Development of malignant melanoma is dependent on a switch in Embryonic Transcription Factors orchestrated by the BRAF-MAPK pathway

Reactivation of master regulators of epithelial to mesenchymal transition (MR-EMT) represents the molecular basis for tumour cell plasticity, malignant transformation and metastases. However, the current evidence on the specific role of MR-EMT in melanomagenesis has not been fully addressed. The purpose of this investigation was to assess the expression and regulation of these factors in malignant melanoma and to evaluate their prognostic and clinical significance. In vitro experiments indicated that a switch in MR-EMT protein expression ZEB2/SNAI2 to ZEB1/TWIST1 is RAS-RAF-MAPK signalling dependent. In addition, evidence supported a MR-EMT interactome, in which transcriptional repression of ZEB2 by Fra-1 resulted in upregulation of ZEB1, independently of miR-200 family. Further in vitro and immunohistochemical (IHC-P) analyses showed that E-cadherin and VDR protein levels were significantly reduced by the presence of ZEB1 in melanoma cells and archive tissues. Motility assays demonstrated that ZEB1 but not ZEB2 enhances cell migration. IHC-P analyses of ZEB2/SNAI2 (n=142/28) showed a statistically significant gradient of stronger staining at superficial sites compared to the deep sites in a select cohort of independent and matched melanoma tumour samples. In contrast, ZEB1 (n=142) and TWIST1 (n=133) showed higher staining in deep sites of primary melanomas and metastases. Trend analyses showed a significant MR-EMT switch in this progression series from high levels of ZEB2/SNAI2 in naevi towards high ZEB1/TWIST1 expression in melanomas. In primary melanomas these factors were also significant in Kaplan Meier survival curves and after two step cluster analysis the combined profile of ZEB1[superscript high]/TWIST1[superscript high]/ZEB2[superscript low] predicted the worse prognosis (P=0.001). Multivariate Cox regression analyses of IHC-P staining indicated that only the gain of ZEB1 (P<0.002, n=98) and superficial TWIST1 (P=0.012) were associated with poor metastasis-free survival and independent of breslow depth. In conclusion, the reversible switch between ZEB1/TWIST1 and ZEB2/SNAI2 is controlled by RAS-RAF-MAPK pathway activity and constitutes an independent factor of poor prognosis in patients with malignant melanoma.