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Mutant B-RAF mediates resistance to anoikis via Bad and Bim

Oncogene volume 27, pages 3301–3312 (2008)Cite this article

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 BimEL, mainly through control of protein turnover. Increased BimEL 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.

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Acknowledgements

We are grateful to Melanie Mayberry and Rong Hu for help with the propidium iodide staining experiments. We also acknowledge Ethan V. Abel for help with the lentiviral system, and the AMC flow cytometry core facility for the use of the FACSCanto and the FlowJo software. We thank Mr Matthew VanBrocklin and Dr George Vande Woude (Van Andel Research Institute) for the Bad plasmids, Dr Hidesuke Fukazawa (National Institute of Infectious Diseases, Tokyo, Japan) for the Bim plasmids and Dr Meenhard Herlyn (Wistar Institute, Philadelphia) for the melanoma cell lines. Additionally, Drs Julio Aguirre-Ghiso, Kevin Pumiglia and Michael DiPersio provided helpful discussions. This work was supported by a grant, GM067893, from the National Institutes of Health.

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  1. Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY, USA

    K Boisvert-Adamo & A E Aplin

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  1. K Boisvert-Adamo
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Correspondence to A E Aplin.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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Boisvert-Adamo, K., Aplin, A. Mutant B-RAF mediates resistance to anoikis via Bad and Bim. Oncogene 27, 3301–3312 (2008). https://doi.org/10.1038/sj.onc.1211003

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