α‐MSH tripeptide analogs activate the melanocortin 1 receptor and reduce UV‐induced DNA damage in human melanocytes

One skin cancer prevention strategy that we are developing is based on synthesizing and testing melanocortin analogs that reduce and repair DNA damage resulting from exposure to solar ultraviolet (UV) radiation, in addition to stimulating pigmentation. Previously, we reported the effects of tetrapeptide analogs of α‐melanocortin (α‐MSH) that were more potent and stable than the physiological α‐MSH, and mimicked its photoprotective effects against UV‐induced DNA damage in human melanocytes. Here, we report on a panel of tripeptide analogs consisting of a modified α‐MSH core His⁶‐d ‐Phe⁷‐Arg⁸, which contained different N‐capping groups, C‐terminal modifications, or arginine mimics. The most potent tripeptides in activating cAMP formation and tyrosinase of human melanocytes were three analogs with C‐terminal modifications. The most effective C‐terminal tripeptide mimicked α‐MSH in reducing hydrogen peroxide generation and enhancing nucleotide excision repair following UV irradiation. The effects of these three analogs required functional MC1R, as they were absent in human melanocytes that expressed non‐functional receptor. These results demonstrate activation of the MC1R by tripeptide melanocortin analogs. Designing small analogs for topical delivery should prove practical and efficacious for skin cancer prevention.     

Endothelin‐1 and α‐melanocortin have redundant effects on global genome repair in UV‐irradiated human melanocytes despite distinct signaling pathways

Human melanocyte homeostasis is sustained by paracrine factors that reduce the genotoxic effects of ultraviolet radiation (UV), the major etiological factor for melanoma. The keratinocyte‐derived endothelin‐1 (End‐1) and α‐melanocyte‐stimulating hormone (α‐MSH) regulate human melanocyte function, proliferation and survival, and enhance repair of UV‐induced DNA photoproducts by binding to the G_q‐ and G_i‐protein‐coupled endothelin B receptor (EDNRB), and the G_s‐protein‐coupled melanocortin 1 receptor (MC1R), respectively. We hereby report that End‐1 and α‐MSH regulate common effectors of the DNA damage response to UV, despite distinct signaling pathways. Both factors activate the two DNA damage sensors ataxia telangiectasia and Rad3‐related and ataxia telangiectasia mutated, enhance DNA damage recognition by reducing soluble nuclear and chromatin‐bound DNA damage binding protein 2, and increase total and chromatin‐bound xeroderma pigmentosum (XP) C. Additionally, α‐MSH and End‐1 increase total levels and chromatin localization of the damage verification protein XPA, and the levels of γH2AX, which facilitates recruitment of DNA repair proteins to DNA lesions. Activation of EDNRB compensates for MC1R loss of function, thereby reducing the risk of malignant transformation of these vulnerable melanocytes. Therefore, MC1R and EDNRB signaling pathways represent redundant mechanisms that inhibit the genotoxic effects of UV and melanomagenesis.     

Significance of the melanocortin 1 receptor in the DNA damage response of human melanocytes to ultraviolet radiation

Activation of the melanocortin 1 receptor (MC1R) by α‐melanocortin (α‐MSH) stimulates eumelanin synthesis and enhances repair of ultraviolet radiation (UV)‐induced DNA damage. We report on the DNA damage response (DDR) of human melanocytes to UV and its enhancement by α‐MSH. α‐MSH up‐regulated the levels of XPC, the enzyme that recognizes DNA damage sites, enhanced the UV‐induced phosphorylation of the DNA damage sensors ataxia telangiectasia and Rad3‐related (ATR) and ataxia telangiectasia mutated (ATM) and their respect‐ive substrates checkpoint kinases 1 and 2, and increased phosphorylated H2AX (γH2AX) formation. These effects required functional MC1R and were absent in melanocytes expressing loss of function (LOF) MC1R. The levels of wild‐type p53‐induced phosphatase 1 (Wip1), which dephosphorylates γH2AX, correlated inversely with γH2AX. We propose that α‐MSH increases UV‐induced γH2AX to facilitate formation of DNA repair complexes and repair of DNA photoproducts, and LOF of MC1R compromises the DDR and genomic stability of melanocytes.     

