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UV Signaling Pathways within the Skin

  • Author Footnotes
    3 These authors contributed equally to this work.
    Hongxiang Chen
    Footnotes
    3 These authors contributed equally to this work.
    Affiliations
    Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Department of Dermatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Author Footnotes
    3 These authors contributed equally to this work.
    Qing Y. Weng
    Footnotes
    3 These authors contributed equally to this work.
    Affiliations
    Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
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  • David E. Fisher
    Correspondence
    Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Building 149, 13th Street Charlestown, Boston, Massachusetts 02129, USA
    Affiliations
    Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
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  • Author Footnotes
    3 These authors contributed equally to this work.
      The effects of UVR on the skin include tanning, carcinogenesis, immunomodulation, and synthesis of vitamin D, among others. Melanocortin 1 receptor polymorphisms correlate with skin pigmentation, UV sensitivity, and skin cancer risk. This article reviews pathways through which UVR induces cutaneous stress and the pigmentation response. Modulators of the UV-tanning pathway include sunscreen agents, melanocortin 1 receptor activators, adenylate cyclase activators, phosphodiesterase 4D3 inhibitors, T-oligos, and microphthalmia-associated transcription factor regulators such as histone deacetylase inhibitors. UVR, as one of the most ubiquitous carcinogens, represents both a challenge and an enormous opportunity in skin cancer prevention.

      Abbreviations

      DOPA
      dihydroxyphenylalanine
      MC1R
      melanocortin 1 receptor
      MITF
      microphthalmia-associated transcription factor
      α-MSH
      alpha melanocyte-stimulating hormone
      PGC-1α
      peroxisome proliferator-activated receptor-γ coactivator-1α
      SUMO
      small-ubiquitin-like modifier
      Tyrp1
      tyrosinase-related protein 1

      Introduction

      The incidence of melanoma and non-melanoma skin cancers has continued to rise over the past few decades. The etiology is multifactorial with discrete genetic pathways and environmental factors. Although genetic factors may contribute significantly, environmental factors can be modified to potentially decrease the risk of developing deadly diseases such as melanoma. Exposure to UVR from sunlight is well established as a significant risk factor for melanoma development. However, indoor tanning is a source of preventable UVR exposure that represents a growing, multi-billion dollar industry (
      • Levine J.A.
      • Sorace M.
      • Spencer J.
      • et al.
      The indoor UV tanning industry: a review of skin cancer risk, health benefit claims, and regulation.
      ). UVR is a major environmental risk factor that contributes to carcinogenesis through DNA damage and immune modulation via inflammatory and immunosuppressive pathways (
      • Tran T.T.
      • Schulman J.
      • Fisher D.E.
      UV and pigmentation: molecular mechanisms and social controversies.
      ;
      • Liu J.J.
      • Fisher D.E.
      Lighting a path to pigmentation: mechanisms of MITF induction by UV.
      ;
      • D'Orazio J.
      • Jarrett S.
      • Amaro-Ortiz A.
      • et al.
      UV radiation and the skin.
      ;
      • Weinstock M.A.
      Epidemiology and UV exposure.
      ). It has long been appreciated that tanning, through increasing epidermal melanin content, is the skin's major photoprotective response against acute and chronic UV damage. DNA damage from UVR induces signaling cascades that ultimately lead to activation of pigmentation machinery to produce the tanning effect. This process can be synthetically perturbed at different points along the pathway to upregulate driver signals or to suppress inhibitory feedback, thereby promoting a UVR-independent protective tanning response. These strategies range from broad, such as transcriptional activators, to narrow, such as molecular analogs. Because the UV-tanning pathway is essential for both melanogenesis and protection from skin cancers, we summarize here the consequences of the UV signaling pathway deficiencies and strategies to regulate the the UV signaling pathway.

