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The Extensive Proliferation of Human Cancer Cells with Ever-Shorter THE EXTENSIVE PROLIFERATION OF HUMAN CANCER CELLS WITH EVER-SHORTER TELOMERES Rebecca Dagg A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Faculty of Medicine The University of Sydney 2017 1 Statement of Originality This is to certify that to the best of my knowledge, the content of this thesis is my own work. This thesis has not been submitted for any degree or other purposes. I certify that the intellectual content of this thesis is the product of my own work and that all the assistance received in preparing this thesis and sources have been acknowledged. Rebecca Dagg i Abstract Cellular immortalisation is currently regarded as an essential step in malignant transformation and is consequently considered a hallmark of cancer. Acquisition of replicative immortality is achieved by activation of a telomere lengthening mechanism (TLM), either telomerase or the alternative lengthening of telomeres (ALT), to counter normal telomere attrition. However, a substantial proportion of glioblastomas, liposarcomas, retinoblastomas, and osteosarcomas are reported to be TLM-negative. The lack of serial untreated malignant human tumour samples over time has made it impossible to examine telomere length over time and therefore determine whether they are truly TLM-deficient, or whether this is the result of false-negative assays. Here we describe a subset (11%) of high-risk neuroblastomas (NB; MYCN-amplified or metastatic disease) that lack evidence of any significant TLM activity despite a 51% 5-year mortality rate. NB cell lines (COG-N-291 and LA-N-6) derived from such tumours proliferated for 500 population doublings (PDs) with ever-shorter telomeres (EST). COG-N-291 and LA-N-6 cells had exceptionally long and heterogeneous telomere lengths as measured by terminal restriction fragment (TRF) analysis (mean TRF of 31 and 37.8 kb, respectively) and telomere fluorescence in situ hybridisation. Both cell lines were telomerase negative during culturing and did not have elevated markers of ALT or associated gene mutations. The telomeres of these cells shortened by 80 and 55 bases/PD, consistent with telomere attrition due to normal cell division, but did not reach senescence after 500 PDs in culture. This is conclusive evidence that cells from highly malignant, lethal tumours are able to undergo continuous proliferation in spite of an EST phenotype. The EST phenotype was rescued by activation of telomerase (via transduction with hTERT expression constructs) or ALT (spontaneous occurrence of a nonsense TP53 mutation, followed by spontaneous activation of ALT after 100 PDs). We also found that NB EST cells are very sensitive to topoisomerase I inhibitors indicating the potential to target the EST phenotype with topoisomerase I inhibitors in high-risk NB. ii Acknowledgements The first person that I need to thank is my supervisor Dr Loretta Lau. I cannot express how grateful I am that you took a chance and employed me eight years ago. I am the scientist that I am today because of you. Thank you for your guidance and support and putting up with short deadlines with my writing. You have been a great teacher, friend and mentor and I couldn’t have asked for a better supervisor. I would also like to thank my co-supervisor Prof. Roger Reddel. I am constantly astounded by your replies to emails at all times of the day and I am so grateful for your guidance and insights. Thank you for always having an open door, because of you I have learnt to be a better scientist and critical thinker. The success of my PhD is in large part due to the support that I have received from both of my supervisors. To all of the members of the CCRU past and present you have made the long hours in the lab far more enjoyable and become some of my dearest friends. I would especially like to thank Vanita for always answering my questions (no matter how many times I had to ask about the same form), Belinda for always offering me advice with a smile, and lunches and dinners spent laughing with Radikha, Jess, Cuc, Kylie, Michael, Greg, Nick, Amanda, Oksana, Natalie, Namratha, Camilla, and Yu Wooi. Yuyan your support has helped me more than I can express and I will miss being able to chat with you at the bench. To Peta, Sarah, Naz, Kaitlin and Amy I can’t believe we decided to start Stress whilst doing our PhD but I’m so proud of what we achieved! Ladies you have become some of my dearest friends and I can’t imagine having survived the last few years without you. I