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Novel Mechanisms of Pten Dysfunction in Pten Hamartoma Tumor Syndromes

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Novel Mechanisms of Pten Dysfunction in Pten Hamartoma Tumor Syndromes NOVEL MECHANISMS OF PTEN DYSFUNCTION IN PTEN HAMARTOMA TUMOR SYNDROMES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Marcus G. Pezzolesi, B.S. ***** The Ohio State University 2008 Dissertation Committee: Approved by Professor Allan J. Yates, Advisor Professor Charis Eng, Co-Advisor _________________________________ Professor Wolfgang Sadee Advisor Integrated Biomedical Science Professor Michael C. Ostrowski Graduate Program Professor Lawrence S. Kirschner Professor Lei Shen ABSTRACT Phosphatase and tensin homolog deleted on chromosome ten (PTEN) encodes a tumor suppressor phosphatase frequently mutated in both sporadic and heritable forms of human cancer. Germline mutations in PTEN are associated with a number of heritable cancer syndromes referred to as the PTEN hamartoma tumor syndromes (PHTS) and includes both Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome (BRRS). Data from our laboratory suggests that alternate mechanisms of PTEN deregulation are likely to, at least in part, contribute to dysfunction in patients with these syndromes, particularly in those for whom germline mutations have yet to be identified. To better understand the mechanism(s) underlying dysregulation of PTEN in these syndromes, we employed a series of genetic and biochemical approaches aimed at investigating novel mechanisms involved in the regulation and deregulation of PTEN. Using a haplotype-based approach, we identified specific haplotypes and rare alleles within the PTEN locus that contribute to disease susceptibility and the phenotypic complexity of this syndrome. Within a haplotype block associated with PTEN-mutation negative patients, we identified a canonical E-box sequence located upstream of PTEN’s minimal promoter. We also investigated the role of microRNAs (miRNAs) in regulating PTEN and in PHTS. We show that miR-519e, a miRNA computationally predicted to target PTEN, ii specifically interacts with the gene’s 3’ untranslated region (UTR) and down-regulates endogenous PTEN expression in vitro. Subsequently, we show that miR-19a and miR-21, two miRNAs previously shown to target and repress PTEN protein levels, are differentially expressed in CS patients, irrespective of the PTEN mutation status. Our data suggest that these miRNA likely contribute to the phenotypic variability commonly seen in PHTS. The findings presented in this dissertation contribute significantly to our understanding of the pathogenesis of PHTS in patients in whom traditional screening methodologies have been unable to uncover a genetic cause. We further show that alternate mechanisms of PTEN dysfunction contribute to its deregulation and also to the variable phenotypic spectrum observed in PHTS. It is our hope that these data may lead to improved diagnostic measures and better predictive testing, and ultimately enable PHTS patients and their family members access to better personalized care. iii DEDICATION To my beautiful wife, Melissa, who has supported me throughout our Ohio adventure, and to my two sons, Quentin and Avery, all three of whom I love so much. And in memory of my father, Gerald G.N. Pezzolesi, who is somewhere smiling proudly. iv ACKNOWLEDGMENTS I would like to thank Dr. Charis Eng for all of her support and mentorship throughout my graduate training. The time spent I’ve in her laboratory has been invaluable. She have been instrumental in helping me to think like a scientist and I am truly grateful for all of the opportunities she has afforded me during this time and for her unending patience. I also want to thank the members of my dissertation committee, Drs. Allan J. Yates, Wolfgang Sadee, Michael C. Ostrowski, Lawrence S. Kirschner, and Lei Shen for their many helpful discussions and for the time that each has devoted in helping to further my scientific education and my research. I especially would like to thank Dr. Yates who, despite his many other commitments, has graciously served as my co-advisor over the past two and a half years. I thank the many member of the Eng laboratory, both past and present, for their scientific support and for their friendships. I especially would like to thank Drs. Kevin M. Zbuk, Kristin A. Waite, and Attila Patocs and Ms. Nita Williams, Ms. Rosemary Teresi, Ms. Patricia Kessler, and Mr. Todd Romigh. I would also like to thank Drs. Andrzej S. Krolewski and James H. Warram for all of their support over many years, without which I most certainly would not be where I am today. v Finally, and most importantly, to wife, Melissa, and my two sons, Quentin and Avery, who have all inspired me throughout my studies. Melissa, I thank you for your continuous support and for your love. You have always been there for me and I am forever grateful. I look forward to enjoying the rest of our lives together. Quentin and Avery, you have both inspired me in life and are both so precious to me; my great hope is that life brings you as much happiness and joy as you have brought me. vi VITA March 6, 1972.