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Investigating the Role of Epithelial-Mesenchymal INVESTIGATING THE ROLE OF EPITHELIAL-MESENCHYMAL PLASTICITY IN PROSTATE CANCER Nataly Stylianou BAppSc(MedSc)(Hons) School of Biomedical Sciences Faculty of Health Queensland University of Technology (QUT) Submitted in fulfilment of the requirement for the degree of Doctor of Philosophy 2017 i Keywords Prostate, prostate cancer, cancer progression, metastasis, epithelial, mesenchymal, epithelial to mesenchymal transition, EMT, mesenchymal to epithelial transition, MET, mesenchymal to epithelial reverting transition, MErT, epithelial- mesenchymal plasticity, EMP, cell plasticity, plasticity, inducible expression models, lentivirus, Snail, Slug, Zeb1, E-cadherin, vimentin, invasion, dormancy, cell cycle, temporal transcriptional profiling, microarray, gene signature, survival, biochemical recurrence, predictive, metabolism, animal models ii Abstract Prostate cancer is the most diagnosed cancer and the second leading cause of cancer- related deaths amongst Australian men. Unfortunately, the majority of deaths occur due to the metastatic spread of cancer cells to distant organs. The propensity of cancer cells to transition between epithelial and mesenchymal phenotypic states via the epithelial-mesenchymal transition (EMT) program is considered to be critical in metastatic processes, cancer progression and treatment resistance. The activation of EMT imparts epithelial cancer cells with invasive characteristics, aiding their dissemination to distant organs. However, recent research suggests that a reversion back to their proliferative epithelial phenotype via a mesenchymal-epithelial reverting transition (MErT) is imperative for the formation of overt metastases. With increasing evidence that cell plasticity plays an important role in cancer progression, more accurate models are required to study these events more thoroughly. To investigate a transient EMT in prostate adenocarcinoma, inducible and reversible models of EMT were generated whereby the expression of EMT inducing transcription factors Snail, Slug, or Zeb1 could be experimentally controlled both in vitro and in vivo via treatment with Doxycycline Hyclate (Dox). Exposure of cells to Dox induced a rapid transition of cells to a mesenchymal-like phenotype, which was confirmed via the reduction in epithelial specific markers and an increase in mesenchymal markers at the mRNA and protein level. Upon removal of Dox from cells having undergone an EMT, the EMT markers returned to their basal levels along with the re-establishment of the epithelial phenotype, overall signifying a MErT. Cells induced into the EMT-state became highly invasive in 3D-on-top Matrigel™ assays, and this was paralleled by a loss of proliferative ability and entry into a dormant-like state. Subsequent removal of Dox saw the re-awaking of these dormant cells which was characterised by the reacquisition of cell proliferation. The reversible EMT models were successfully grown in the prostate of mice and the expression of the EMT transcription factors could be regulated by Dox treatment. While their use for in vivo studies requires further optimisation, this study is the first to generate orthotopic models whereby the induction and reversal of the EMT program can be experimentally controlled. iii Comprehensive transcriptional profiling of a reversible EMT using a custom made 180k probe Agilent microarray elucidated for the first time the temporal dynamics of the less characterised MErT process. This revealed that the transcriptional profile of MErT is not a simple mirror image of EMT. The MErT not only consisted of dynamically reversible transcripts but also transcripts that remained persistently altered following an EMT or became uniquely activated with MErT. Both reversible and novel transcriptional subprograms were enriched in samples of lethal metastatic castration resistant prostate cancer (mCRPC), supporting the clinical role of a reversible EMT in metastasis. From this enrichment, a metastasis-derived gene signature (MPS) was identified to predict more rapid cancer relapse and reduced survival in not only prostate cancer patients but also across a number of other human carcinoma types. Interestingly, The MPS was found to have a high enrichment of metabolism related genes. As there is little evidence supporting the involvement of EMT in cancer metabolism, further investigation revealed that the metabolism-related genes were also predictive of poor patient outcome when examined in isolation. Furthermore, functional testing of the effect of EMT on cell metabolism showed the EMT program to regulate the metabolic phenotype of prostate cancer cells. Cells actively undergoing EMT entered a metabolically quiescent state. Surprisingly, this study provides prototypical evidence that MErT not only relieved this effect, but resulted in a metabolically more energetic phenotype than that observed prior to EMT. Cumulatively, this study provides first-in-field evidence to support the association of epithelial plasticity with poor clinical outcomes in multiple human carcinoma types. Furthermore, the identified novel signatures are promising candidates for discovering 1) biomarkers that can identify these transitions in clinical samples; and 2) drivers of these transitions that could be used to develop better targeted therapies; something that is currently lacking in the field. iv Table of Contents Keywords ....................................................................................................................ii Abstract ..................................................................................................................... iii Table of Contents ....................................................................................................... v Table of Tables ........................................................................................................... x Table of Figures ........................................................................................................xii List of Abbreviations .............................................................................................. xvi QUT Confidentiality Undertaking......................................................................... xix Statement of Original Authorship .......................................................................... xx List of Publications and Awards ............................................................................ xxi Acknowledgments .................................................................................................. xxv Chapter 1: Literature Review ................................................................................... 1 1.1. Prostate cancer: incidence, detection, and management. .................................. 2 1.2 Epithelial-mesenchymal plasticity (EMP) and prostate gland development...... 4 1.3 EMP in cancer. ................................................................................................... 6 1.4 Regulation of the Epithelial to Mesenchymal Transition (EMT). ..................... 8 1.4.1 EMT markers. ........................................................................................... 8 1.4.2 EMT regulators. ...................................................................................... 10 1.4.2.1 The Snail family. ........................................................................ 10 1.4.2.2 The Zeb family. .......................................................................... 12 1.4.2.3 The basic/helix-loop-helix (bHLH) family. ............................... 12 1.4.3 EMT inducers. ........................................................................................ 13 1.5 The role of EMP in the metastatic cascade. ..................................................... 15 1.5.1 EMP and tumour heterogeneity. ............................................................. 16 1.5.2 EMP and metastatic dissemination. ........................................................ 18 1.5.3 EMP and the metastatic niche. ................................................................ 20 v 1.5.4 EMP and metastatic colonisation. ........................................................... 21 1.6 EMT and therapy resistance. ............................................................................ 23 1.7 EMT and cancer stem cells (CSCs). ................................................................. 24 1.8 Study aims ........................................................................................................ 25 1.8.1 Rationale ................................................................................................. 25 1.8.2 Hypotheses .............................................................................................. 26 1.8.3 Aims ........................................................................................................ 26 Chapter 2: Materials and Experimental Methods ................................................ 27 2.1 Tissue culture. .................................................................................................. 28 2.2 pINDUCER20 vector system. .......................................................................... 28 2.3 HEK293T transfection and lentivirus production. ........................................... 33 2.4 Cell transduction and infection......................................................................... 33 2.5 Three-dimensional (3D)-on-top Matrigel™ assays. ........................................ 33 2.6 Immunofluorescence
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