The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 12-2019 Uncovering the ZEB1 Interactome to Identify Novel Regulators of Metastatic Non-small Cell Lung Cancer Roxsan Manshouri Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Biology Commons, Cancer Biology Commons, Cell Biology Commons, and the Medicine and Health Sciences Commons Recommended Citation Manshouri, Roxsan, "Uncovering the ZEB1 Interactome to Identify Novel Regulators of Metastatic Non- small Cell Lung Cancer" (2019). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 976. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/976 This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. Uncovering the ZEB1 Interactome to Identify Novel Regulators of Metastatic Non-small Cell Lung Cancer by Roxsan Manshouri, M.S. APPROVED: ______________________________ Don L. Gibbons, M.D./Ph.D. Advisory Professor ______________________________ Lauren A. Byers, M.D. ______________________________ Andrew Gladden, Ph.D. ______________________________ Michelle C. Barton, Ph.D. ______________________________ Pierre McCrea, Ph.D. APPROVED: ____________________________ Dean, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences i Uncovering the ZEB1 Interactome to Identify Novel Regulators of Metastatic Non-small Cell Lung Cancer A DISSERTATION Presented to the Faculty of The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY by Roxsan Manshouri, M.S. Houston, Texas August 23, 2019 ii Acknowledgements I would like to thank my thesis advisor, Dr. Don Gibbons, who has been a wonderful mentor throughout these past six years. He has continuously challenged, encouraged, and amused me. Thank you for taking the chance on me. Thank you to all my committee members Drs. Michelle Barton, Andrew Gladden, Lauren Byers, and Pierre McCrea for their constructive feedback and support. I would also like to acknowledge Dr. Amin, who has been instrumental to my education at MD Anderson. His mentorship throughout my formative years was extremely influential to my decision to pursue my studies at GSBS and I will always be grateful. I’d like to thank the members of the Gibbons’ lab for their support. I would specifically like to thank Dr. Samrut Kundu who patiently trained me and has continued to help me develop this project and Drs. Christin Ungewiss and Jonathon Roybal for answering every foolish question — admittedly there were quite a few. Also, thank you to the entire Gibbon’s laboratory for guiding me scientifically and making every day at work too much fun. And most importantly, I would like to acknowledge my family and Alex. Thank you to my parents and brother who encouraged me to pursue a PhD. Thank you to my father who has been an attentive study partner and sounding board whenever the situation required. Lastly, thank you Alex who has stabilized me throughout the stressful moments and has provided the laughter to revitalize me after draining work days. Thank you all for being there for me. iii Uncovering the ZEB1 Interactome to Identify Novel Regulators of Metastatic Non-small Cell Lung Cancer Roxsan Manshouri, M.S. Advisory Professor: Don L. Gibbons, M.D./Ph.D. Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death in the United States, due in part to the robust affinity of lung cancer cells to metastasize. Understanding the processes that contribute to metastasis provides promise for the discovery of novel therapeutic targets. Epithelial-to- mesenchymal transition (EMT) is a proposed model for the initiation of metastasis. During EMT cell adhesion and polarity is reduced, allowing epithelial cancer cells to dissociate from the primary tumor and invade distant organs. The transcription factor zinc finger E-box-binding homeobox 1 (ZEB1) has been reported to uniquely correlate with NSCLC disease progression and to confer therapy resistance in multiple tumor types. Additionally, depletion of ZEB1 has been found to reverse therapy resistance, hence uncovering regulators of ZEB1 provides promise for innovative therapeutic strategies that may improve lung cancer patient outcome. Recent publications demonstrate that ZEB1 undergoes post-translational modification, suggesting a method for regulating ZEB1 function; however, the extent to which ZEB1 is modified as well as the