A retro‐inverso α‐melanocyte stimulating hormone analog with MC1R‐binding selectivity

α‐melanocyte stimulating hormone (α‐MSH) is a tridecapeptide fragment of pro‐opiomelanocortin (POMC) with broad effects on appetite, skin pigmentation, hormonal regulation, and potential roles in both inflammation and autoimmunity. The use of this peptide as an anti‐inflammatory agent is limited by its low selectivity between the melanocortin receptors, susceptibility to proteolytic degradation, and rapid clearance from circulation. A retro‐inverso (RI) sequence of α‐MSH was characterized for receptor activity and resistance to protease. This peptide demonstrated surprisingly high selectivity for binding the melanocortin receptor 1 (MC1R). However, RI‐α‐MSH exhibited a diminished binding affinity for MC1R compared to α‐MSH. Mapping of the residues critical for agonist activity, receptor binding, and selectivity by alanine scanning, identified the same critical core tetrapeptide required for the native peptide. Modest improvements in affinity were obtained by conservative changes employing non‐natural amino acids and substitution of the C‐terminal sequence with a portion of a MC1R ligand peptide previously identified by phage display. Recombination of these elements yielded a peptide with an identical K_i as α‐MSH at MC1R and a lower EC₅₀ in Mel‐624 melanoma cells. A number of other structural modifications of the RI peptide were found to differ in effect from those reported for the L ‐form α‐MSH, suggesting a significantly altered interaction with the MC1R. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Melanin content and MC1R function independently affect UVR‐induced DNA damage in cultured human melanocytes

Malignant transformation of melanocytes leads to melanoma, the most fatal form of skin cancer. Ultraviolet radiation (UVR)‐induced DNA photoproducts play an important role in melanomagenesis. Cutaneous melanin content represents a major photoprotective mechanism against UVR‐induced DNA damage, and generally correlates inversely with the risk of skin cancer, including melanoma. Melanoma risk is also determined by susceptibility genes, one of which is the melanocortin 1 receptor (MC1R) gene. Certain MC1R alleles are strongly associated with melanoma. We hereby present experimental evidence for the role of two melanoma risk factors, constitutive pigmentation, as assessed by total melanin, eumelanin and pheomelanin contents, and MC1R genotype and function, in determining the induction and repair of DNA photoproducts in cultured human melanocytes after irradiation with increasing doses of UVR. We found that total melanin and eumelanin contents (MC and EC) correlated inversely with the extent of UVR‐induced growth arrest, apoptosis and induction of cyclobutane pyrimidine dimers (CPD), but not with hydrogen peroxide release in melanocytes expressing functional MC1R. In comparison, melanocytes with loss‐of‐function MC1R, regardless of their MC or EC, sustained more UVR‐induced apoptosis and CPD, and exhibited reduced CPD repair. Therefore, MC, mainly EC, and MC1R function are independent determinants of UVR‐induced DNA damage in melanocytes.     

Agouti protein,mahogunin, and attractin in pheomelanogenesis and melanoblast‐like alteration of melanocytes: a cAMP‐independent pathway

Melanocortin‐1 receptor (MC1R) and its ligands, α‐melanocyte stimulating hormone (αMSH) and agouti signaling protein (ASIP), regulate switching between eumelanin and pheomelanin synthesis in melanocytes. Here we investigated biological effects and signaling pathways of ASIP. Melan‐a non agouti (a/a) mouse melanocytes produce mainly eumelanin, but ASIP combined with phenylthiourea and extra cysteine could induce over 200‐fold increases in the pheomelanin to eumelanin ratio, and a tan‐yellow color in pelletted cells. Moreover, ASIP‐treated cells showed reduced proliferation and a melanoblast‐like appearance, seen also in melanocyte lines from yellow (A^y/a and Mc1r^e/ Mc1r^e) mice. However ASIP‐YY, a C‐terminal fragment of ASIP, induced neither biological nor pigmentary changes. As, like ASIP, ASIP‐YY inhibited the cAMP rise induced by αMSH analog NDP‐MSH, and reduced cAMP level without added MSH, the morphological changes and depigmentation seemed independent of cAMP signaling. Melanocytes genetically null for ASIP mediators attractin or mahogunin (Atrn^{mg‐3J/mg‐3J} or Mgrn1^{md‐nc/md‐nc}) also responded to both ASIP and ASIP‐YY in cAMP level, while only ASIP altered their proliferation and (in part) shape. Thus, ASIP–MC1R signaling includes a cAMP‐independent pathway through attractin and mahogunin, while the known cAMP‐dependent component requires neither attractin nor mahogunin.     

