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E3 Ligase Identified by Differential Display (EDD) Enhances Cell Survival and Cisplatin Resistance in Epithelial Ovarian Cancer and Oral Squamous Cell Carcinoma

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E3 Ligase Identified by Differential Display (EDD) Enhances Cell Survival and Cisplatin Resistance in Epithelial Ovarian Cancer and Oral Squamous Cell Carcinoma Medical University of South Carolina MEDICA MUSC Theses and Dissertations 2014 E3 Ligase Identified yb Differential Display (EDD) Enhances Cell Survival and Cisplatin Resistance in Epithelial Ovarian Cancer and Oral Squamous Cell Carcinoma Amber Thompson Bradley Medical University of South Carolina Follow this and additional works at: https://medica-musc.researchcommons.org/theses Recommended Citation Bradley, Amber Thompson, "E3 Ligase Identified yb Differential Display (EDD) Enhances Cell Survival and Cisplatin Resistance in Epithelial Ovarian Cancer and Oral Squamous Cell Carcinoma" (2014). MUSC Theses and Dissertations. 148. https://medica-musc.researchcommons.org/theses/148 This Dissertation is brought to you for free and open access by MEDICA. It has been accepted for inclusion in MUSC Theses and Dissertations by an authorized administrator of MEDICA. For more information, please contact [email protected]. E3 Ligase Identified by Differential Display (EDD) Enhances Cell Survival and Cisplatin Resistance in Epithelial Ovarian Cancer and Oral Squamous Cell Carcinoma By: Amber Thompson Bradley A dissertation submitted to the faculty of the Medical University of South Carolina in partial fulfillment of the requirement for the degree of Doctor of Philosophy in the college of Graduate Studies Department of Cell and Molecular Pharmacology and Experimental Therapeutics 2014 Approved by: ________________________ Scott T. Eblen Chairman, Advisory Committee ________________________ Joe B. Blumer ________________________ Steven A. Rosenzweig ________________________ Robin C. Muise-Helmericks ________________________ Bryan P. Toole I dedicate this to my loving husband, Andy Bradley, for his patience and support. Without his love and understanding I could not have completed my work for this dissertation. You believed in me when I did not have the courage to believe in myself. You encouraged me to be persistent in my efforts. I know that you have spent many hours praying for my experiments, exams, presentations, and my sanity. I appreciate your notes and emails of encouragement. You always know how to make me laugh and relax after a long day in lab. I would also like to thank my parents, Wayne and Donna Thompson, for pushing me to achieve as much as possible and enabling me to have all of the opportunities that I have had in my life. You both provided a strong foundation to allow me to pursue my education. Thank you for the cards of support and encouragement when I needed them most. ii Acknowledgements I am truly grateful to my mentor, Dr. Scott Eblen, for teaching me and allowing me to perform research in his lab. I appreciate you always taking the time to explain experimental protocols and clarifying results. You actively perform research which facilitated your knowledge of experiments and troubleshooting, which was extremely valuable to me on a regular basis. You have equipped me with the necessary tools to be an effective scientist. I appreciate the willingness of Dr. Joe Blumer, Dr. Steven Rosenzweig, Dr. Robin Muise-Helmericks, and Dr. Bryan Toole to serve on my committee and guide me throughout my graduate training. I am thankful for our productive discussions and your leadership. I am also grateful to Dr. Blumer, and members of his lab, for teaching me new experimental protocols and for analysis of my results. I appreciate Dr. Lauren Ball for serving as graduate coordinator to direct my studies and offer me as many opportunities as possible. Thank you to previous members of the Eblen lab for your advice, patience, and guidance as I acquired the skills that I needed in the lab. I am also truly grateful to members of Dr. Rosenzweig’s, Dr. Neumann’s, and Dr. Blumer’s labs for your friendship and valuable conversations. I will cherish the moments we have shared. iii Table of Contents Acknowledgements . iii List of Figures and Tables . v List of Abbreviations. viii Abstract . xi Chapter 1: Introduction . 