UNIVERSITY OF CINCINNATI _____________December 6 , 20 _____ 00 I,________________________________Randall Glenn Marsh ______________, hereby submit this as part of the requirements for the degree of: ________________________Doctorate of Philosophy (Ph.D.) ________________________ in: ________________________Cell and Molecular Biology ________________________ It is entitled: ________________________Characterization of a new role for plakoglobin________________________ in ________________________suppressing epithelial cell translocation________________________ ________________________________________________ ________________________________________________ Approved by: ________________________Dr. Robert Brackenbury, Ph.D. (Chair) ________________________Dr. Wallace Ip, Ph.D. ________________________Dr. Randal Morris, Ph.D. ________________________Dr. James Greenberg, M.D. ________________________ CHARACTERIZATION OF A NEW ROLE FOR PLAKOGLOBIN IN SUPPRESSING EPITHELIAL CELL TRANSLOCATION A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) in the Department of Cell Biology, Neurobiology, and Anatomy of the College of Medicine 2000 by Randall Glenn Marsh B.Sc., University of Alberta, 1989 M.Sc., University of Alberta, 1992 Committee Chair: Dr. Robert Brackenbury, Ph.D. ii ABSTRACT Despite advances in the treatment of cancer patients, spread, or metastasis, of tumor cells remains a major cause of death. Although metastasis is a complex process, an important component is cell motility. Acquisition of motility allows tumor cells to breach normal barriers and thereby disseminate. Regulation of motile behavior is poorly understood, but evidence suggests the cadherin family of adhesion molecules plays an important role. The goal of my thesis was to investigate regulation of epithelial cell translocation by cadherins. For my studies, I focused on PAM212 keratinocytes. These cells express high levels of E-cadherin and show suppression of translocation upon contact (STUC). Using repeated light trypsinization and assessment of colony morphology, I selected for variants having a scattered, motile phenotype. As hoped, some variants had reduced expression of E-cadherin (ED cells). Surprisingly, however, some variants retained relatively normal E-cadherin expression but had drastically reduced plakoglobin levels (PD cells). Re-expression of E-cadherin in ED cells, or plakoglobin in PD cells, restored STUC, demonstrating a causal role for each protein. Time- lapse analyses revealed that E-cadherin mediated initial cell-cell contacts, while plakoglobin mediated a subsequent long-term stabilization event. Plakoglobin has known adhesive and signaling functions and could therefore suppress translocation through an adhesive or signaling mechanism. Strong support for a signaling mechanism was provided by: (a) adhesion assays, which revealed no significant difference in adhesiveness of parental PAM212 versus PD cells; (b) time-lapse analyses, which revealed no difference in the ability of parental versus PD cells to form contacts, and; (c) analysis of a deletion-mutant of plakoglobin, which restored STUC despite being unable to participate in desmosome assembly or cadherin adhesion. The most likely candidates for mediating signaling iii downstream of plakoglobin were TCF/LEF transcription factors, but these were ruled out by: (a) analysis of a dominant-negative TCF/LEF, which failed to render parental cells motile, and; (b) the effects of a deletion-mutant of the plakoglobin-related protein β-catenin, which restored STUC in PD cells despite being unable to bind these factors. These results reveal a new role for plakoglobin in suppressing epithelial cell translocation, and show that an undescribed signaling pathway underlies this suppression. iv v ACKNOWLEDGEMENTS I would like to thank: My thesis advisor – Dr. Robert Brackenbury – for his advice, kindness, and support over the last five years. His friendship and fun-loving spirit have helped me through many rough spots in the lab and in life. The other members of my thesis committee – Drs. Kathleen Green, James Greenberg, Wallace Ip, William Larsen, and Randal Morris – for their valuable advice and support. The Albert J Ryan Foundation for financial support and wonderful trips to Squam Lake, New Hampshire. My colleagues in the Brackenbury lab, past and present – Drs. Mary Chaiken, Hiayan Chen, Charlie Li, Joan Morris, Nancy Paradies, MaryAnn Szegedy, Mrs. Amy Koshoffer, and Mr. Bradford Mallory – for their help, friendship, and comradery. My fellow graduate students for friendship, good times, and fond memories. My fellow microanatomists - Drs. John Michaels and Robert Cardell, and Mrs. Emma Lou Cardell – for the