      Features of UVR And UV-induced mutagenesis

      UVR, spanning the 200 to 400 nm wavelengths of the electromagnetic spectrum, is a high-energy component of solar radiation. UVR is divided into three categories based on wavelength: UVA (400–320 nm), UVB (320–290 nm), and UVC (290–200 nm). Over 95% of UVA and 1–10% of UVB radiation reaches the earth’s surface, whereas almost 100% of solar UVC is absorbed by the atmosphere and the ozone layer. Thus, most of the research on the effects of UVR has focused on UVA and UVB. A history of sunburn in childhood and continued unprotected exposure to UVR through adolescence and adulthood contribute to skin cancer risk. However, many adolescents and adults continue to seek a tan, either from direct sun exposure or from tanning beds.
      UVR directly targets macromolecules in the skin such as proteins, lipids, and nucleic acids, with the latter resulting in signature mutations characteristically found in melanomas and other skin cancers. When these mutations occur within genes regulating apoptosis, cell cycle progression, and genetic repair machinery, they may initiate oncogenic transformation (
      • Schulman J.M.
      • Fisher D.E.
      Indoor ultraviolet tanning and skin cancer: health risks and opportunities.
      ;
      • Fisher D.E.
      • James W.D.
      Indoor tanning–science, behavior, and policy.
      ). UVR photoexcitation of the direct chromophore DNA produces excited electron states and toxic by-products, leading to direct and indirect DNA damage. This often produces signature mutations dependent on the insult and mechanism of damage. We will focus on mutations resulting from UVA and UVB specifically.
      UVA radiation, upon exciting endogenous chromophores, can generate reactive oxygen species capable of causing oxidative DNA damage. Through generation of singlet oxygen (1O2) or type-1 photosensitization reactions, UVA is able to cause oxidative base modifications, predominately at guanine bases. This process leads to generation of 7,8-dihydro-8-oxoguanine lesions, which have been shown to induce specific DNA mutations if not repaired. (
      • Garibyan L.
      • Fisher D.E.
      How sunlight causes melanoma.
      ).The major UVA-induced mutations are G→T transversions and G→A transitions. Like UVB, UVA may also trigger DNA damage through cyclobutane pyrimidine dimer (CPD) formation.
      UVB contact with DNA activates a photochemical reaction that usually occurs between adjacent pyrimidine nucleotides and leads to formation of photoproducts known as CPDs and pyrimidine 6-4 pyrimidones. After the formation of CPDs and pyrimidine 6-4 pyrimidone photoproducts, either spontaneous reversion may occur (for CPDs), or DNA repair enzymes participate in the correction of the damage. Incorrect repair of these damaged DNA lesions leads to mutations in epidermal cells that may initiate oncogenesis. When UVB-induced CPDs and pyrimidine 6-4 pyrimidones are incorrectly resolved, certain signature mutations may form, including C→T and CC→TT transition mutations (
      • Tran T.T.
      • Schulman J.
      • Fisher D.E.
      UV and pigmentation: molecular mechanisms and social controversies.
      ;
      • Garibyan L.
      • Fisher D.E.
      How sunlight causes melanoma.
      ).
      These characteristic mutations are not exclusively induced by UVR from sunlight. DeMarini and colleagues compared the mutagenic effects of radiation from three common sources using Salmonella assays and determined that mutagenic ability was most potent in radiation from tanning salon beds, followed by sunlight. White fluorescent light represented the least mutagenic source of radiation. The most common mutations were G:C→A:T transitions. The CC→TT transitions characteristic of UVB exposure represented 83% of mutations induced by tanning bed radiation exposure, demonstrating that both solar and non-solar sources of UV radiation are capable of inflicting signature UV mutations (
      • DeMarini D.M.
      • Shelton M.L.
      • Stankowski Jr, L.F.
      Mutation spectra in Salmonella of sunlight, white fluorescent light, and lightfrom tanning salon beds: induction of tandem mutations and role of DNA repair.
      ;
      • Besaratinia A.
      • Pfeifer G.P.
      Sunlight ultraviolet irradiation and BRAF V600 mutagenesis in human melanoma.
      ).
      Although UVB mutations have comprised the majority of the traditional UVR-associated mutations, little overlap exists between these mutations and those observed in codon V600 of the BRAF gene, the most common location of the well-established BRAF mutations in melanoma. BRAF V600 variants can be attributed to G→A transitions and T→A, T→G, and G→T transversions (
      • Thomas N.E.
      • Berwick M.
      • Cordeiro-Stone M.
      Could BRAF mutations in melanocytic lesions arise from DNA damage induced byultraviolet radiation.
      ;
      • Besaratinia A.
      • Pfeifer G.P.
      Sunlight ultraviolet irradiation and BRAF V600 mutagenesis in human melanoma.
      ). In contrast, traditional UVB-induced mutations from exposure to sunlight are characterized by single or tandem C→T transitions at dipyrimidine nucleotides. Damage from UVA radiation has been characterized more recently, with one mechanism being the generation of DNA cross-links and lesions through oxidative damage from UVA-induced photosensitization reactions. Certain of these UVA-induced DNA lesions resemble mutations in BRAF V600 variants from sun-exposed melanomas, suggesting a greater role for UVA in melanomagenesis than traditionally thought (
      • Garibyan L.
      • Fisher D.E.
      How sunlight causes melanoma.
      ). Importantly, BRAF V600 mutations may also occur in non-sun-exposed malignancies, such as colon, lung, and thyroid, potentially consistent with oxidative damage as a common carcinogenic mechanism (in those cases independent of UVA). Other important melanoma-associated genes such as INK4A, PTEN, FGFR2, and N-RAS may also possess mutations attributable to UVR (
      • Mar V.J.
      • Wong S.Q.
      • Li J.
      • et al.
      BRAF/NRAS wild-type melanomas have a high mutation load correlating with histologic and molecular signatures of UV damage.
      ).

