Mutant B-RAF mediates resistance to anoikis via Bad and Bim.
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.
Boisvert-Adamo
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Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY 12208, USA.
Aplin
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D023102:Anoikis / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism / Q000502:physiology*; D000072224:Bcl-2-Like Protein 11; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020930:MAP Kinase Kinase Kinases / Q000502:physiology; D008545:Melanoma / Q000235:genetics; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D050505:Mutant Proteins / Q000502:physiology; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D014407:Tumor Cells, Cultured; D051025:bcl-Associated Death Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D008565:Membrane Proteins; D050505:Mutant Proteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D051025:bcl-Associated Death Protein; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020930:MAP Kinase Kinase Kinases; D046988:Proteasome Endopeptidase Complex