Melanin content and MC1R function independently affect UVR-induced DNA damage in cultured human melanocytes

Malignant transformation of melanocytes leads to melanoma, the most fatal form of skin cancer. Ultraviolet radiation (UVR)-induced DNA photoproducts play an important role in melanomagenesis. Cutaneous melanin content represents a major photoprotective mechanism against UVR-induced DNA damage, and generally correlates inversely with the risk of skin cancer, including melanoma. Melanoma risk is also determined by susceptibility genes, one of which is the melanocortin 1 receptor (MC1R) gene. Certain MC1R alleles are strongly associated with melanoma. We hereby present experimental evidence for the role of two melanoma risk factors, constitutive pigmentation, as assessed by total melanin, eumelanin and pheomelanin contents, and MC1R genotype and function, in determining the induction and repair of DNA photoproducts in cultured human melanocytes after irradiation with increasing doses of UVR. We found that total melanin and eumelanin contents (MC and EC) correlated inversely with the extent of UVR-induced growth arrest, apoptosis and induction of cyclobutane pyrimidine dimers (CPD), but not with hydrogen peroxide release in melanocytes expressing functional MC1R. In comparison, melanocytes with loss-of-function MC1R, regardless of their MC or EC, sustained more UVR-induced apoptosis and CPD, and exhibited reduced CPD repair. Therefore, MC, mainly EC, and MC1R function are independent determinants of UVR-induced DNA damage in melanocytes.     

Cutaneous pharmacologic cAMP induction induces melanization of the skin and improves recovery from ultraviolet injury in melanocortin 1 receptor‐intact or heterozygous skin

Homozygous loss of function of the melanocortin 1 receptor (MC1R) is associated with a pheomelanotic pigment phenotype and increased melanoma risk. MC1R heterozygosity is less well studied, although individuals inheriting one loss‐of‐function MC1R allele are also melanoma‐prone. Using the K14‐Scf C57BL/6J animal model whose skin is characterized by lifelong retention of interfollicular epidermal melanocytes like that of the human, we studied pigmentary, UV responses, and DNA repair capacity in the skin of variant Mc1r background. Topical application of forskolin, a skin‐permeable pharmacologic activator of cAMP induction to mimic native Mc1r signaling, increased epidermal eumelanin levels, increased the capacity of Mc1r‐heterozygous skin to resist UV‐mediated inflammation, and enhanced the skin's ability to clear UV photolesions from DNA. Interestingly, topical cAMP induction also promoted melanin accumulation, UV resistance, and accelerated clearance in Mc1r fully intact skin. Together, our findings suggest that heterozygous Mc1r loss is associated with an intermediately melanized and DNA repair‐proficient epidermal phenotype and that topical cAMP induction enhances UV resistance in Mc1r‐heterozygous or Mc1r‐wild‐type individuals by increasing eumelanin deposition and by improving nucleotide excision repair.     

Alpha-melanocyte-stimulating hormone suppresses oxidative stress through a p53-mediated signaling pathway in human melanocytes

Epidermal melanocytes are skin cells specialized in melanin production. Activation of the melanocortin 1 receptor (MC1R) on melanocytes by α-melanocyte-stimulating hormone (α-MSH) induces synthesis of the brown/black pigment eumelanin that confers photoprotection from solar UV radiation (UVR). Contrary to keratinocytes, melanocytes are slow proliferating cells that persist in the skin for decades, in an environment with high levels of UVR-induced reactive oxygen species (ROS). We previously reported that in addition to its role in pigmentation, α-MSH also reduces oxidative stress and enhances the repair of DNA photoproducts in melanocytes, independent of melanin synthesis. Given the significance of ROS in carcinogenesis, here we investigated the mechanisms by which α-MSH exerts antioxidant effects in melanocytes. We show that activation of the MC1R by α-MSH contributes to phosphorylation of p53 on serine 15, a known requirement for stabilization and activation of p53, a major sensor of DNA damage. This effect is mediated by the cAMP/PKA pathway and by the activation of phosphoinositide 3-kinase (PI3K) ATR and DNA protein kinase (DNA-PK). α-MSH increases the levels of 8-oxoguanine DNA glycosylase (OGG1) and apurinic apyrimidinic endonuclease 1 (APE-1/Ref-1), enzymes essential for base excision repair. Nutlin-3, an HDM2 inhibitor, mimicked the effects of α-MSH resulting in reduced phosphorylation of H2AX (γ-H2AX), a marker of DNA damage. Conversely, the p53 inhibitor pifithrin-α or silencing of p53 abolished the effects of α-MSH and augmented oxidative stress. These results show that p53 is an important target of the downstream MC1R signaling that reduces oxidative stress and possibly malignant transformation of melanocytes.     