      UV signaling pathways for tanning

      The core component of the skin response to sunlight is the epidermal melanin unit, comprised of the melanocyte and its associated keratinocytes. UV exposure induces DNA damage in keratinocytes and results in stabilization of the p53 tumor suppressor protein. This promotes p53 transcriptional activation of proopiomelanocortin, which is enzymatically cleaved to produce α–melanocyte-stimulating hormone (α-MSH). α-MSH is released by keratinocytes and binds the MC1R on melanocytes. MC1R activation by α-MSH triggers an increase in cAMP levels within the melanocytes, which increase transcription of microphthalmia-associated transcription factor (MITF) via CRE-binding protein/activating transcription factor 1. Binding of MITF to the E-box sequences in promoter regions triggers transcription of numerous pigmentation genes (
      • Tran T.T.
      • Schulman J.
      • Fisher D.E.
      UV and pigmentation: molecular mechanisms and social controversies.
      ;
      • Hearing V.J.
      Determination of melanin synthetic pathways.
      ,
      • Hearing V.J.
      Milestones in melanocytes/melanogenesis.
      ). These genes act to synthesize, mature, and traffic melanin, the most common types of which are brown-black eumelanin and yellow-red pheomelanin. The melanin is packaged in melanosomes which are exported to keratinocytes, where they localize over the nucleus and may protect the genomic material from further UVR-induced damage (Figure 1 and Figure 2).
      Figure thumbnail gr1
      Figure 1The epidermal melanin unit and tanning response to UV radiation. UV radiation induces DNA damage, which leads to activation of p53. In turn, p53 stimulates transcriptional upregulation of the proopiomelanocortin (POMC) gene, which is posttranslationally processed to adrenocorticotrophic hormone (ACTH), α-melanocyte-stimulating hormone (α-MSH), and β-endorphin. Secreted α-MSH binds to the melanocortin 1 receptor (MC1R) on melanocytes, leading to production of melanin. The melanin is packaged within melanosomes and transported back to keratinocytes, where they localize over the nucleus as part of the protective tanning response to UV radiation.
      Figure thumbnail gr2
      Figure 2Melanin synthesis and strategies to regulate the tanning response. Secreted α-melanocyte-stimulating hormone (α-MSH) from keratinocytes binds melanocortin 1 receptor (MC1R) on melanocytes, leading to upregulation of cAMP, which stimulates expression of microphthalmia-associated transcription factor (MITF). MITF then transcriptionally activates expression of enzymatic machinery including tyrosinase and tyrosinase-related protein 1 (Tyrp1), which are critical in the synthesis of melanin within melanosomes. Tyrosinase catalyzes the initial conversion of tyrosine to dihydroxyphenylalanine (DOPA) and dopaquinone. Dopaquinone may then combine with cysteine to form the pheomelanin precursor cysteinyldopa, or it may enter a separate pathway catalyzed in part by Tyrp1 to produce the eumelanin precursor. The matured melanin is then transported in vesicles called melanosomes to the overlying epidermal keratinocytes. Strategies such as MC1R activators, adenylate cyclase activators, phosphodiesterase 4D3 inhibitors, and MITF regulators are shown to regulate the UV-tanning response by targeting different components of this pathway.

