Prominent role of cyclic adenosine monophosphate signalling pathway in the sensitivity of (WT)BRAF/(WT)NRAS melanoma cells to vemurafenib.

Vemurafenib improves survival in patients with melanoma bearing the (V600E)BRAF mutation, but it did not show any benefit in clinical trials focusing on wild type tumours while it may well inhibit (WT)BRAF considering the dosage used and the bioavailability of the drug. As tumours may contain a mixture of mutant and wild type BRAF cells and this has been also put forward as a resistance mechanism, we aimed to evaluate the sensitivity/resistance of six, randomly selected, (WT)BRAF/(WT)NRAS lines to vemurafenib and found four sensitive. The sensitivity to the drug was accompanied by a potent inhibition of both phospho-ERK and phospho-AKT, and a significant induction of apoptosis while absent in lines with intrinsic or acquired resistance. Phospho-CRAF expression was low in all sensitive lines and high in resistant ones, and MEK inhibition can effectively potentiate the drug effect. A possible explanation for CRAF modulation is cyclic adenosine monophosphate (cAMP), a mediator of melanocortin receptor 1 (MC1R) signalling, since it can actually inhibit CRAF. Indeed, we measured cAMP and found that all four sensitive lines contained significantly higher constitutive cAMP levels than the resistant ones. Consequently, vemurafenib and cAMP stimulator combination resulted in a substantial synergistic effect in lines with both intrinsic and acquired resistance but only restricted to those where cAMP was effectively increased. The use of a cAMP agonist overcame such restriction. In conclusion, we report that (WT)BRAF/(WT)NRAS melanoma lines with low phospho-CRAF and high cAMP levels may be sensitive to vemurafenib and that CRAF inhibition through cAMP stimulation may overcome the resistance to the drug.