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Purpose: Metastatic melanoma is characterized by extremely poor survival rates and hence novel therapies are urgently required. The ability of many anticancer drugs to activate autophagy, a lysosomal-mediated catabolic process which usually promotes cell survival, suggests targeting the autophagy pathway may be a novel means to augment therapy., Experimental Design: Autophagy and apoptosis were assessed in vitro in human melanoma cell lines in response to clinically achievable concentrations of the endoplasmic reticulum (ER) stress-inducing drugs fenretinide or bortezomib, and in vivo using a s.c. xenograft model., Results: Autophagy was activated in response to fenretinide or bortezomib in B-RAF wild-type cells, shown by increased conversion of LC3 to the autophagic vesicle-associated form (LC3-II) and redistribution to autophagosomes and autolysosomes, increased acidic vesicular organelle formation and autophagic vacuolization. In contrast, autophagy was significantly reduced in B-RAF-mutated melanoma cells, an effect attributed partly to oncogenic B-RAF. Rapamycin treatment was unable to stimulate LC3-II accumulation or redistribution in the presence of mutated B-RAF, indicative of de-regulated mTORC1-dependent autophagy. Knockdown of Beclin-1 or ATG7 sensitized B-RAF wild-type cells to fenretinide- or bortezomib-induced cell death, demonstrating a pro-survival function of autophagy. In addition, autophagy was partially reactivated in B-RAF-mutated cells treated with the BH3 mimetic ABT737 in combination with fenretinide or bortezomib, suggesting autophagy resistance is partly mediated by abrogated Beclin-1 function., Conclusions: Our findings suggest inhibition of autophagy in combination with ER stress-inducing agents may represent a means by which to harness autophagy for the therapeutic benefit of B-RAF wild-type melanoma., (©2011 AACR.)

Exploiting vulnerabilities in the intracellular signaling pathways of tumor cells is a key strategy for the development of new drugs. The activation of cellular stress responses mediated by the endoplasmic reticulum (ER) allows cancer cells to survive outside their normal environment. Many proteins that protect cells against ER stress are active as protein disulfide isomerases (PDI) and the aim of this study was to test the hypothesis that apoptosis in response to ER stress can be increased by inhibiting PDI activity. We show that the novel chemotherapeutic drugs fenretinide and velcade induce ER stress-mediated apoptosis in melanoma cells. Both stress response and apoptosis were enhanced by the PDI inhibitor bacitracin. Overexpression of the main cellular PDI, procollagen-proline, 2-oxoglutarate-4-dioxygenase beta subunit (P4HB), resulted in increased PDI activity and abrogated the apoptosis-enhancing effect of bacitracin. In contrast, overexpression of a mutant P4HB lacking PDI activity did not increase cellular PDI activity or block the effects of bacitracin. These results show that inhibition of PDI activity increases apoptosis in response to agents which induce ER stress and suggest that the development of potent, small-molecule PDI inhibitors has significant potential as a powerful tool for enhancing the efficacy of chemotherapy in melanoma.