University of Central Florida STARS Electronic Theses and Dissertations, 2004-2019 2008 Role Of Transient Receptor Potential Canonical-6 (trpc6) Channel In Metastasis Of Glioblastoma Multiforme Rajarajeshwari Venkataraman University of Central Florida Part of the Cancer Biology Commons, Microbiology Commons, Molecular Biology Commons, and the Oncology Commons Find similar works at: https://stars.library.ucf.edu/etd University of Central Florida Libraries http://library.ucf.edu This Masters Thesis (Open Access) is brought to you for free and open access by STARS. It has been accepted for inclusion in Electronic Theses and Dissertations, 2004-2019 by an authorized administrator of STARS. For more information, please contact [email protected]. STARS Citation Venkataraman, Rajarajeshwari, "Role Of Transient Receptor Potential Canonical-6 (trpc6) Channel In Metastasis Of Glioblastoma Multiforme" (2008). Electronic Theses and Dissertations, 2004-2019. 3714. https://stars.library.ucf.edu/etd/3714 ROLE OF TRANSIENT RECEPTOR POTENTIAL CANONICAL-6 (TrpC6) CHANNEL IN METASTASIS OF GLIOBLASTOMA MULTIFORME By RAJARAJESHWARI VENKATARAMAN B.Sc. Mysore University, India, 1996 M.Sc. Maniple Academy of Higher Education, India, 1999 A thesis submitted in partial fulfillment of the requirements For the degree of Master of Science In the Department of Molecular Biology and Microbiology In the Brunet School of Biomedical sciences In the College of Medicine At the University of Central Florida Orlando, Florida Fall Term 2008 ABSTRACT Glioblastoma multiforme (GBM) is one of the extremely fatal brain tumors. The main reason that makes it so lethal is its capability to invade and spread to other parts of CNS producing secondary tumors. Among other factors hypoxia, reduced oxygen availability, is linked to higher metastatic potential of cancers. Hypoxia causes numerous changes in genome and proteome of the cell. These changes help a normal cell to adapt to nutritional deficiency, but the same changes can increase the malignancy and metastasis in tumor cells. Extensive research by a number of curious scientists reveal that various pathways involving numerous proteins cross-talk and interact with each other and execute a response to hypoxia. We are trying to establish the link between two such pathways – HIF1-alpha pathway and Notch pathway. Both, HIF1-alpha, which is a transcription factor that becomes active in hypoxic conditions and Notch, which is an evolutionarily conserved cell-fate determinant, are implicated in hypoxia-induced metastasis of cancer. In this given project, we confirm the cross talk between Notch and HIF1-alpha pathway and further continue our study to show that TrpC6 is the downstream mediator of this pathway, leading to metastasis of GBM. Expression analysis of hypoxia-induced U373 cells (Grade 3 glioblastoma cells), using Real-time PCR, western blot and immunocytochemistry, revealed elevated levels of Notch, Hif1 and TrpC6 indicating that these proteins might be important for the cellular response to hypoxia. Blocking Notch and/or HIF1-alpha, either by DAPT or HIF1- inhibitor, confirmed the communication between these two pathways. Role of TrpC6 in ii metastasis was demonstrated by knocking down this gene using siRNA against TrpC6. Inhibition of TrpC6 markedly decreased cell proliferation, migration, angiogenesis and tumorigenesis in these hypoxia-induced Glioblastoma cells. In summary, all these results reveal that TrpC6 is indeed an important member of the Notch-mediated metastasis of Glioblastoma under hypoxic conditions. This role of TrpC6 can therefore be utilized for pharmacological intervention to prevent hypoxia-induced metastasis in GBM. iii Dedicated to my family and friends Your support, confidence and love carried me iv ACKNOWLEDGMENTS I would like to thank God Almighty for allowing me the privilege and opportunity to obtain higher education. I thank Dr. Kiminobu Sugaya, for giving me the opportunity to conduct research in his lab. His commitment to training students, dedication to biomedical research and high standards has guided me throughout my graduate career. I would also like to thank my committee members, Dr. Sic.L.Chan, Dr. Steven Ebert and Dr. Dinender Singla, for their assistance in completion of my thesis and encouragement. Special thanks go to my spouse Sathyashankar Somasekar for his patience and understanding. I would like to recognize Dr. Srinivas for assistance and guidance with the project. Sincere thanks goes to all my lab sisters and brothers, especially Stephanie, Serene, Rasha and Manny. To my children Shankari and Vignesh, I thank you for providing me with unconditional love and motivation. Finally I would like to thank my Parents and my family members, you have made it possible. I love you all. v TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................................... viii LIST OF TABLES ......................................................................................................................... ix LIST OF ABBREVIATIONS ......................................................................................................... x CHAPTER 1: INTRODUCTION ................................................................................................... 1 1.1 Glioblastoma Multiforme – Background and pathophysiology............................................ 5 1.2. Signs and Symptoms of the disease ..................................................................................... 6 1.3. Treatment ............................................................................................................................. 6 1.4 Molecular Genetics of GBM ................................................................................................. 7 1.5 Metastasis .............................................................................................................................. 9 1.6 Hypoxia and Metastasis ...................................................................................................... 11 1.7 Research in our lab.............................................................................................................. 12 1.8 Hypoxia Induced Factor 1 ................................................................................................... 12 1.8.1 HIF-1 – Structure and Function ................................................................................... 12 1.8.1A Structure of HIF1: ................................................................................................. 12 1.8.1B Regulation of alpha subunit: ................................................................................. 13 1.8.1C Function of HIF1: .................................................................................................. 14 1.8.2 Role of HIF-1 in Cancer .............................................................................................. 14 1.9 Notch ................................................................................................................................... 15 1.9.1. Structure and Function ................................................................................................ 15 1.9.1A Structure of notch protein: .................................................................................... 15 1.9.1B Processing of notch protein: .................................................................................. 16 1.9.1C Functions of Notch Protein: .................................................................................. 16 1.9.2. Role of Notch in Cancer: ............................................................................................ 17 1.10 TrpC6 ................................................................................................................................ 20 1.10.1 Structure and function of TrpC6 channels ................................................................. 20 1.10.1A Structure of TrpC6: ............................................................................................. 20 1.10.1B Function of TrpC6: .............................................................................................. 21 1.10.2 Role of TrpC6 in Cancer ............................................................................................ 21 1.11 Hypothesis......................................................................................................................... 22 1.12 Aims of the project............................................................................................................ 23 1.13 Experimental design.......................................................................................................... 23 1.13.1 Aim 1: Expression of Notch, TrpC6 and Hif-1 increases during hypoxia ................ 23 1.13.2 Aim 2: HIF1 and Notch pathways crosstalk under hypoxia ...................................... 24 1.13.3 Aim 3: TrpC6 acts as the downstream effector of Notch and HIF1 and is responsible for the effect of these proteins on metastasis of Glioblastoma. ............................................ 25 CHAPTER 2: MATERIALS AND METHODS .......................................................................... 26 2.1 Cell Culture – .................................................................................................................. 26 2.2 Inducing Hypoxia –........................................................................................................