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Graduate Program in Biochemistry RHAMM PROMOTES NEOPLASTIC CONVERSION AND PROGRESSION THROUGH THE REGULATION OF ERK1,2 ACTIVITY AND AP-1 MEDIATED TRANSCRIPTION (Spine title: Rhamm Regulates ERK1,2 and AP-1 Mediated Transcription) (Thesis format: Integrated-Article) by Sara Rae Hamilton Graduate Program in Biochemistry A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Faculty of Graduate Studies The University of Western Ontario London, Ontario, Canada September, 2007 © Sara R. Hamilton 2007 Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-39274-4 Our file Notre reference ISBN: 978-0-494-39274-4 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Plntemet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. Canada THE UNIVERSITY OF WESTERN ONTARIO FACULTY OF GRADUATE STUDIES CERTIFICATE OF EXAMINATION Supervisor Examiners Dr. Eva A. Turley Dr. David Litchfield Supervisory Committee Dr. David Rodenhiser Dr. Eric Ball Dr. Lynne-Marie Postovit Dr. David Litchfield Dr. Linda Pilarski The thesis by Sara Rae Hamilton entitled: Rhamm Promotes Neoplastic Conversion and Progression Through the Regulation of ERK1,2 Activity and AP-1 Mediated Transcription is accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Date Chair of the Thesis Examination Board ii ABSTRACT Rhamm (Receptor for Hyaluronic Acid Mediated Motility) is a cell surface hyaluronan receptor and intracellular mitotic spindle protein with limited expression in normal tissues but high expression in aggressive tumours. Cell surface Rhamm regulates signaling by the Ras GTPase in cellular transformation and migration, while intracellular Rhamm regulates mitotic spindle stability and genomic instability. This thesis investigates the role of cell surface and intracellular Rhamm in tumourigenesis and identifies the signaling pathways necessary for Rhamm transformation. Analysis of motogenic signaling in aggressive breast cancer cells revealed a novel autocrine mechanism used to sustain their high basal rates of motility. This involved increased production of hyaluronan and increased surface display of CD44 and Rhamm. Cell surface Rhamm was identified as a coreceptor for CD44 and together they act coordinately to promote ERK (Extracellular Regulated Kinase) 1,2-regulated motogenic signaling. An oncogenic Rhamm isoform that is highly expressed in many human tumours was previously found to transform immortalized fibroblasts. In this thesis, the involvement of Ras-regulated pathways in Rhamm-mediated transformation was assessed. Rhamm actions upstream and downstream of Ras were collectively required for transformation. Intracellular Rhamm binds directly to ERK1 leading to increased activation and nuclear localization of ERK 1 and increased activation of AP (Activator Protein)-1-regulated transcription through RSK (Ribosomal S6 kinase) 1,2 phosphorylation and c-fos protein stabilization. Microarray analysis of Rhamm- transformed fibroblasts compared to parental fibroblasts revealed altered expression of iii genes involved in all stages of tumourigenesis, including genes involved in Rhamm's previously reported functions. However, the majority of genes with altered expression were involved in the regulation of self-sufficiency in growth and insensitivity to growth inhibition, suggesting that Rhamm's role in tumourigenesis is more complex than previously thought. This thesis provides the first evidence for a physical and functional association between Rhamm and CD44. Extracellular Rhamm may contribute to tumourigenesis by enhancing the latent rumour-promoting activities of CD44. These studies provide the first evidence for a role of intracellular Rhamm in activating Ras-regulated signaling in cellular transformation, and for Rhamm in regulating the expression of multiple genes involved in all stages of tumour progression. Understanding the complex role of Rhamm in tumourigenesis may lead to the development of better therapies for the treatment of cancer. KEYWORDS: Rhamm, CD44, Hyaluronan, Fibroblasts, Motility, Transformation, Microarray iv CO-AUTHORSHIP The following thesis contains material from a