The role of the relaxin receptor RXFP1 in brain cancer By Usakorn Kunanuvat A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfilment for the requirements of the degree of MASTER OF SCIENCE Department of Human Anatomy and Cell Science University of Manitoba Winnipeg Copyright © 2012 by Usakorn Kunanuvat ABSTRACT Relaxin (RLN2) promotes cell migration/invasion, cell growth, and neoangiogenesis through binding to the relaxin receptor RXFP1 in many types of cancers. However, there have been no studies to determine the role of this system in brain tumors, especially in Glioblastoma Multiforme (GB), the most lethal primary brain tumor in adults. GB is a systemic brain disease and aggressively invades brain tissue. In this study, we have identified RXFP1 receptor, but not RLN2, in GB cell lines and primary GB cells from patients. RLN2 treatment resulted in a significant increase in migration of GB cell line and primary GB cells. To determine molecular mechanisms that facilitate RXFP1-mediated migration in GB cells, we employed a pseudopodia assay and 2D LC-MS/MS to investigate the protein composition at cell protrusions (pseudopodia) during GB cell migration. We also observed the expression of known mediators promoting tissue invasion upon RLN2 treatment. We identified PGRMC1, a candidate protein from 2D LC-MS/MS as a novel relaxin target protein in RXFP1-expressing brain tumor cells. RLN2 treatment also caused an increase in cathepsin (cath)-B and -L and enhanced production of as the small Rho-GTPases Rac1 and Cdc42 in GB cells. Collectively, these findings indicate that RXFP1-induced cell migration is mediated by the upregulation and intracellular actions of Rac1, Cdc42 and by cath-B and cath–L who serve as matrix modulating factors to facilitate brain tumor cells migration. PGRMC1 also contributes to RXFP1- mediated cell migration through an as yet unknown mechanism. RLN2 is not present in the brain. We determined the role of a peptide ligand of RXFP1, the newly discovered C1q/TNF related peptide (CTRP)8-derived P74 peptide, in promoting migration in GB cells. Similar to relaxin, P74 was found to have pro-migratory effects on GB cells. The biological activity of this peptide was also similar to relaxin and caused the upregulation of cath-B, cath-D and cath-L in the primary GB cells, thus, indicating that P74 might serve as a novel RXFP1 activating peptide ligand. We conclude that RXFP1 receptor signaling plays a key role in brain tumors cell migration and invasion. i ACKNOWLEDGEMENTS In the first place I am heartily thankful to my supervisor, Dr. Thomas Klonisch for bringing me to Winnipeg and giving me a great opportunity to work on my Master’s Degree. You are just a great supervisor. Your supervision, advice, and guidance from the very early stage of this research gave me extraordinary experiences throughout the work. I have encountered many obstacles performing experiments as is true for every student. Fortunately, you provided me with unflinching encouragement and support in various ways. Sometimes, things did not turn out exactly the way I intended to and I felt depressed and downhearted. You always encouraged me with our lab’s motto “Never ever give up”. That made me converts the negative energies of my struggles into building a solid foundation and continue research. Thank you for continuously supporting me and pushing me to my limits to complete my Master’s studies, and for all the patience and great support you gave me since the beginning. I would like to express my gratitude to Dr. Sabine Hombach-Klonisch. I am much indebted to you for your valuable advice in science discussions, supervision in the lab, using your precious time to help me to overcome problems and challenges I faced many times during my lab experiments. Without your help I would not be this far. Thank you for believing in me, helping me to believe in myself and encouraging me to pursue excellence. I would like to acknowledge the advice and guidance of my committee members, Dr. John A. Wilkins and Dr. Rajinder Bhullar. Thank you for using your precious time reading this thesis and for your valuable and critical comments that were essential in further improving my work. I thank you both for providing the necessary infrastructure, resources and expertise to accomplish parts of my research work. You have been always very generous to me. ii I express my sincere thanks to my lab members; Aleksandra, Adrian, Suchitra and Manoj. I had the fortune of working at this exciting lab. While we are multicultural, we get along together very well and are friends. You guys were fabulous. I had a great time and learned a great deal by working together with you. Thank you for being good friends who are always so supportive and