Functional Analysis of FOXC1 in TGF‐β Mediated Epithelial to Mesenchymal Transition

Abstract

Elevated expression of FOXC1 has been detected in many metastatic cancers characterised by mesenchymal characteristics. Furthermore in development FOXC1 functions in the formation mesenchymal derived tissues including the axial skeleton and eye that are derived through transforming growth factor beta (TGFβ) signalling pathways. However it is not known whether FOXC1 functions in initiation of EMT by TGFβ signalling. We utilized the mouse mammary epithelial cell line NMUMG, to test what role FOXC1 plays in EMT. Treatment of NMUMG cells with 5 ng/ml of TGFβ induced EMT and was accompanied by a down regulation of the epithelial marker E‐cadherin and an upregulation of mesenchymal markers N‐cadherin and Vimentin and Snail1. Elevated levels of Foxc1 mRNA were detected at 24 and 48 hours of treatment. To test whether this upregulation of Foxc1 was necessary for initiation of EMT we used RNA interference to reduce levels of by 60‐85%. When treated with TGFβ, knock down of Foxc1 had no effect on EMT progression based on morphological assessment, and through changes in gene expression monitored by quantitative PCR and immunofluorescence assays. Elevated N‐cadherin mRNA expression was detected in Foxc1 knock down cells prior to the initiation of EMT. In wound closure assays Foxc1 knock down cells demonstrated an increase in cell migration compared to control cells. We next created an NMUMG cell line expressing Foxc1 under the control of a tetracycline inducible system. When Foxc1 levels were elevated in the absence of TGFβ, NMUMG cells continued to express epithelial markers and an induction in the expression mesenchymal markers (N‐cadherin, Vimentin, Snail1) was not observed. Our data suggest that Foxc1 is not necessary or sufficient for the initiation of EMT by TGFβ in murine cells lines.