Targeting MC1R depalmitoylation to prevent melanomagenesis in redheads

Abstract

Some genetic melanocortin-1 receptor (MC1R) variants responsible for human red hair color (RHC-variants) are consequently associated with increased melanoma risk. Although MC1R signaling is critically dependent on its palmitoylation primarily mediated by the ZDHHC13 protein-acyl transferase, whether increasing MC1R palmitoylation represents a viable therapeutic target to limit melanomagenesis in redheads is unknown. Here we identify a specific and efficient in vivo strategy to induce MC1R palmitoylation for therapeutic benefit. We validate the importance of ZDHHC13 to MC1R signaling in vivo by targeted expression of ZDHHC13 in C57BL/6J-MC1R^RHC mice and subsequently inhibit melanomagenesis. By identifying APT2 as the MC1R depalmitoylation enzyme, we are able to demonstrate that administration of the selective APT2 inhibitor ML349 treatment efficiently increases MC1R signaling and represses UVB-induced melanomagenesis in vitro and in vivo. Targeting APT2, therefore, represents a preventive/therapeutic strategy to reduce melanoma risk, especially in individuals with red hair.

Fig. 1

Clinical relevance of ZDHHC13 and melanocortin-1 receptor signaling in human melanomas. a, b Pearson’s correlation between ZDHHC13 and MITF (a) or DCT (b) in The Cancer Genome Atlas (TCGA) SKCM. Plots show the Spearman’s correlation and MITF (left) or DCT (right) with ZDHHC13 mRNA level from RNA-seq data in TCGA melanoma calculated by GEPIA (Gene Expression Profiling Interactive Analysis). R and p values are shown. c Melanoma patient survival analysis based on ZDHHC13 expression (cutoff = 50%). All patients in the TCGA melanoma study were divided according to the expression level of ZDHHC13 (higher or lower level than median expression value of all patients)

Fig. 2

Transgenic ZDHHC13 increases MC1R^R151C signaling in vivo. a Images of mice with the indicated genotypes. All mice are on the C57/BL6J background. b Measurement of eumelanin and pheomelanin content in the whole back skin collected from the indicated mice shown in a. Eumelanin and pheomelanin were quantified by high-performance liquid chromatography based on the level of pyrrole-2,3,5-tricarboxylic acid (PTCA) by alkaline hydrogen peroxide oxidation of eumelanin and 4-amino-3-hydroxyphenylalanine (4-AHP) by hydriodic acid reductive hydrolysis of pheomelanin, respectively. Final results were determined by a conversion as described (eumelanin = 45 × PTCA, pheomelanin = 9 × 4-AHP). Three independent mice were used. c Tails and d ears of the indicated mice as shown in a. e Fontana–Masson staining of the indicated ear sections as shown in d. f Melanin quantification of the indicated tail and ear samples as shown in c, d. Three independent mice were used. g Melanocytes were isolated from the dorsolateral skin of 3.5-day postnatal mice. Total protein was extracted from mouse primary melanocytes and was then used for immunoprecipitation (IP) with specific anti-melanocortin-1 receptor antibodies. Acyl biotin exchange reaction and immunoblot analysis using the indicated antibodies were performed subsequently. *p < 0.05, ***p < 0.001, unpaired Student’s t test. Error bars represent ± s.d.

Fig. 3

Transgenic ZDHHC13 inhibits ultraviolet B (UVB)-induced melanomagenesis in redheads in vivo. a Schematic for UVB-induced melanoma development in mice. b Kaplan–Meier plot showing melanoma-free survival of the indicated mice. Ninety days after the final UV radiation, melanoma was diagnosed in 92% (11/12), 64% (9/14), and 19% (3/16) of mice with MC1R^C315S, MC1R^R151C, and MC1R^WT. Melanoma incidence was much lower in the Tg-ZDHHC13/MC1R^R151C mice than in the MC1R^R151C mice (27% (4/15) vs. 64% (9/14), log-rank test, p = 0.0457). c Hematoxylin and eosin staining of histological sections and immunohistochemistry staining of S100 of representative cutaneous melanomas. Genotypes were as indicated

Fig. 4

Targeted inhibition of APT2 inhibits ultraviolet B (UVB)-induced melanomagenesis in vivo. a Illustration of the dynamic melanocortin-1 receptor (MC1R) palmitoylation/depalmitoylation cycle. b cAMP levels in human primary melanocytes (HPMs) after treatment with increasing concentrations of the indicated inhibitors. HPMs in which endogenous MC1R is stably depleted using shMC1R were infected with Flag-MC1R^R151C and then treated with 1 μM α-melanocyte-stimulating hormone (α-MSH) and indicated inhibitors for 3.5 h. The resulting cells were harvested for a cAMP immunoassay. The data were compiled from five independent experiments. c MC1R-depleted HPMs were infected with the indicated Flag-MC1R-encoding retroviruses and then pretreated with 1 μM α-MSH and 100 nM ML349 for 30 min followed by 100 J/m² UVB irradiation. The resulted cells were harvested for immunoprecipitation, acyl-biotin exchange, and immunoblotting analysis with the specific antibodies as indicated 3 h after UVB exposure. d cAMP levels in MC1R-depleted HPMs expressing the indicated Flag-MC1R encoding retroviral constructs and pretreated with 1 μM α-MSH and 100 nM inhibitors for 30 min followed by 100 J/m² UVB irradiation. The resulted cells were harvested for cAMP immunoassay 3 h after UVB exposure. Data were compiled from five independent experiments. e–g Growth curves (e), dissected tumors (f), and tumor weight (g) for the xenograft experiments. MC1R-depleted hTERT/p53DD/CDK4(R24C)/BRAF^V600E melanocytes were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were preincubated with 1 μM α-MSH and 100 nM inhibitors for 30 min before being irradiated with 20 J/m² UVB. After 24 h, 3 × 10⁶ cells were inoculated subcutaneously into each flank of nude mice. Visible tumors were measured at the indicated days. h Melanoma-free survival of the indicated mice. In the UVB radiation period, 5 mg/kg ML349 was injected intraperitoneally into mice prior to the treatment with UV. Ninety days after the final UVR, melanoma was diagnosed in 23% (3/13) or 64% (7/11) of mice with or without ML349 treatment, respectively (p = 0.0366). **p < 0.01, ***p < 0.001, unpaired Student’s t test. Error bars represent ± s.d.