      Consequences of UV signaling pathway deficiency

      The loss of p53

      As an important regulator of the genotoxic response, p53 is a key tumor suppressor gene that is mutated frequently in human cancer, including skin cancers. The p53 protein regulates multiple signaling pathways in response to stimuli such as DNA damage, oxidative stress, hypoxia, heat shock, membrane compromise, and other stresses (
      • Cui R.
      • Widlund H.R.
      • Feige E.
      • et al.
      Central role of p53 in the suntan response and pathologic hyperpigmentation.
      ). p53 is thought to participate in DNA repair via multiple mechanisms, including control of cell cycle checkpoint activity as well as regulation of the DNA repair machinery. Lesions with mutant p53 are readily found in UV-exposed hairless mouse skin and sun-exposed healthy human skin. These mutations tend to be localized to dipyrimidine sequences and consist of C→T or CC→TT transitions (
      • Beaumont K.A.
      • Shekar S.N.
      • Cook A.L.
      • et al.
      Red hair is the null phenotype of MC1R.
      ;
      • Weinstock M.A.
      Epidemiology and UV exposure.
      ).

      MC1R mutations in skin cancers

      The MC1R gene is highly polymorphic in humans, with over 80 variants identified. Certain variants are closely associated with red hair color (RHC) phenotype, which is accompanied by fair skin, poor tanning ability, high sunburn risk, and the highest risk of melanoma for any skin pigmentation type. Other MC1R polymorphic variants with weaker melanoma associations are known as “non-red hair color” variants. Three RHC variants of MC1R that are associated with fair skin and poor tanning are Arg151Cys, Arg160Trp, and Asp294His (
      • Han J.
      • Kraft P.
      • Colditz G.A.
      • et al.
      Melanocortin 1 receptor variants and skin cancer risk.
      ). The 151Cys variant was associated with increased risks of the three types of skin cancer after controlling for hair color, skin color, and other skin cancer risk factors. Women with medium or olive skin color carrying one non-red hair color allele and one red hair color allele had the highest risk of melanoma (
      • Han J.
      • Kraft P.
      • Colditz G.A.
      • et al.
      Melanocortin 1 receptor variants and skin cancer risk.
      ;
      • Fargnoli M.C.
      • Gandini S.
      • Peris K.
      • et al.
      MC1R variants increase melanoma risk in families with CDKN2A mutations: ameta-analysis.
      ).
      One mechanism by which MC1R polymorphisms affect melanoma risk may be through repair of DNA damage (
      • Kadekaro A.L.
      • Kavanagh R.
      • Kanto H.
      • et al.
      α-Melanocortin and endothelin-1 activate anti-apoptotic pathways and reduce DNA damage in human melanocytes.
      ). Human melanocyte cultures exposed to varying levels of UVR were found to have CPD levels that correlated with MC1R genotype and function. In the melanocytes with non-functional MC1R, treatment with forskolin to directly activate adenylate cyclase appeared to enhance CPD repair (
      • Hauser J.E.
      • Kadekaro A.L.
      • Kavanagh R.
      • et al.
      Melanin content and MC1R function independently affect UVR-induced DNA damage in cultured human melanocytes.
      ).

