Negative regulation of Bmf and Bim by MAPK signaling promotes melanoma cell survival

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The purpose of this study was to define the mechanism(s) by which the mitogen-activated protein kinase (MAPK) pathway promotes survival in melanoma cells in order to identify molecular targets for therapeutic intervention. Mutually exclusive mutations in NRAS and BRAF result in constitutive activation of MAPK in the majority of melanomas. We and others have demonstrated that inhibition of MEK efficiently induces apoptosis in most melanoma cells in vitro and in vivo. The highly variable apoptotic sensitivity of melanoma cell lines to MEK inhibition has largely been attributed to NRAS or BRAF mutation status although no mechanism has been established. We analyzed the endogenous expression, post-translational modification, and localization of Bcl-2 family member proteins in a large panel of human melanoma cell lines treated with the small molecule MEK inhibitor PD184352 over time. Comparing sensititve (high degree of apoptosis) and resistant (low degree of apoptosis) fractionated cell lysates revealed a dramatic difference in Bmf localization. Bmf mRNA expression increased in all melanoma cells in response to MEK inhibition, but Bmf cytosolic localization correlated with apoptotic sensitivity; high amounts of Bmf were observed in the cytosolic fraction of sensitive cells while little or no Bmf was observed in this fraction in resistant cells. Moreover, suppression of Bmf expression by RNA interference (RNAi) efficiently protected sensitive melanoma cells from PD184352-induced apoptosis. Surprisingly, dramatic Bim expression and mitochondrial localization occurred in all melanoma cell lines tested but had minimal impact on apoptosis. Overexpression of Bcl-xL, which directly interacts with and antagonizes both Bmf and Bim, provided complete rescue from apoptosis induced by MEK inhibition, while Bfl-1 (which antagonizes Bim, but not Bmf) had minimal impact. Therefore, apoptotic sensitivity of melanoma cell lines to MEK inhibition has less to do with NRAS or BRAF status as it does with the overreliance on MAPK for BH3-only suppression. Those cell lines that suppress Bim and Bmf by MAPK are sensitive to MEK inhibition while those cell lines that have an alternative means of suppressing Bmf survive.