Deciphering Twist functions in breast cancer

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Twist is a bHLH (basic helix loop helix) protein that acts as a master regulator of mesenchymal patterning in embryo development where it functions both as a positive and negative regulator of gene transcription. Recently, it was demonstrated that overexpression of Twist in breast epithelial cells not only induces an epithelial to mesenchymal-like transition but could also be a predictor of metastatic potential. In addition, Twist expression has been shown to induce genomic instability both in-vitro and in breast cancer patient samples. These results indicate that Twist over-expression in breast epithelial cells can induce neoplastic transformation: functions associated with oncogenesis. To fully characterize the effects of Twist on breast epithelial cells, microarray analyses was performed on Twist overexpressing cells and data mined to identify downstream target genes. From the genes dysregulated by Twist overexpression, E-cadherin was identified as a potential candidate gene. Loss of E-cadherin expression in breast cancer has been associated with poor prognosis and increased metastasis. Work by other investigators has indicated that Snail (a downstream target of Twist) along with Twist could functionally regulate E-cadherin expression. However, a systematic study to characterize the effect of Twist on E-cadherin has not been performed. In this study, we demonstrate that Twist can transcriptionally repress E-cadherin expression, both in-vitro and in-vivo. Initial analyses of the E-cadherin promoter sequence revealed the presence of numerous E-boxes to which Twist can potentially bind. Using promoter assays, we show that Twist can downregulate expression of the reporter gene by up to four folds. This was further supported by immunoblot analyses which indicated that overexpression of Twist can decrease E-cadherin protein levels in breast cancer cells. Subsequent confirmation in Hs 578t breast cancer cells (with high endogenous Twist expression) showed that an inverse correlation exists between Twist and E-cadherin levels. Chromatin immunoprecipitation was performed on Hs 578t and MCF-7/Twist cells which showed Twist binding to the E-cadherin promoter. Finally, the functional relevance of this regulation was verified by measuring Twist and E-cadherin transcript levels from a cohort of breast cancer patient samples by quantitative real-time PCR. The data demonstrated that the decrease in E-cadherin expression was associated with Twist overexpression and tumor grade. Taken together, we have shown that Twist mechanistically downregulates E-cadherin expression both in cell culture and in patient samples and that this may be one of the pathways by which E-cadherin expression is lost in breast tumors. This finding along with the other known functions of Twist in neoplastic transformation underscores the need for further investigations into the many functions of Twist in breast cancer development and progression.