Proteomic Identification of Mediators Implicated in the Metastatic Progression of Ovarian Cancer by Natasha Musrap A thesis submitted in conformity with the requirements for the degree of Doctorate of Philosophy Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Natasha Musrap 2015 Proteomic Identification of Mediators Implicated in the Metastatic Progression of Ovarian Cancer Natasha Musrap Doctor of Philosophy Laboratory Medicine and Pathobiology University of Toronto 2015 Abstract Ovarian cancer (OvCa) is the leading cause of death among gynecological malignancies, and is characterized by peritoneal metastasis and increased resistance to chemotherapy. Acquired drug resistance is often attributed to the formation of multicellular aggregates (MCAs) in the peritoneal cavity, which seed abdominal surfaces, particularly, the mesothelial lining of the peritoneum. Given that the presence of metastatic implants is a predictor of poor survival, a better understanding of the underlying biology surrounding OvCa metastasis may lead to the identification of key molecules that are integral to the progression of the disease, which therefore, may serve as practicable therapeutic targets. To that end, in vitro cell line models of cancer-peritoneal interaction and aggregate formation were used to identify proteins that are differentially expressed during cancer progression, using mass spectrometry-based approaches. First, we performed a proteomics analysis of a co-culture model of ovarian cancer and mesothelial cells, in which we identified numerous proteins that were differentially regulated during cancer-peritoneal interaction. We further validated one protein, MUC5AC, and confirmed its expression at the cancer-peritoneal interface. Next, we conducted a quantitative proteomics analysis of a cell line grown as a monolayer and as MCAs. After identifying a subset of overexpressed proteins, we determined that CLCA1 plays a role in MCA formation. ii Additional studies using a CLCA1 blocker, as well as siRNA knockdown of CLCA1 in OvCa cells, resulted in a decreased ability of cancer cells to aggregate, a reduced ability to adhere to extracellular matrix components, and decreased cell viability. Moreover, we demonstrated that CLCA1 is able to regulate MUC5AC expression, as CLCA1-knockdown cells exhibited reduced expression of MUC5AC. In summary, the research presented in this thesis adds to our current understanding about ovarian cancer progression, and identifies two proteins that play a role in OvCa progression, which may serve as novel therapeutic targets. iii Acknowledgements This thesis would not have been possible without the help and support of many individuals around me, with whom I am very grateful to share this journey. First and foremost, I would like to express my sincere gratitude to my supervisor, Dr. Eleftherios P. Diamandis, for his continuous support and guidance throughout my PhD. I would like to thank him for giving me the opportunity and freedom to pursue my scientific interests, and for his patience and mentorship that led me to complete this work. He taught me that success takes hard work and is an excellent role model of that principle. I would also like to extend my gratitude and thanks to members of my advisory committee, Dr. Bharati Bapat and Dr. Patricia Shaw, for providing valuable advice and feedback throughout the years and challenging me to take my project as far as possible. Also, I would like to thank members of my final oral examination committee, Dr. Ivan Blasutig and Dr. James Scholey, for their time and providing feedback. I am also very grateful to my external examiner, Dr. Barbara Vanderhyden, for dedicating her time in reviewing the thesis and being a key part of my defense. Also, I would like to express my thanks to all past and present members of the Advanced Centre for the Detection of Cancer. You have all made my time here enjoyable and I will definitely miss each and every one of you. Not only have you all helped me grow as a researcher; you have also helped me grow as a person. Thank you to my friends for always supporting me, and thank you to Vince for cheering me on and for reassuring me when I needed it the most. Thanks to members of the Department of Laboratory Medicine and Pathobiology, including Rama Ponda, Ferzeen Sammy, and Dr. Harry Elsholtz, for administrative help in scheduling the iv defense. I would also like to acknowledge the funding I received from the LMP department and the Canadian Institutes of Health Research. Last but not least, none of this work would have been possible without the love and support of my family. Thank you to my brother, Chris, and more importantly, my parents, Joanne and Ramatali, for always believing in me, and encouraging me to do my best – this thesis is dedicated to you. v Table of Contents Abstract ..................................................................................................................................... ii Acknowledgements .................................................................................................................. iv Table of Contents ..................................................................................................................... vi List of Abbreviations ................................................................................................................ x List of Tables ......................................................................................................................... xiii List of Figures ......................................................................................................................... xiv Chapter 1: Introduction ........................................................................................................... 1 1.1 Ovarian Cancer .............................................................................................................. 2 1.1.1 Ovarian cancer statistics and epidemiology ............................................................... 2 1.1.2 Classification and histological subtypes .................................................................... 3 1.1.3 Ovarian cancer diagnosis and treatment .................................................................... 5 1.1.3.1 Diagnosis ............................................................................................................. 5 1.1.3.2 Treatment ............................................................................................................ 6 1.2 The Role of the Tumor Microenvironment during Ovarian Cancer Progression and its Clinical Implications ......................................................................................... 9 1.2.1 Inflammatory cytokine and chemokine networks in ovarian cancer ....................... 11 1.2.2 The role of tumor-associated stromal cells .............................................................. 14 1.2.2.1 Tumor-associated macrophages ........................................................................ 15 1.2.2.2 Cancer-associated fibroblasts ............................................................................ 18 1.2.2.3 Omentum-derived adipocytes ........................................................................... 22 1.2.2.4 Mesenchymal stem cells ................................................................................... 23 1.2.3 Targeting extracellular matrix components ............................................................. 25 1.3 Ovarian Cancer Metastasis ......................................................................................... 27 1.3.1 Ovarian cancer progression to the peritoneum ........................................................ 27 1.3.2 Ovarian cancer spheroid formation .......................................................................... 28 1.4 Proteomic Approaches to Study Ovarian Cancer Biology ....................................... 30 1.4.1 Proteomics initiatives in OvCa ................................................................................ 30 1.4.2 Quantitative proteomics ........................................................................................... 32 1.4.2.1 Labeling approaches ......................................................................................... 33 1.4.2.2 Label-free quantitation ...................................................................................... 33 1.5 Rationale and Goals ..................................................................................................... 35 1.5.1 Rationale .................................................................................................................. 35 1.5.2 Hypothesis ................................................................................................................ 35 1.5.3 Objectives ................................................................................................................ 36 Chapter 2: Proteomic analysis of cancer and mesothelial cells reveals an increase in Mucin 5AC during ovarian cancer and peritoneal interaction ...................................... 38 2.1 Introduction .................................................................................................................. 39 2.2 Materials and Methods ................................................................................................ 42 vi 2.2.1 Cell lines .................................................................................................................