opportunity to teach Microscopic Anatomy to the medical students, and for providing me with an educational and enjoyable experience. The administrative staff of the Department and of the Graduate Program in Cell and Molecular Biology – Mrs. Barb Carter, Mrs. Susan Eder, Miss Kimberly Fry, Ms. Peggy Grause, Mr. Wade Hedgren, Mrs. Linda Moeller, Mrs. Felicia Romine, and Mrs. Susan Seiler – for their help with more things than I can possibly remember. My parents and family for their love and encouragement, even though they were never quite sure what I was working on. My dogs – Krista, Bandit, Gizmo, and Taffy – for their unconditional love and companionship. My wife, Heather, for her continual love and support. Without her patience and understanding, this work would not have been possible. vi TABLE OF CONTENTS ABSTRACT …………………………………………...…………………………………… iii ACKNOWLEDGEMENTS …………………………………………………………..……... vi TABLE OF CONTENTS …………………………………………………………………..……… 1 LIST OF FIGURES ………………………………………………………………………..………. 3 LIST OF TABLES ……………………………………………………………………….………... 5 LIST OF ABBREVIATIONS ……………………………………………………………….…….. 6 CHAPTER 1 – Introduction ………………………………………………….…………... 8 Metastasis ……………….………………………………….……… 9 Cell Motility ………….………………………………….………… 15 Cell Adhesion ……………….…………………………….……….. 23 Role of Cadherins and Catenins in Invasion and Metastasis ……...… 37 References …………………………………………………………. 52 CHAPTER 2 – E-cadherin and Plakoglobin Mediate Sequential Steps in Suppression of Epithelial Cell Translocation ……………………...… 77 Abstract ……………………………………………………………. 77 Introduction ………………………………………………………... 78 Materials and Methods …………………….………………………. 80 Results ……………………………………………………………... 86 Discussion …………………………………………………………. 121 Acknowledgments …………………………………………………. 131 References …………………………………………………………. 131 CHAPTER 3 – Preliminary Analysis of Gene Expression Regulated By Plakoglobin …………………………………………………………. 138 Abstract ……………………………………………………………. 138 Introduction ………………………………………………………... 139 Materials and Methods …………………….………………………. 144 1 Results ……………………………………………………………... 146 Discussion …………………………………………………………. 149 Acknowledgments …………………………………………………. 157 References …………………………………………………………. 157 CHAPTER 4 – Summary of Findings and Future Directions ……………………………. 159 References …………………………………………………………. 172 2 LIST OF FIGURES FIGURE 1 – The multi-step process of metastasis …………………………………………. 10 FIGURE 2 – The four steps in translocation of a cell over a substratum …………………... 18 FIGURE 3 – Structural organization of a classical cadherin and its associated catenins …... 28 FIGURE 4 – Plakoglobin structure and binding partners ………………………………...… 42 FIGURE 5 – Role of plakoglobin and β-catenin in Wnt signaling cascade …………..……. 45 FIGURE 6 – Colony morphology and motile behavior of PAM212 keratinocytes ………… 87 FIGURE 7 – PAM212 variants grow as scattered colonies ………………………………… 91 FIGURE 8 – Expression of E-cadherin and catenins in parental PAM212 cells and in variants generated by repeated light trypsinization …………………... 94 FIGURE 9 – Re-expression of plakoglobin in the plakoglobin-deficient variants restores compact colony morphology …………………………………….…. 98 FIGURE 10 – Motile behavior of plakoglobin-deficient and plakoglobin- reconstituted variants …………………………………………………….... 101 FIGURE 11 – E-cadherin-deficient and plakoglobin-deficient variants differ in formation of initial contacts and in stability of pre-existing contacts ……... 104 FIGURE 12 – Initial contacts do not become stabilized in plakoglobin-deficient cells …..… 106 FIGURE 13 – Plakoglobin-deficient PAM39 cells form desmosomes …………..…………. 111 FIGURE 14 – Expression vectors encoding variants of plakoglobin, β-catenin, and TCF/LEF ……………………………………………………….……... 114 FIGURE 15 (TOP) – The adhesive activity of plakoglobin is not required for suppression of motility ……………………………………….…….. 116 3 FIGURE 15 (BOTTOM) – TCF/LEF signaling is not required for suppression of motility ………………………………………….…………. 116 FIGURE 16 – Two step model for suppression of translocation upon contact (STUC), showing initial role of E-cadherin and subsequent role of plakoglobin in this process ……………………………………………………….……. 122 FIGURE 17 – Laser scan of a gene chip, showing fluorescent signals for the arrayed elements ………………………………………………………….. 140 4 LIST OF TABLES TABLE 1 – Relative expression of E-cadherin and catenins in parental PAM212 cells and in variants generated by repeated light trypsinization ……………... 96 TABLE 2 – Relative adhesiveness of PAM212 parental, PAM39 and PAM41 plakoglobin- deficient, and PAM13 and PAM17 E-cadherin-deficient cells ……..………. 110 TABLE 3 – Example of differential gene expression results obtained from a gene chip, showing upregulated and