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Abstract

Over the last decades, novel therapies against melanoma have allowed for a prolonged survival rate of malignant melanoma while not allowing for a cure because of its aggressiveness and death resistance. Therefore, adaptation of existing and development of innovative therapeutic interventions for melanoma are required. One promising option could be discovery of drugs that are able to induce programmed cell death in melanoma. Another strategy is to inhibit molecules and cell signalling that are relevant for cell death resistance of melanoma to therapies. Thus, the detailed understanding of cell death regulation and potential resistance mechanisms of melanoma are indispensable. IAPs exert their inhibitory effect on cell death through impairing caspase activities and ubiquitination targeted proteins. In our study, the negative regulatory role of IAPs in CD95L-mediated cell death was confirmed by using IAP antagonist in a panel of human melanoma cell lines in vitro. Furthermore, we found that CD95L-induced cell death in absence of IAPs in melanoma cells was dominantly mediated by caspase-dependent apoptosis but not necroptosis, which suggested the resistance of melanoma to the necroptotic signalling. When we analysed expression of necroptosis-related proteins, we found that the protein expression of RIPK3 was low or absent in melanoma cell lines compared with cultured keratinocytes, primary melanocytes and nevus cells, which resulted from absence of RIPK3 mRNA expression. Ectopical expression of RIPK3 in melanoma uncovered the CD95L-mediated necroptosis which was correlated with MLKL phosphorylation. The indispensability of RIPK3 in necroptosis was further confirmed by mutation of RIPK3 kinase domain and RIPK3 specific inhibitors. Hypermethylation of RIPK3 DNA promoter was identified as the reason for RIPK3 absence in melanoma cell lines. Restoration of RIPK3 expression was successful via demethylation reagent. Unexpectedly, demethylation-mediated RIPK3 reconstitution failed to induce necroptosis in treated melanoma cells. NF-κB activation mainly as a pro-survival signalling initiates a variety of genes induction in melanomas. In our study, NF-κB activation led to expression or upregulation of cIAPs and cFLIP which rendered cell death resistance to melanoma. Cell death assay results confirmed the negative regulatory role of NF-κB in TNF-mediated cell death. Unusually, NF-κB activation resulted in spontaneous cell sensitivity to IAP antagonist via promotion of RIPK1-dependent complex formation in IKK2 constitutively activated melanomas. Our data verified the negatively regulatory effect of IAPs and NF-κB activation on cell death in melanoma cells. RIPK3 was also identified as essential link for necroptotic cell death in melanomas. Therefore, overcoming cell death resistance by antagonizing IAP activity is a promising strategy for melanoma treatment. In addition, reconstitution of RIPK3 to induce necroptosis in melanoma is as an alternative option for melanoma therapy.