A Dissertation Submitted To

The role and function of the Ras-related protein TC21 in Neurofibromatosis type 1 A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirement for the degree of Doctor of Philosophy in the Department of Cancer and Cell Biology of the College of Medicine by Deanna M Patmore BS, Voorhees College June 2007 Dissertation Committee: Nancy Ratner, PhD (Chair) Vaughn Cleghon, PhD George Thomas, PhD Susanne Wells, PhD Yi Zheng, PhD ABSTRACT Neurofibromatosis type 1 is a common autosomal dominant disorder affecting 1in 3500 individuals worldwide. Neurofibromin, the protein mutated in NF1 disease, is a GTPase activating protein (GAP) for Ras proteins, inactivating the Ras proteins H-Ras, N-Ras, K-Ras, M-Ras, R-Ras, and TC21. Missense mutations in the GAP related domain of neurofibromin cause NF1 disease, indicating that increased Ras activity is likely critical for disease pathogenesis. Loss of NF1 in Schwann cells causes formation of benign tumors known as neurofibromas. These tumors can become malignant forming malignant peripheral nerve sheath tumors (MPNSTs), which are a major source of morbidity for NF1 patients. TC21 is a member of the R-Ras family of small Ras GTPases. TC21 is an oncogene able to transform epithelial and fibroblast cell lines, and it is also capable of inducing tumors in vivo . PI3K is the predominant effector of TC21 transforming activity, and studies support the idea that not all features of NF1 mutant cells can be ascribed to the activation of the classical Ras proteins (e.g. H-, N-, K-Ras). We hypothesize that the effects of neurofibromin mutation that are unrelated to classical Ras-GTP may be explained by activation of the non-classical Ras protein TC21. The signaling pathway of TC21 is unclear. We activated all Ras proteins in vivo by deletion of Nf1 and using these mice along with mice deficient for TC21 -/-, we examined the role and function of TC21 in development and tumorigenesis. Additionally, we used NF1 -/- human MPNST cell lines with an acute loss of TC21 by shRNA to examine tumor growth in xenograft mice. We found that TC21 loss delayed benign neurofibroma formation in Nf1fl/fl;DhhCre mice. Nf1 loss increased mRNA encoding the cytokine transforming growth factor-beta (TGF-beta) and rendered Schwann cell progenitors i insensitive to TGF-beta; these phenotypes could be rescued by TC21 and were mediated through TGF-beta receptors. Conversely, growth of Nf1;Trp53 brain tumors and NF1 -/- MPNST sarcomas were accelerated by TC21 loss. MPNST from Nf1;Trp53 mice and NF1 -/- MPNST xenografts had increased levels of TGF-beta mRNA and protein and blocking TGF-beta decreased sarcoma size induced by shTC21. The results are important because TGF-beta acts as a tumor suppressor in numerous types of benign tumors and is also known for its oncogenic role in cellular transformation and tumor progression. Our data elucidates TGF-beta as a therapeutic target in NF1 malignancy. ii iii ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my mentor, Dr. Nancy Ratner, for her continuous support during the past five years of my graduate studies. Dr. Ratner’s leadership and guidance has significantly helped in molding me as a scientist. Her mentorship allowed me to equip myself with the scientific knowledge and skills necessary to establish a life-long career in basic research. During my graduate studies, I have developed a keen appreciation for cancer research and particularly pediatric cancer research. Additionally, I would like to thank my thesis committee members, Dr. Vaughn Cleghon, Dr. George Thomas, Dr. Susanne Wells and Dr. Yi Zheng for their insightful ideas, comments and advice throughout the development of my thesis project, as well as their significant contribution to my scientific training. I would also like to acknowledge and thanks the present and former members of the Ratner lab for their technical guidance and support, as well as their valued time spent in discussion of data and analytical interpretation. Last but not least, I would like to give my deepest gratitude to my family and friends who have always believed in and supported me. Without you these past five years would have been impossible. I dedicate this thesis study to my mother, Mrs. iv Yvette Lemonius. Your unconditional love and support, your determination and faith have made me the person I am today. I love you always. v TABLE OF CONTENTS ABSTRACT……………………………………………………………….……………..i ACKNOWLEDGEMENTS.......................................................................................iv TABLE OF CONTENTS .........................................................................................vi LIST OF FIGURES .................................................................................................viii LIST OF TABLES....................................................................................................x CHAPTER 1: INTRODUCTION OVERVIEW……………………………………………………………………….1 NEUROFIBROMATOSIS TYPE 1: THE DISEASE………………………….2 NF1 MOUSE MODELS………………………………………………………….5 THE NF1 GENE……………………………………………………………..