The master-regulators of EMT and E-cadherin constitute a novel pathway in malignant melanoma

The master-regulators of an epithelial-mesenchymal transition (MR-EMT) have a pivotal role in the regulation of carcinoma development, promoting transformation and generating a migratory and invasive phenotype. Within epithelial cells, the ZEB proteins are co-regulated, jointly repressed by the miR-200 family of microRNAs. However, here it is demonstrated that the expression and regulation of the MR-EMT in malignant melanoma cell lines appears to be fundamentally different, with a hierarchical organisation identified. ZEB2 and SNAIL2 were found to be expressed in melanocytes, whilst ZEB1 and TWIST1 expression was acquired by a sub-set of malignant melanoma cell lines. Melanoma-initiating mutations within B-RAF and NRAS were shown to reversibly promote expression of ZEB1 and TWIST1 at the expense of ZEB2 and SNAIL2. Additionally, ZEB2 and SNAIL2 were identified up-stream of ZEB1 and TWIST1 within the MAPK signalling cascade, with ZEB2 functioning as a repressor of ZEB1. Furthermore, ZEB2 and SNAIL2 were found to positively regulate expression of MITF, a marker of melanocyte differentiation. In contrast, ZEB1 repressed expression of MITF and was the primary transcriptional repressor of E-cadherin, an adhesion molecule vital for the interaction between differentiated melanocytes and keratinocytes. Previously, within epithelial cell lines, all the MR-EMT have been identified as transcriptional repressors of E-cadherin. However, ZEB2 and SNAIL2 were co-expressed with E-cadherin within melanocytes and melanoma cell lines and, along with TWIST1, were not able to independently induce E-cadherin re-activation following repression. Surprisingly, ZEB2 became a repressor of E-cadherin in conjunction with ZEB1. Finally, E-cadherin expression was also shown to be controlled in a ZEB1-dependent manner by the transcriptional co-repressor BRG1, the ATPase subunit of the SWI/SNF chromatin remodelling complex, and by the presence of DNA methylation at the E-cadherin promoter. Indeed, DNA methylation was identified as a possible factor controlling the success rate of metastatic colonisation in melanoma cells, allowing for the dynamic re-expression of E-cadherin at the secondary site. These data demonstrate that in malignant melanoma the expression and regulation of the MREMT is fundamentally different to that of epithelial tumours, with the MR-EMT structured hierarchically, with opposing regulatory functions.