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FC2 Oncogenic B-RAF signalling confers the resistance of metastatic melanoma to autophagy

Armstrong, J.L.a; Corazzari, M.b; Piacentini, M.b; Lovat, P.E.a

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doi: 10.1097/01.cmr.0000382804.37451.8c
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Introduction Cutaneous metastatic melanoma remains largely untreatable due to the notorious resistance of these tumours to apoptosis. Oncogenic activating mutations in the B-RAF protein kinase occur in approximately 70% of melanomas and contribute to tumour progression and resistance to apoptosis. Recent research demonstrates autophagy is a highly efficient mode of cell death by excessive self-digestion, and as such pro-autophagic chemotherapy represents an alternative approach for apoptosis-resistant tumours. We have recently identified fenretinide, a synthetic retinoid and bortezomib, a 26S proteosome inhibitor as novel agents able to induce potent cell death of melanoma cells through the activation of endoplasmic reticulum (ER) stress culminating in apoptosis [1,2]. Since both these agents are also known to induce autophagy in other tumour types, the aim of the current study was to determine the influence of oncogenic B-RAF mutations on the cytotoxic response to fenretinide and bortezomib and the relative contribution of autophagy to this process.

Methods B-RAF wild-type and mutant metastatic melanoma cell lines and xenograft tumours were treated with fenretinide or bortezomib. Autophagy was assessed by western blotting for LC3-II induction, and electron microscopy to demonstrate autophagosome formation and the presence of autophagic vacuoles. Activation of BRAF signalling through transient over-expression of activated B-RAF V600E or down-regulation through the use of a MEK specific inhibitor U0126 was used to determine the involvement of B-RAF signalling in the autophagic response to both agents, while RNAi mediated knockdown of Beclin-1 was used to establish the relationship between autophagy and ER stress-induced apoptosis.

Results Showed fenretinide and bortezomib are potent activators of autophagy, demonstrated by increased LC3-II induction and extensive autophagic vacuolization in vitro and in vivo. However, melanoma cells harbouring B-RAF mutations were inherently less sensitive to autophagy or apoptosis induction in response to either agent. Transient over-expression of BRAF V600E, or treatment with U0126, confirmed the involvement of B-RAF signalling in autophagy resistance. In addition, knockdown of Beclin-1 resulted in diminished cell death in response to both fenretinide and bortezomib supporting a positive relationship between autophagy and ER stress-induced apoptosis and suggesting both processes are required for effective cellular demise.

Conclusion Taken together these data suggest oncogenic RAS/RAF signalling results in the resistance of melanoma to therapy-induced autophagy and cell death. Combining fenretinide or bortezomib treatment with strategies to relieve the inhibitory effect of B-RAF activation may therefore represent a means by which to harness autophagy for the therapeutic benefit of metastatic melanoma.

© 2010 Lippincott Williams & Wilkins, Inc.