IDENTIFYING THE MOLECULAR MEDIATORS OF VN AND THE IGF:VN COMPLEX-STIMULATED BREAST CANCER CELL SURVIVAL Ali Shokoohmand School of Biomedical Sciences, Faculty of Health Queensland University of Technology, Australia A thesis submitted for the degree of Doctor of Philosophy of the Queensland University of Technology 2015 QUT Verified Signature Acknowledgements Commencing, pursuing and completing this dissertation like any other project, required abundant resources as well as strong motivation, which wouldn’t have been possible without the people who provided me with the much needed encouragement, support, scientific advice and help with experiments. Therefore, I would like to express my gratitude to the people below. I would like to thank my principal supervisor ‘Dr Mr’ Hollier whose advice, guidance and encouragement has been always available for me through my PhD journey. Thanks for encouraging me to work hard at all times and keeping me motivated during my PhD. Your work ethic and your scientific expertise will always inspire me and I truly learned a lot from you. Abhi, I have always been appreciating to have you beside me. Your encouragement and support was a very great thing to me. I have learnt many deals from you. Your encouragement and support always helped me to work harder. Above all, I always enjoyed talking with you about our cultures, people and countries. I am sure we still have many things to talk about! Zee, thanks for the support you have given throughout my PhD. Without your support, this journey could have been harder for me. Derek, I would like to thank you for listening to me sometimes and being here for me. I would like to thank all members of the TRR team for being helpful, considerate and supportive. It has been a pleasure working with you all. Special shout outs to Chen, Lipsa, Leo, Raju and Dominic. Dom, I would also thank you for helping me in generating heatmaps during my PhD studies, thank you ‘king of heatmaps’. I would also thank our laboratory technician Dod for providing me with reagents and equipment in the least possible time. Good friends are the most important ingredient for any achievement. I would like to thank my great friends back home Morteza, Masoud, Mehdi and especially Amirhossien “Sardar” for their immense support and constant encouragement. Thank you for all those encouraging words and online sooshi breaks! ii Joony June, I don’t have words great enough to convey how much I have appreciated your love and care over the past three years. You have been my backbone and I was lucky enough to have you doing your PhD at the same time as mine. I am highly grateful for your support. I think this line summarises your impact on my life: “Thank you so much for all you do; you’re truly a delight; when my life overwhelms, you make everything all right”. You are one the most peaceful and down-to-earth human beings I have seen throughout my life! Finally, to my family, I greatly appreciate your support throughout not only this entire PhD journey but the last 7 years that I have been away from home. You didn’t once complain about me being overseas and you always encouraged me to achieve my goals, even if that meant I was thousand miles away from you all. Mum and Dad, thanks for being great parents. You both are my biggest role models. Mum, you are just brilliant and the most amazing and big-hearted person I have ever known. “Life is hard only for its first hundred years” H.Karaminina In loving memory of my dear grandmother iii Keywords Attachment, breast, breast cancer, cancer progression, connective tissue growth factor, cysteine rich angiogenic inducer 61, extracellular matrix, gene knockdown, growth factors, growth factor binding proteins, insulin-like growth factor, integrin, integrin-mediated signalling, lentivirus, mediated down-regulation, metastasis, migration, molecular therapeutic targets, shRNA proliferation, three-dimensional cultures, survival, viability, vitronectin, vitronectin-binding integrins. iv Abstract A key step in the cancer metastatic process is the ability of the cancer cells to survive in foreign tissue microenvironments. Two factors thought to be pivotal in cancer metastasis are altered cellular interactions with extracellular matrix (ECM) proteins within the tumour microenvironment and exposure to elevated levels of mitogenic hormones and growth factors. A compelling body of evidence suggests that the ECM protein, vitronectin (VN), can modulate tumour metastasis by providing traction and directionality to migratory cancer cells in addition to its effect on promoting cancer cell survival. While an increased deposition of VN has been reported at the leading edge of breast tumours, few studies have investigated the downstream VN-induced transcriptional changes responsible