MEK-independent regulation of FRA-1 and implications for EMT-TF reprogramming

The main drivers of cutaneous malignant melanoma (MM) are mutations in BRAF and NRAS oncogenes, which stimulate MEK-ERK signalling. FRA-1, an effector of the NRAS and BRAF pathways, is a modulator of cellular plasticity and epithelial mesenchymal transition (EMT)-like processes in MM. FRA-1 harbours a C-terminal destabiliser that is responsible for ubiquitin-independent proteasomal degradation of the protein in epithelial cells. The activity of this motif is neutralised by phosphorylation of two Serines, Ser-252 and Ser-265, by RSK and ERK respectively.

Here I demonstrate that an uncharacterised pathway acts in parallel with NRAS/BRAF/MEK/ERK signalling to regulate FRA-1 abundance in MM cells. Indeed, although FRA-1 expression statistically correlates with P-ERK in clinical samples of MM, there are many examples of tumour areas showing lack of such correlation. The activity of this novel pathway depends on cell density; in sparse melanoma cell cultures, FRA-1 protein is present at much higher levels than in dense cultures, whereas levels of mRNA are cell density-independent. Our immunofluorescence data showed that subcellular localisation of FRA-1 is influenced by cell density. FRA-1 was predominantly nuclear in dense cultured cells. We gained mechanistic insight into this aspect of FRA-1 regulation in melanoma cells in vitro. By using FRA-1 mutants where the C-terminus is inactivated, we showed that the latter does not participate in cell density dependent expression of the protein. By using lysine-deficient FRA-1 mutants, we found that in dense MM cell culture, FRA-1 is degraded by a proteasome via ubiquitin-dependent mechanism. Degradation is MEK/ERK independent and requires native N-terminal protein domain. Therefore, in contrast to epithelial cells, there are two protein modules determining FRA-1 abundance in MM cells, a C-terminal MEK/ERK-dependent destabiliser, and an extra N-terminal regulatory element. These two protein elements promote FRA-1 degradation in ubiquitination-dependent and –independent modes respectively. Experiments with the conditioned media demonstrated that dense MM cell cultures secrete soluble factors, including IGFBP-7, which reduce FRA-1 abundance in sparse cell cultures. We propose that IGFBP-7 may participate in the activation of an E3 ligase which leads to ubiquitination of FRA-1. In the future, our data will open new opportunity for therapeutic targeting FRA-1 in MM to tackle plasticity of tumour cells.