ROLE OF PTPRT IN OBESITY AND ITS SUBSTRATE PAXILLIN TYROSINE-88 IN COLORECTAL CANCER by ANTHONY SCOTT Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Genetics and Genome Sciences CASE WESTERN RESERVE UNIVERSITY January 2015 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Anthony Scott candidate for the degree of Doctor of Philosophy*. Committee Chair Hua Lou Committee Member (Advisor) Zhenghe John Wang Committee Member Sanford Markowitz Committee Member Clark Distelhorst Committee Member Alex Huang Date of Defense 7/17/2014 *We also certify that written approval has been obtained for any proprietary material contained therein. 2 Table of Contents List of Tables 8 List of Figures 9 Acknowledgements 11 List of Abbreviations 12 Abstract 15 Chapter 1: Background and Significance 17 Colorectal Cancer 18 Etiology, staging, and therapy 18 Molecular basis of colorectal cancer 20 Genomics of colorectal cancer 21 PTPRT: Structure and Function 24 PTPRT’s Domain Structure 25 PTPRT’s role in neurological development 26 PTPRT as a tumor suppressor 26 Other Type IIB RPTPs as tumor suppressors 28 3 PTPRT’s role in cell adhesion 29 PTPRT in cancer signaling pathways 30 Paxillin, a substrate of PTPRT 31 Summary of PTPRT structure and function 34 Obesity 36 Diet and obesity 36 Obesity-related pathology 37 Central nervous system and obesity 38 Summary 39 Chapter 2: Identification of paxillin Y88 as a direct target of Src kinase 41 Abstract 42 Introduction 43 Results 45 Src regulates paxillin Y88 phosphorylation 45 Src directly phosphorylates paxillin Y88 47 PY88 paxillin regulates PI3-Kinase activation 48 4 PY88 paxillin does not correlate with clinical 50 characteristics of CRC Paxillin serves as a predictive factor for dasatinib sensitivity 52 Discussion 53 Materials and Methods 56 Chapter 3: PTPRT regulates high-fat diet-induced 59 obesity and insulin resistance Abstract 60 Introduction 61 Results 63 Ptprt-/- mice are resistant to high-fat diet-induced obesity 63 Ptprt-/- mice have less body fat by percentage than 64 wild type littermates Ptprt-/- reduces food intake 66 Ptprt-/- mice have reduced energy expenditure than WT mice 68 Ptprt-/- mice resist high-fat diet-induced hyperglycemia 69 and insulin resistance 5 Metabolic differences between Ptprt+/+ and Ptprt-/- littermates 72 Phospho-STAT3 increased in the hypothalamus of Ptprt-/- mice 72 Discussion 74 Materials and Methods 76 Chapter 4: Discussion and Future Directions 81 Summary 82 PTPRT and obesity: Future Directions 82 What is the role of PTPRT in glucose and lipid metabolism? 82 How does PTPRT affect the relationship between NPY, 90 stress and obesity? What are other implications of hypothalamic 91 phospho-STAT3? What is the in vivo role of PTPRT? 94 Paxillin and cancer: Future Directions 96 Why is pY88 paxillin important therapeutically? 96 How does pY88 paxillin affect p130cas phosphorylation? 98 6 What are alternative pathways through which 100 pY88 paxillin can act? Bibliography 103 7 List of Tables Chapter 2 Table 2-1. Clinical characteristics of colorectal carcinoma samples. 50 8 List of Figures Chapter 1 Figure 1-1. A multiple-stage colorectal progression model. 21 Figure 1-2. Effect of PTPRT mutations on its function. 24 Figure 1-3. Schematic of neurohormonal/nutrient feedback loop. 39 Chapter 2 Figure 2-1. Src phosphorylates paxillin at Y88 in cell lines. 46 Figure 2-2. Src directly phosphorylates paxillin at Y88. 47 Figure 2-3. PY88 paxillin regulates p130CAS-p85 interaction. 49 Figure 2-4. PY88 paxillin is upregulated in colorectal cancer tissues 51 Figure 2-5. PY88 paxillin levels predict sensitivity to dasatinib. 53 Chapter 3 Figure 3-1. PTPRT KO mice demonstrate slightly lower body weight than wild type littermates on normal chow diet. 63 Figure 3-2. PTPRT KO mice are resistant to high-fat diet- induced body composition changes. 65 Figure 3-3. PTPRT KO mice eat less but do not absorb dietary fats differently. 67 9 Figure 3-4. PTPRT KO mice do not have different circulating levels of leptin. 67 Figure 3-5. PTPRT KO mice have decreased NPY levels before high-fat diet. 67 Figure 3-6. PTPRT KO mice utilize more glucose and expend less energy than wild type mice. 69 Figure 3-7. PTPRT KO mice have less insulin resistance than wild type mice after high-fat diet. 71 Figure 3-8. PTPRT KO mice demonstrate better insulin regulation than wild type mice. 73 Figure 3-9. PTPRT KO mice have different blood chemistry values after high-fat diet. 73 Figure 3-10. PTPRT regulates STAT3 phosphorylation in mouse hypothalamus. 74 Chapter 4 Figure 4-1. Summary of PTPRT KO phenotypes after 14 weeks on a high-fat diet. 83 Figure 4-2. Diagram of hepatic insulin signaling. 85 Figure 4-3. Effect of PTPRT KO on end organs. 