encouraging. I do appreciate your help in everything. Special thanks go to Dana Henderson and Neha Khanna for taking time to check my English in my thesis and polish my thesis until it was in the stage that it was to my satisfaction. I extend my warm thanks to Peyman Ezzati who always kindly granted me his time, helped me and answered the many questions on Proteomics and the analysis of my data. I would also like to extend a huge and warm thank you to all Thai students in Winnipeg. All of you make me feel that I am not alone while I was far away from home. You guys have made a mini Thai community where we helped and supported each other in every way despite that we knew each other only when we came here. Special thanks to Ruedee and family, Narison and Thatchawan who always helped and supported me and stayed at my side no matter what. I felt so lucky to know each of you. Thank you for being “big sisters” to me. Likewise, my appreciation goes to my friends in Thailand who always encouraged me when I felt bad and laughed with me on the days of my successes. Most importantly, you had the faith in me that I will overcome all the challenges. I will forever be thankful to my former supervisors, Dr.Tewin Tencomnao and Dr. Wanida Laiwatttanapaisal. You have been helpful in providing advice many times during my undergraduate school and also when I was here. Thank you both for giving me the opportunity to get to know Dr. Klonisch and his team which then paves the way of my life to the success. iii Last but not least, my deepest gratitude goes to my family for their unconditional love and support throughout my life; this thesis would have been impossible without the love and patience of my family. This thesis is dedicated to all members of my family who have been a constant source of love, support and strength for all these years. To Mom, I have no suitable word that can fully describe your everlasting love to me. Thank you for a long distance calls every day and every time I was facing problems. You are always by my side even though we are a thousand miles apart. To Dad, thank you for your unequivocal support and everlasting love for me. To both of you, thank you both for your warm and constant support for giving me strength and confidence during my work. To my sister, Saranrat and my brother, Dhanachart , thank you for taking good care of Mom and Dad while I was here and thank you for your love and support in everything that I do. To Grandpa, this work is dedicated to you. I promised you that I will fight to get my degree from North America. Notwithstanding, you are not with me anymore but I do believe that you know I did it. To Grandma who lovingly supports me, regardless of where in the world I am. To my aunt, thank you for so many encouraging emails that always make me feel positive every time I read them. Your optimistic thinking always makes me feel that tomorrow everything will be better when things go wrong no matter what I do. Finally, I would like to thank everybody who has helped me directly or indirectly and so was important to the successful realization of this thesis, as well as expressing my apologies that I could not mention personally one by one. Usakorn Kunanuvat iv CONTENTS LIST OF ABBREVIATIONS ……………………………………………………………………...vii LIST OF TABLES……………………………………………………………………..…………….xi LIST OF FIGURES………………………………………………………..…………….……..…...xii LIST OF COPYRIGHTED MATERIAL………………………………………………………….xiv CHAPTER 1 INTRODUCTION…………………………..……………………….….………….…..1 1.1 The relaxin family of peptides…………………………………..……………………..….1 1.1.1 The discovery of relaxin…………………………………….….………..….2 1.2 Relaxin family peptide receptors (RXFPs)…………..………………..………..................4 1.2.1 Relaxin family peptide receptor1 (RXFP1)……………………………………..6 1.3 Function of Relaxin-RXFP1 system……………………………...……………………...13 1.3.1 Physiological functions………………………………………………..……….13 1.3.2 Disease related functions…………………………………………....................15 1.4 P74 peptide and Complement C1q tumor necrosis factor (TNF) related protein 8 (CTRPs) (C1q/TNF8 or CTRP8)…………………………………………………………24 1.4.1 P74 peptide is a short, linear peptide derived from the collagen domain Of CTRP8………………………………………………………….………...…24 1.4.2 CTRPs family and CTRP8………………………..……………..……..............26 1.5 Brain cancer………………………………..………………………………..…………...28 1.6 Rational for this study……………………………………………………………………33 1.7 Hypothesis and objectives………………….……..………………………………….…..33 1.7.1 Hypothesis…………………………………………………………………..….33 1.7.2 Objectives……………………………..……………………………….............34 v CHAPTER 2 MATERIALS AND METHODS…………………………………………….……….35 CHAPTER 3 RESULTS AND FIGURES….……………………………………………..……….50 3.1 RXFP1 is expressed in human primary brain tumor cells and GB cell lines……………50 3.2 Recombinant human RLN2