1 1.1. Ovarian Cancer. 2 1.2. Oral Squamous Cell Carcinoma . 11 1.3. Ubiquitin/Proteasome Pathway. 14 1.4. EDD. 23 1.5. EDD in Cancer. 34 1.6. Apoptosis. 38 Statement of the Hypothesis and Specific Aims. 41 Chapter 2: EDD Regulates Cellular Survival in Ovarian Cancer Cells. 42 2.1. Introduction. 43 2.2. Materials and Methods . 45 2.3. Results. 49 2.4. Discussion. 65 Chapter 3: EDD Increases Cisplatin Resistance in Ovarian Cancer Cells . 67 3.1. Introduction. 68 3.2. Materials and Methods . 70 3.3. Results. 74 3.4. Discussion. 88 Chapter 4: EDD Regulates Cisplatin Resistance in Oral Squamous Cell Carcinoma. 93 4.1. Introduction. 94 4.2. Materials and Methods . 96 4.3. Results. 99 4.4. Discussion. 122 Chapter 5: Conclusion . 127 5.1. Conclusion . 128 5.2. Context in the Field . 135 5.3. Future Directions . 139 References . 154 iv List of Figures and Tables Chapter 1 Table 1.1. General Mechanisms of Resistance to Platinum Chemotherapy or Paclitaxel. 8 Figure 1.1. MAP kinase signaling pathways. 10 Figure 1.2. Ubiquitination. 17 Figure 1.3. E3 ubiquitin ligases. 20 Figure 1.4. Domains of EDD. 25 Figure 1.5. C-Terminal HECT domain of EDD. 25 Figure 1.6. UBA domain of EDD. 25 Figure 1.7. Phosphorylation sites of EDD . 26 Figure 1.8. Cellular functions of EDD. 33 Figure 1.9. Separate roles of EDD – dependent or independent of ubiquitin ligase function . 33 Figure 1.10. Allelic imbalance of EDD . 35 Figure 1.11. EDD levels are regulated at the mRNA level through upregulation or downregulation. 35 Chapter 2 Figure 2.1. EDD is overexpressed in ovarian cancer cell lines and EDD knockdown induces apoptosis. 51 Figure 2.2. EDD knockdown reduces cellular survival . 53 Figure 2.3. EDD downregulation decreases Mcl-1 protein levels, whereas Mcl-1 overexpression inhibits apoptosis upon EDD knockdown . 58 Figure 2.4. EDD regulates Mcl-1 levels through a GSK-3β-independent mechanism . 61 Figure 2.5. EDD regulates Mcl-1 levels through transcriptional regulation. 63 v Chapter 3 Figure 3.1. Transient EDD knockdown may sensitize cells to cisplatin . 76 Figure 3.2. Stable EDD knockdown increases cisplatin sensitivity. 78 Figure 3.3. Normal Mcl-1 expression in EDD stable knockdowns . 80 Figure 3.4. EDD overexpression in apoptosis assay and EDD localization . 82 Figure 3.5. EDD overexpression is sufficient to induce cisplatin resistance, dependent on its ubiquitin ligase activity . 83 Figure 3.6. DOPC nanoparticle delivery of EDD siRNA in vivo reduces tumor burden . 86 Figure 3.7.Model for EDD regulation of survival and cisplatin resistance . 91 Table 3.1. Cisplatin sensitivities across ovarian cancer cell lines . 92 Chapter 4 Figure 4.1. SiRNA mediated EDD knockdown causes apoptosis in some oral squamous cell carcinoma cell lines. 101 Figure 4.2. Transient knockdown of EDD may increase cisplatin sensitivity in some oral squamous cell carcinoma cell lines. 105 Figure 4.3. Inducible stable EDD knockdown in oral squamous cell carcinomas. 108 Figure 4.4. UM74B stable EDD knockdown cells demonstrate reduced colony size in cisplatin sensitivity colony formation assays . 112 Figure 4.5. UM74B EDD stable knockdown cells show a reduction in proliferation . 116 Figure 4.6. UM22B stable EDD knockdown cell lines demonstrate increased cisplatin sensitivity. 120 Table 4.1. Effects of transient and stable EDD knockdown on apoptosis and cisplatin sensitivity. 126 vi Chapter 5 Figure 5.1. Stable GFP-EDD overexpressing cell lines. 143 Figure 5.2. Mass spectrometry analysis of ubiquitinated proteins from EDD overexpressing cell lines . 144 Figure 5.3. Drug screen for compounds that increase cisplatin sensitivity . 148 Figure 5.4. In vivo model to test increased cisplatin sensitivity due to EDD stable knockdown. 151 vii List of Abbreviations ACVRL1 - activin receptor-like kinase 1 Akt - protein kinase B (PKB) AP-1 - Activator protein 1 APC - adenomatous polyposis coli ATM - ataxia telangiectasia mutated Bcl2 - B cell lymphoma-2 Bcl-xL - B cell.
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