      Eumelanin and pheomelanin synthesis contributes to melanomagenesis

      In addition to its direct effects on DNA damage repair, MC1R may also affect oncogenic drivers through regulation of pigmentation. MC1R signaling and cysteine availability govern the balance in production of eumelanin and pheomelanin. The amino acid cysteine is required for pheomelanin synthesis but not eumelanin synthesis. When MC1R signaling is strong, cysteine stores are insufficient to keep pace with the rate of generation of pigment precursors, and eumelanin production is favored. When the MC1R signal is weak as in redhead melanocytes, cysteine stores keep pace with the slower formation of pigment precursors, leading to formation of cysteine-containing pheomelanin. In our 2012 study using redhead mice with inactivating MC1R mutations, UVR was not necessary for increased melanoma development in these mice when compared with black mice expressing an activating BRAF mutation in their melanocytes. This study supports carcinogenic potential of the pheomelanin synthetic pathway through an UVR-independent mechanism.
      Oxidative stress appeared to have a role in pheomelanin-mediated melanomagenesis (
      • Mitra D.
      • Luo X.
      • Morgan A.
      • et al.
      An ultraviolet-radiation-independent pathway to melanoma carcinogenesis in thered hair/fair skin background.
      ). We hypothesize two possible mechanistic pathways to explain the observed pheomelanin-dependent oxidative DNA damage that drives melanomagenesis. First, pheomelanin might generate reactive oxygen species that directly or indirectly cause oxidative DNA damage. Second, pheomelanin synthesis might consume cellular antioxidant stores and make the cell more vulnerable to other endogenous reactive oxygen species (
      • Morgan A.M.
      • Lo J.
      • Fisher D.E.
      How does pheomelanin synthesis contribute to melanomagenesis?: two distinctmechanisms could explain the carcinogenicity of pheomelanin synthesis.
      ).

      Strategies to regulate the pigmentation signaling pathway

      The UV signaling pathway can be synthetically perturbed at different points to regulate the activity of MC1R, adenylate cyclase, cAMP, and MITF. Such strategies could induce a UV-independent tanning response, potentially conferring a photoprotective effect against UVR-mediated melanomagenesis. Here, we will discuss targetable processes at each level in detail.

      MC1R activators (analogs of α-MSH)

      In addition to the use of sunscreen agents, one strategy for melanoma prevention is based on analogs of α-MSH that function as MC1R agonists (
      • Marwaha V.
      • Chen Y.H.
      • Helms E.
      • et al.
      T-oligo treatment decreases constitutive and UVB-induced COX-2 levels throughp53- and NFkappaB-dependent repression of the COX-2 promoter.
      ). These include products such as melanotan I, melanotan II, afamelanotide, Ac-His-D-Phe-Arg-Trp-NH2, and n-Pentadecanoyl- and 4-Phenylbutyryl-His-D-Phe-Arg-Trp-NH2. Those analogs were more potent than α-MSH itself in stimulating melanogenesis, as well as reducing apoptosis, decreasing release of hydrogen peroxide, and enhancing repair of DNA photoproducts in melanocytes exposed to UVR. The photoprotective and other biological effects of α-MSH analogs await full determination (
      • Hadley M.E.
      • Hruby V.J.
      • Blanchard J.
      • et al.
      Discovery and development of novel melanogenic drugs. Melanotan-I and -II.
      ;
      • Langan E.A.
      • Nie Z.
      • Rhodes L.E.
      Melanotropic peptides: more than just 'Barbie drugs' and ‘sun-tan jabs’.
      ;
      • Miller A.J.
      • Tsao H.
      New insights into pigmentary pathways and skin cancer.
      ;
      • Schulze F.
      • Erdmann H.
      • Hardkop L.H.
      • et al.
      Eruptive naevi and darkening of pre-existing naevi 24h after a single mono-dose injection of melanotan II.
      ).
      Some pathologic processes can alter levels of α-MSH and indirectly affect melanogenesis. α-MSH, like ACTH and thyroid-stimulating hormone, is secreted by the anterior pituitary gland. In Addison’s disease (chronic adrenal insufficiency), lack of negative feedback from cortisol induces the anterior pituitary to produce greater levels of ACTH. As a by-product, more MSH is also produced, leading to hyperpigmented lesions in these patients. The classical hypothalamic-pituitary-adrenal axis is a negative feedback neuroendocrine pathway that is essential for the systemic response to external or internal stress. Emerging evidence has indicated that a fully functional cutaneous equivalent participates in the response of skin to local stress as well as other homeostatic contexts (
      • Slominski A.
      • Wortsman J.
      Neuroendocrinology of the skin.
      ;
      • Zbytek B.
      • Wortsman J.
      • Slominski A.
      Characterization of a ultraviolet B-induced corticotropin-releasinghormone-proopiomelanocortin system in human melanocytes.
      ;
      • Slominski A.
      • Wortsman J.
      Neuroendocrinology of the skin.
      ,
      • Slominski A.
      • Wortsman J.
      • Tuckey R.C.
      • et al.
      Differential expression of HPA axis homolog in the skin.
      ,
      • Slominski A.T.
      • Zmijewski M.A.
      • Skobowiat C.
      • et al.
      Sensing the environment: regulation of local and global homeostasis by the skin'sneuroendocrine system.
      ). This local system can modulate the function of skin and follicular melanin units following UVR exposure and maintain or restore immune privilege in hair follicles. In the tanning pathway, the epidermal melanin unit comprised of the keratinocyte and melanocyte can be recognized as a functional equivalent of the hypothalamic-pituitary-adrenal axis in the skin.

