Characterizing the Role of Protein Tyrosine Kinase 7 (PTK7) in Cancer Development and Cellular Fitness by Sachin Anand Kumar A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto © Copyright by Sachin Anand Kumar (2015) Characterizing the Role of Protein Tyrosine Kinase 7 (PTK7) in Cancer Development and Cellular Fitness Sachin Anand Kumar Master of Science Department of Molecular Genetics University of Toronto 2015 Abstract Protein Tyrosine Kinase 7 has been recently affiliated with the development and progression of various cancers. Contradictory results about its expression, localization and processing across various cancer types has left a major gap in understanding its molecular mechanism and function. In my thesis, I utilized large data sets from essentiality screens and RNAseq, as well as antibody-related tools, to investigate the role of PTK7 in cellular fitness. I observed a negative correlation between PTK7 processing and its degree of essentiality. Furthermore, I demonstrated that the PTK7 intracellular domain (ICD) is integral to its function. Treatment of mouse xenografts with PTK7 antibodies, capable of blocking PTK7 processing, inhibited tumour growth in an ICD-expression dependent manner. Lastly, mRNA profiling of PTK7-knockdown cells and ICD over-expression cells provided a list of putative downstream gene targets under PTK7 regulation. Collectively, these findings implicate PTK7 in cellular transformation and provide insight into its signaling mechanism. II Acknowledgments First and foremost, I would like to thank my supervisor Dr. Jason Moffat. You gave me an opportunity to step outside of my comfort zone and partake in research that pushed the boundary of science, technology and discovery. Your mentorship and creativity have been an inspiration to me – teaching me how to be a critical scientist, think outside of the box and perseverant throughout the hardships of research. Thank you for always being candid with me about my capabilities, strengths and weaknesses and pushing me to achieve more than I knew I was capable of. From you I have learned the tools and foundation I need to pursue my dream career as a physician scientist. To my committee members Dr. Jeff Wrana and Dr. Igor Stagljar, thank you for your guidance, wealth of knowledge and constructive criticism. The rigorous perspective I have developed in my scientific questioning and approach were an integral part of my success, and will only flourish going forward. Thank you to my colleagues in the Moffat Lab, past and present. To Andrea, thank you for setting a rigorous example of what scientific questioning should be. Regardless of the ups and downs over the past years, what I have learned and taken away from this experience is invaluable. A special thank you to Peter Xu, who kept me motivated when the multi-day experiments failed and provided insight when I was out of ideas. The late night hours of lab work wouldn’t have been the same. To Megha, Kristin, Michael, Taras, Carly, Hayoung, Clara and Zvezden, thank you for providing countless memories and hilarious discussions – these are the moments that make graduate school a truly wonderful experience. Kevin and Traver, you always indulged my curiosity and pushed me to learn coding to test every single theory I had – you’ve made me a better scientist. Last but not least, to Patti and Christine, thank you for keeping our lab functioning. Whether ordering things last minute or rolling up your sleeves to help us get stuff done, I truly appreciate your commitment to our success and the wealth of knowledge you both have about pretty much every technique ever. To my friends and supporters (you know who you are), thank you for keeping my spirits high. Research finds unique ways to push you and test your limits. You were always there to help me push back. Last, but certainly not least, thank you to my family. To Mom & Dad, you never III really understood what I was working on, but let me bore you with the details anyways because you knew how passionate I was. That support is a huge factor in making me who I am, and where I am, today. To my brother Rishi, you always knew I could achieve anything I put my mind to. Some days that’s the only thing that kept me going. Thank you. IV Table of Contents Acknowledgments .......................................................................................................................... III Table of Contents ............................................................................................................................ V List of Tables ............................................................................................................................... VII List of Figures ............................................................................................................................. VIII List of Abbreviations ...................................................................................................................... X Chapter 1 Introduction .....................................................................................................................1 1.1 Signaling and activation of receptor tyrosine kinases ..........................................................1 1.2 Receptor tyrosine kinases in the development and progression of cancer ...........................5 1.3 Screening for genetic-based fitness defects in cancer ..........................................................6 1.4 Protein tyrosine kinase 7 (PTK7) .......................................................................................10 1.4.1 PTK7 in development and Wnt signaling ..............................................................11 1.4.2 Post-translational processing of PTK7 ...................................................................13 1.4.3 PTK7 as a prognostic biomarker in cancer ............................................................15 1.5 Thesis objectives ................................................................................................................15 Chapter 2 The intracellular domain of PTK7 is integral for proliferation .....................................17 2 Acknowledgements ...................................................................................................................17 2.1 Materials and Methods .......................................................................................................17 2.1.1 Cell Culture ............................................................................................................17 2.1.2 Effect of PTK7 knockdown on proliferation .........................................................18 2.1.3 Western Blot ..........................................................................................................19 2.1.4 Over-expression of PTK7 ......................................................................................20 2.1.5 Anchorage independent growth .............................................................................20 2.1.6 Immunofluorescence of PTK7 fragments ..............................................................21 2.1.7 ELISA and Cytometric Bead Array .......................................................................21 V 2.1.8 Tumour xenografts and antibody treatment ...........................................................23 2.1.9 Cleavage assessment of recombinant PTK7 protein ..............................................23 2.1.10 Cellular RNA isolation and sequencing .................................................................24 2.2 Results ................................................................................................................................25 2.2.1 PTK7 knockdown negatively affects cancer cell fitness .......................................25 2.2.2 PTK7 full-length rescues this proliferation defect .................................................28 2.2.3 PTK7 intracellular domain localizes to the nucleus ..............................................30 2.2.4 PTK7 intracellular domain is sufficient to partially rescue the fitness defect observed upon knockdown ....................................................................................32 2.2.5 Processing of PTK7 negatively-correlates to its essentiality .................................32 2.2.6 PTK7 antibodies demonstrate tumour growth inhibition potential in vivo ............34 2.2.7 PTK7 intracellular domain rescues the antibody effect in vivoError! Bookmark not defined. 2.2.8 Antibodies block cleavage of PTK7 in vitro and in vivo .......................................36 2.2.9 RNAseq of PTK7 knockdown and ICD over-expression identify putative targets for PTK7 associated function .....................................................................39 2.3 Discussion ..........................................................................................................................43 Chapter 3 Summary and Future Directions ...................................................................................49 3 Summary ...................................................................................................................................49 3.1 Future Directions ...............................................................................................................50 3.1.1 Determining protein interactors and transcriptional targets of the PTK7 intracellular domain ...............................................................................................50