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Id3 Induces an Elk-1- and Caspase-8-Dependent Apoptotic Pathway In

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ID3 INDUCES AN ELK-1- AND CASPASE-8-DEPENDENT APOPTOTIC PATHWAY IN SQUAMOUS CARCINOMA 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 & Cellular Biology By You-shin Chen, M.S. Washington, DC November 18, 2014 Copyright 2014 by You-shin Chen All Rights Reserved ii ID3 INDUCES AN ELK-1- AND CASPASE-8-DEPENDENT APOPTOTIC PATHWAY IN SQUAMOUS CARCINOMA CELLS You-shin Chen, M.S. Thesis Advisor: Dean S. Rosenthal, Ph.D. ABSTRACT Inhibitors of differentiation/DNA binding (Id) proteins are helix-loop-helix (HLH) transcription factors. The Id protein family (Id1-Id4) mediates tissue homeostasis by regulating cellular processes including differentiation, proliferation, and apoptosis. Previously, we found that Id3 induced apoptosis in immortalized human keratinocytes (Simbulan-Rosenthal et al., 2006), consistent with its role as a tumor suppressor (Richter et al., 2012; Schmitz et al., 2012). To investigate the role of Id3 in malignant SCC cells (A431), a tetracycline-regulated inducible system was used to induce Id3 in cell culture and mouse xenograft models. We found that upon Id3 induction, there was a decrease in cell number under low serum conditions, as well as in soft agar. Microarray, RT-PCR, immunoblot, siRNA, and inhibitor studies revealed that Id3 induced expression of Elk-1, an ETS-domain transcription factor, inducing procaspase-8 expression and activation. Id3 deletion mutants revealed that 80 C-terminal amino acids, including the HLH, are important for Id3-induced apoptosis. In a mouse xenograft model, Id3 induction decreased tumor size by 30%. Using immunofluorescence analysis, we determined that the tumor size decrease was also mediated through apoptosis. Further, we show that Id3 synergizes with 5-FU and cisplatin therapies for non-melanoma skin cancer cells. Our studies have shown that Id3 induces apoptosis in SCC cells via an Elk-1- and caspase-8-dependent pathway, and this information can provide some strategic insights for new SCC treatments. iii Acknowledgements First, I would like to sincerely thank my thesis advisor Dr. Dean Rosenthal for giving me the opportunity to work in his lab. Without his guidance, I couldn’t have completed the research and thesis writing. Besides, I feel fortunate to be given the opportunities to learn how to lead a research team comprised of people from different scientific and cultural background. The past years in the Rosenthal lab have been very productive, and I feel blessed to have this kind of training experience. I want to thank my committee members: Drs. Albanese, Chirikjian, Kasid, and Vasudevan and the Department Chair Dr. Crooke for their advice and feedback throughout the years. Special thanks to Dr. Cynthia Rosenthal, from whose life experience I find inspiring and for giving me the opportunities to develop teaching skills. I would also like to thank my colleagues along the way: Valerie, Kyle, Lucia, Virginia, Anirudh, and Maryam, for their constant encouragement and help in the lab. The good memories with them will always be remembered. Last but not least, I would like to thank my family and friends. My father Chin-tu, mother Tung-chin, sister You-jen, and brother Yu-fu form strongest support team to help me in any possible ways in the past years to complete my degree. My boyfriend Derrick has been my best friend, top cheerleader, and supporter. Derrick has offered me his warmest hands during my down times, without whom, the completion of my research would not be possible. I also want to thank my best friends, Lynn, Peiyu, and Tina who never abandon me even when I miss their important life events while pursuing this degree. Many thanks, You-shin iv Table of Contents Table of Abbreviations and Definitions ....................................................................................... viii List of Figures ............................................................................................................................... xv List of Tables ............................................................................................................................. xviii Chapter 1. Introduction ................................................................................................................... 1 1.1. Hypothesis, Rationale, and Research Aims .............................................................. 1 1.2. Introduction to Squamous Cell Carcinoma (SCC) .................................................... 2 1.2.1. Skin Cancer Statistics .................................................................................... 2 1.2.2. Risk Factors for Squamous Cell Carcinoma ................................................. 3 1.2.3. SCC Progression ........................................................................................... 4 1.2.4. Current Treatment Options for SCC ............................................................. 5 1.3. Introduction to Id Proteins ......................................................................................... 9 1.3.1. Id Genetic and Protein Information ............................................................... 9 1.3.2. Ids as HLH Transcription Factors ............................................................... 11 1.3.3. Ids as Regulators of Differentiation and Proliferation ................................ 17 1.3.4. Ids as Regulators of Apoptosis .................................................................... 21 1.3.5. Ids as Potential Tumor Suppressors ............................................................ 22 1.3.6. Ids and SCC ................................................................................................. 25 1.3.7. Ids and TCF Transcription Factors .............................................................. 27 1.4. Introduction to Apoptosis ........................................................................................ 34 1.4.1. Caspases ...................................................................................................... 34 1.4.2. Bcl-2 Family Proteins .................................................................................. 37 1.4.3. Apoptotic Signaling Pathways .................................................................... 39 1.4.4. Ids and Apoptosis ........................................................................................ 41 Chapter 2. Materials and Methods ................................................................................................ 43 2.1. Cell Culture ............................................................................................................. 43 2.2. Tet-inducible System ............................................................................................... 44 2.3. Plasmids .................................................................................................................. 47 2.4. Inducible Cell Lines ................................................................................................ 48 2.5. Fluorescently-labeled Cell Lines ............................................................................. 49 v 2.6. Growth Curve .......................................................................................................... 49 2.7. Cell Cycle Analysis ................................................................................................. 50 2.8. Cell Invasion Assay ................................................................................................. 51 2.9. Microarray Analysis ................................................................................................ 51 2.10. siRNA Transfection ................................................................................................. 51 2.11. Tumor Xenografts ................................................................................................... 52 2.12. Immunoblot Analysis .............................................................................................. 52 2.13. Immunostaining ....................................................................................................... 53 2.14. RT-PCR/qRT-PCR .................................................................................................. 53 2.15. Soft Agar Growth Assay ......................................................................................... 54 2.16. 5-FU and Cisplatin Treatment ................................................................................. 54 2.17. Statistical Analysis .................................................................................................. 55 Chapter 3. Results ......................................................................................................................... 56 3.1. Establishment of A431/Id3 and A431/Vc Cell Lines .............................................. 56 3.1.1. Endogenous Levels of Id3 are Down-regulated in A431 Cells ................... 56 3.1.2. Verification of Inducible A431/Id3 Clones ................................................. 57 3.2. Study of the Effects of Id3 in vitro .......................................................................... 59 3.2.1. Id3 Does Not Affect Cell Growth in Normal Serum, in Matrigel, or Invasion ....................................................................................................... 59 3.2.2. Id3 Reduces Viable Cell
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