An ultraviolet-radiation-independent pathway to melanoma carcinogenesis in the red hair/fair skin background

Abstract

People with pale skin, red hair, freckles and an inability to tan--the 'red hair/fair skin' phenotype--are at highest risk of developing melanoma, compared to all other pigmentation types. Genetically, this phenotype is frequently the product of inactivating polymorphisms in the melanocortin 1 receptor (MC1R) gene. MC1R encodes a cyclic AMP-stimulating G-protein-coupled receptor that controls pigment production. Minimal receptor activity, as in red hair/fair skin polymorphisms, produces the red/yellow pheomelanin pigment, whereas increasing MC1R activity stimulates the production of black/brown eumelanin. Pheomelanin has weak shielding capacity against ultraviolet radiation relative to eumelanin, and has been shown to amplify ultraviolet-A-induced reactive oxygen species. Several observations, however, complicate the assumption that melanoma risk is completely ultraviolet-radiation-dependent. For example, unlike non-melanoma skin cancers, melanoma is not restricted to sun-exposed skin and ultraviolet radiation signature mutations are infrequently oncogenic drivers. Although linkage of melanoma risk to ultraviolet radiation exposure is beyond doubt, ultraviolet-radiation-independent events are likely to have a significant role. Here we introduce a conditional, melanocyte-targeted allele of the most common melanoma oncoprotein, BRAF(V600E), into mice carrying an inactivating mutation in the Mc1r gene (these mice have a phenotype analogous to red hair/fair skin humans). We observed a high incidence of invasive melanomas without providing additional gene aberrations or ultraviolet radiation exposure. To investigate the mechanism of ultraviolet-radiation-independent carcinogenesis, we introduced an albino allele, which ablates all pigment production on the Mc1r(e/e) background. Selective absence of pheomelanin synthesis was protective against melanoma development. In addition, normal Mc1r(e/e) mouse skin was found to have significantly greater oxidative DNA and lipid damage than albino-Mc1r(e/e) mouse skin. These data suggest that the pheomelanin pigment pathway produces ultraviolet-radiation-independent carcinogenic contributions to melanomagenesis by a mechanism of oxidative damage. Although protection from ultraviolet radiation remains important, additional strategies may be required for optimal melanoma prevention.

Figure 1. Without UV radiation, BRaf^CA red mice have an increased rate of melanoma development relative to black and albino BRaf^CA animals

(a) C57BL/6 pigmentation variants with epidermal melanocytes (K14-SCF). From left to right: black (wild-type), red (Mc1r^e/e), and albino (Tyr^c/c). (b) Genotype of animals used for experimental studies. (c) Percent survival of pigmentation variants not carrying the K14-SCF transgene, i.e. no epidermal melanocytes. (n_black=28, n_red=40, n_albino=48) p_black-albino=0.250, p_black-red=0.003, p_albino-red=0.003. (d) Percent survival of pigmentation variants carrying the K14-SCF transgene, i.e. epidermal melanocytes (n_black=49, n_red=77, n_albino=41) p_black-albino=0.103, p_black-red=0.009, p_albino-red<0.0001.

Figure 2. Melanomas on all three pigmentation variants are morphologically similar and exhibit common histologic features

Melanomas on (a) black, (b) albino, and (c) red mice are grossly amelanotic. Histologically, (d) black, (e) albino, and (f) red melanomas are also mostly amelanotic though superficial tumor cells in black-BRaf^V600E tumors carry melanin. (g) Red-BRaf^V600E melanomas also can carry pigment (arrows). (h) Further magnification of two red melanomas also illustrates pigmented tumor cells. (i) Forskolin induces epidermal pigmentation (arrowheads) and mild tumor cell pigmentation (arrows). (j) Tumor cells stain positive for S100. (k) Skin-draining lymph nodes carry gp100+ cells (red).

Figure 3. Tumor cells from a red-BRaf^CA animal behave like classic BRAF^V600E melanomas after cAMP upregulation or BRAF inhibition

(a) 20 μM forskolin upregulates expression of melanocytic markers (n=4). (b) MAPK inhibition by PLX4720 or U0126 decreases melanoma cell proliferation (GI⁵⁰_PLX=0.5 μM, GI⁵⁰_U0126=2 μM) (n=3). (c) PLX4720 blocks melanoma growth in vivo (n=3). (d) 2 μM PLX4720 upregulates expression of melanocytic markers. mRNA expression normalized to 18s rRNA and 0 h time-point. Error bars denote s.e.m.

Figure 4. The UV-independent propensity of red BRaf^CA mice to develop melanoma is dependent on pigment production

(a) Genotypes of mice studied. (b) The albino allele protects Mc1r^e/e mice from melanoma development (n_red=40, n_albino=48, n_albino-red=90) p_{albino/albino-red}=0.308, p_albino/red<0.0001, p_{red/albino-red}<0.0001. (c) ROS reacts with purine nucleosides to produce 8,5′-cyclopurine lesions (cdA shown). (d) Selected-ion chromatograms for DNA from albino-Mc1r^e/e mouse skin. The insets show the positive-ion MS³ spectra for unlabeled and labeled S-cdA. (e) Both diastereomers of cdA and cdG are significantly higher in red-Mc1r^e/e skin. (n=3) *=p<0.05; **=p<0.01; ***=p<0.001. (f) Lipid peroxide levels are significantly higher in red-Mc1r^e/e skin (n=3). p<0.0001.