Tumor cells often utilize developmental processes in order to progress towards advanced disease. The E-box transcription factor TWIST1 is essential to epithelial-mesenchymal transition and cell migration in the developing neural crest. In melanoma, which derives from the neural crest cell lineage, enhanced TWIST1 expression has been linked to worse clinical prognosis. However, mechanisms underlying TWIST1 expression and whether aberrant TWIST1 levels promote steps in melanoma progression remain unknown. Here, we report that elevated TWIST1 mRNA/protein expression is dependent on ERK1/2 signaling, which is hyperactive in the majority of melanomas. We show that TWIST1 protein levels are especially high in melanoma cell lines generated from invasive, pre-metastatic stage tumors. Furthermore, TWIST1 expression is required and sufficient to promote invasion through Matrigel and spheroid outgrowth in three-dimensional dermal-mimetic conditions. Alterations to spheroid outgrowth were not as a result of altered cell death, cell cycle profile, or paradigm EMT protein changes. Importantly, we identify matrix metalloproteinase-1 (MMP-1) as a novel downstream target of TWIST1. We have determined that TWIST1 acts, in a dose-dependent manner, as a mediator between hyperactive ERK1/2 signaling and regulation of MMP-1 transcription. Together, these studies mechanistically demonstrate a previously unrecognized interplay between ERK1/2, TWIST1, and MMP-1 which is likely significant in the progression of melanoma towards metastasis.

Activating mutations in B-RAF and N-RAS occur in B60 and B15% of melanomas, respectively. The most common mutation in B-RAF is V600E, which activates B-RAF and the downstream MEK-ERK1/2 pathway. Thus, B-RAF V600E is a viable therapeutic target. PLX4720 is a selective inhibitor of mutant B-RAF and its analog, PLX4032, is currently undergoing clinical trials in melanoma. However, the effects of PLX4720 across the genotypic spectrum in melanoma remain unclear. Here, we describe that PLX4720 treatment rapidly induces hyperactivation of the MEK-ERK1/2 pathway in mutant N-RAS melanoma cells. Furthermore, we demonstrate that C-RAF is the major RAF isoform involved in this process. Importantly, PLX4720-induced hyperactivation of the MEK-ERK1/2 pathway promotes resistance to apoptosis in both non-invasive and invasive mutant N-RAS melanoma cells but does not enhance cell cycle properties. These findings underscore the need to genotypically stratify melanoma patients before enrollment on a mutant B-RAF inhibitor trial.