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Effects of Tnfaip8 Knockdown on Egfr and Igf-1R Signaling and Cytotoxicities of Targeted Drugs in Non-Small Cell Lung Cancer Cells

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Effects of Tnfaip8 Knockdown on Egfr and Igf-1R Signaling and Cytotoxicities of Targeted Drugs in Non-Small Cell Lung Cancer Cells EFFECTS OF TNFAIP8 KNOCKDOWN ON EGFR AND IGF-1R SIGNALING AND CYTOTOXICITIES OF TARGETED DRUGS IN NON-SMALL CELL LUNG CANCER CELLS 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 Biochemistry and Molecular Biology By Timothy Francis Day, B.S. Washington, DC February 25, 2015 Copyright 2015 by Timothy Francis Day All Rights Reserved ii EFFECTS OF TNFAIP8 KNOCKDOWN ON EGFR AND IGF-1R SIGNALING AND CYTOTOXICITIES OF TARGETED DRUGS IN NON-SMALL CELL LUNG CANCER CELLS Timothy Francis Day, B.S. Thesis Advisor: Usha N. Kasid, Ph.D. ABSTRACT Lung cancer is the most commonly diagnosed cancer and leading cause of cancer-related deaths worldwide. Non-small cell lung carcinoma (NSCLC) accounts for 85% of lung cancer cases. Molecular therapies targeting epidermal growth factor receptor (EGFR) have been developed for NSCLC. However, resistance of NSCLC to various therapies is a major clinical challenge. Previous studies from our laboratory and others have shown that tumor necrosis factor-α-inducible protein 8 (TNFAIP8), the first discovered member of a highly conserved TNFAIP8 family, is an oncogenic and metastatic molecule. TNFAIP8 (T8) expression is induced by NF-κB and is high in many cancers including NSCLC. This thesis project investigates the effects of T8 shRNA (shT8) on EGFR signaling in NSCLC cells. Since acquired resistance to EGFR-targeted tyrosine kinase inhibitors (TKIs) has been associated with activation of insulin-like growth factor-1 receptor (IGF-1R), the effects of shT8 on IGF-1R signaling were also studied. The shRNA silencing of T8 resulted in inhibition of growth factor (EGF, IGF-1, FGF-1, VEGF)-stimulated cell migration in A549 lung cancer cells. T8 knockdown cells showed increased expression of sorting nexin 1 (SNX1), a regulator of EGFR expression through endosomal trafficking, and siRNA silencing of SNX1 partially restored EGFR expression in shT8 cells. Decreased levels of EGF-inducible pEGFR and pERK were observed in shT8 cells as compared to control cells. T8 knockdown cells also showed increased expression of IGF-1 iii binding protein 3 (IGFBP3), a negative regulator of IGF-1R signaling. Consistently, IGF-1- inducible expression of pIGF-1R and pAKT was decreased, and siRNA silencing of IGFBP3 resulted in increased pIGF-1R and pAKT levels in shT8 cells. In preliminary studies, cytotoxic effects of EGFR and IGF-1R TKIs, gefitinib and AG-1024, were enhanced in shT8 cells. Aberrant regulations of EGFR and IGF-1R signaling are hallmarks of several other tumors including breast, melanoma and pancreatic cancers. Present studies suggest that TNFAIP8 plays an important role in EGFR and IGF-1R signaling in lung cancer cells, and offer a rationale for development of T8 knockdown models of a wide range of TKI-resistant tumor cells. Ultimately, these efforts may lead to a new strategy for preventing or delaying resistance to EGFR and IGF- 1R-targeted therapeutics. iv ACKNOWLEDGEMENTS THIS WORK WAS SUPPORTED, IN PART, BY MY PRE-DOCTORAL FELLOWSHIP AWARD W81XWH-10-1-0107 FROM THE DEPARTMENT OF DEFENSE. I WOULD LIKE TO THANK MY MENTOR, DR. USHA KASID. HER GREAT PATIENCE AND GUIDANCE HAS HELPED ME BECOME A MORE THOUGHTFUL AND ANALYTICAL SCIENTIST. I WOULD LIKE TO THANK MY THESIS COMMITTEE MEMBERS, DRS. DANIEL DJAKIEW, CAROLYN HURLEY, MARY BETH MARTIN, AND RAJA MAZUMDER. I HAVE BENEFITED GREATLY FROM EVERY INTERACTION WITH MY COMMITTEE, WHETHER IT WAS ADVICE ON AN EXPERIMENT OR HOW TO THINK ABOUT MY WORK, THEIR HELP HAS GUIDED ME TOWARD BEING A BETTER SCIENTIST. I WOULD LIKE TO THANK MY PARENTS, JIM AND CONNIE, FRIENDS, AND CO-WORKERS FOR THEIR SUPPORT, INTERACTIONS, AND OCCASIONAL DISTRACTIONS. YOUR UNYIELDING CONFIDENCE IN MY GOALS HAS NEVER WAVERED. FINALLY, I WOULD LIKE TO THANK MY WIFE IKUKO. YOU HAVE BEEN MY FOUNDATION THROUGHOUT MY DOCTORAL STUDIES. TOGETHER WE HAVE HAD SO MUCH JOY AND CELEBRATION, AND, WITH OUR WONDERFUL DAUGHTER ANNABELLE, I CANNOT THINK A MORE PERFECT FAMILY. THANK YOU. Many thanks, Timothy Francis Day v TABLE OF CONTENTS ABSTRACT .........................................................................................................................iii ACKNOWLEDGMENTS ...................................................................................................v TABLE OF CONTENTS ....................................................................................................vi LIST OF FIGURES ...................................................................................................x LIST OF TABLES .....................................................................................................xv