RHAMM, CD44 EXPRESSION AND ERK ACTIVATION ARE LINKED IN MALIGNANT HUMAN BREAST CANCER CELLS AND ARE ASSOCIATED WlTH CELL MOTlLlTY Frouz Frozan Paiwand A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Laboratory Medicine and Pathobiology University of Toronto O Copyright by Frouz Frozan Paiwand 1999 National Library Bibliothèque nationale 1*1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Sewîes senrices bibliographiques 395 Wellington Street 395. rua Ws(lingt0ri OaawaON K1AW OFtswaON K1AONI canada CaMde The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de rnicrofiche/nlm, de reproduction sur papier ou sur format électronique. The author retains owaership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. I dedicate this Master's thesis to my rnother, who is the source of my inspiration, and to rny family for their never-ending love and support. RHAMM, CD44 Expression and ERK Activation are Linked in Human Breast Cancer Cells and are Associated with Cell Motility Frouz Frozan Paiwand Master of Science, 1999 Department of Laboratory Medicine and Pathobiology University of Toronto ABSTRACT We assessed CD44 RHAMM,erk, and ras expression in human breast cancer ce11 lines that Vary as xenografts in nude mice. Compared to MCF-7 cells, MDA-MB-231 cells expressed higher levels of CD44, RHAMM,erk, and ras, showed higher levels of ce11 surface RHAMM, and displayed greater motility that was reduced by anti-RHAMM or anti-CD44 antibodies, or by a MEK inhibitor. These inhibitors also reduced the motility of mutant active ras-transfected MCF-IOA cells which, relative to wild-type ras or empty vector transfected cells, were more motile and had increased RHAMM, CD44 and activated erk expression. MDA-MB-23 1 cells and mutant ras-transfected MCF-1OA cells demonstrated nuclear CO-localizationof RHAMM and erk, whereas RHAMM and CD44 CO-localizedto perinuclear regions or to ce11 processes. Co-immunoprecipitation of RHAMM with both CD44 and erk was also observed. These resuIts suggest that RHAMM and CD44 may coordinate signaling through the ras-MAP kinase pathway to control ce11 motility. iii This thesis could not have been completed without the help and support of numerous coworkers, colleagues, and friends. 1 am most thanldùl to my supervisor and mentor, Dr. Eva Turley, who not only provided me with the material resources, technical training, and scientific guidance needed to undertake the studies in which 1 participated, but who was also a source of encouragement far me to continuously srrive for excellence. 1 am also indebted to al1 the members of Dr. Turley's research group, past and present. My thanks extend especially to Lisa CoUis, Rene Hamson, JingBo A, Shwin Zhang and Judy Edwards - with whorn I shared formative experiences as a graduate student - for their advice and support on issues both scientific and personal. For their friendship, stimulating discussions, and knowledge, L would also Iike to thank ail the other students, postdoctoral fellows, and associates who are the present members of the Iaboratory. 1 would like to express my sincere thanks to the members of my thesis advisory cornmittee - Drs. Marlene Rabinovitch and Gabrielle Boulianne - for their invaluable guidance on the direction of my research project and maintainhg the standard of the M.Sc. degree at the University of Toronto. As well, 1 thank the other members of my original examination committee - Dr. Fred Keeley and Dr. Howard Lipshitz - for their advice in regard to my previous research project. To my mother and sister Leeza, and to al1 the members of my farnily, 1 owe gratitude for their continuous patience and support throughout the years of my univeaity education. It is to them that 1dedicate this thesis. Foremost among my fiiends deserving acknowledgement is Michael Levesque, whose daily encouragement and advice nourished me in mcuIt times and inspired me to wnte this thesis in order to progress frorn Muate studies to the educational. professional, and personal oppoctunities that Lie beyond. Finally, 1would like to acknowledge the financial support of Hyal Pharrnaceutical Corporation, Mississauga, Ontario, Canada. The studies described in this thesis were performed at the Department of Cardiovascular Research at The Hospital for Sick Children, under the aegis of the Graduate Department of Laboratory Medicine and Pathobiology at the University of Toronto, during the years 1997- 1999. DEDICATION .................................. .., ...................................... ABSTRACT .............................0................................................ ACKNOWLEDGEMENTS ............................................................. TABLE OF CONTENTS ................................................................ LIST OF TABLES ........................................................................ LIST OF FIGURES ................................. -... .............................. LIST OF ABBREVIATIONS .......................................................... CEIAPTER 1. INTRODUCTION ................................. .... .......................... 1. 1 . Extracellular Matrix and Tumorigenesis ............................. 1. 1.1. Tumorigenesis and Metastasis ..................... .... .. 1. 1.2. HA and Tumorigenesis ....................................... 1.2. The HA Binding Receptors: RHAMM and CD44 .................... 1.2.3. Domains of CD44 Related to Ce11 Motility and Celi Cycle Control ............................................. 1.2.5. RHAMM ........................................................ 1. 2 . 6 . RHAMM Isoforms ................... ..,. ........ .......... 1.2.7. Domains of RHAMM Related to Ce11 Motility and Ce11 Cycle Control ......................................... 1.3. Tumor Progression is Associated with Elevated Expression of CD44 and RHAMM ....................... .,. ...... .............. 1.4. Hypothesis. Rationale. and Objectives of this Smdy ................. 23 I. MATERIALS AND METKODS .......................................... 25 II .1 . Cell Culture ......................... ,., .....~.O........................26 II .2 . Antibodies ..................... ,. .... ... ..~........................... 27 II .3 . Western Irnmunoblotting and Immunopr~cipitation ................ 28 II .4 . Time-lapse Cinemicrography ......... .. ..... .......................... 30 II. 5. Flow Cytometry (FACS) .. - .. - .- - - - .. .. .. .. II. 6. Immunofluorescence Staining .. - .. - .. - - .. .-. .. .. .- - III. RESULTS .......................................................................... III. 1. Antibodies Specifically Detect RHAMM .. .. - - - .. .. LII. 2. CeU Surface RHAMM Expression Varies with Time on the Surface of Human Breast Cancer Cells .. .. .. -. III. 3. Breast Cancer CeUs Express Several RHAMM bofonns .. ... .... III. 4. RHAMM Overexpression is Associated with the Presence of Mutant Active ras .. .. .. ... .. -. - - -.-. .... -... .. .- -. .-.. III. 5. RIIAMM Overexpression in MDA-MB-231, MCF-7,or MCF- 1OA CelIs Correlates with the Overexpression of ras, Presence of Active erk, and with High LRvels of CD44 .. .. .. .. ... .. III. 6. RHAMM Co-distributes with erk and CD44 in Breast Cancer CeIls .. .. .. .- - - .. -. -. -.. III. 7. RHAMM Co-irnmunoprecipitates with erk and CD44 in MDA-MB-23 1, MCF-7,and in ras-transfected Breast Epithelial Cells .. - .. -. .. .. - .- - - -..-. -. - .. - .- - - .. m. 8. RHAMM and CD44 are Required for the Locomotion of MDA-MB-23 Ceils and Ceils Transfected with Mutant Active ras .. -. - .. -. - .-. III. 9. Figures of Chapter III .. .. ....... .-. -. - N. DISCUSSION .- .. - .. - .- .. - .. - -.- - - - - .- - - .- .. N.1. Ras and raderk Signaling Cascade in Breast Cancer Development .. .. .. .... .. ....... .. .. .. 69 N.2. CD44 and RHAMM, Co-receptors that Mediate Tumor Ce11 Motility through the ras/MAP Kinase Pathway ... .. .. .. ... 73 W.3. RHAMM and CD44 Expression are Linked to ras Overexpression and erk Activation in Breast Cancer Cells and Breast Epithelial Cells Transfected with Mutant Active ras .. .. .. .. .. .. - .. .. .. .. 80 W.4. RHAMM CO-associateswith erk in MDA-MB-23 1 Cells and MCF-1OA Cells Transfected with Mutant Active ras .. 8 1 IV. 5. RHAMM Co-associates with CD44 in Breast Cancer Cells and Breast Epitheliai CeiIs Transfected with Mutant Active ras .. .. 83 IV. 6. A Mode1 of HA and its Receptoa in raslerk Signaling and Breast Cancer Development .. 84 IV. 7. Future Studies .. ... .. - .. .. .. 88 vii V. REF'ERENCES . .. .. .. .. .. .. .. .. 91 VI. APPENDLX ...... ..*.....**.. ..-....... .-......,...... .. .. 11 1 The DrosophUn RHAMM Homologue LIST OF TABLES CHAPTER 1. Table 1.1. HA Binding Pro teins .. - .. - - - .. .. 8 CaAPTER IV. Table N. 1. Characteristics of Human Breast Epithelial Cell Lines .. ... 76 LIST OF FIGURES Fig. 1.1. The Basic Repeating Disaccharide Sequence in HA