Paracrine regulation of melanocyte genomic stability: a focus on nucleotide excision repair

UV radiation is a major environmental risk factor for the development of melanoma by causing DNA damage and mutations. Resistance to UV damage is largely determined by the capacity of melanocytes to respond to UV injury by repairing mutagenic photolesions. The nucleotide excision repair (NER) pathway is the major mechanism by which cells correct UV photodamage. This multistep process involves the basic steps of damage recognition, isolation, localized strand unwinding, assembly of a repair complex, excision of the damage‐containing strand 3′ and 5′ to the photolesion, synthesis of a sequence‐appropriate replacement strand, and finally ligation to restore continuity of genomic DNA. In melanocytes, the efficiency of NER is regulated by several hormonal pathways including the melanocortin and endothelin signaling pathways. Elucidating molecular mechanisms by which melanocyte DNA repair is regulated offers the possibility of developing novel melanoma‐preventive strategies to reduce UV mutagenesis, especially in UV‐sensitive melanoma‐prone individuals.     

Rate Limiting Factors in Melanocortin 1 Receptor Signalling Throughthe cAMP Pathway

The melanotropic actions of α‐melanocyte‐stimulating hormone (α‐MSH) and other melanocortins are mediated by activation of the melanocortin 1 receptor (MC1R). This G protein‐coupled receptor is positively coupled to Gs and triggers the cyclic adenosine mono‐phosphate (cAMP) pathway. Mutations of the MC1R gene are associated with skin type and pigmentation phenotypes, and with increased risk of skin cancers. Genetic studies have demonstrated an heterozygote carrier effect for these associations, suggesting the importance of variant allele dosage. This could be accounted for, at least partially, if the number of MC1R molecules, rather than the Gs protein or the effector enzyme, adenylyl cyclase, is limiting for the activation of the signalling pathway. However, the nature of the limiting factor(s) in MC1R signalling has not been investigated. We addressed this question by comparing the cAMP output of clones of human melanoma cell lines enriched in MC1R by stable transfection. We also analysed heterologous cell systems widely used for functional studies of MC1R. We show that cAMP production in clones of Chinese hamster ovary cells stably expressing the MC1R is a linear function of receptor number up to high, supraphysiological levels of approximately 50 000 α‐MSH binding sites per cell. Enrichment of human melanoma cell lines with MC1R also results in increased cAMP levels, with a small leftward shift of the agonist dose–response curves. Therefore, at physiological expression levels second‐messenger generation is dependent on receptor density. Within melanoma cells and also likely in normal melanocytes, MC1R appears the limiting factor controlling the output of the cAMP signalling pathway.     

High diversity in functional properties of melanocortin 1 receptor (MC1R) in divergent primate species is more strongly associated with phylogeny than coat color

We have characterized the biochemical function of the melanocortin 1 receptor (MC1R), a critical regulator of melanin synthesis, from 9 phylogenetically diverse primate species with varying coat colors. There is substantial diversity in melanocyte-stimulating hormone (MSH) binding affinity and basal levels of activity in the cloned MC1Rs. MSH binding was lost independently in lemur and New World monkey lineages, whereas high basal levels of MC1R activity occur in lemurs and some New World monkeys and Old World monkeys. Highest levels of basal activity were found in the MC1R of ruffed lemurs, which have the E94K mutation that leads to constitutive activation in other species. In 3 species (2 lemurs and the howler monkey), we report the novel finding that binding and inhibition of MC1R by agouti signaling protein (ASIP) can occur when MSH binding has been lost, thus enabling continuing regulation of the melanin type via ASIP expression. Together, these findings can explain the previous paradox of a predominantly pheomelanic coat in the red ruffed lemur (Varecia rubra). The presence of a functional, MSH-responsive MC1R in orangutan demonstrates that the mechanism of red hair generation in this ape is different from the prevalent mechanism in European human populations. Overall, we have found unexpected diversity in MC1R function among primates and show that the evolution of the regulatory control of MC1R activity occurs by independent variation of 3 distinct mechanisms: basal MC1R activity, MSH binding and activation, and ASIP binding and inhibition. This diversity of function is broadly associated with primate phylogeny and does not have a simple relation to coat color phenotype within primate clades.     

