Regulation of MITF and Brn2 in melanoma

Melanoma is the most aggressive skin cancer with high recurrence and low survival rate. In addition to genetic mechanisms, resistance also arises from phenotypic heterogeneity in which a proportion of cells, the so-called melanoma stem or initiating cells, survive therapy. Due to a lack of reliable markers, however, there is still debate about the existence of these cells in melanoma. Consistent with phenotypic heterogeneity, previous observations in our laboratory have demonstrated that cells in melanoma can reversibly segregate in vivo into different subpopulations with different properties, such as differentiation or increased invasive capacity (potentially attributed to the existence of de-differentiated stem-like cells). To characterise these cells, a dual reporter lentiviral system was engineered, expressing fluorescent proteins under cell stage/phenotype-specific promoters. The promoters for the transcription factors POU3F2 (Brn2) (to mark de-differentiated cells) and the microphthalmia-associated transcription factor (MITF) (to mark proliferating and differentiated cells) were chosen. Lentivirally-transduced cells were used to screen a library of kinase inhibitors for their potential to affect promoter activity in vitro. The RhoA/ROCK pathway, known to contribute to invasion and metastases, was identified to play a role in Brn2 promoter activity and exhibited differential effects on both the MITF and Brn2 promoters in 501mel and SKmel28 cell lines. Through investigation of other signalling pathways involved in melanoma metastasis, we also identified the co-activator Mastermind-like 1 (MAML1), previously reported to act in the Notch pathway, as an activator of the Brn2 promoter via the transcription factor TCF3, and the MITF promoter through the lymphoid-enhancer binding factor 1 (LEF1). The effects of MAML1 on Brn2 and MITF promoter activity were potentiated by β-catenin. These findings provide new opportunities for the identification of therapeutic targets to prevent metastases formation in melanoma.