purpose of ZEB1 modification has not been fully elucidated. Here, we apply two independent screens- BioID and an Epigenome shRNA dropout screen- to define ZEB1 interactors that regulate post-translational modification and are critical to metastatic NSCLC. These screens revealed an interaction amongst ZEB1 and the histone deacetylase (HDAC)-containing nucleosome and deacetylase remodeling complex (NuRD). Through treatment with class I HDAC inhibitors Trichostatin A, we identified that ZEB1 homodimerizes and determine that acetylation at lysine residue 811 regulates this association. Furthermore, we identify the NuRD complex as a novel ZEB1 co-repressor and the Rab22 GTPase activating protein TBC1D2b as a ZEB1/NuRD complex target. We find that TBC1D2b suppresses E-cadherin internalization, thus hindering cancer cell invasion and metastasis. Ultimately, this project provides a novel regulatory node for ZEB1 function and insight to the role of EMT in endocytosis. iv Table of Contents Approvals i Titles ii Acknowledgements iii Thesis Abstract iv Table of Contents v Chapter 1: Introduction 1 Lung cancer 2 Post-transcriptional regulation of E-cadherin 4 Transcriptional regulation of E-cadherin 5 BioID screening method 7 Transcription factor dimerization 9 Chapter 2: Materials and Methods 11 Cell Culture 12 BioID 12 Epigenome Screen 13 Quantitative Real-Time PCR Analysis 17 Antibodies and Immunofluorescence 17 Proximity Ligation Assay 17 Gel Filtration 18 Immunoprecipitation 18 RNA Interference 18 Luciferase Reporter Assays 19 Chromatin Immunoprecipitation 19 Migration and Invasion Assay 20 Biotin Internalization Assay 21 v In vivo tumor and metastasis experiments 21 Biotin Internalization Assay 21 3D Culture 22 Lambda Phosphatase incubation 22 Chapter 3: ZEB1/NuRD Complex Suppresses TBC1D2b to Stimulate 23 E-cadherin Internalization to Promote Invasion and Metastasis in Lung Cancer BioID screen reveals ZEB1 interactome 24 Loss-of-function screen identifies vulnerabilities in NSCLC 27 ZEB1 interacts with the NuRD complex 33 The CHD4-containing NuRD complex is a ZEB1 co-repressor 37 Identifying targets of a ZEB1/NuRD complex 42 TBC1D2b/Rab22 axis mediate E-cadherin endocytosis 51 TBC1D2b hinders NSCLC metastasis 62 Chapter 4: ZEB1 acetylation precludes homodimerization 72 Class I HDAC inhibitors regulate ZEB1 dimerization 73 ZEB1 dimerization is regulated by acetylation 77 Chapter 5: Discussion 86 Chapter 6: Future directions 82 Bibliography 97 Vita 103 vi CHAPTER 1: Introduction 1 Lung cancer Lung cancer is the leading cause of all cancer related death in the United States and worldwide1. The two major types of lung cancer are non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)2. NSCLC constitutes approximately 85% of lung cancers and is comprised of three subtypes: squamous cell carcinoma, large cell carcinoma and the most common histologic type, adenocarcinoma. Poor patient prognosis is primarily due to the affinity of tumors to metastasize leading to advanced disease at the time of diagnosis2. Nearly two-thirds of lung cancer patients are diagnosed with metastatic disease. Despite ongoing research the 5 year survival rate of lung cancer patients has changed little in the last decade due in part to the incomplete understanding of the metastatic process. To study the biologic processes driving lung cancer progression, several genetically engineered mouse models have been generated3. The somatic activation of the KrasG12D allele (KrasLA1) was found to contribute to the development spontaneous lung adenocarcinoma; however these models lacked metastatic potential4. Introduction of a mutant p53 allele (p53R172H∆G)- commonly found in Li-Fraumeni syndrome patients- to the KrasLA1 mouse model (denoted as KrasLA1p53R172HDG or KP mouse model) was found to recapitulate features of metastasis-prone lung adenocarcinoma cancer patients. To understand the molecular mechanisms driving metastatic disease our group derived a panel of cell lines from the primary and various metastatic tumor sites of the KP model 4. These cell lines were subcutaneously injected into syngeneic mice to evaluate the propensity to metastasize. The cell line 393P (derived from primary lung tumor) was defined as a metastasis incompetent cell line, 344SQ (subcutaneous