Summary Inappropriate drug delivery, secondary toxicities and persistent chemo- and immuno-resistance have traditionally compromised treatment response in melanoma. Using cellular systems and genetically engineered mouse models, we show that melanoma cells retain an innate ability to recognize cytosolic dsRNA and mount persistent stress response programs able to block tumor growth, even in highly immunosuppressed backgrounds. The dsRNA mimic polyinosine-polycytidylic acid (pIC), coadministered with polyethyleneimine (PEI) as a carrier, was identified as an unanticipated inducer of autophagy downstream of an exacerbated endosomal maturation program. A concurrent activity of the dsRNA helicase MDA-5 driving the proapoptotic protein NOXA resulted in an efficient autodigestion of melanoma cells. These results reveal tractable links for therapeutic intervention among dsRNA helicases, endo/lysosomes and apoptotic factors.

In human and mouse, cAMP plays a key role in the control of pigmentation. cAMP, through the activation of protein kinase A, increases the expression of microphthalmia-associated transcription factor (MITF), which in turn stimulates tyrosinase gene expression, to allow melanin synthesis. Beyond this simplified scheme, cAMP inhibits phosphatidylinositol 3-kinase (PI3K), and inhibition of PI3K, by a specific inhibitor, stimulates melanogenesis. However, the link between the PI3K pathway and melanogenesis remained to be elucidated. In this report, we showed that cAMP, through a protein kinase A-independent mechanism, led to inhibition of AKT phosphorylation and activity. Consistent with the role of AKT in the regulation of glycogen synthase kinase 3␤ (GSK3␤), cAMP decreased the phosphorylation of GSK3␤ and stimulated its activity. Further, experiments were performed to investigate the role of GSK3␤ in the regulation of MITF expression and function. We observed that GSK3␤ regulated neither MITF promoter activity nor the intrinsic transcriptional activity of MITF but synergized with MITF to activate the tyrosinase promoter. Additionally, lithium, a GSK3␤ inhibitor, impaired the response of the tyrosinase promoter to cAMP, and cAMP increased the binding of MITF to the M-box. Taking into account that GSK3␤ phosphorylates MITF and increases the ability of MITF to bind its target sequence, our results indicate that activation of GSK3␤ by cAMP facilitates MITF binding to the tyrosinase promoter, thereby leading to stimulation of melanogenesis.

In melanocytes and melanoma cells α-melanocyte stimulating hormone (α-MSH), via the cAMP pathway, elicits a large array of biological responses that control melanocyte differentiation and influence melanoma development or susceptibility. In this work, we show that cAMP transcriptionally activates Hif1a gene in a melanocyte cell–specific manner and increases the expression of a functional hypoxia-inducible factor 1α (HIF1α) protein resulting in a stimulation of Vegf expression. Interestingly, we report that the melanocyte-specific transcription factor, microphthalmia-associated transcription factor (MITF), binds to the Hif1a promoter and strongly stimulates its transcriptional activity. Further, MITF “silencing” abrogates the cAMP effect on Hif1a expression, and overexpression of MITF in human melanoma cells is sufficient to stimulate HIF1A mRNA. Our data demonstrate that Hif1a is a new MITF target gene and that MITF mediates the cAMP stimulation of Hif1a in melanocytes and melanoma cells. Importantly, we provide results demonstrating that HIF1 plays a pro-survival role in this cell system. We therefore conclude that the α-MSH/cAMP pathway, using MITF as a signal transducer and HIF1α as a target, might contribute to melanoma progression.

IGF1 plays a key role in the development and growth of multiple tumors and in the prevention of apoptosis. In melanoma cells, IGF1 has been shown to mediate resistance to anoikis-induced apoptosis. However, the effect of IGF1 on other proapoptotic stimuli has never been reported. Further, the molecular mechanisms by which IGF1 mediates its prosurvival properties in melanoma cells remain unknown. Here, we demonstrate that IGF1 impairs the onset of tumor necrosis factor-related apoptosis-inducing ligand and staurosporine-induced apoptosis in melanoma cells expressing either wild-type or oncogenic B-Raf. Further, we show that IGF1 inhibits mitochondrial damage that occurs during apoptosis, thereby indicating that IGF1 acts at the level of mitochondria to mediate its antiapoptotic stimuli. Accordingly, IGF1 increases the mRNA levels and protein expression of antiapoptotic members of the BCL2 family--BCL2 and BCL-X(L)--and that of the inhibitor of apoptosis protein, survivin. Further, their specific silencing by small interfering RNA prevents the protective effect of IGF1. These findings therefore delineate the molecular mechanisms by which IGF1 mediates its prosurvival properties and provide a basis for clinical strategies designed to neutralize IGF1 or its target genes.