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MOLECULAR CHARACTERIZATION OF ANIMAL MODELS OF PHEOCHROMOCYTOMA A Dissertation submitted to the Faculty of the Graduate School of Arts and Sciences of Georgetown University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Tumor Biology By Edwin W. Lai, B.S. Washington D.C. April 15, 2009 Copyright 2009 by Edwin W. Lai All Rights Reserved MOLECULAR CHARACTERIZATION OF ANIMAL MODELS OF PHEOCHROMOCYTOMA Edwin W. Lai, B.S. Thesis Advisors: Karel Pacak, M.D., Ph.D., D.Sc. and Chris Albanese, Ph.D. ABSTRACT Pheochromocytoma remains one of the most misunderstood and under-diagnosed human cancers. Tumors primarily arise from chromaffin cells of the adrenal medulla, but also from extra-adrenal sympathetic ganglia termed “extra-adrenal paraganglioma.” Despite advances in diagnosis and localization of pheochromocytoma, the majority of patients with pheochromocytoma have no known genetic background and the etiology of these tumors is unknown. Another difficulty is that there are currently no biochemical or molecular markers to predict metastatic disease. Metastatic disease is only characterized by localization of tumors where chromaffin cells are normally absent. Significant emphasis has recently been placed on developing and testing novel experimental therapeutic options, especially targeted therapy which exploit specific biochemical properties of this tumor. However, unlike other diseases, few biologically relevant animal models have been developed and no human pheochromocytoma cell lines have been successfully established. Therefore, the paucity of effective and accurate preclinical models remains an obstacle. iii Enhanced or dys-regulated signaling by the receptor tyrosine kinase ErbB-2 (HER2/Neu) has been associated with a wide variety of diseases. In our studies, we implicated ErbB-2 expression may be linked to pheochromocytoma and enhanced ErbB-2 signaling may play an important role in the pathogenesis of this tumor. We also identify prognostic markers such as positive nuclear cyclin D1 staining or loss of 4E-BP1, and associate signal pathway targets for small molecule inhibition, such as mTOR. In vitro studies for pheochromocytoma are often performed in the mouse pheochromocytoma cell line. Our group established in vivo animal models generated by subcutaneous or tail vein injecting mouse pheochomocytoma cells. These models are useful, especially for studying metastatic pheochromocytoma. Microarray analysis of liver metastases and subcutaneous tumors revealed differential gene expression. In conjunction with human microarray, potential target genes have been identified. Increased expression of interleukin (IL)-13Ralpha2 in pheochromocytoma was identified through human and mouse microarray analysis as a potential cell surface receptor target for immunotoxins. To target pheochromocytoma cells overexpressing IL-13Ralpha2, an immunotoxin consisting of IL-13 and truncated Pseudomonas exotoxin A (IL-13PE) was tested in vitro and in vivo. IL-13PE directed therapy provides a unique opportunity for cancer therapy. iv ACKNOWLEDGEMENTS I would like to acknowledge my mentors Karel Pacak and Chris Albanese for their generous support and guidance in my research. Karel, I am grateful for your kindness and for seeing potential in me. You have given me the opportunities that have led me to where I am. I have learned many valuable lessons, which I will use throughout my career. To Chip, you have always been available for advice and support and have been willing to lead me through my Ph.D and beyond. Your enthusiasm to include pheochromocytoma to your interests is a shared success. I would not have been able to navigate through this system without your efforts. This thesis is the result of the combined effort of many people, and would not have been possible without their help. I would like to thank all the members of my thesis committee, Anna Riegel, Susette Mueller, Leena Hilakivi-Clarke, and Graeme Eisenhofer for their advice and time in the preparation for this thesis. Thank you for your critical review and direction to make this happen. In particular, to Graeme who has taught me biochemical analysis by HPLC and is truly another mentor to me. I have to thank all our collaborators who, on many occasions directed and taught me, several of whom are listed as co-authors on publications. In particular, Abdel Elkahloun, Alan L.Y. Pang, Art Tischler, Bob A. Wesley, Irina A. Lubensky, John C. Morris, and Mones Abu-Asab were always available to answer my questions. I also have to recognize Bharat H. Joshi and Raj K. Puri and the members of the Center for Biologics v Evaluation and Research of the Food and Drug Administration who have been a tremendous help for the IL-13PE study. To the members of