      Adenylate cyclase activation

      Another strategy to promote the tanning response is through direct stimulation of adenylate cyclase activity downstream of MC1R. UVR-induced tanning is defective in numerous fair-skinned individuals, some of whom possess functional disruption of the MC1R. Although UVR is capable of inducing α-MSH production in keratinocytes, loss of MC1R function in red-haired mouse models results in inability to produce a tanning response upon UV exposure. However, pigmentation can be rescued by topical application of the cAMP agonist forskolin. This process can occur without UVR, demonstrating that the pigmentation machinery is available despite the absence of functional MC1R (
      • D'Orazio J.A.
      • Nobuhisa T.
      • Cui R.
      • et al.
      Topical drug rescue strategy and skin protection based on the role of Mc1r inUV-induced tanning.
      ).

      Alternative strategies

      cAMP is an ATP-derived secondary messenger that functions in signal transduction for a variety of intracellular pathways. Levels of cAMP are controlled by its production, catalyzed by adenylate cyclase, and its hydrolysis, catalyzed by the phosphodiesterase class of enzymes. Phosphodiesterase 4D3 was identified as a direct target of the MSH/cAMP/MITF pathway (
      • Khaled M.
      • Levy C.
      • Fisher D.E.
      Control of melanocyte differentiation by a MITF-PDE4D3 homeostatic circuit.
      ). Its activation creates a negative feedback loop that induces refractoriness to chronic stimulation of the cAMP pathway in melanocytes. This highlights a potent mechanism controlling melanocyte differentiation that may be amenable to pharmacologic manipulation (
      • Khaled M.
      • Levy C.
      • Fisher D.E.
      Control of melanocyte differentiation by a MITF-PDE4D3 homeostatic circuit.
      ). Telomere-related oligonucleotides also have shown promise in augmenting the tanning pathway while bypassing UV-stimulation to confer a protective effect on skin (
      • Arad S.
      • Konnikov N.
      • Goukassian D.A.
      • et al.
      T-oligos augment UV-induced protective responses in human skin.
      ). This strategy was born from an understanding of telomeric-derived oligonucleotides as inducers of DNA repair responses in melanocytes, as well as concomitant inducers of melanogenesis (
      • Atoyan R.Y.
      • Sharov A.A.
      • Eller M.S.
      • et al.
      Oligonucleotide treatment increases eumelanogenesis, hair pigmentation andmelanocortin-1 receptor expression in the hair follicle.
      ;
      • Gilchrest B.A.
      • Eller M.S.
      • Yaar M.
      Telomere-mediated effects on melanogenesis and skin aging.
      ).

      Regulation of MITF through direct targeting and modification of posttranscriptional processes

