Ultraviolet radiation (UVR) is the principal causal factor for melanoma; albeit the underlying mechanisms remain unclear. While the mutagenic properties of UVR are irrefutable, the role of UVR-induced mutations in the initiation of melanoma is controversial which highlights the gap in our knowledge of the initial critical molecular mechanisms of UVR-induced melanomagenesis. To investigate the potential non-mutational mechanisms of UVR-induced melanomagenesis, we studied the role of UVR in modulating DNA methylation changes in melanocytes via next-generation sequencing-based methodologies. Here we show that UVR directly causes stable changes in the DNA methylome and transcriptome, one month after exposure. Genomic features associated with transcription were protected from 5mC alterations whereas CpG sites found in intergenic regions were more likely to be affected. Additionally, the long-term effects of UVR seem to perturb signaling pathways important for melanocyte biology. Interestingly, UVR-sensitive CpG sites were found to be prognostic of overall patient survival and highlighted a subset of CpG sites that may be relevant in melanomagenesis.

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Interferon-gamma (IFNG) has long been regarded as the flag-bearer for the anti-cancer immunosurveillance mechanisms. However, relatively recent studies have suggested a dual role of IFNG, albeit there is no direct experimental evidence for its potential pro-tumor functions. Here we provide in vivo evidence that treatment of mouse melanoma cell lines with physiological levels of Ifng enhances their tumorigenicity and metastasis in lung colonization allograft assays performed in immunocompetent syngeneic host mice, but not in immunocompromised host mice. We also show that this enhancement is dependent on downstream signaling via Stat1 but not Stat3, providing evidence of an oncogenic function of Stat1 in melanoma. The experimental results suggest that melanoma cell-specific Ifng signaling modulates the tumor microenvironment and its pro-tumorigenic effects are dependent on the γδ T cells, as Ifng-enhanced tumorigenesis was inhibited in the TCR-δ knockout mice. Overall, these results show that Ifng signaling may have tumor-promoting effects in melanoma by modulating the immune cell composition of the tumor microenvironment.

Melanogenesis (melanin pigment production) in melanocytes is canonically stimulated by the alpha melanocyte stimulating hormone (αMSH), which activates the cyclic-AMP-mediated expression of the melanocyte inducing transcription factor (MITF) and its downstream melanogenic genes, including the principal rate-limiting melanogenic enzyme tyrosinase (TYR). Here, we report that interferon-gamma (IFNG; type II interferon), but not interferon-alpha (a type I interferon), induces a noncanonical melanogenic pathway in mouse and human melanocytic cells. Inhibition of IFNG pathway by the JAK1/2 inhibitor ruxolitinib or knocking out Stat1 gene abrogated the IFNGinduced melanogenesis. Interestingly, IFNG-induced melanogenesis was independent of MITF. IFNG markedly increased the TYR protein expression but did not affect the mRNA expression, suggesting a post-translational regulatory mechanism. In contrast, IFNG had no effect on the expression of other melanogenesis-related proteins, for example, tyrosinase-related protein 1 (TYRP1) and dopachrome tautomerase (DCT).Glycosidase digestion assays revealed that IFNG treatment increased the mature glycosylated form of TYR, but not its de novo synthesis. Moreover, cycloheximide chase assay showed that degradation of TYR was decreased in IFNG-treated cells. These results suggest that the IFNG-STAT1 pathway regulates melanogenesis via regulation of the post-translational processing and protein stability of TYR.

Melanogenesis (melanin pigment production) in melanocytes is canonically stimulated by the alpha-melanocyte stimulating hormone (αMSH), which activates the cyclic-AMP-mediated expression of the melanocyte inducing transcription factor (MITF) and its downstream melanogenic genes, including the principal rate-limiting melanogenic enzyme tyrosinase (Tyr). Here we report that interferon-gamma (IFNG; type II interferon), but not IFN-alpha (a type I interferon), induces a noncanonical melanogenic pathway. Inhibition of IFNG pathway by the JAK inhibitor ruxolitinib or knocking out Stat1 abrogated the IFNG-induced melanogenesis. Interestingly, IFNG-induced melanogenesis was independent of MITF. IFNG markedly increased the Tyr protein expression but did not affect the mRNA expression, suggesting a post-translational regulatory mechanism. In contrast, IFNG had no effect on the expression of other melanogenesis-related proteins, e.g. tyrosinase-related protein 1 (Tyrp1) and dopachrome tautomerase (Dct). Glycosidase digestion assays revealed that IFNG treatment increased the mature glycosylated form of Tyr, but not its de novo synthesis. Moreover, cycloheximide chase assay showed that degradation of Tyr was decreased in IFNG-treated cells. These results suggest that the IFNG-STAT1 pathway regulates melanogenesis via modulation of the post-translational processing and protein stability of Tyr.