……………………………….. Born – Leominster, Massachusetts 1994.…………………...…………………….. B.S. Exercise Science, University of Massachusetts, Amherst. 2003 – 2005…………………………………...Graduate Research Associate The Ohio State University. 2005 – 2008………………………………….. Pre-Doctoral Research Fellow Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic. PUBLICATIONS Research Publications 1. Differential expression of PTEN-targeting microRNAs, miR-19a and miR-21, in Cowden syndrome. Pezzolesi MG, Platzer P, Waite KA, Eng C (In Preparation) 2. High-density SNP genome wide linkage scan for susceptibility genes for diabetic nephropathy in type 1 diabetes: Discordant sib-pair approach. Rogus JJ, Poznik GD, Pezzolesi MG, Smiles AM, Dunn JS, Walker W, Wanic K, Canani LH, Araki H, Makita J, Warram JH, Krolewski AS (Diabetes. Submitted) 3. Cowden syndrome-affected patients with PTEN promoter mutations demonstrate abnormal protein Translation. Teresi RE, Zbuk KM, Pezzolesi MG, Waite KA, Eng C (Am J Hum Genet. Oct:81(4):756-767) 4. Comparative genomic and functional analyses reveal a novel cis-acting PTEN regulatory element as a highly conserved functional E-box motif deleted in Cowden syndrome. Pezzolesi MG, Zbuk KM, Waite KA, Eng C (Hum Mol Genet. 2007 May:16(9):1058-1071) vii 5. Mutation-positive and mutation-negative patients with Cowden and Bannayan- Riley-Ruvalcaba syndromes associated with distinct 10q haplotypes. Pezzolesi MG, Li Y, Zhou XP, Pilarski R, Shen L, Eng C (Am J Hum Genet. 2006 Nov;79(5):923- 934) 6. Examination of PPP1R3B as a candidate gene for the type 2 diabetes and MODY loci on chromosome 8p23. Dunn JS, Mlynarski WM, Pezzolesi MG, Borowiec M, Powers C, Krolewski AS, Doria A (Ann Hum Genet. 2006 Sep;70(Pt 5):587-293) 7. A newly identified mutation in an ipf1 binding site of the insulin gene promoter may predispose to type 2 diabetes mellitus. Malecki MT, Lebrun P, Pezzolesi M, Warram JH, Krolewski AS, Jhala US (Diabetologia 2006 Aug; 49(8):1985-1987) 8. Examination of candidate chromosomal regions for type 2 diabetes mellitus (T2DM) reveals a susceptibility locus on human chromosome 8p23.1. Pezzolesi MG, Nam M, Nagase T, Klupa T, Dunn JS, WM Mlynarski, Rich SS, Warram JH, Krolewski AS (Diabetes. 2004 Feb; 53(2):486-491) 9. Genetic modifiers of the age at diagnosis of diabetes (MODY3) in carriers of hepatocyte nuclear factor-1alpha mutations map to chromosomes 5p15, 9p22, and 14q24. Kim SH, Ma X, Klupa T, Powers C, Pezzolesi M, Warram JH, Rich SS, Krolewski AS, Doria A. (Diabetes. 2003 Aug; 52(8):2182-2186) 10. Determinants of the development of diabetes (maturity-onset diabetes of the young-3 [MODY3] in carriers of HNF-1a mutations. Klupa T, Warram JH, Antonellis A, Pezzolesi M, Nam M, Malecki MT, Doria A, Rich SS, Krolewski AS (Diabetes Care. 2002 Dec; 25(12):2292-2301) 11. A method for developing high density snp maps and its application at the type 1 angiotensin ii receptor (AGTR1) gene locus. Antonellis A, Rogus JJ, Canani LH, Makita Y, Pezzolesi MG, Nam M, Ng D, Moczulski D, Warram JH, Krolewski AS (Genomics. 2002 Mar; 79(3):326-332) 12. Further evidence for a susceptibility locus for type 2 diabetes mellitus on chromosome 20q13.1-q13.2. Klupa T, Malecki MT, Pezzolesi M, Ji L, Curtis S, Rich SS, Warram JH, Krolewski AS (Diabetes. 2000 Dec; 49(12):2212-2216) viii FIELDS OF STUDY Major Field: Integrated Biomedical Science ix TABLE OF CONTENTS P a g e ABSTRACT........................................................................................................................ii DEDICATION.................................................................................................................. iv ACKNOWLEDGMENTS ..................................................................................................v VITA................................................................................................................................ vii LIST OF TABLES...........................................................................................................xiii LIST OF FIGURES .........................................................................................................xiv CHAPTER 1 INTRODUCTION ........................................................................................1 1.1 COWDEN SYNDROME AND PTEN .........................................................................1 1.2 PTEN BIOCHEMISTRY..............................................................................................4
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