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Elucidating the Mechanism of NUAK1 Function in Ovarian Cancer Metastasis Jamie Fritz the University of Western Ontario

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Elucidating the Mechanism of NUAK1 Function in Ovarian Cancer Metastasis Jamie Fritz the University of Western Ontario Western University Scholarship@Western Electronic Thesis and Dissertation Repository August 2019 Elucidating the mechanism of NUAK1 function in Ovarian Cancer Metastasis Jamie Fritz The University of Western Ontario Supervisor Dr. Trevor Shepherd The University of Western Ontario Graduate Program in Anatomy and Cell Biology A thesis submitted in partial fulfillment of the requirements for the degree in Master of Science © Jamie Fritz 2019 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Cancer Biology Commons Recommended Citation Fritz, Jamie, "Elucidating the mechanism of NUAK1 function in Ovarian Cancer Metastasis" (2019). Electronic Thesis and Dissertation Repository. 6364. https://ir.lib.uwo.ca/etd/6364 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract Epithelial ovarian cancer (EOC) has a unique mode of metastasis, where upon shedding from the primary tumor, cells form spheroids and spread through the peritoneal cavity. There is a need to elucidate pathways driving spheroid formation to identify novel therapeutic targets. We previously showed that LKB1 is required for EOC metastasis. Using multiplex inhibitor bead-mass spectrometry, we identified NUAK1 as a top LKB1 substrate candidate. We confirmed LKB1 maintains NUAK1 phosphorylation and expression. NUAK1KO cells had lower cell adhesion and generated spheroids with reduced integrity. We identified a cell attachment pathway that was enriched in parental compared with NUAK1KO spheroids. The FN1 gene, encoding fibronectin, exhibited the greatest differential expression in NUAK1KO spheroids. Parental spheroids have enhanced fibronectin expression, which was undetectable in NUAK1KO spheroids. Treatment of NUAK1KO cells with fibronectin restored a compact spheroid phenotype. We have identified a novel mechanism where NUAK1 promotes spheroid formation through fibronectin deposition. Keywords Liver Kinase B1, AMPK-related kinase, NUAK1, spheroid, fibronectin, adhesion, epithelial ovarian cancer, metastasis ii Co-Authorship Statement OVCAR8 STK11KO, NUAK1KO, HEYA8 and OVCAR3 NUAK1 overexpressing cells were established by Dr. Trevor Shepherd and Olga Collins. iOvCa series, ascites-derived cell lines, were established by Dr. Gabriel DiMattia. Multiplex inhibitor bead-mass spectrometry preparation and analysis was completed by Dr. Adrian Buensuceso, Dr. Trevor Shepherd, Kyle Francis, Kevin Brown, Karen Colwill, Anne- Claude Gingras, and Robert Rottapel. Parima Saxena completed the phostagTM western blot analysis. Tumor samples used for this study were collected from an EOC xenograft study completed by Dr. Adrian Buensuceso. Experiments to assess single cell adhesion, morphology of NUAK1KO spheroids, and live cells in NUAK1KO spheroids labelled with GFP were completed by Olga Collins. Immunofluorescence was completed by Olga Collins. Experiments that measured the viability of spheroids with NUAK1 knocked down were completed by Parima Saxena. Western blots confirming NUAK1 knockdown were completed by Parima. Experiments measuring the doubling time of cells treated with WZ4003 were completed by Parima. Experiments that measured viability and doubling time of NUAK1KO spheroids and spheroids overexpressing NUAK1 were completed by Olga Collins. Tritiated thymidine incorporation assay was performed in collaboration with Pirunthan Perampalam and Dr. Fred Dick. Dr. Trevor Shepherd wrote the method sections describing the generation of NUAK1KO and NUAK1 overexpressing cells. Parima Saxena wrote the method sections for experiments she completed, including the phostagTM western blot, spheroid viability assay, and tritiated thymidine incorporation assay. Dr. Karen Colwill wrote the method sections describing Multiplexed Inhibitor Bead (MIB) Chromatography, Mass Spectrometry Analysis, and Mass Spectrometry Data Analysis. iii Acknowledgments First, I am sincerely thankful to my supervisor, Dr. Trevor Shepherd, for his support, encouragement, and guidance throughout my master’s program. I would also like to thank Dr. Gabriel DiMattia for providing valuable insight into my project and for allowing me to use cells from the repository of patient ascites-derived cell lines. I would also like to acknowledge my advisory committee, Dr. Paul Walton, Dr. Silvia Penuela, and Dr. Chris Pin for all of their guidance during my master’s degree. Finally, I would like to thank all members of the Translational Ovarian Cancer Research Unit, including Adrian, Olga, Yudith, Bart, and Jeremi for helping me learn lab techniques, providing technical assistance, and creating an enjoyable lab environment. iv Table of Contents Abstract .......................................................................................................................... ii Co-Authorship Statement............................................................................................... iii Acknowledgments ......................................................................................................... iv Table of Contents ............................................................................................................v List of Figures .............................................................................................................. vii List of Supplementary Figures ..................................................................................... viii List of Supplementary Tables ........................................................................................ ix List of Abbreviations .......................................................................................................x Chapter 1 .........................................................................................................................1 1 Introduction ................................................................................................................1 1.1 Ovarian cancer epidemiology, clinical presentation, and diagnosis .......................1 1.2 Treatment for advanced-stage ovarian cancer .......................................................2 1.3 High-grade serous ovarian cancer .........................................................................3 1.4 Ovarian cancer metastasis and experimental model system...................................4 1.5 Ovarian cancer spheroid formation .......................................................................8 1.6 The role of LKB1 in ovarian cancer metastasis.....................................................9 1.7 NUAK1: Structure and Regulation ..................................................................... 13 1.8 The role of NUAK1 in tumor progression .......................................................... 16 1.9 Research goal, hypothesis, and objectives .......................................................... 20 1.10 References ......................................................................................................... 21 Chapter 2 ....................................................................................................................... 30 2 A novel role for the LKB1 target NUAK1 in promoting ovarian cancer spheroid formation through regulation of fibronectin deposition .............................................. 30 2.1 Introduction ....................................................................................................... 30 2.2 Materials and Methods ....................................................................................... 33 2.2.1 Antibodies and Reagents ........................................................................ 33 2.2.2 Cell culture and treatments ..................................................................... 33 2.2.3 Generation of OVCAR8 STK11-KO and OVCAR8 NUAK1-KO Cells .. 34 2.2.4 Generation of HEYA8 and OVCAR3 NUAK1 overexpressing cells ........ 35 2.2.5 Immunoblot analysis .............................................................................. 35 2.2.6 PhostagTM Western Blot ......................................................................... 36 2.2.7 Immunofluorescence .............................................................................. 37 2.2.8 Quantitative RT-PCR ............................................................................. 37 2.2.9 Tandem Ubiquitin Binding Entities (TUBEs) ......................................... 37 2.2.10 Determination of Doubling Time ............................................................ 38 2.2.11 IncuCyte Zoom Live Cell Analysis System to image spheroid morphology in real-time ............................................................................................. 39 2.2.12 Phase contrast images of spheroids ......................................................... 39 2.2.13 Spheroid viability assay .......................................................................... 39 2.2.14 Timed adhesion assay ............................................................................. 39 2.2.15 Small-interfering RNA Transfection ....................................................... 40 2.2.16 Tritiated Thymidine Incorporation Assay................................................ 40 2.2.17 Multiplexed Inhibitor Bead (MIB) Chromatography ............................... 41 2.2.18 Mass Spectrometry Analysis ..................................................................
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