95 Figure 4-4. Extended pY88 paxillin signaling model. 101 10 Acknowledgements First, I would like to acknowledge the hard work that my adviser Dr. Zhenghe John Wang put in to train me. Specifically, a key scientific skill I needed to work on when I entered the lab was the organization and presentation of my research. Regardless of whether it was a lab journal club or departmental seminar, Dr. Wang emphasized the importance of presenting research in a succinct and easily digestible manner, poring over my and my labmates' presentations to continually improve them. I attribute my three CWRU Biomedical Graduate Student Symposium poster awards, which are indicative of my progress to these ends, to his diligence in training me. I also would like to thank all of my labmates past and present, especially Dr. Yujun Hao and Dr. Xiujing Feng who were with me for all four years. They were both very gracious in providing me with help in lab and Xiujing particularly with conducting mouse studies for our obesity work. Next, I very much appreciate the time put in by my thesis committee, Dr. Hua Lou (chair), Dr. Sanford Markowitz, Dr. Clark Distelhorst and Dr. Alex Huang in preparing me to produce this body of work. Also, I would like to thank my parents and my girlfriend, Dr. Katie Linder, for their support as well. 11 List of Abbreviations ACTH Adrenocorticotropic Hormone AOM Azoxymethane ATP Adenosine Triphosphate BMI Body Mass Index CRC Colorectal Carcinoma Csk C-terminal Src Kinase DMEM Dulbecco's Modified Essential Medium DNA Deoxyribonucleic Acid EDTA Ethylenediaminetetraacetic acid EE Energy Expenditure FAP Familial Adenomatous Polyposis FBS Fetal Bovine Serum FFA/NEFA Free Fatty Acids/Non-Essential Fatty Acids FN Fibronectin G6P Glucose 6-Phosphate GAPDH Glyceraldehyde 3-phosphate dehydrogenase GTT Glucose Tolerance Test HNPCC Hereditary Non-Polyposis Colorectcal Carcinoma HOMA-IR Homestatic Model Assessment-Insulin Resistance HRP Horseradish Peroxidase Ig Immunoglobulin IPTG Isopropyl β-D-1-thiogalactopyranoside 12 ITT Insulin Tolerance Test MAM Meprin/A5-protein/PTPmu NPY Neuropeptide Y p Phospho- p130cas p130-Crk Associated Substrate PAGE Polyacrylamide Gel Electrophoresis PDK1 Phosphoinositide-Dependent Kinase PI3K Phosphoinositide 3-kinase PIP2 Phosphatidylinositol-4,5-bisphosphate PIP3 Phosphatidylinositol-3,4,5-triphosphate PKC Protein Kinase C POMC Proopiomelanocortin PTK Protein Tyrosine Kinase PTP Protein Tyrosine Phosphatase PTPN14 Protein Tyrosine Phosphatase, Non-receptor type 14 PTPRT Protein Tyrosine Phosphatase, Receptor type, T PTPRT KO Protein Tyrosine Phosphatase, Receptor type, T homozygous knockout mice PXN Paxillin pY Phosphotyrosine pY88 Phosphotyrosine-88 (paxillin) RIPA Radioimuniprecipitation assay RPTP Receptor-type Protein Tyrosine Phosphatase RQ Respiratory Quotient 13 SDS Sodium Dodecyl Sulfate SFK Src Family Kinase SH Src Homology SHP-2 Src Homology-2 domain containing phosphatase-2 shRNA Short Hairpin RNA SNP Single Nucleotide Polymorphism STAT3 Signal Transducer and Activator of Transcription 3 VLDL Very Low Density Lipoprotein Y88F Paxillin Tyrosine-88-to-Phenylalanine knock-in mutation 14 Role of PTPRT in Obesity and its Substrate Paxillin Tyrosine-88 in Colorectal Cancer Abstract By ANTHONY SCOTT Regulation of protein tyrosine phosphorylation is important in maintaining appropriate cellular homeostasis. Accordingly, protein tyrosine phosphatases are frequently mutated in cancer. The most commonly mutated protein tyrosine phosphatase in colorectal cancer is PTPRT. Follow-up studies validated it as a tumor suppressor, especially through its activity on its substrates STAT3 phosphotyrosine-705 and paxillin phosphotyrosine-88. While the latter substrate is well characterized, further study is needed into pY88 paxillin. Since previous studies show that PTPRT is inactivated in colorectal carcinoma, understanding what kinase directly phosphorylates its substrates is an important question to investigate. Here, we show that paxillin Y88 is directly targeted by Src kinase for phosphorylation. Consequently, this finding has implications for cells that express high levels of pY88 paxillin, as they become sensitive to dasatinib treatment. Moreover, although prior work demonstrated that pY88 paxillin impacts Akt signaling, how this signal was transduced was not immediately clear. We show that pY88 paxillin promotes 15 interaction between p130Cas and the p85alpha regulatory subunit of PI3K. Therefore, we shed further light into how PTPRT affects colorectal cancer tumorigenesis. Another important aspect of this and many other cancers is the role of obesity in tumor development. One of PTPRT’s substrates, pY705 STAT3, plays a crucial role in energy