LIST OF SUPPLEMENTAL FIGURES ...................................................................xvi LIST OF SUPPLEMENTAL TABLES .....................................................................xvi LIST OF ABBREVIATIONS AND PHRASES .......................................................xvii CHAPTER 1. INTRODUCTION ......................................................................................1 1.1 Hypothesis, rationale, and specific aims .............................................................1 1.2. Introduction to non-small cell lung carcinoma (NSCLC) ..................................2 1.2.1. Representative targetable pathways in NSCLC ...................................4 1.3. Introduction to T8 ...............................................................................................5 1.3.1. T8 protein family: an overview. ...........................................................5 1.3.2. TIPE1, an apoptosis inducer regulating the Rac1 pathway. ...............11 1.3.3. TIPE2, a negative regulator of immune homeostasis ..........................11 1.3.4. TIPE3, a novel phosphoinositides transfer protein that promotes cancer .............................................................................................................12 1.3.5. T8 is a novel prosurvival, oncogenic and metastatic molecule: implications in cancer prognosis and therapy response ................................14 vi 1.4. Introduction to shRNA and siRNA silencing of gene expression ......................18 1.5. Introduction to EGFR-induced signal transduction and endocytic mechanisms of EGF-induced internalization and degradation of EGFR...................21 1.5.1. EGFR structure, ligand-induced activation, and signal transduction ...................................................................................................21 1.5.2. Regulation of EGFR expression via endocytic trafficking and other mechanisms....................................................................................25 1.6. EGFR targeted therapies and mechanisms of drug resistance ............................31 1.6.1. EGFR targeted therapies .....................................................................31 1.6.2. Mechanisms of resistance to EGFR targeted therapies .......................35 1.7. Introduction to IGF-1R structure and signal transduction ..................................41 1.8. IGFBP3-mediated regulation of IGF/IGF-1R signal transduction .....................46 1.9. Acquired resistance to EGFR TKIs is associated with activation of IGF-1R pathway: implications of combined targeting of EGFR and IGF-1R in treatment of resistant NSCLC cells ............................................................................................48 CHAPTER 2. MATERIALS AND METHODS ...............................................................54 2.1. Cell lines and culture conditions .........................................................................54 2.2. Antibodies, growth factors, reagents and chemicals ...........................................54 2.3. Plasmid DNA preparations .................................................................................56 2.4. T8 shRNA transient transfections .......................................................................57 2.5. T8 shRNA lentivirus and stable transductions....................................................61 2.6. SNX1 and IGFBP3 siRNA transfections ...........................................................63 2.7. Growth factor treatments ....................................................................................64 2.8. A549 scr and A549 shT8 culture medium treatments ........................................64 vii 2.9. Wound healing assay for cell migration .............................................................66 2.10. RNA isolation, RT-PCR and qRT-PCR ...........................................................66 2.11. Immunoblotting.................................................................................................70 2.12. Immunofluorescence .........................................................................................72 2.13. XTT assay for cell viability ..............................................................................73 2.14. Statistical analysis .............................................................................................74 CHAPTER 3. RESULTS ....................................................................................................75 3.1. Study of the effects of T8 knockdown on EGF-induced signal transduction in non-small cell lung cancer cells ........................................................75 3.1.1. Analysis of T8 transcripts and protein isoforms in various tumor cells.....................................................................................75
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