Akt3 and Mutant V600EB-Raf Cooperate to Promote Early Melanoma Development

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Abstract
B-Raf is the most mutated gene in melanoma; however, the mechanism through which it promotes early melanomas remains uncertain. Most nevi contain activated V600EB-Raf but few develop into melanoma, and expression in melanocytes is inhibitory with low protein levels present in surviving cells, suggesting unknown cooperative oncogenic events are necessary for melanoma development. Because many melanomas have V600EB-Raf and active Akt3, it is possible that these proteins cooperatively facilitate melanocyte transformation. In this study, Akt3 is shown to phosphorylate V600EB-Raf to lower its activity as well as that of the downstream mitogen-activated protein kinase (MAPK) pathway to levels promoting early melanoma development. Expression of active Akt3 in early melanoma cells containing V600EB-Raf reduced MAPK signaling and promoted anchorage-independent growth. Furthermore, expression of both V600EB-Raf and active Akt3 in melanocytes promoted a transformed phenotype. Mechanistically, aberrant Akt3 activity in early melanomas serves to phosphorylate Ser364 and Ser428 on V600EB-Raf to reduce activity of V600EB-Raf to levels that promote rather than inhibit proliferation, which aids melanocytic transformation. Inhibition of V600EB-Raf or Akt3 in advanced melanoma cells in which both pathways were active reduced anchorage-independent growth and tumor development in a cooperatively acting manner. Inhibition of Akt3 alone in these cells led to increased MAPK signaling. In summary, these results suggest that activating B-Raf mutations initially promote nevi development, but the resulting high, intense activation of the MAPK pathway inhibits further tumor progression requiring Akt3 activation to bypass this barrier and aid melanoma development. [Cancer Res 2008;68(9):3429–39]