Cowden Syndrome and Pten Promoter Regulation
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COWDEN SYNDROME AND PTEN PROMOTER REGULATION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Rosemary Elaine Teresi ***** The Ohio State University 2008 Dissertation Committee: Professor Allen Yates, Advisor Approved by Professor Charis Eng, Co-Advisor Professor Ching-Shih Chen _________________________________ Professor Denis Guttridge Advisor Integrated Biomedical Sciences Professor Matthew Ringel Graduate Program Professor Kristin Waite ABSTRACT Germline mutations of PTEN (phosphatase and tensin homolog deleted on chromosome ten) are associated with the multi-hamartomatous disorder Cowden syndrome (CS). We show here that the PPARγ agonist Rosiglitazone, along with Lovastatin, Simvastatin, Pravastatin and Fluvastatin can induce PTEN expression by inducing PTEN transcription. Additionally, we observed, for the first time, that upregulation of SREBP protein, known to induce PPARγ expression, can increase PTEN expression. Our results indicate that Rosiglitazone, and SREBP utilizes PPARγ’s transcriptional activity to induce PTEN transcription, while the statins signal through PPARγ’s protein activity to upregulate PTEN expression. Studing the full-length PTEN identified a region between -854 and -791 that binds an as yet unidentified transcription factor, through which the statins induce PTEN expression. We examined the downstream effect of five PTEN promoter variants (-861G/T, - 853C/G, -834C/T, -798G/C, and -764G/A) that are not within any known cis-acting regulatory elements. We demonstrated that protein binding to the PTEN promoter (-893 to -755) was not altered in the five variants, when compared to the wild-type (WT) promoter. However, three of the variants (-861G/T, -853C/G, and -764G/A) demonstrated ~50% decrease in luciferase activity compared to the WT construct. PTEN ii mRNA levels were not altered in these variants, while secondary structure predictions indicated that different PTEN 5’UTR transcript folding patterns exist in three variants, suggesting an inhibition of protein translation. This was confirmed by PTEN protein analysis. These data indicate that variants causing large mRNA secondary structure alterations result in an inhibition of protein translation and a decrease in PTEN protein expression. These data emphasize the importance of PTEN promoter nucleotide variations and their ability to lead to CS progression by a novel regulatory mechanism. Importantly, these patients have a high prevalence of breast, thyroid and endometrial malignancies, thus understanding of the mechanism of PTEN dysfunction in these patients will lead to more sensitive molecular diagnostic and predictive testing and ultimately, to rational targeted therapies to treat or prevent malignancy. iii DEDICATION This dissertation is dedicated to the men in my life: Paul Killian, Terry Roof, Stephen Teresi and Rigley Teresi. To my grandfather, Paul, who emphasized the importance of education and always taught me to “keep my eyes on the books and not on the boys”. To my father, Terry, who has been my role model for hard work, persistence and personal sacrifices, and who instilled in me the motivation to set high goals and the confidence to achieve them. To my husband, Stephen, who has been my emotional anchor and provided unending support throughout my doctoral career. Last but not least, to my son Rigley, who is the light of my life. I would also like to dedicate this work to the rest of my family and friends. You’ve been there for me through both the good and the bad. I will forever be grateful for your continuous encouragement. iv ACKNOWLEDGMENTS My thanks and appreciation to Dr. Charis Eng for persevering with me as my advisor through my time at The Ohio State University and the Cleveland Clinic Foundation. Her drive and encouragement pushed me to where I am today. I am grateful as well to Drs. Allen Yates and Kristin Waite for coordinating and overseeing my research over the last five years. Additionally to the members of my dissertation committee, Drs. Ching-Shih Chen, Denis Guttridge, Christoph Plass, and Matt Ringel who have generously given their time and expertise to better my work. I thank them all for their many contributions and support. I would also like to acknowledge all the members of Dr. Eng’s laboratory that I’ve had the privilege of working with over the last several years; it has been an honor to work with each of you. I would specifically like to acknowledge Drs. Marcus Pezzolesi, Yufang Tang, Kevin Zbuk, Sarah Planchon-Pope, Guillaume Assié, and Frank Weber, as well as Michelle Sinden, Nita Williams, and Pat Kessler for many helpful discussions over the years. Moreover, I would like to thank Drs. Jodi Bubenik, Donna