Agonist‐Independent,High Constitutive Activity of the Human Melanocortin 1 Receptor

The melanocortins (α‐melanocyte‐stimulating hormone and adrenocorticotropin) act on epidermal melanocytes to increase melanogenesis, the eumelanin/pheomelanin ratio and dendricity. These actions are mediated by the heptahelical melanocortin 1 receptor (MC1R), positively coupled to adenylyl cyclase. Gain‐of‐function mouse Mc1r alleles are associated with a dark, eumelanic coat. Conversely, loss‐of‐function variants, or overexpression of agouti, a natural melanocortin antagonist, yield yellow, pheomelanic furs. In humans, loss‐of‐function MC1R variants are associated with fair skin, poor tanning, propensity to freckle and increased skin cancer risk. Therefore, MC1R is a key regulator of mammalian pigmentation. Several observations such as induction of constitutive pigmentation in amelanotic mouse melanoma cells following expression of MC1R indicate that the receptor might display agonist‐independent activity. We report a systematic and comparative study of MC1R and Mc1r constitutive activity. We show that expression of MC1R in heterologous systems leads to an agonist‐independent increase in cyclic adenosine monophophate (cAMP). Basal signalling is a function of receptor expression and is two to fourfold higher for MC1R than for Mc1r. Moreover, it is observed in human melanoma cells over‐expressing the MC1R. Constitutive signalling is abolished or reduced by point mutations of MC1R impairing the response to agonists, and is only doubled by the Lys94Glu mutation, mimicking the constitutively active mouse E^so‐3J allele. Stable or transient expression of wild‐type MC1R, but not of loss‐of‐function mutants, potently stimulates forskolin activation of adenylyl cyclase, a common feature of constitutively active Gs‐coupled receptors. Therefore, human MC1R displays a strong agonist‐independent constitutive activity.     

Purification and growth of melanocortin 1 receptor (<Emphasis Type="Italic">Mc1r</Emphasis>)-defective primary murine melanocytes is dependent on stem cell factor (SFC) from keratinocyte-conditioned media

The melanocortin 1 receptor (MC1R) is a transmembrane G_s-coupled surface protein found on melanocytes that binds melanocyte-stimulating hormone and mediates activation of adenylyl cyclase and generation of the second messenger cyclic AMP (cAMP). MC1R regulates growth and differentiation of melanocytes and protects against carcinogenesis. Persons with loss-of-function polymorphisms of MC1R tend to be UV-sensitive (fair-skinned and with a poor tanning response) and are at high risk for melanoma. Mechanistic studies of the role of MC1R in melanocytic UV responses, however, have been hindered in part because Mc1r-defective primary murine melanocytes have been difficult to culture in vitro. Until now, effective growth of murine melanocytes has depended on cAMP stimulation with adenylyl cyclase-activating or phosphodiesterase-inhibiting agents. However, rescuing cAMP in the setting of defective MC1R signaling would be expected to confound experiments directly testing MC1R function on melanocytic UV responses. In this paper, we report a novel method of culturing primary murine melanocytes in the absence of pharmacologic cAMP stimulation by incorporating conditioned supernatants containing stem cell factor derived from primary keratinocytes. Importantly, this method seems to permit similar pigment expression by cultured melanocytes as that found in the skin of their parental murine strains. This novel approach will allow mechanistic investigation into MC1R’s role in the protection against UV-mediated carcinogenesis and determination of the role of melanin pigment subtypes on UV-mediated melanocyte responses.     

