Understanding mechanisms that contribute to tumorigenesis and metastasis is important for developing more effective cancer therapies. Epithelial-mesenchymal transition (EMT) is associated with loss of the cell surface protein E-cadherin, increased tumour cell metastasis and acquisition of a cancer stem cell (CSC) phenotype. Whilst the process of EMT and aberrant E-cadherin expression during metastasis have been studied in detail, the role of exogenous inhibition of E-cadherin protein alone in epithelial cells remains to be elucidated. In this study the E-cadherin neutralising antibody SHE78.7 (nAb) and a peptide inhibitor of E-cadherin (nPep) have been used to assess how alterations in cell surface E-cadherin affect adenocarcinoma cell line MCF-7. MCF-7 cells exhibit an epithelial phenotype characterised by cell surface E-cadherin expression and lack of EMT marker expression, such as N-cadherin and Vimentin. Exogenous inhibition of cell surface E-cadherin using nAb in MCF-7 cells is a reversible process which induces increased cell numbers due to decreased apoptosis and cell proliferation. However, nAb treatment was insufficient to induce EMT or a CSC phenotype. Treatment of MCF-7 cells with nPep also induced increased cell numbers, but this was due to increased proliferation of the cells, with no changes in apoptosis observed. Microarray analysis of nAb-treated MCF-7 cells revealed >1000 gene transcript alterations compared to control Ab-treated cells, with changes associated with a wide range of cellular functions. Using an in silico network analysis approach, E-cadherin was identified as a positive regulator of the histone acetyltransferase p300. Exposure of MCF-7 cells to the p300 inhibitor garcinol resulted in increased CD44 and Slug, and decreased CD24 CSC associated transcripts. nPep treatment of MCF-7 cells and subsequent high content screening (HSC) analysis following exposure to a panel of cancer therapeutics demonstrated increased drug efficacy in combination with nPep for most of the therapeutics. Furthermore, nPep-treated MCF-7 cells exhibited a significantly altered plasma membrane-associated protein profile compared to control cells. Together, these results show that exogenous inhibition of E-cadherin in MCF-7 cells is a reversible event associated with increased proliferation, reduced apoptosis and significantly altered protein and transcript expression that may contribute to neoplasm formation in vivo. Furthermore, I show for the first time that inhibition of p300-dependent gene transactivation induces a CSC gene transcript expression profile in MCF-7 cells in vitro.
|Date of Award||31 Dec 2015|
- The University of Manchester
|Supervisor||Christopher Ward (Supervisor) & Catherine Merry (Supervisor)|