Sirna Screening of the Kinome Identified Aurora a Kinase As a Therapeutic Target Gene in Cervical Cancer Fawzi Faisal A
Total Page:16
File Type:pdf, Size:1020Kb
siRNA screening of the kinome identified Aurora A kinase as a therapeutic target gene in cervical cancer Fawzi Faisal A. Bokhari, M.Sc. A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 The University of Queensland Diamantina Institute Abstract HPV oncogenes disable a number of tumour suppressor pathways, including p53 and Rb, contributing to the transformed phenotype. Loss of these critical host cell functions may also provide an opportunity to selectively target the destruction of HPV-transformed cells. We have performed an siRNA screen using the kinome (779 genes) library to identify genes that when depleted are synthetically lethal with HPV transformation. The primary and validations screens have confirmed Aurora A kinase (AURKA) as a potential synthetic lethal target selective for HPV transformed cells. AURKA has been further investigated using the selective small molecule inhibitor MLN8237. We found that MLN8237 was significantly more potent towards the HPV transformed cells. The effect was not a consequence of targeting mitosis as two other mitotic inhibitors, PLK1 inhibitor (BI2536) and taxol, demonstrated no selectivity. Analysis of the nuclear structure and DNA content showed that Aurora A inhibition promoted a high level of polyploidy in non-HPV treated cells whilst this same degree of polyploidy was associate with apoptosis in the HPV-transformed cell lines. Whereas Bcl-2 over expression in HeLa cells had no effect on sensitivity to MLN8237, Mcl-1 overexpressing HeLa cells were less sensitive to the MLN8237 in comparison to the parental cell line, which may suggests the involvement of Noxa or Puma pro- apoptotic proteins in the induction of the apoptosis in the HPV-transformed cells. The transfection of the non-HPV C33A cervical cancer and SCC25 squamous cell carcinoma cell lines with the HPV16 oncogenic E7 increased sensitivity to MLN8237 between 3 >10 fold suggesting that the sensitivity to MLN8237-dependent killing was a direct consequence of HPV E7 expression. Xenograft experiments with cervical cancer cell lines in immunodeficient mice showed MLN8237 inhibited growth of HPV and non-HPV xenografts during treatment with 30mg/kg MLN8237 once a day for 10 consecutive days. However, outgrowth of tumour was noticed from the second day post-treatment in the non-HPV tumour group whereas the HPV-induced tumour group did not show cancer recurrence for 50 days post-treatment. These findings suggest that MLN8237 represent a promising novel therapeutic targeted agent against HPV-transformed cervical cancer. ii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iii Publications during candidature Blake, SJ., Bokhari, FB., McMillan, NA. (2012). RNA interference for viral infections. Curr Drug Targets, Oct; 13(11):1411-20 Publications included in this thesis No publications included. iv Contributions by others to the thesis Technical assistance from Mr Max Ranall (ARVEC Unit UQDI) for the design and optimisation of the kinome screen. Statement of parts of the thesis submitted to qualify for the award of another degree None. v Acknowledgements “In the name of Allah, the most gracious, the most merciful” Thank you Allah for helping me to finish this PhD thesis. I would like to start the first acknowledgment in this section with the person who pushed me to go throughout all the postgraduate studies Masters and PhD. The person whom I will never forget and I wish he is now with me sharing with me these moments. But many times what we wish never comes to reality. To the person that I have lost in the mid of my PhD, to his soul; to my Father: I have made it to the end of the journey Dad. I hope this thesis will make your soul as happy as you would have been with me today. I owe you a lot and thank you is not even enough to what you have done for me; and all words will not be adequate to describe my feelings. I love you Dad. This PhD work would have not been done without the financial support I have received from the generous government of the Kingdom of Saudi Arabia and the support I had from the funding of Associate Professor Brian Gabrielli at the UQDI and Professor Nigel McMillan at Griffith University. Thank you all for your great support. Honestly I do not know how to thank my great supervisors, both Associate Professor Brian Gabrielli and Professor Nigel McMillan. I owe you a huge thank you for all what you have done with me. I HAVE LEARNT SO MUCH FROM YOU BOTH. Thank you for your usual support, understandings, guiding me throughout my PhD, and providing me with opportunities to learn and develop my skills to become a scientist. Your doors have been always open for guidance, discussions, ideas, and questions. Thank you so much. Special thanks also go to my previous associate supervisor Professor Tom Gonda who guided me throughout the beginning of this project. Your feedback and guidance have been always appreciated. To all the Gabrielli lab members, you have been great friends and colleagues to be with. I would like to specifically thank Dr Loredana Spoerri for her helps in reviewing my Materials and Methods vi Chapter of this thesis and for her continuous advices. Also, I will never forget to say thank you McMillan lab members past and present, I have enjoyed being with you all. Any work in this life has ups and downs and moments where you need special squad to make you feel you are still alive. To my amazing friends in Australia and in Saudi Arabia who helped me overcome all the hectic moments I have been through during this project (YES! Those Thursday nights at the La Dolce Vita Café having coffee, helped me so much). Last, but of course not least, this section is devoted to those who really being part of my life. Firstly, to my Mother, any word in this life will not be enough to convey my feelings towards you. Thank you for being with me all the time, for the support you gave me, for the advices that always put me on top of this life, thank you for everything you have given me. Without you, not only my PhD but also my whole life will be tasteless. I love you Mom. Secondly, my wife Nahla, the person who left everything behind to join me in this journey; thank you for all the cares you gave me, helps and encouragements. I would thank Allah everyday throughout my life for giving me such a great person like you. I owe you so much Nahla and, of course, I love you. To my brothers and sisters, Bassem, Ashwaq, AbdulRahman, and Ghazal. You are the great companion gifts from Allah to me in this life. Your support and helps were key players in finishing this work; thank you so much, I love you all. To my sons, who are my real eyes in this life, Faisal and AbdulRahman. All the pain, stress, and challenges resolve when I see your great smiles and hug you. You have been always and will remain the bright side of my life. vii Keywords siRNA, AURKA, AURKB, Haspin, GSG2, cervical cancer, HPV, synthetic lethality. Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 111207, Molecular Targets, 50% ANZSRC code: 111201, Cancer Cell Biology, 30% ANZSRC code: 111203, Cancer Genetics, 20% Fields of Research (FoR) Classification For code: 0604, Genetics 50% FoR code: 0601, Biochemistry and Cell Biology, 25% FoR code: 1112, Oncology and Carcinogenesis, 25% viii Table of Contents Abstract ............................................................................................................................................... ii Acknowledgements............................................................................................................................ vi List of Tables ................................................................................................................................... xiv List of Figures ................................................................................................................................... xv List of Abbreviations ..................................................................................................................... xvii 1 Literature Review .......................................................................................................................