In Melanoma, RAS Mutations Are Accompanied by Switching Signaling from BRAF to CRAF and Disrupted Cyclic AMP Signaling

Abstract

Melanocytes require the RAS/RAF/MEK/ERK and the cyclic AMP (cAMP) signaling pathways to maintain the fine balance between proliferation and differentiation. We have investigated how cross-talk between these pathways affects melanoma progression. We show that cAMP suppresses CRAF activity in melanocytes and that this is essential to suppress the oncogenic potential of CRAF in these cells. As a consequence, BRAF alone is responsible for signaling to MEK. However, when RAS is mutated in melanoma, the cells switch their signaling from BRAF to CRAF. This switch is accompanied by dysregulated cAMP signaling, a step that is necessary to allow CRAF to signal to MEK. Thus, a fundamental switch in RAF isoform usage occurs when RAS is mutated in melanoma, and this occurs in the context of disrupted cAMP signaling. These data have important implications for the development of therapeutic strategies to treat this life-threatening disease.

c-Kit mutants require hypoxia-inducible factor 1α to transform melanocytes

“…We have already shown that Ras mutations are not equivalent to B-Raf mutations in melanoma cell lines as they do not activate the Ras/Raf/Mek/Erk pathway in the same way. B-Raf mutations constitutively activate Mek/ Erk, whereas Ras mutation induces a switch in signaling from B-Raf to C-Raf to activate Mek/Erk (Dumaz et al, 2006). Our current results on c-Kit mutants show that they do not activate Erk in the absence of HIF-1a but instead use the PI3K/Akt pathway for melanocyte transformation, pointing out the diversity in the mechanism of melanocyte transformation.…”

“…Therefore all the c-Kit constructs used in this study were devoid of the 12 bases coding for these four amino acids. Wild-type c-Kit, kindly provided by Dr Ro¨nnstrand (Lund University, Malmo, Sweden), was subcloned in the pMCEF vector (Dumaz et al, 2006). Mutations were introduced by site-directed mutagenesis and confirmed by sequence analysis.…”

“…Before SCF (Peprotech, Neuilly-sur-Seine, France) treatment, cells were maintained for 16 h in PMA-free medium (melan-a cells) or serum-free medium (501mel). To measure proliferation in normoxia, DNA synthesis was determined using thymidine as previously described (Dumaz et al, 2006). To create a hypoxic environment, cells were grown in a hypoxic chamber in 1% oxygen in a 37 1C incubator.…”

Targeted Therapy for Cutaneous Melanoma: Beyond BRAF...

“…While sorafenib was initially investigated in melanoma primarily as a therapeutic strategy for tumors with BRAF V600 mutations, it is a more potent inhibitor of CRAF (IC50 6 nmol) than it is of either wild-type (22 nmol) or mutant (38 nmol) BRAF proteins. 19 As preclinical studies support that NRAS is more dependent upon CRAF than other RAF isoforms to activate the RAS-RAF-MAPK pathway, 20 sorafenib may be effective in melanomas with activating NRAS mutations. This hypothesis is supported by a recent analysis of the results of the ECOG 2603 trial, which was a randomized trial of paclitaxel and carboplatin (TC) with or without sorafenib in treatment-naïve metastatic melanoma patients.…”

Signal control through Raf: in sickness and in health

“…Activation of MAPK pathway by cAMP occurs in cells that are neural crest-derived, such as melanocytes and the neuroendocrine pheochromocytoma PC12 cell line [67,68].…”