Driscoll, Don Luse, and Kwaku Dayie for their insight and Robert Pilarski and Jennifer Stein for aid in patient recruitment. v VITA July 13, 1980. Canton, OH Summer 2001. Cell Biology Internship University of Cincinnati Cincinnati, OH Mentor: Dr. Karen Knudsen May 12, 2002. Bachelor of Science in Biology Bachelor of Science in Chemistry Heidelberg College; Tiffin, OH Mentor: Dr. Robert Murray 2002 – 2005. Graduate Research Assistant The Ohio State University Columbus, OH Mentor: Dr. Charis Eng 2005 – 2007. Predoctoral Fellow Cleveland Clinic Foundation Cleveland, OH Mentor: Dr. Charis Eng PUBLICATIONS 1. Van Brocklyn J.R,, Young N., Roof R. Sphingsine-1-phosphate stimulates motility and invasiveness of human glioblastoma multiforme cells. Cancer Lett 2003 Sep 10; 199(1):53-60. 2. Teresi, R.E., Chaiu, C.W., Chen, C.S., Chatterjee, K.V., Waite, K.A., Eng, C. PTEN’s Transcription Regulated by PPARγ. Int J Cancer 2006 May 15;118(10):2390-8. vi 3. Weber, F, Teresi, R.E., Broelsch, C.E., Frilling, A, and Eng, C. A Limited Set of Deregulated Human microRNA's Contribute to Follicular Thyroid Carcinogenesis. J Clin Endocrinol Metab 2006 Sep 91(9):3584-91. 4. Teresi R.E., Zbuk K.M., Pezzolesi M.G., Waite K.A., and Eng C. Cowden Syndrome-Affected Patients with PTEN Promoter Mutations Demonstrate Abnormal Protein Translation. Am J Hum Genet. 2007 Oct;81(4):756-67. 5. Teresi R. E., Planchon S.M., Waite K.A., Eng C. Regulation of the PTEN Promoter by Statins and SREBP. Hum Mol Genet, in press (12-04-2007). 6. Teresi R. E., and Waite K.A. PTEN, PPARγ and the Fight Against Cancer. PPAR Research, submitted (02-2008) FIELDS OF STUDY Major Field: Integrated Biomedical Sciences vii TABLE OF CONTENTS P a g e Abstract..........................................................................................................................ii Dedication ..................................................................................................................... iv Acknowledgments ...........................................................................................................v Vita ................................................................................................................................vi List of Tables..................................................................................................................xi List of Figures ...............................................................................................................xii List of Abbreviations................................................................................................... xiv Chapter 1 INTRODUCTION .........................................................................................1 1.1 History ......................................................................................................................1 1.2 Cowden Syndrome ....................................................................................................1 1.2.1 Phenotype ..................................................................................................1 1.2.2 PTEN..........................................................................................................4 1.3 PTEN Hamartoma Tumor Syndrome ........................................................................4 1.3.1 Bannayan-Riley-Ravalcaba Syndrome .......................................................5 1.3.2 Others .........................................................................................................5 1.4 PTEN ........................................................................................................................6 1.4.1 Mutations....................................................................................................6 1.4.2 PTEN Protein Domains...............................................................................8 viii 1.5 PTEN Functions ......................................................................................................10 1.5.1 Nuclear Compartmentalization .................................................................10 1.5.2 Phosphatase Activity.................................................................................10 1.6 PTEN Regulation.....................................................................................................13 1.6.1 Degradation ..............................................................................................13 1.6.2 Transcription ............................................................................................14 1.6.2.1 Promoter....................................................................................14 1.6.2.2 Transcription Factors .................................................................16