ADissertation entitled The Role of mDia2 in Adherens Junctions in Epithelial Ovarian Cancers by Yuqi Zhang Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences Kathryn Eisenmann, Ph.D., Committee Chair Rafael Garcia-Mata, Ph.D., Committee Member Randall Ruch, Ph.D., Committee Member Ivana de la Serna, Ph.D., Committee Member Eda Yildirim-Ayan, Ph.D., Committee Member Dr. Cyndee Gruden, Dean College of Graduate Studies The University of Toledo February 2019 Copyright 2019, Yuqi Zhang This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of The Role of mDia2 in Adherens Junctions in Epithelial Ovarian Cancers by Yuqi Zhang Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences The University of Toledo February 2019 Epithelial ovarian cancer (EOC) cells disseminate within the peritoneal cavity, in part, via the peritoneal fluid as single cells, clusters, or spheroids. Initial single cell egress from a tumor can involve disruption of cell-cell adhesions as cells are shed from the primary tumor into the peritoneum. In epithelial cells, Adherens Junc- tions (AJs) are characterized by homotypic linkage of E-cadherins on the plasma membranes of adjacent cells. AJs are anchored to the intracellular actin cytoskele- tal network through a complex involving E-cadherin, p120 catenin, β-catenin, and αE-catenin. However, the specific players involved in the interaction between the junctional E-cadherin complex and the underlying F-actin network remains unclear. Recent evidence indicates that mammalian Diaphanous-related (mDia) formins plays a key role in epithelial cell AJ formation and maintenance through generation of lin- ear actin filaments. We previously demonstrated that loss of the formin mDia2 was specifically associated with invasive single cell egress from EOC spheroids through disruption of junctional F-actin. In this work, we show that mDia2 has a role at AJs in ovarian cancer (OVCA) 429 cells and human embryonic kidney (HEK) 293 cells through its association with αE-catenin and β-catenin. mDia2 depletion in EOC cells leads to reduction in actin polymerization and disruption of cell-cell junctions with decreased interaction between β-catenin and E-cadherin. In summary, our findings indicate an essential role for mDia2 in AJ formation and stability in EOC cells. These iii effects are likely achieved through its interactions with and regulation of α-andβ- catenin. Our findings support a novel mechanism for EOC dissemination that should be considered in development of targeted therapy against this deadly disease. iv Acknowledgments IwouldliketothankmyadvisorDr.KathrynEisenmannforgivingmetheopportu- nity to work with her and her mentorship over the past several years. Her guidance and assistance were essential to the successful completion of the experiments pre- sented in this work. I also thank my committee members for their helpful insights. I am also grateful for the members of the Eisenmann lab and all my friends and teach- ers in the Cancer Biology department for supplying me with the technical toolbox to execute these experiments. Last but not least, I thank my parents for believing in me all these years and lending me the strength to get to where I am today. v Contents Abstract iii Acknowledgments v Contents vi List of Figures xi List of Abbreviations xiii 1 An Introduction to Epithelial Ovarian Cancer, Adherens Junctions, and mDia Formins 1 1.1 An Introduction to Epithelial Ovarian Cancer (EOC) and its treatment 1 1.1.1 The epidemiology of EOC . 1 1.1.2 Symptoms and Diagnosis . 3 1.1.3 Prognosis and Treatment . 4 1.1.3.1 TreatmentofchemoresistantEOC . 5 1.2 Molecular mechanisms of EOC development and metastasis . 6 1.2.1 Molecular characteristics of EOC . 6 1.2.1.1 Low-grade serous ovarian cancer . 6 1.2.1.2 High-gradeserousovariancancer . 7 1.2.2 Origins of EOC . 9 1.2.3 Mechanisms of EOC metastasis . 11 1.2.3.1 HematogenousDissemination . 11 vi 1.2.3.2 Passive Dissemination . 12 1.2.3.3 RoleofAscitesinEOCMetastasis . 13 1.2.3.4 EOC seeding within the abdominal cavity . 14 1.2.3.5 EMT in EOC metastasis . 14 1.2.3.6 Potential therapeutic targets in EMT . 16 1.2.3.7 EMT and MET in EOC metastasis and chemoresistance 17 1.2.3.8 PartialEMT/METinEOC . 18 1.2.3.9 EMTandchemoresistance . 19 1.3 EMT and the Adherens Junction (AJ) . 20 1.3.1 AJformationandstabilization: anoverview . 20 1.3.2 The cytoskeleton in AJ stability and cell motility . 23 1.3.2.1 The cytoskeleton is necessary for AJ formation and stability . 23 1.3.2.2 The actin cytoskeleton . 24 1.3.2.3 The microtubule cytoskeleton . 30 1.3.2.4 Intermediate filaments . 34 1.3.3 MechanismsofJunctionDestabilization. 34 1.3.4 Cell motility is achieved through regulation of the cytoskeleton 35 1.3.4.1 Overview of membrane protrusions . 36 1.3.5 AJ regulators: cadherins, catenins, and vinculin . 38 1.3.5.1 Cadherins . 