISCUSSION .............................................................................................................. 112 MATERIALS AND METHODS ................................................................................. 116 BIBLIOGRAPHY ............................................................................................................ 149 viii PRIOR PUBLICATIONS Elkin SR, Kumar A, Price CW, Columbus L. A broad specificity nucleoside kinase from Thermoplasma acidphilum. Proteins. 2013; 81(4):568-82. ix LIST OF FIGURES CHAPTER ONE FIGURE ONE: Schematic representation of the different endocytic pathways described in mammalian cells. ................................................................................................................. 13 CHAPTER TWO FIGURE 2-1: Ikarugamycin inhibits clathrin-mediated endocytosis .................................. 26 FIGURE 2-2: Ikarugamycin is selective for CME ........................................................ 28 FIGURE 2-3: Ikarugamycin inhibition of CME is rapid and partially reversible ......... 29 FIGURE 2-4: Ikarugamycin disrupts clathrin coated pit distributions .......................... 31 FIGURE 2-5: Ikarugamycin alters Golgi morphology .................................................. 34 FIGURE 2-6: The effect of ikarugamycin on cell viability ............................................ 36 FIGURE 2-7: Ikarugamycin inhibition of CME is rapid and partially reversible at high concentrations ................................................................................................................. 37 FIGURE 2-S1: Ikarugamycin affects clathrin coated pit morphology ........................... 40 FIGURE 2-S2: Ikarugamycin does not affect the morphology of other cellular organelles 41 FIGURE 2-S3: Ikarugamycin does not activate Caspase cleavage in cells .................... 42 CHAPTER THREE FIGURE
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