for its effect on cell survival and metastasis. In order to fill this knowledge gap, this project aimed to identify specific downstream mediators of VN induced in breast cancer cells with functional roles in cell survival and metastasis. In addition to VN, insulin-like growth factors (IGFs) are known to play a critical role in the growth and development of the non-malignant cells as well as malignant cell types. The IGF system is complex and the biological effects of the IGFs are determined by their diverse interactions between many molecules, including their interactions with ECM proteins. Studies over the past decade have demonstrated that IGF ligands associate with VN, forming IGF:VN complexes that are capable of stimulating biological functions, namely survival and migration in both normal and malignant cell types. Considering the important role of IGF-I in the progression of breast cancer and its association with VN, this project also aimed to identify specific molecular mediators that are important in IGF-I:VN-mediated survival. In order to identify the molecular mediators underpinning VN- and the IGF- I:VN complex-stimulated breast cancer cell survival, transcriptional profiling was performed using DNA microarrays to identify the transcriptional responses of MCF- 7 breast cancer cells stimulated with VN and the IGF-I:VN complex. Bioinformatics analysis identified a substantial number of differentially expressed transcripts in response to each treatment, whereby there was a considerable overlap observed between the transcripts differentially regulated by VN and the IGF:VN complexes. v To select transcripts for functional validation studies, we employed a strategy focused on selecting candidates to target not only VN, but potentially IGF-I:VN complex-mediated biological actions. Gene ontology and functional pathway enrichment were used to identify candidates that were regulated by both VN and the IGF-I:VN complex with a bias toward biological functions relevant to breast cancer cell survival. Based on published literatures, we identified a number of genes differentially expressed in common by VN and the IGF-I:VN complex with important roles in cell survival, such as TGFβ2, Smad6, S100A4, S100P, ANXA2, CCN1/Cyr61, CCN2/CTGF, RASGRP1, c-FOS, c-JUN, CAV1, snail 2 (slug), mir21 and LAMB1. Among the candidates identified, CCN family members, Cysteine-rich angiogenic inducer 61 (CCN1/Cyr61) and Connective Tissue Growth Factor (CCN2/CTGF), were selected to investigate their role in VN- and IGF-I:VN-induced breast cancer cell survival. It was demonstrated that VN and the IGF-I:VN complex increase Cyr61 and CTGF expression in a number of breast cancer cell types ranging from the weakly metastatic luminal-like to the highly metastatic basal-like breast cancer subtypes. Studies using functional blocking antibodies and chemical inhibitors confirmed the critical involvement of VN-binding αv-subunit containing integrins and the activation of the intracellular Src complex, PI3-K/AKT and ERK/MAPK signal transduction pathways. Furthermore, the functional role of Cyr61 and CTGF was assessed using shRNA-mediated gene suppression, which reduced VN- and IGF-I:VN-stimulated MCF-7 cell viability in both 2-dimentional and 3- dimentional cultures. Moreover, Cyr61 was observed to be important for the increased viability induced by VN and the IGF-I:VN complex in the highly metastatic MDA-MB-231 cell line, whereas CTGF had a more prominent role in the invasive and migratory phenotype. In summary, this PhD project has identified for the first time the changes in global gene expression profiles underpinning VN- and the IGF:VN complex- mediated cancer cell survival. In addition, the outcomes of this study provide valuable new information on the mechanistic events underpinning VN- and IGF- I:VN-mediated effects on breast cell functions. Importantly, the up-regulation of Cyr61 and CTGF mediated by VN and the IGF:VN complex was determined to have an important functional role in breast cancer cell growth, survival and migration. Targeting Cyr61 and CTGF proteins or associated signalling pathways may disrupt vi IGF- and VN-mediated cancer progression, and with further development have the potential to be prognostic indicators and therapeutics for cancer patients. vii Table of Contents Statement of Original Authorship .............................................................................. i Acknowledgements .................................................................................................. ii Keywords ................................................................................................................ iv Abstract ...................................................................................................................