…....6 THE RAS SUPER-FAMILY PROTEINS……………...……………………......8 THE RAS-RELATED PROTEIN, TC21……………………………………..…9 INDIVIDUAL FUNCTIONS OF RAS PROTEINS………………………..…...10 TRANSFORMING GROWTH FACTOR BETA…………………………...…...12 CROSSTALK BETWEEN RAS AND TGF-BETA PATHWAYS………..…..14 CHAPTER 2: IN VIVO REGULATION OF TGF-BETA BY THE RAS PROTEIN TC21, REVEALED THROUGH THE LOSS OF THE NF1 RAS-GAP ABSTRACT……………………………………………………………………....20 BACKGROUND………………………………………………………………….21 MATERIALS AND METHODS…………………………………………………24 RESULTS………………………………………………………………………....30 vi DISCUSSION……………………………………………………………………..44 CHAPTER 3: DISCUSSION AND FUTURE DIRECTIONS……………………........69 REFERENCES……………………………………………………………………………78 vii LIST OF FIGURES FIGURE 1.1: RAS EFFECTOR PATHWAYS………………………………….18 FIGURE 1.2: TGF-BETA SIGNALING…………………………………………..19 FIGURE 2.1: INSERTION INTO THE MOUSE TC21 GENE CAUSES A NULL ALLELE……………………………………………………………………………..48 FIGURE 2.2: LOSS OF TC21 EXTENDS SURVIVAL OF NEUROFIBROMA-BEARING MICE BUT DECREASES SURVIVAL OF NPCis MICE……………………….51 FIGURE 2.3: Nf1 -/- TUMOR INITIATING CELLS ARE DECREASED WHEN TC21 IS ABSENT WHILE RESULTING TUMORS SHOW NEUROFIBROMA HITOLOGY, TUMOR SIZE AND TUMOR NUMBER…………………………………………..52 FIGURE 2.4: A TGF-BETA AUTOCRINE LOOP IN Nf1 -/- SCHWANN CELL PRECURSORS……………………………………………………………………..54 FIGURE 2.5: TGF-BETA EXPRESSION IN SCIATIC NERVES AND NEUROFIBROMAS………………………………………………………………..56 FIGURE 2.6: SURVIVAL OF NF1 MUTANT SCHWANN CELL PRECURSORS IS DEPENDENT ON TGF-BETA AND AKT………………………………………..57 FIGURE 2.7: Nf1-/- SCHWANN CELL PRECURSORS EXPRESS HIGH LEVELS OF PHOSPHO-AKT BUT NOT PHOSPHO-SMAD2/3……………………………..59 FIGURE 2.8: LOSS OF TC21 DECREASES THE SURVIVAL OF NPCis MICE BY INCREASING BRAIN TUMORS………………………………………………….61 FIGURE 2.9: LOSS OF TC21 IN MPNST CELLS INCREASE SARCOMA GROWTH…………………………………………………………………………….62 viii FIGURE 2.10: TGF-BETA MEDIATES AGGRESSIVE GROWTH OF MPNST XENOGRAFTS……………………………………………………………………………..64 FIGURE 2.11: TGF-BETA LIGANDS AND RECEPTOR EXPRESSION IN MPNST CELLS……………………………………………………………………………………….66 FIGURE 2.12: MPNST CELLS EXPRESS TGF-BETA AND LOSE TGFbRII............67 APPENDIX A: TGFb2 mRNA EXPRESSION IN MPNST CELLS AFTER INHIBITION OF JNK OR sFOS…………………………………………………………………………77 ix LIST OF TABLES TABLE 1: LOSS OF TC21 PARTIALLY RESCUES Nf1 MUTANT EMBRYONIC LETHALITY……………………………………….…………………………………….50 x Chapter 1 Introduction Overview The neurofibromatoses are a set of distinct genetic disorders that cause tumors to grow along or within various types of nerves. These disorders include neurofibromatosis type 1 (NF1), neurofibromatosis type 2 (NF2) and schwannomatosis. Together these disorders affect approximately 100,000 persons in the US. The most common of these disorders is NF1, which affects 1 in 3000 live births (1) while NF2 affects 1:25,000 births and Schwannomatosis affects an estimated 1:40,000 births (2). These disorders each predispose affected individuals to develop Schwann cell tumors and they arise from mutations in different genes, each of which plays a key role in regulating Schwann cell function. The NF1 gene on human chromosome 17q11, encodes an intracellular signaling molecule that functions as a GTPase activating protein for Ras proteins (3). Loss of NF1 results in activation of at least seven Ras proteins. In contrast, the NF2 gene on human chromosome 22q12 encodes a cytoskeletal-membrane linking protein (4). A candidate gene, INI1, has been recently identified in Schwannomatosis and investigating its role and function in this disorder is progressing (5). In these studies we focus on NF1, a known inhibitor of Ras proteins. The loss of the NF1 gene in neurofibromatosis type 1 is correlated with increased activation of Ras proteins. However, not all the phenotypes of the disease can be explained by the activation of the commonly studied canonical Ras proteins (H-, N- and K-Ras). We 1 therefore investigated the role of the little studied Ras related protein, TC21 in NF1 development and tumorigenesis. We show that TC21 is an important Ras protein in neurofibromatosis type 1. Our studies examine the role of TC21 in NF1, showing that if regulates the cytokine, TGF-beta, elucidating a role for TGF-beta as a therapeutic target particularly in malignant NF1. Neurofibromatosis Type 1: The Disease Formally known as Von Recklinghausen disease, NF1 is an autosomal dominant disorder affecting the nervous system, brain and bones (6). The disorder

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