Western University Scholarship@Western Electronic Thesis and Dissertation Repository 6-21-2017 12:00 AM NUAK1 Has Tumor Suppressive Activity in Epithelial Ovarian Cancer Parima Saxena 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 equirr ements for the degree in Master of Science © Parima Saxena 2017 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Cancer Biology Commons, and the Cell Biology Commons Recommended Citation Saxena, Parima, "NUAK1 Has Tumor Suppressive Activity in Epithelial Ovarian Cancer" (2017). Electronic Thesis and Dissertation Repository. 4598. https://ir.lib.uwo.ca/etd/4598 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 NUAK1, a downstream substrate of the stress metabolism regulator Liver kinase B1 (LKB1), has been implicated as an oncogene and tumor suppressor, with distinct roles in cell cycle, senescence and cell adhesion. Epithelial ovarian cancer (EOC) spheroids remain dormant during intraperitoneal metastasis with reduced proliferation and metabolism to survive metabolically harsh conditions, possibly implicating a role of NUAK1 in EOC spheroid biology. I hypothesize that NUAK1 is regulated by LKB1 to promote dormancy in EOC. I demonstrate that NUAK1 expression and phosphorylation is regulated by LKB1 in EOC cell lines. NUAK1 is largely under-expressed in many established and new ascites-derived EOC cell lines; in fact, further NUAK1 knockdown increases spheroid cell viability, size, and reattachment capability. Pharmacologic NUAK1/2 inhibition increased EOC cell growth and clonogenicity. Collectively, my data supports NUAK1 as having tumour suppressive activity downstream of LKB1 in EOC. Keywords Liver Kinase B1, AMPK-related kinase, NUAK1, spheroid, tumor suppressor, epithelial ovarian cancer, metastasis Co-Authorship Statement OVCAR8 STK11KO cells were established by Dr. Trevor Shepherd using CRISPR-Cas9 technology to lack LKB1 expression. iOvCa series, ascites-derived cell lines, were established by Dr. Gabriel DiMattia. siARK screen was performed by Yudith Ramos-Valdes. qPCR for ARK mRNA levels was conducted by Elizabeth Kobylecky. Tritiated thymidine incorporation assay was performed in collaboration with Piru Perampalam and Dr. Fred Dick i Acknowledgments I would like to acknowledge my supervisor, Dr. Trevor Shepherd, for allowing me to take on this interesting and engaging project and for his support throughout the course of my project. I would also like to thank my committee members for all of their insightful guidance which they have provided me, which has been critical for driving this project forward. I would also like to thank Dr. Gabriel DiMattia for all of his contributions towards my project, valuable insight into my work, and for generously allowing me to use the repository of ascites-derived cell lines. Furthermore, I would like to thank my past and present lab mates, Yudith, Adrian, Olga, Jessica and Samah, for not only helping me learn scientific techniques but for also providing an excellent work environment. ii Table of Contents Abstract ................................................................................................................................. Co-Authorship Statement..................................................................................................... i Acknowledgments............................................................................................................... ii Table of Contents ............................................................................................................... iii List of Tables ..................................................................................................................... vi List of Figures ................................................................................................................... vii List of Abbreviations ......................................................................................................... ix Chapter 1 ............................................................................................................................. 1 1 Introduction .................................................................................................................... 1 1.1 Epidemiology, Clinical Presentation and Treatment of Epithelial Ovarian Cancer 1 1.2 Origin and Subtypes of EOC .................................................................................. 3 1.3 EOC Metastasis and Experimental Model Systems................................................ 6 1.4 LKB1-AMPK in EOC Metastasis ........................................................................... 9 1.5 AMPK-Related Kinases and Their Potential Importance in EOC Metastasis ...... 12 1.6 NUAK1 Regulation and Function ........................................................................ 14 1.7 Research Goal, Hypothesis and Experimental Objectives .................................... 17 Chapter 2 ........................................................................................................................... 18 2 Materials and Methods ................................................................................................. 18 2.1 Cell Culture ........................................................................................................... 18 2.2 Western Blot ......................................................................................................... 20 2.3 Phostag™ Western Blot ........................................................................................ 22 2.4 siRNA-mediated knockdown ................................................................................ 22 2.5 Reattachment Assays ............................................................................................ 23 2.6 Clonogenic Assays ................................................................................................ 23 iii 2.7 CellTiter-Glo® Viability Assay ............................................................................ 23 2.8 CyQUANT Viability Assay .................................................................................. 24 2.9 Trypan Blue Exclusion Viability Assay ............................................................... 24 2.10 IC50 Determination .............................................................................................. 25 2.11 Incucyte ZOOM Growth Curves .......................................................................... 26 2.12 Quantitative Real-time PCR ................................................................................. 26 2.13 Tritiated Thymidine Incorporation Assay ............................................................. 29 2.14 Spheroid size determination .................................................................................. 29 2.15 Graphing and Statistical Analysis ......................................................................... 29 Chapter 3 ........................................................................................................................... 30 3 Results .......................................................................................................................... 30 3.1 NUAK1 has limited profile in EOC...................................................................... 30 3.2 NUAK1 expression decreases in spheroid culture................................................ 33 3.3 NUAK1 is regulated by LKB1 ............................................................................. 33 3.4 NUAK1 knockdown increases EOC spheroid cell viability ................................. 36 3.5 NUAK1 and NUAK2 are unable to functionally compensate for each other ....... 38 3.6 NUAK1 inhibition increases EOC cell growth and clonogenicity ....................... 44 3.7 Specific NUAK1 inhibition increases EOC cell growth ....................................... 45 3.8 NUAK1 inhibition is insufficient to induce platinum sensitivity ......................... 51 3.9 NUAK1 inhibition does not affect thymidine incorporation ................................ 55 Chapter 4 ........................................................................................................................... 59 4 Discussion .................................................................................................................... 59 4.1 Summary of findings............................................................................................. 59 4.2 Potential growth suppressive of NUAK1 in EOC ................................................ 60 4.3 Therapeutic implications of results ....................................................................... 63 4.4 NUAK1 and LKB1 ............................................................................................... 63 iv 4.5 Limitations of current study .................................................................................. 64 4.6 Future Work .......................................................................................................... 65 4.7 Overall Conclusion ..............................................................................................