38 1.3.5.2 α-catenins . 39 1.3.5.3 β-catenin . 39 1.3.5.4 p120 catenin . 40 1.3.5.5 Vinculin......................... 40 1.4 Formin proteins: an introduction . 41 1.4.1 Anoverviewofformins. 41 vii 1.4.2 Structure of mDia formins . 41 1.4.3 LocalizationandfunctionofmDiaformins . 43 1.4.3.1 Formin function determines their intracellular local- ization . 43 1.4.3.2 ForminsinAJs ..................... 45 1.4.3.3 Integrative functions of mDia formins. 47 1.4.3.4 Regulation by Rho-GTPases . 48 1.4.3.5 Dia-interacting protein (DIP) and the cell cycle in mDia2 regulation . 50 1.5 Formins in development and disease . 50 1.5.1 Forminsindevelopment . 50 1.5.2 Forminsinimmunityandhomeostasis . 51 1.5.3 Formins in cancer . 52 1.5.4 Formins as therapeutic targets . 55 1.5.4.1 Formin inhibition . 55 1.5.4.2 Formin activation . 57 1.6 SummaryofPastFindingsandGapsintheKnowledge . 58 1.7 Hypothesis................................. 59 2 Results 61 2.1 Introduction . 61 2.2 Results . 65 2.2.1 mDia2 is essential for junction integrity in spheroids. 65 2.2.2 RoleofmDia2inAJformation . 67 2.2.3 mDia2 interacts with β-andα-catenin but not E-cadherin . 69 2.2.4 mDia2 co-precipitates with β-andα-catenin in HEK293 cells 72 2.2.5 mDia2 affects junctional stability . 73 viii 2.2.6 mDia2 expression affects interactions between junctional proteins 76 2.2.7 Actin disruption does not inhibit interactions between mDia2 and α-andβ-catenin . 78 2.3 Discussion . 80 2.4 Conclusions . 84 2.5 Methods . 85 2.5.1 Cell lines and reagents . 85 2.5.2 Western blotting . 85 2.5.3 Transfection and knockdown . 86 2.5.4 Immunoprecipitation . 86 2.5.5 ImmunofluorescenceandImageAnalysis . 87 2.5.6 InSituProximityLigationAssay(PLA) . 88 2.5.7 HangingDropAssay ....................... 89 2.5.8 CalciumSwitchAssay ...................... 90 2.5.9 Statistics . 90 2.6 Acknowledgements ............................ 90 2.7 Funding . 91 3 Discussion 92 3.1 mDia2 interacts with both α-andβ-catenin . 92 3.2 Biomechanics of EOC . 93 3.3 Limitations . 93 3.4 Future Directions . 94 3.5 Summary of findings . 94 References 96 A Investigation of the respiratory diaphragm as a key site of EOC ix invasionandmetastasis 149 A.1 Introduction................................ 149 A.2 Methods: Development of an ex vivo diaphragm stretch model . 150 A.3 Results................................... 151 x List of Figures 1-1 Two models for anchorage of the E-cadherin/catenin complex to F-actin. 22 1-2 Formins mediate linear actin polymerization. 26 1-3 Arp2/3/mediatesbranchedactinpolymerization. 27 1-4 mDia formin domains. 42 2-1 Analysis of mDia2 in a functional cell-cell adhesion assay. 66 2-2 Quantification of functional cell-cell adhesion assay . 67 2-3 Effect of mDia2 on adherens junction formation. 68 2-4 mDia2 interacts with β-catenin and αE-catenin but not E-cadherin. 70 2-5 mDia2 interacts with β-catenin and αE-catenin but not E-cadherin. 71 2-6 mDia2 does not interact with E-cadherin. 72 2-7 mDia2 co-precipitates with αE- and β-catenininHEK293cells. 73 2-8 mDia2 expression affects junctional stability. 74 2-9 Two models for anchorage of the E-cadherin/catenin complex to F-actin. 75 2-10 mDia2 expression affects interactions between junctional proteins. 76 2-11 Quantification of interactions between junctional proteins. 77 2-12 Actin disruption does not inhibit interactions between mDia2 and α or βcatenin. 79 2-13 Quantification of interactions between mDia2 and α or βcatenin. 80 A-1 Spheroid invasion at 48 hours on stretched diaphragm explant. 151 A-2 Spheroid invasion at 48 hours on unstretched diaphragm explant. 152 xi A-3 Normalized areas of GFP-SKOV3 invasion on stretched and unstretched diaphragms. 153 A-4 GFP-SKOV3 spheroids demonstrate collagen alignment and egress from the spheroid as single cells and clusters. 153 A-5 X-Z projections of GFP-SKOV3 spheroids seeded on stretched and un- stretched diaphragm explants. 154 xii List of Abbreviations Arp ....................... actin-related proteins AJ ........................ adherens junction BD . basic domain BMP . bone morphogenetic protein CC . coiled coil DAD ...................... diaphanous auto-regulatory domain DD . dimerization domain DID . diaphanous inhibitory domain DIP . Dia-interacting protein DRFs ..................... Diaphanous-related formins EGFR .................... Epidermal Growth Factor Receptor EOC ...................... epithelial ovarian cancer ESPs ..................... early serous proliferations EMT . epithelial-to-mesenchymal FA ........................ focal adhesion FSH ...................... follicle-stimulating hormone FH ........................ formin homology GBM . glioblastoma GAP ...................... GTPase-activating protein GDI . guanine nucleotide displacement inhibitor GEF . guanine nucleotide exchange factors GFP ...................... Green Flourescent Protein IL .