ONCOSTATIN M CONTRIBUTES TO BREAST TUMOR METASTASIS by Hunter J. Covert A Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomolecular Sciences Boise State University May 2017 © 2017 Hunter J Covert ALL RIGHTS RESERVED BOISE STATE UNIVERSITY GRADUATE COLLEGE DEFENSE COMMITTEE AND FINAL READING APPROVALS of the dissertation submitted by Hunter J. Covert Dissertation Title: Oncostatin M Contributes To Breast Tumor Metastasis Date of Final Oral Examination: 14 April 2017 The following individuals read and discussed the dissertation submitted by student Hunter J. Covert, and they evaluated his presentation and response to questions during the final oral examination. They found that the student passed the final oral examination. Cheryl Jorcyk, Ph.D. Chair, Supervisory Committee Julia Thom Oxford, Ph.D. Member, Supervisory Committee Kenneth A Cornell, Ph.D. Member, Supervisory Committee Daniel Fologea, Ph.D. Member, Supervisory Committee Grant Trobridge, PhD External Examiner The final reading approval of the dissertation was granted by Cheryl Jorcyk, Ph.D., Chair of the Supervisory Committee. The dissertation was approved by the Graduate College. DEDICATION To my wife who has offered me so much support and convinced me to not give up numerous times during graduate school. This is also dedicated to my two young sons who keep me focused on the goals at hand and cheer me up whenever I am down. iv ACKNOWLEDGEMENTS I will start by acknowledging my mentor, Dr. Cheryl Jorcyk for letting me enter her lab in 2009 during my final semester as an undergraduate. Dr. Jorcyk taught me so many things during the time I spent in her lab. She taught me how become a better critical thinker in order to answer difficult scientific questions. She was always available for meetings to ensure progress was being made during my graduate studies. She has always been very supportive even when my ideas seemed a little far-fetched. The biggest thing that I am thankful for is Dr. Jorcyk helped build my confidence as a graduate student by always being very supportive. I very much would like to thank the members of my committee, Dr. Julie Oxford, Dr. Ken Cornell, and Dr. Daniel Fologea, who have contributed great ideas over the years. I would also like to thank Dr. Liliana Mellor, in Dr. Julie Oxford’s laboratory, who initially loaned our lab the antibody used for all of the CD44 western blotting, which began us on the trail of exploring CD44 in breast cancer. She also answered any questions we had throughout the last 5 years. I also want to acknowledge past and present members of the Jorcyk laboratory. Dr. Celeste Bolin, who taught me many molecular techniques and how to be a better critical thinker. Ken Tawara, M.S., who taught me everything about in vivo work and answered every question I’ve needed help with the last 8 years. Jake, Dollie, Jordan, and Danielle, who not only were tremendous team members in the lab, but also great friends. Hannah and Jackie deserve acknowledgement for helping with in vivo studies and also performing a lot of statistical analysis on large amounts of raw data. Nichole Ankenbrandt was extremely v helpful as an undergrad researcher who prepared a lot of the immunofluorescence used in my early graduate work. Raquel Brown at the Biomolecular Research Center for helping with confocal microscopy. Dr. Allan Albig for trusting me to use a lot of his equipment well after normal work hours. I also want to acknowledge Dr. Denise Wingett who is the director of the Biomolecular Science Graduate Program, as she has helped myself and others in our inaugural class, and the program in many ways. Finally, I would like to thank Beth Gee who is the Biomolecular Sciences Graduate Program coordinator. Being a member of the first class of a graduate program has been a learning experience for everyone involved, and Beth has always been there to support students and answer questions when needed. vi ABSTRACT The process of breast tumor metastasis has been examined to a great extent in recent years, but connecting specific protein-protein interactions to respective metastatic events remains challenging. The primary cause of death in patients with breast cancer is not from the primary tumor itself but rather from tumor metastases, which cause over 90% of all mortalities. In order for tumor cells to metastasize, they must undergo a phenotypic change known as the epithelial-mesenchymal transition (EMT). An EMT allows for the intravasation of tumor cells into nearby blood vessels or lymphatic channels. Once the primary tumor cells are within the network of vessels, they undergo a homing process to specific organs within the body. For breast cancer, secondary metastatic locations include the liver, brain, lung, and bone. It is known that signaling by proteins called cytokines influences breast tumor cells to adopt a more metastatic and aggressive phenotype. Cytokine signaling often changes protein functioning in the surrounding tumor environment and can lead to increased vasculature, which provides nutrients to a growing tumor, or phenotypic changes to individual cells on the invasive edge of a proliferating tumor. Transmembrane proteins, such as cluster of differentiation 44 (CD44), contribute to cell migration, cell detachment, and cellular docking during metastasis. The upregulation of CD44 expression by cytokine signaling is observed in less aggressive tumor cells, while more aggressive tumor cells constitutively express high basal levels of CD44. Both EMT and CD44 induced by cytokines are involved in metastasis during specifically timed events in the metastatic cascade. vii TABLE OF CONTENTS DEDICATION ................................................................................................................... iv ACKNOWLEDGEMENTS .................................................................................................v ABSTRACT ...................................................................................................................... vii LIST OF TABLES ............................................................................................................. xi LIST OF FIGURES .......................................................................................................... xii LIST OF ABBREVIATIONS .......................................................................................... xvi CHAPTER ONE: INTRODUCTION ..................................................................................1 Breast Cancer ...........................................................................................................1 Breast Cancer Subtypes ...............................................................................1 Metastatic Breast Cancer .............................................................................2 Oncostatin M (OSM) ...................................................................................3 Cluster of Differentiation 44 (Cd44) ............................................................5 Cancer Stem Cells (CSCs) and Breast Cancer Stem Cells (BCSCs) ...........7 Circulating Tumor Cells (CTCs) .................................................................8 In Vivo Models of Human Breast Cancer ....................................................8 Patient survival.............................................................................................9 Summary ..................................................................................................................9 CHAPTER TWO: OSM CONTRIBUTES TO BREAST CANCER METASTATIC POTENTIAL THROUGH CD44-INDUCED CELL MIGRATION AND DETACHMENT ................................................................................................................14 viii Abstract ..................................................................................................................15 Introduction ............................................................................................................16 Materials and Methods ...........................................................................................19 Cell Culture and Cytokine Stimulation ......................................................19 Immunofluorescence Staining and Microscopy.........................................19 qRT-PCR....................................................................................................20 Western Blot Analysis ...............................................................................20 Flow Cytometry .........................................................................................21 shRNA and Chemical Inhibitors ................................................................21 Detachment Assay .....................................................................................22 Scratch Assay .............................................................................................22 Results ....................................................................................................................23 OSM Induces Both CD44 Expression and Cleavage .................................24 OSM-Induced EMT is Independent of CD44 ............................................26 OSM-Induced Tumor Cell Detachment is Dependent on CD44 ...............27 OSM-Induced Tumor Cell Migration is Dependent on CD44...................28 Discussion ..............................................................................................................28