BIOLOGICAL MODELS OF COLORECTAL CANCER METASTASIS AND TUMOR SUPPRESSION PROVIDE MECHANISTIC INSIGHTS TO GUIDE PERSONALIZED CARE OF THE COLORECTAL CANCER PATIENT By Jesse Joshua Smith Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University In partial fulfillment of the requirements For the degree of DOCTOR OF PHILOSOPHY In Cell and Developmental Biology May, 2010 Nashville, Tennessee Approved: Professor R. Daniel Beauchamp Professor Robert J. Coffey Professor Mark deCaestecker Professor Ethan Lee Professor Steven K. Hanks Copyright 2010 by Jesse Joshua Smith All Rights Reserved To my grandparents, Gladys and A.L. Lyth and Juanda Ruth and J.E. Smith, fully supportive and never in doubt. To my amazing and enduring parents, Rebecca Lyth and Jesse E. Smith, Jr., always there for me. .my sure foundation. To Jeannine, Bill and Reagan for encouragement, patience, love, trust and a solid backing. To Granny George and Shawn for loving support and care. And To my beautiful wife, Kelly, My heart, soul and great love, Infinitely supportive, patient and graceful. ii ACKNOWLEDGEMENTS This work would not have been possible without the financial support of the Vanderbilt Medical Scientist Training Program through the Clinical and Translational Science Award (Clinical Investigator Track), the Society of University Surgeons-Ethicon Scholarship Fund and the Surgical Oncology T32 grant and the Vanderbilt Medical Center Section of Surgical Sciences and the Department of Surgical Oncology. I am especially indebted to Drs. R. Daniel Beauchamp, Chairman of the Section of Surgical Sciences, Dr. James R. Goldenring, Vice Chairman of Research of the Department of Surgery, Dr. Naji N. Abumrad, Chairman of the Department of Surgery and Dr. John L. Tarpley, Program Director and Professor of the Department of Surgery for constant support, encouragement and guidance. I would especially like to thank Mrs. Donna Putnam, Dr. Natasha G. Deane, Associate Research Professor, Department of Surgical Oncology, Dr. Nancy J. Brown, Director, Department of Clinical Pharmacology, Dr. Yu Shyr, Professor and Chief, Department of Cancer Biostatistics and Dr. Terence Dermody, Professor and Director of the Vanderbilt Medical Scientist Training Program, who have supported my career goals and who worked tirelessly to provide me with the resources, protected time and encouragement to pursue my dreams and goals. I would also like to thank two individuals who have been taken from us, Niki Carey and Dr. Neville Grant, who provided measures of heroism and inspiration during my early residency and graduate training. I am thankful to Fei Wu, M.D., Ph.D. for getting the 34-gene story off to a great start. I thank Drs. J. Chad Johnson, Carl Schmidt, Nipun B. Merchant, Steven Eschrich, Timothy J. Yeatman and Martin J. Heslin for initial efforts to organize and secure the colorectal tissues used for the microarray analysis in this work. I am grateful to all of those with whom I have had the pleasure to work during the last 4 years in the laboratory. Each of my Dissertation Committee members has provided excellent professional iii guidance and spurred me on to pursue science and research at a new level. Notably, Drs. Mark de Caestecker and Ethan Lee had tremendous roles in my development as a scientist. I am thankful for their guidance, time, patience and generosity with reagents. I would especially like to thank Dr. Bob Coffey and Dr. Steve Hanks who have exemplified a steady hand in my graduate school career and who have provided me with much wisdom and encouragement. I would like to thank my medical school mentors, Drs. Hazim J. Safi, Charles C. Miller and Anthony L. Estrera for continued support and encouragement in my studies, my life and my career. Dr. Safi has been a stalwart and was the first to introduce me to the wonderful world of medicine and science. I am forever grateful for his inspiration and encouragement. Dr. Miller taught me how to think like a clinical scientist and kept me sane during medical school. Dr. Estrera encouraged me during my pursuit of surgical residency and taught me how to round on patients as a first year medical student. I am thankful to R. Daniel Beauchamp, M.D. and Maximilian Buja, M.D. for giving me a chance. Nothing has been more important to me than my faith and my family as a bedrock of support during my training. My mom and dad, in-laws, my brother Nate and extended family have provided much grace and love to Kelly and myself. Most importantly, I wish to thank my dear wife Kelly and our yet unknown children for unending love, support and help during my training. This work was supported by the following grants from the National Institutes of Health: 1) CA69457; DK52334; CA068485; CA077839 (R. Daniel Beauchamp, M.D.); 2) TL1 RR024978; CA106183 (Institutional support for J. Joshua Smith); 3) CA46413; CA95103; CA084239 (R. J. Coffey, M.D.); 4) CA112215 (Timothy J. Yeatman, M.D.); 5) CA126588; CA128323 (Natasha G. Deane, Ph.D.); Other sources of funding: Society of University Surgeons-Ethicon Scholarship Award (J.J.S.). iv TABLE OF CONTENTS Page DEDICATION........................................................................................................................................ ii ACKNOWLEDGEMENTS .....................................................................................................................iii LIST OF TABLES................................................................................................................................. ix LIST OF FIGURES ............................................................................................................................... x LIST OF ABBREVIATIONS..................................................................................................................xii Chapter I. INTRODUCTION TO THE PROBLEM OF COLORECTAL CANCER...............................................1 Epidemiology and Pathogenesis.......................................................................................................1 Genomic Instability............................................................................................................................2 Mutational Inactivation of Tumor-Suppressor Genes........................................................................2 Oncogenic Pathway Activation..........................................................................................................3 Developmental Biology and Carcinogenesis in Intestinal Epithelial Cells .........................................5 Overview of Prominent Signaling Pathways.................................................................................5 Metastasis and Epithelial-Mesenchymal Transition (EMT): Parallels in Developmental Biology and Cancer......................................................................................................................5 Transforming Growth Factor- (TGF) Superfamily Signaling in Epithelial Biology.....................7 TGF and Smad4 in Gastrointestinal Homeostasis and Cancer.............................................9 Bone Morphogenetic Proteins in the Intestinal Tract.............................................................11 TGF Superfamily Signaling and EMT..................................................................................12 Wnt Signaling in Intestinal Growth, Differentiation and Colorectal Cancer.................................13 Adherens junctions, Wnt and EMT........................................................................................15 Tumor Biology at the Invasive Front......................................................................................17 Other Contributing Pathways in the Pathogenesis of Colorectal Cancer ........................................18 Predictive and Prognostic Markers and Molecular Detection in Colorectal Cancer.........................20 Biological Models of Colorectal Cancer...........................................................................................21 Summary.........................................................................................................................................23 II. AN EXPERIMENTALLY DERIVED METASTASIS GENE EXPRESSION PROFILE PREDICTS RECURRENCE AND DEATH IN COLON CANCER PATIENTS ................................25 Abstract...........................................................................................................................................25 Introduction .....................................................................................................................................26 Materials and Methods....................................................................................................................27 v Cell Culture and Mouse Model Overview...................................................................................27 Detailed Cell Culture and Mouse Methods............................................................................28 Human Tissue and Microarray Platforms...................................................................................30 Statistical Methods and Identification of VMC High-Risk Patients..............................................30 Clinical Outcomes and Testing of the 34-gene Based Metastasis Score...................................32 Vanderbilt Cox Model (Training Dataset) ..............................................................................32 Moffitt Cox Model (Test Dataset)...........................................................................................33