Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Anatomy and Cell Biology

Supervisor

Dr. Trevor Shepherd

Abstract

Epithelial-mesenchymal transition (EMT) serves as a key mechanism driving tumour cell migration, invasion, and metastasis in many carcinomas. Transforming growth factor-beta (TGFβ) signalling is implicated in several steps during cancer pathogenesis and acts as a classical inducer of EMT. Since epithelial ovarian cancer (EOC) cells have the potential to switch between epithelial and mesenchymal states during metastasis, we predicted that modulation of TGFβ signalling would significantly impact EMT and the malignant potential of EOC spheroid cells. Ovarian cancer patient ascites-derived cells naturally underwent an EMT response when aggregating into spheroids, and this was reversed upon spheroid re-attachment to a substratum. CDH1/E-cadherin expression was markedly reduced in spheroids compared with adherent cells, in concert with an up-regulation of several transcriptional repressors, i.e., TWIST1/2, ZEB2, and most notably SNAI1/Snail. Treatment of EOC spheroids with the TGFβ type I receptor inhibitor, SB-431542, severely reduced Snail expression and potently blocked the endogenous activation of EMT in spheroids. Furthermore, treatment of spheroids with SB-431542 upon re-attachment enhanced the epithelial phenotype of dispersing cells and significantly decreased cell motility and Transwell migration. Spheroid formation was significantly compromised by exposure to SB-431542 that correlated with a reduction in cell viability particularly in combination with carboplatin treatment. Thus, our findings are the first to demonstrate that intact TGFβ signalling is required to control EMT in EOC ascites-derived cell spheroids and it promotes the malignant characteristics of these structures. Next, we sought to investigate the function of Snail, the most robustly-upregulated EMT marker in EOC spheroids, since its role in EOC metastasis particularly in spheroids is largely unknown. Knockdown of SNAI1 encoding Snail led to a modest reduction in spheroid formation and cell viability, but we observed a significant decrease in the ability of spheroids to migrate due to loss of Snail expression. Further to this, we retrovirally-transduced the Hey ovarian cancer cell line to ectopically express Snail. HeySNAIL cells exhibited a strong EMT phenotype in adherent culture, a result which correlated with significantly enhanced spheroid cell migration, and adhesion and invasion of spheroids to fibronectin and collagen matrices. Furthermore, using an ex ovo chick embryo chorioallantoic membrane assay, Snail-expressing EOC spheroids possessed significantly enhanced tumour-forming potential, invasion, and liver metastasis. These results highlight the therapeutic potential for targeting the Snail/EMT/TGFβ signalling pathway in patients with late-stage ovarian cancer.

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