also need to thank the past and present members of the cancer research and telomere length regulation units at CMRI for all of the advice and assistance I’ve received which have made this a better thesis. During my PhD I have spent more time in the lab than anywhere else and I would like to thank all of my friends and family for being so understanding about the events that I’ve missed, left early or been late to, or had to reschedule around experiments. You have all been so understanding and I iii cannot say how much I love you and appreciate your support. I would especially like to thank Chris and my grandparents for being an unending source of support, you have helped me become who I am today and I love you more than I can say. To Mum and Katherine, I don’t think that there are words to say what you mean to me, but to start with I love you. You have seen me at my best and worst during the PhD and done everything you can to make sure that I’m still sane at the end of it. Thank you for always listening, being there when I need you, and doing everything from eating cake with me to getting posters printed when I was on the other side of the world. I never doubted that I would get to the end of this because of your unending love, support and encouragement. In many ways, I think of this as something that we have achieved together, so thank you for getting onto this rollercoaster with me and helping me become the person that I am today. iv Publications Dagg RA, Pickett HA, Neumann AA, Napier CE, Henson JD, Teber ET, Arthur JW, Reynolds CP, Murray J, Haber M, Lau L MS, Reddel RR (2017). Extensive proliferation of human cancer cells with ever-shorter telomeres. Cell Reports. Vol 20;19(12): 2544-2556. Hayward NK, Wilmott JS, Waddell N, Johansson PA, Field MA, Nones K, Patch A, Kakavand H, Alexandrov LB, Burke H, Jakrot V, Kazakoff S, Holmes O, Leonard C, Sabarinathan R, Mularoni L, Wood S, Xu Q, Waddell N, Tembe V, Pupo GM, Paoli-Iseppi RD, Vilain RE, Shang P, Lau L MS, Dagg RA, Schramm S, Pritchard A, Dutton-Regester K, Newll F, Fitzgerald A, Shang CA, Grimmond SM, Pickett HA, Jean Y. Yang JY, Stretch JR, Behren A, Kefford RF, Hersey P, Long GV, Cebon J, Shackleton M, Spillane AJ, Saw R PM, Lopez-Bigas N, Pearson JV, Thompson JF, Scolyer RA, Mann GJ (2017). Whole genome landscapes of major melanoma subtypes. Nature. Vol 11;545(7653): 175-180. Bradbury P, Turner K, Mitchell C, Griffin K, Lau L, Dagg RA, Taran E, Cooper-White J, Fabry B, O’Neill GM (2017). The focal adhesion targeting (FAT) domain of p130 Crk associated substrate (p130Cas) confers mechanosensing function. Journal of Cell Science. Vol 1;130(7): 1263-1273. Scarpa A, Chang DK, Nones k, Corbo V, Patch A, Bailey P, Lawlor RT, Johns AL, Miller DK, Mafficini A, Rusev B, Scardoni M, Antonello D, Barbi S, Sikora KO, Cingarlini S, Vicentini C, Simpson S, Quinn MCJ, Bruxner TJC, Christ AN, Harliwong I, Idrisoglu S, Manning S, Nourse C, Nourbakhsh E, Wilson PJ, Anderson MJ, Fink JL, Newell F, Waddell N, Holmes O, Kazakoff SH, Leonard C, Wood S, Xu Q, Nagaraj SH, Amato E, Dalai I, Bersani S, Cataldo I, Capelli P, Davì MV, Landoni L, Malpaga A, Miotto M, Whitehall V, Leggett B, Khanna KK, Harris J, Jones MD, Humphris J, Chantrill LA, Chin V, Nagril A, Pajic M, Scarlett v CJ, Pinho A, Rooman I, Toon C, Wu J, Pinese M, Cowley M, Giry-Laterriere M, Mawson A, Humphrey ES, Colvin EK, Chou A, Lovell JA, Jamieson NB, Duthie F, Gingras M, Muzny D, Dagg RA, Lau LMS, Lee M, Pickett HA, Reddel RR, Samra JS, Kench JG, Merrett ND, Epari K, Nguyen NQ, Zeps N, Falconi M, Simbolo M, Butturini G, Fassan M, Australian Pancreatic Cancer Genome Initiative, Gill AJ, Wheeler DA, Gibbs RA, Musgrove EA, Bassi C, Tortora G, Pederzoli P, Pearson JV, Waddell N, Biankin AV and Grimmond SM (2017). Whole-genome landscape of pancreatic neuroendocrine tumours. Nature. Vol 543: 65–71. Henson JD, Lau L MS, Koch S, Martin La Rotta N, Dagg RA, Reddel RR (2016). The C-circle assay for Alternative-Lengthening-of-Telomeres activity. Methods. http://dx.doi.org/10.1016/j.ymeth.2016. 08.01 Farooqi AS, Dagg RA, Choi LM R, Shay JW, Reynolds P, Lau L MS (2014). Alternative Lengthening of Telomeres in neuroblastoma cell lines is associated with a lack of MYCN genomic amplification and with p53 pathway aberrations. Journal of Neuro-Oncology. Vol 119(1): DOI:10.1007/s11060-014- 1456-8. Lee M, Hills M, Conomos D, Stutz M, Dagg RA, Lau L MS, Reddel RR, Pickett HA (2014). Telomere extension by telomerase and ALT generates variant repeats by mechanistically distinct processes.
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