manuscript published previously in the Journal of Biological Chemistry (Chapter 2). Copyright permission is not required by this journal (see: https://www.jbc.org/misc/Copyright_Permission.shtml). This thesis also contains a manuscript submitted for publication in Molecular and Cellular Biology (Chapter 3). Chapter 2 has been previously published as "The hyaluronan receptors CD44 and Rhamm (CD 168) form complexes with ERK1,2, which sustain high basal motility in breast cancer cells" by S.R. Hamilton, S.F. Fard , F.F. Paiwand , C. Tolg , M. Veiseh, C. Wang, J.B. McCarthy, M.J. Bissell, J. Koropatnick and E.A. Turley in The Journal of Biological Chemistry. S.F. Fard, F.F. Paiwand and C. Tolg are co-second authors and contributed equally with each other in the research and preparation of this manuscript. The measurement of hyaluronan production by the breast cancer cell lines (Figure 2.1 A) was done by C. Wang. The migration data using the hyaluronan binding (HABP) peptide in Figure 2.IB, as well as the characterization of total cellular CD44 expression (Figure 2.2A), total cellular Rhamm expression (Figure 2.3), and total cellular ERK1,2 expression (Figure 2.9A) were done by F.F. Paiwand. The migration data in Figure 2.1C (in response to exogenous hyaluronan), in Figure 2.8 (effect of anti-CD44 and/or anti- Rhamm blocking antibodies), and in Figure 2.12B (effect of blocking MEK and Rhamm function) were done jointly by me and C. Tolg. The remaining experiments were done by me. I wrote the manuscript under the supervision of E.A. Turley with the help of S.F. Fard and C. Tolg. v Chapter 3 has been submitted for publication to Molecular and Cellular Biology as "Rhamm/HMMR Transforms Fibroblasts via ERK1 and AP-1 Mediated Transcription" by S.R. Hamilton, S. Zhang, C. Tolg, S. Crump, J.B. McCarthy, and E.A. Turley. The foci formation and growth data seen in Figures 3.4, 3.5A, and 3.1 IB, C were done by S. Zhang. The flow data showing cell surface Rhamm expression seen in Figure 3.3A was done by J. Bo. The foci formation and growth in soft agar data seen in Figures 3.2A, B and summarized in Figure 3.1, the kinase assays seen in Figures 3.10 and 3.11 A, and the in vitro binding data seen in Figure 3.7 and 3.8B were done by both S. Zhang and me. I performed the majority of the work and wrote the manuscript under the supervision of E.A Turley. vi Dedication Julie and Ted, "I get by with a little help from my friends." - John Lennon Thank You. "The woods are lovely dark and deep, but I have promises to keep, and miles to go before I sleep, and miles to go before I sleep." - Robert Frost vii ACKNOWLEDGEMENTS I would first like to express my sincerest gratitude to my thesis supervisor and mentor, Dr. Eva Turley for your unwavering encouragement and guidance along the way. You have taught me to be an independent, critical and creative thinker, as well as a leader and communicator. These are skills that will help me in the years to come. All of this would not have been possible without my friends and lab mates: Dr. Cornelia Tolg, Jenny Ma, Sarah Crump, Max Qi, Anu Bhalla, and Beatrice Kowalska. Conny, I will always value your insightful comments and our many discussions. Life in the Turley lab would not have been the same without you. I would also like to thank Dr. Shiwen Zhang and Frouz Paiwand as your work was instrumental to my own. I would like to acknowledge members of my advisory committee, Drs. David Litchfield and Eric Ball, for their valuable feedback, direction and support. To Drs. Joe Mymryk, Tracey Jason, and Karen Morley, I cannot thank you enough for your critical review of this thesis. I would like to thank all of my friends and family who have supported me throughout this journey. To my Mom for encouraging me to find my own path and for teaching me that we have no limits. To my sisters, Kelley and Elizabeth and my brothers, Chris and Jonny, for all of the love, support and encouragement they have given me over the years. Nobody knows me better! For the opportunities that they have provided me, I would also like to thank my Grandparents, Glenna and John.
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