the Program in Reproductive Biology and Endocrinology at the National Institutes of Health (NIH), including Lucia Martiniova, Thanh-Truc Huynh, Stephanie Fliedner, Bas Havekes, Shiromi Perera, Shoichiro Ohta, Kyle Horak as well as the members of the Albanese lab, especially Olga Rodriguez, Sarada Vassapragata, Patricia Salinas, and Paul Siarajuddin who all deserve my gratitude and whom without their efforts this research would not be possible. They also make coming in early to the lab or staying late worthwhile. I also have to thank my classmates in the NIH Graduate Partnerships Program who have helped me through my coursework and who helped me make it through, Kelly Jean Thomas, Kate Callahan, and Jason Warfel. Also, thanks to Alison McBride and Mark Cookson the other NIH-Georgetown students for their support and encouragement. This research benefits the patients who generously provided clinical samples and are an inspiration to us. I hope this body of work repays them. There was also substantial support from our clinical staff that sustains clinical research and often goes unrecognized, Karen T. Adams and the many nurses, technicians, and physicians of the NIH Clinical Center. Finally, I would like to thank my family and friends from California, Michigan, Maryland and everywhere in between for their continual guidance, prayer, and support – they make this journey meaningful and worthwhile. vi TABLE OF CONTENTS ABSTRACT.....................................................................................................................III ACKNOWLEDGEMENTS ............................................................................................ V TABLE OF CONTENTS ............................................................................................. VII LIST OF FIGURES ........................................................................................................XI LIST OF TABLES .......................................................................................................XIII LIST OF ABBREVIATIONS .....................................................................................XIV 1. INTRODUCTION...................................................................................................... 1 1.1 PHEOCHROMOCYTOMA: CLINICAL PRESENTATION, DIAGNOSIS AND MANAGEMENT 2 1.1.1 Clinical presentation ........................................................................................ 2 1.1.2 Diagnosis and management ............................................................................. 3 1.2 CURRENT RESEARCH OBJECTIVES IN PHEOCHROMOCYTOMA ................................... 7 1.2.1 To determine perturbations in the downstream PI3K/PTEN/mTOR signaling during the development of pheochromocytoma .......................................................... 7 1.2.2 To determine expression and utilization of IL-13receptor in mouse models of pheochromocytoma for IL-13 immunotoxin targeted therapy .................................... 8 1.3 OUTLINE OF THESIS.................................................................................................. 9 2. TECHNIQUES AND METHODS APPLIED IN THIS THESIS ........................ 11 2.1 ANIMAL MODELS OF TUMOROGENESIS IN PHEOCHROMOCYTOMA .......................... 12 vii 2.1.1 Cell cultures ................................................................................................... 12 2.1.2 Cell viability assay ......................................................................................... 12 2.1.3 Transgenic and xenograph animal models .................................................... 13 2.2 SMALL ANIMAL IMAGING....................................................................................... 14 2.2.1 Micro computed tomography ......................................................................... 14 2.2.2 Magnetic resonance imaging ......................................................................... 15 2.2.3 Small animal positron emission tomography ................................................. 15 2.3 MOLECULAR BIOLOGY AND BIOCHEMISTRY........................................................... 16 2.3.1 SuperArray ..................................................................................................... 16 2.3.2 Reverse transcriptase-polymerase chain reaction and quantitative real-time polymerase chain reaction........................................................................................ 17 2.3.3 Immunohistochemistry, Nuance and spectral semi-quantitative analysis ..... 19 2.3.4 Western blot.................................................................................................... 20 2.3.5 High pressure liquid chromatography ..........................................................