Characterization of survival pathways in melanoma

A hallmark feature of cancer is resistance to anoikis, apoptosis induced when cells either lose contact with or encounter an inappropriate extracellular matrix. Melanoma is inherently associated with a high degree of resistance to apoptosis. Over the past 30 years, the development of new therapeutics for melanoma treatment has been unsuccessful. Therefore, a better understanding of the apoptotic mechanisms functioning in melanoma cells is critical. Mutations in B-RAFV600E are prevalent in melanoma and promote constitutive MEK-ERK1/2 signaling and cell transformation. Acquisition of B-RAF mutations correlates with vertical phase growth when melanoma cells invade into the dermis, a collagen-rich environment that also contains fibronectin matrix. In addition, alterations in PI-3 kinase signaling that lead to activation of AKT are detected in advanced melanomas. Here we show that depletion of B-RAF expression by siRNA or pharmacological inhibition of MEK rendered melanoma cells susceptible to anoikis. Furthermore, adhesion to fibronectin but not collagen protected melanoma cells from anoikis through a PI-3 kinase-dependent pathway. Therefore, melanoma cells require either B-RAF or PI-3 kinase activation for protection from anoikis. Notably, AKT signaling in melanoma cells is substrate-specific. These findings demonstrate that melanoma cells utilize multiple signaling pathways to provide resistance to apoptosis.; Understanding the mechanism underlying both the B-RAF-MEK and adhesion mediated, PI-3 kinase dependent survival pathways in melanoma may allow for the development of targeted therapies for melanoma in the future. Therefore, we also provide evidence that B-RAF-MEK signaling blocks anoikis through the inhibition of two pro-apoptotic proteins: Bad and Bim. B-RAF-MEK signaling regulates phosphorylation of the inhibitory serine-75 residue of Bad, and decreases Bad expression. RNA interference and over-expression experiments demonstrate that Bad contributes to the susceptibility of B-RAF-depleted cells to anoikis. Additionally, B-RAF-MEK signaling regulates the expression of BimEL, mainly though control of protein turnover. Bim is also partially required for anoikis in B-RAF depleted cells and ectopic expression of Bim promotes apoptosis in non-adherent melanoma cells. Notably, depletion of Bim together with Bad has an additive effect on protecting B-RAF knockdown cells from anoikis. Together, our data show that Bad and Bim are major B-RAF responsive proteins regulating apoptosis in melanoma cells. We also provide evidence in support of a potential role for the anti-apoptotic proteins Bcl-xL and Mcl-1 in the PI-3 kinase-dependent, adhesion-mediated survival pathway. Here we show that both Bcl-xL and Mcl-1 are up-regulated upon adhesion to fibronectin but not collagen and that their expression is dependent upon PI-3 kinase signaling.; Together, this work describes two pathways functioning in melanoma cells to promote survival. One pathway is dependent on B-RAF-MEK signaling and functions through the inhibition of two pro-apoptotic proteins Bad and BimEL. The other pathway is mediated by adhesion to fibronectin but not collagen and is dependent on PI-3 kinase signaling and may function through the anti-apoptotic proteins, Mcl-1 and/or Bcl-xL.