Surgery (St Vincent's) - Theses

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Subject: breast cancer × Author: Soo, Eliza Ting Li ×

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    Epithelial-to-Mesenchymal Transition (EMT) in human breast cancer: investigating microRNAs in breast cancer EMT
    Soo, Eliza Ting Li ( 2015)
    Breast cancer is the most common malignancy among women worldwide, with mortality primarily associated with metastasis. In recent years, microRNAs (miRNAs) have emerged as a new class of master regulatory molecules with the potential to influence carcinoma progression. They are a class of small RNA molecules that regulate gene transcript stability and processing by binding to discreet motifs in the 3’ and 5’ untranslated regions of mRNAs. They regulate important mechanisms in development, including epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET), which have also been associated with cancer metastasis. miRNA profiling of control and EMT-induced PMC42-ET, PMC42-LA and MDA-MB-468 cultured human cell lines using microarray and Next Generation Sequencing (NGS) was undertaken. Several miRNAs were reproducibly up- or downregulated between the untreated cells, and in response to epidermal growth factor. Variations in miRNA expression were also assessed bioinformatically using publically available data from >50 human breast cancer cell lines. Whilst a number of these have already been implicated in cancer, other novel miRNAs consistently associated with EMT/MET were also identified. The expression levels of >20 miRNAs were experimentally validated and stable miRNA manipulations (overexpression and knockdown) in cell lines with low endogenous expression were achieved by lentiviral transduction. Recent evidence has demonstrated a crucial role for the miR-200 family in carcinoma progression, tumourigenesis and metastasis in various cancers including breast cancer. miR-200 regulation of EMT and MET was identified in our microarray and NGS datasets and was characterised in the two EMT models commonly used in our laboratory – the PMC42 and MDA-MB-468 human breast cancer cell lines. In vitro functional changes upon miR-200 manipulation showed a strong correlation between high miR-200 levels and the epithelial phenotype. In vivo changes mirrored the in vitro changes, where miR-200c knockdown resulted in reduced primary tumour growth and increased axillary lymph node metastasis in the MDA-MB-468 xenograft model. Transcriptionally, it was observed that the MDA-MB-468 miR-200c knockdown tumours showed upregulation of epithelial genes (such as E-cadherin, Grhl2, EpCAM) and a corresponding downregulation of mesenchymal genes (such as BNIP3, FN1), suggesting that the MDA-MB-468 miR-200c knockdown cells that were able to survive and eventually form a tumour had to transcriptionally activate epithelial-associated genes to achieve tumour development. Despite this, circulating tumour cells in the peripheral blood of MDA-MB-468 miR-200c knockdown tumour-bearing mice showed indications of a hybrid state, trending towards a more mesenchymal profile. Apart from the miR-200 family, eight other miRNAs—namely miR-744, miR-153, miR-708, miR-483, miR-100, miR-34b/c, miR-146a and miR-29a, were short-listed as candidate EMT-associated miRNAs identified from microarray, NGS and bioinformatic analyses. Collectively our findings suggest that no one particular miRNA alone was capable of functionally altering MDA-MB-468 breast cancer cells. Interestingly, when the cells were driven to a more mesenchymal state, there was a trend towards increased lymph node metastasis, suggesting that a mesenchymal profile is advantageous in that process. The overall findings of this thesis suggest that miRNAs do play a key role in the area of breast cancer EMT and each step in the metastasis cascade can be regulated by different miRNAs. A cooperative network of miRNA changes results in downstream changes in tumourigenesis and metastasis. These studies highlight the need to further investigate context-dependent miRNA manipulations and validate miRNAs that play critical regulatory roles to properly understand the complex role of various important miRNAs in breast cancer EMT.