RARRES1 MODULATION OF TUBULIN DEGLUTAMYLATION REGULATES METABOLISM AND CELL SURVIVAL 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 Cell Biology By Sara Maimouni, B.A. Washington, D.C. August 20, 2018 Copyright 2018 by Sara Maimouni All Rights Reserved ii RARRES1 Modulation of Tubulin Deglutamylation Regulates Metabolism and Cell Survival Sara Maimouni, B.A. Thesis Advisor: Stephen Byers, Ph.D. ABSTRACT One of the driving mechanisms of cancer progression is the reprogramming of metabolic pathways in intermediary metabolism. Cancers increase their energy expenditure by increasing ATP production for utilization in anabolic pathways to increase production of proteins, nucleic acids and lipids. The Warburg Effect, where cancer cells predominantly use aerobic glycolysis rather than oxidative phosphorylation to produce ATP, was long thought to be the main initiating pathway in increasing tumor burden. However, compelling new evidence shows that there exists metabolic heterogeneity among and within tumors. Mitochondrial respiration often plays a major role in tumor progression, as many different cancers contain a subpopulation of slow-cycling tumor-initiating cells that are multidrug-resistant and dependent on oxidative phosphorylation. These cells represent a target for cancer therapy. However, the identification of endogenous regulators of mitochondrial respiration is understudied. Depletion of (RARRES1) occurs in many cancers, including melanoma, colon, prostate and breast cancers. Interestingly, in cancers associated with fibrosis, such as triple negative breast cancer, pancreatic and hepatocellular carcinomas, RARRES1 is overexpressed. Its role in metabolism might explain the duality of this protein in cancer. Our data show that RARRES1 and its target CCP2 regulate mitochondrial bioenergetics and subsequently alter energy homeostasis by modulating the function of the mitochondrial voltage-dependent anion channel (VDAC). The changes in energy homeostasis rewire glucose for biosynthetic pathways, such as de novo lipogenesis, that drive the pathogenicity iii and survival of cancer. These data lay the foundation for metabo-therapy of the many tumor types that exhibit RARRES1 depletion or overexpression and may have the added benefit of targeting drug-resistant tumor-initiating cells. i v Dedication This thesis is dedicated to my parents, Ihsan Lemtouni and Rachid Maimouni, who have supported me and sacrificed everything to support my endeavors in the United States. I would have never gotten this far in my career without you. My grandparents, Aboubakr Lemtouni and Rabia Temsamani, who have always echoed to me that “No matter what, education comes first”. To my brother, Karim Maimouni and my sister, Dania Maimouni, who have always believed in me and reminded me why I chose this path in the first place. Acknowledgments I would like to thank Dr. Stephen Byers for his invaluable guidance during my thesis work and his advice and support in my career development. Thank you to Dr. Jessica Jones, Dr. Rebecca Riggins and Dr. Kathryn Sandberg for their mentorship and support. I would like to thank my thesis committee members, Dr. Aykut Uren, Dr. Partha Banerjee, Dr. Amrita Cheema and Dr. Maria Laura Avantaggiati for their guidance during my predoctoral training. Thank you to my lab members and collaborators who have contributed to my research: Naiem Issa, Yong-Sik Bong, Ivana Peran, and Becky Hoxter. Thank you to the students I have mentored and have contributed to my research: Selina Cheng and Asha Krishnakumar. A special thank you to my college advisor, Dr. Kristina Blake-Hodek, who inspired me to pursue my Ph.D. v Table of Contents Introduction ......................................................................................................................................1 RARRES1- A Carboxypeptidase Inhibitor ..................................................................................4 RARRES1 and CCP2, Regulators of the Tubulin Code ............................................................10 The C-Terminal Tail of Tubulin Modulates Mitochondrial VDAC ..........................................14 RARRES1 and CCP2 in Metabolic Diseases ............................................................................18 The Role of RARRES1 and CCCP2 in Tumorigenesis .............................................................19 Statement of Purpose .................................................................................................................24 Specific Aims .............................................................................................................................26 Chapter 1 Tumor Suppressor RARRES1 Links Tubulin Deglutamylation to Metabolic Reprogramming and Cell Survival ...............................................................................................27 Introduction ................................................................................................................................27 Methods......................................................................................................................................28 Results ........................................................................................................................................35 Discussion ..................................................................................................................................62 Chapter 2 RARRES1, A Novel Regulator of Fatty Acid Metabolism .........................................67 Introduction ................................................................................................................................67 Methods......................................................................................................................................68 Results ........................................................................................................................................81 Discussion ..................................................................................................................................98 Chapter 3 Endogenous Transcriptional Regulation of RARRES1 .............................................102 Introduction ..............................................................................................................................102 Methods....................................................................................................................................105 Results ......................................................................................................................................107 Discussion ................................................................................................................................120 Conclusion ..................................................................................................................................125 RARRES1 and Stem Cell Metabolism........................................................................................126 vi RARRES1 and Obesity ..............................................................................................................127 Oncogene or Tumor Supressor?- RARRES1 and Its Role in Fibrosis.....................................129 Role of RARRES1 and CCP2 in Cardiovascular Diseases......................................................130 Other Targets of RARRES1?....................................................................................................132 Appendix: Chapter 3 Supplementary Figures .............................................................................134 Bibliography ...............................................................................................................................143 vii List of Figures Figure 1.1. RARRES1 and Latexin Structure Modeling .................................................................6 Figure 1.2. Phylogenetic Tree of RARRES1 Across All Species .....................................................8 Figure 1.3 RARRES1 (TIG1A) and Its Variant RARRES1 Short (TIG1B) ...................................9 Figure 1.4. The Human Tubulin Isotypes ......................................................................................11 Figure 1.5. The Tubulin Code ........................................................................................................12 Figure 1.6. The Post Translational Modification of GEEY Motif on Tubulin ........................14 Figure 1.7. Multi-Substrate Transport and Gate of Death .............................................................17 − Figure 1.8. Comparison of RARRES1 Across Twelve Analyses of Breast Cancer Stroma, TNBC, Pancreatic Cirrhosis/Cancer, and Liver Cirrhosis Datasets ...........................................................22 Figure 2.1. CCP2 Expression in Tumorigenic and Non-Tumorigenic Cell Lines .........................37 Figure 2.2. CCP2 Splice Variants and Primer Profiling ................................................................38 Figure 2.3. Immunocytochemistry of Polyglutamylated Tubulin .................................................38 Figure 2.4. RARRES1, CCP2 and Retinoic Acid Regulate Tubulin Glutamylation .....................39 Figure 2.5. RARRES1and CCP2 Regulate Apoptosis but Not Cell Proliferation .........................42 Figure 2.6. FACS Analysis