MC1R and the response of melanocytes to ultraviolet radiation

The constitutive color of our skin plays a dramatic role in our photoprotection from solar ultraviolet radiation (UVR) that reaches the Earth and in minimizing DNA damage that gives rise to skin cancer. More than 120 genes have been identified and shown to regulate pigmentation, one of the key genes being melanocortin 1 receptor (MC1R) that encodes the melanocortin 1 receptor (MC1R), a seven-transmembrane G protein-coupled receptor expressed on the surface of melanocytes. Modulation of MC1R function regulates melanin synthesis by melanocytes qualitatively and quantitatively. The MC1R is regulated by the physiological agonists alpha-melanocyte-stimulating hormone (alphaMSH) and adrenocorticotropic hormone (ACTH), and antagonist agouti signaling protein (ASP). Activation of the MC1R by binding of an agonist stimulates the synthesis of eumelanin primarily via activation of adenylate cyclase. The significance of cutaneous pigmentation lies in the photoprotective effect of melanin, particularly eumelanin, against sun-induced carcinogenesis. Epidermal melanocytes and keratinocytes respond to UVR by increasing their expression of alphaMSH and ACTH, which up-regulate the expression of MC1R, and consequently enhance the response of melanocytes to melanocortins. Constitutive skin pigmentation dramatically affects the incidence of skin cancer. The pigmentary phenotype characterized by red hair, fair complexion, inability to tan and tendency to freckle is an independent risk factor for all skin cancers, including melanoma. The MC1R gene is highly polymorphic in human populations, and allelic variation at this locus accounts, to a large extent, for the variation in pigmentary phenotypes and skin phototypes (SPT) in humans. Several allelic variants of the MC1R gene are associated with the red hair and fair skin (RHC) phenotype, and carrying one of these variants is thought to diminish the ability of the epidermis to respond to DNA damage elicited by UVR. The MC1R gene is considered a melanoma susceptibility gene, and its significance in determining the risk for skin cancer is of tremendous interest.     

Effects of Melanogenesis‐Inducing Nitric Oxide and Histamine on the Production of Eumelanin and Pheomelanin in Cultured Human Melanocytes

Melanin pigments produced in human melanocytes are classified into two categories; black coloured eumelanin and reddish‐yellow pheomelanin. Stimulation of melanocytes with α‐melanocyte‐stimulating hormone (α‐MSH), one of several melanogenic factors, has been reported to enhance eumelanogenesis to a greater degree than pheomelanogenesis, which contributes to hyperpigmentation in skin. Nitric oxide (NO) and histamine are also melanogenesis‐stimulating factors that are released from cells surrounding melanocytes following ultraviolet (UV) irradiation. In this study, the effects of NO and histamine on the ratio of eumelanin and pheomelanin were examined in human melanocytes, and then compared with that of α‐MSH. The amounts of eumelanin and pheomelanin were quantified using high‐performance liquid chromatography analysis after oxidation and hydrolysis of melanin. Melanogenesis was induced by the addition of α‐MSH, NO, or histamine to melanocytes. The amount of eumelanin production significantly increased with independent stimulation by these melanogenic factors, especially histamine, while that of pheomelanin significantly increased with α‐MSH and NO, but only slightly with histamine. As a result, the ratio of eumelanin and pheomelanin increased significantly with the addition of NO or histamine. These results suggest that NO and histamine, as in the case of α‐MSH, may contribute to UV‐induced hyperpigmentation by enhancing eumelanogenesis.     

In vitro behavior and UV response of melanocytes derived from carriers of CDKN2A mutations and MC1R variants

Coinheritance of germline mutation in cyclin‐dependent kinase inhibitor 2A (CDKN2A) and loss‐of‐function (LOF) melanocortin 1 receptor (MC1R) variants is clinically associated with exaggerated risk for melanoma. To understand the combined impact of these mutations, we established and tested primary human melanocyte cultures from different CDKN2A mutation carriers, expressing either wild‐type MC1R or MC1RLOF variant(s). These cultures expressed the CDKN2A product p16 (INK4A) and functional MC1R. Except for 32ins24 mutant melanocytes, the remaining cultures showed no detectable aberrations in proliferation or capacity for replicative senescence. Additionally, the latter cultures responded normally to ultraviolet radiation (UV) by cell cycle arrest, JNK, p38, and p53 activation, hydrogen peroxide generation, and repair of DNA photoproducts. We propose that malignant transformation of melanocytes expressing CDKN2A mutation and MC1RLOF allele(s) requires acquisition of somatic mutations facilitated by MC1R genotype or aberrant microenvironment due to CDKN2A mutation in keratinocytes and fibroblasts.