Abstract
Normal cells undergo anoikis when they lose adhesion to or encounter an inappropriate extracellular matrix. By contrast, oncogenic signaling in tumor cells enables resistance to anoikis, a trait that contributes to tumor progression. The B-RAF serine-threonine kinase is mutated in multiple cancers and functions as an oncogene in melanoma. Previously, we demonstrated that B-RAF and downstream mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) signaling are necessary for protection from anoikis in mutant B-RAF-expressing melanoma cells. Regulation of Bcl-2 family members in melanoma and their role in B-RAF-mediated survival is poorly defined. Here, we provide evidence that B-RAF-MEK signaling protects against anoikis through alterations in two proapoptotic Bcl-2 family proteins: Bcl-xL/Bcl-2-associated death promoter (Bad) and Bcl-2-interacting mediator of cell death (Bim). B-RAF-MEK signaling regulates phosphorylation of the inhibitory serine-75 residue of Bad, and decreases Bad mRNA expression. RNA interference and overexpression experiments demonstrate that Bad contributes to the susceptibility of B-RAF-depleted cells to anoikis. Additionally, B-RAF-MEK signaling regulates the expression of Bim(EL), mainly through control of protein turnover. Increased Bim(EL) levels induce apoptosis in suspended cells and are required for anoikis in B-RAF-depleted cells. 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.