UCL-University College London Institute of Ophthalmology Faculty of Brain Sciences Roles of the small leucine-rich repeat proteoglycans OMD and PRELP in development and cancer By Vasiliki Papadaki Thesis submitted to University College London for the degree of Doctor of Philosophy 2014 1 Declaration I, Vasiliki Papadaki confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Also, the current thesis does not exceed the maximum limit of 100,000 words. 2 Abstract Osteomodulin (OMD) and Proline/arginine-rich and Leucine-rich Repeat protein (PRELP) belong to the small leucine-rich repeat proteoglycan (SLRP) family and as extracellular matrix components have the ability to influence various cellular functions, including cell growth, migration and proliferation, while their mutation or aberrant expression can cause developmental disorders and cancer. This thesis extends previous work in further understanding the roles of OMD and PRELP in cancer and also during mouse development. In the current project OMD and PRELP are overexpressed in a bladder cancer cell line where they are found to alter cell morphology, reduce cell invasion and anchorage-independent growth, while they also inhibit tumour growth in xenograft mouse models. Additionally, we show that OMD and PRELP mediate their effects by cross-regulating different signalling pathways and also by affecting tight junction formation. Furthermore, novel knock-out mouse models of OMD and PRELP were generated, where a Lac-Z cassette has been inserted in the coding regions of the two genes, allowing us to follow their expression under X-gal staining. Therefore a detailed analysis of expression patterns was initially conducted. OMD and PRELP were both expressed in skeletal elements during mouse development, while in adult mice they were differentially expressed in neurons and epithelia of the brain and the eye, and in the urothelium of the bladder. Finally, the novel knock-out mice were used to assess any cancer-related aberrant phenotypes arising from OMD and PRELP deficiency. The bladders of the knock-out mice presented with early stages of urothelial papillary formations, where the junctional complexes were also disrupted, suggesting that lack of OMD and/or PRELP is permissive for cancer initiation. Overall, with our current findings we hope to improve the understanding of SLRP biology in carcinogenesis, and we would like to propose OMD and PRELP as potential targets for the development of cancer therapies. 3 Publications Part of the work outlined in this thesis is associated with the following publications and presentations: Papadaki V*, Hamamoto R*, Tamura T*, Watson J*, Dellett M, Sasai N, Nik- Zainal S, Longbottom R, Nakakido M, Saloura V, Veerakumarasivam A, Neal D, Murphy G, Ponder B, Tsumoto K, Nakamura Y, Kelly J, Ohnuma S, "Osteomodulin and PRELP are novel redundant tumor-suppressor genes involved in bladder cancer initiation", Currently ready for submission to Cell Papadaki V*, Dellett M*, Hu W*, Ohnuma S: ˝Small leucine rich proteoglycan family regulates multiple signalling pathways in neural development and maintenance˝, Review, Dev Growth Differ. 2012 Apr;54(3):327-40 Poster presentation: ˝Roles of SLRP proteins in neural development and function˝, Vasiliki Papadaki (presenting author), Margaret Dellett, Shin-ichi Ohnuma. Society for Neuroscience-SFN 2013 Annual Meeting (November 9-13, 2013), San Diego, California *These authors made an equal contribution to this paper 4 Acknowledgments Firstly, I would like to thank my supervisor, Prof. Shin-ichi Ohnuma, for providing me with the opportunity to undertake my PhD in his lab and also for his support and guidance. I would also like to thank all the members of the Ohnuma laboratory. I am extremely grateful to Dr. Margaret Dellett who stood both like a supervisor and a friend to me. Her advice and help was crucial (especially with all the mouse techniques) and she managed to always lift my confidence and motivation when needed. A big thank you goes to Kristin Lühders as well, who was always supporting me through my difficulties and for being the best person to travel with. Our first conference trip together to San Diego couldn’t have been better. My gratitude also goes to Dr. Toshiya Tamura, whose experience and scientific knowledge were vital for the continuation of my project, and also for his help with my initial experiments. Further thanks go to the other lab members Nasrin, Michelle, Wanzhou and Steve for being always kind and helpful. An extra thank you goes to Steve for all his help with the mice. I would like to also mention the girls from the 4th floor, Laura, Ewa, Asma, Lucy and also Su, with whom the lunchtime discussions were always a welcome distraction. I am also grateful to Peter Munro and Mark Hayes for their help with the confocal and EM. Thank you to UCL and especially Takeda Pharmaceutical Company for providing the funding for this research and also the knock-out mice. Finally, thank you to all my friends outside of the lab Michalis, Ioanna, Kostas, Anastasis, Justyna for being so supportive and understanding over the past three years. Also, many thanks go to my neighbour and friend Vanessa, our evening walks with Sally often helped me to clear my mind during writing. Special thanks to my flatmate Constantinos and also my best friend Lia for her massive support throughout the last year. Last but not least, a huge thank you to my parents and brother for their love and encouragement in every decision of my life. 5 Table of Contents Abstract ....................................................................................................................... 3 Publications ................................................................................................................. 4 Acknowledgments ...................................................................................................... 5 List of Figures ........................................................................................................... 12 List of Tables ............................................................................................................ 18 Abbreviations ........................................................................................................... 19 Chapter 1 .................................................................................................................. 21 1.1 Preface to the Introduction .......................................................................... 22 1.2 Cancer .......................................................................................................... 22 1.2.1 Molecular basis of cancer – Oncogenes and tumour-suppressor genes23 1.2.1.1 Oncogenes ..................................................................................... 24 1.2.1.2 Tumour-suppressor genes ............................................................. 25 1.2.2 Extracellular matrix and tumour microenvironment ............................ 26 1.3 Small leucine rich repeat proteoglycans ...................................................... 27 1.3.1 Structural biology ................................................................................. 27 1.3.2 SLRPs expression, functions and signalling pathways ........................ 31 1.3.2.1 Structure and adhesion .................................................................. 32 1.3.2.2 Signalling pathways and other biological functions ..................... 33 1.3.3 SLRPs in Cancer .................................................................................. 35 1.3.3.1 Expression patterns and clinical significance ............................... 36 1.3.3.2 Knockout mice and functions related to cancer ............................ 37 1.3.3.3 Mechanisms of action ................................................................... 39 1.3.4 OMD and PRELP in cancer ................................................................. 44 1.4 Project hypothesis and aims ...................................................................... 45 Chapter 2 .................................................................................................................. 47 2.1 Materials ...................................................................................................... 48 2.1.1 Standard Solutions ............................................................................... 48 2.1.2 Cell lines and media ............................................................................. 49 2.2 Methods ....................................................................................................... 50 6 2.2.1 DNA/RNA Methods ............................................................................ 50 2.2.1.1 Vectors .......................................................................................... 50 2.2.1.2 Plasmids transformation and selection ......................................... 50 2.2.1.3 DNA purification – Mini preps – Maxi preps .............................. 51 2.2.1.4 Gel electrophoresis ....................................................................... 51 2.2.1.5 Restriction enzyme digestion ........................................................ 52 2.2.1.6 Ligation ......................................................................................... 52 2.2.1.7 Quantitative RT-PCR .................................................................... 53