      Finally, strategies to regulate the tanning response may focus on MITF, which is required for melanocyte development and is an amplified oncogene in a fraction of human melanomas. In addition to its control of critical pigmentation genes, MITF also regulates target genes essential to cell cycle progression, apoptosis, and differentiation (
      • Levy C.
      • Khaled M.
      • Fisher D.E.
      MITF: master regulator of melanocyte development and melanoma oncogene.
      ). Therefore, pharmacologic suppression of MITF is of potential interest in a variety of clinical settings. However, MITF is not known to contain intrinsic catalytic activity amenable to direct small-molecule inhibition (
      • Flaherty K.T.
      • Hodi F.S.
      • Fisher D.E.
      From genes to drugs: targeted strategies for melanoma.
      ). An alternative drug-targeting strategy is to identify and interfere with lineage-restricted mechanisms required for MITF expression. Multiple histone deacetylase inhibitor drugs potently suppress MITF expression in melanocytes, melanoma, and clear cell sarcoma cells (which are sometimes pigmented). Although histone deacetylase inhibitors may affect numerous cellular targets, they have been shown to suppress skin pigmentation upon topical application in mice (
      • Yokoyama S.
      • Feige E.
      • Poling L.L.
      • et al.
      Pharmacologic suppression of MITF expression via HDAC inhibitors in themelanocyte lineage.
      ). High throughput screens to identify additional small molecules capable of modulating MITF activity are currently being conducted in the authors’ lab, and candidate leads are under development.
      A germline missense substitution in MITF (Mi-E318K) was found to occur in families with high incidences of melanoma in Australia, United States, Great Britain, and France (
      • Bertolotto C.
      • Lesueur F.
      • Giuliano S.
      • et al.
      A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma.
      ;
      • Yokoyama S.
      • Woods S.L.
      • Boyle G.M.
      • et al.
      A novel recurrent mutation in MITF predisposes to familial and sporadic melanoma.
      ). Codon 318 is located in a small-ubiquitin-like modifier (SUMO) consensus site (PsiKXE), (
      • Miller A.J.
      • Levy C.
      • Davis I.J.
      • et al.
      Sumoylation of MITF and its related family members TFE3 and TFEB.
      ) and Mi-E318K ablated that SUMOylation event on MITF. The Mi-E318K mutation measurably increases MITF’s transcriptional activity. An additional key posttranslational modification on MITF is its phosphorylation by mitogen-activated protein kinase (
      • Hemesath T.J.
      • Price E.R.
      • Takemoto C.
      • et al.
      MAP kinase links the transcription factor microphthalmia to c-Kit signaling in melanocytes.
      ), which subsequently targets MITF for ubiquitination and proteolysis (
      • Wu M.
      • Hemesath T.J.
      • Takemoto C.M.
      • et al.
      c-Kit triggers dual phosphorylations, which couple activation and degradation of the essential melanocyte factor Mi.
      ). More recently, it was shown that MITF is targeted by the de-ubiquitinase USP13, a theoretically drug-able protease whose suppression results in strong downregulation of MITF protein levels (
      • Zhao X.
      • Fiske B.
      • Kawakami A.
      • et al.
      Regulation of MITF stability by the USP13 deubiquitinase.
      ).

      Other MITF gene regulators and MITF gene co-factors

      In addition to directly targeting MITF, potential strategies to regulate the tanning response can target factors upstream of MITF or genes that serve as co-factors for MITF. The peroxisome proliferator-activated receptor gamma coactivator proteins PGC-1α and PGC-1β are key mediators of α-MSH activation of MITF. PGC-1α and PGC-1β are stabilized through α-MSH signaling via phosphorylation by protein kinase A. The PGC-1 proteins subsequently activate MITF transcription, and inhibition of the proteins blocks expression of MITF and its target genes in the tanning pathway.
      Recent studies in humans revealed polymorphisms in PGC-1β that associated with ability to tan and protection against melanoma (
      • Shoag J.
      • Haq R.
      • Zhang M.
      • et al.
      PGC-1 coactivators regulate MITF and the tanning response.
      ). YY1, which functions as both a transcriptional repressor and activator, also cooperates with melanocyte-specific isoform MITF to regulate the expression of the piebaldism gene KIT and multiple additional pigmentation genes (
      • Li J.
      • Song J.S.
      • Bell R.J.
      • et al.
      YY1 regulates melanocyte development and function by cooperating with MITF.
      ).

      Conclusions

      Tanning represents increased melanization of the epidermis following UV exposure. The UV-tanning pathway is a DNA damage-related stress and injury response. Targeting components of the UV-tanning pathway through small molecules such as α-MSH analogs may be one strategy to modulate skin pigmentation. α-MSH analogs would likely be less potent on the MC1R loss-of-function variants that are most frequently found in melanoma patients, but they might still function. The strategies targeting components downstream of MC1R show potential in rescuing deficiencies of the UV-tanning pathway. These include adenylate cyclase activators, phosphodiesterase 4D3 inhibitors, and telomere-derived oligonucleotides. Additional interventions which may suppress key melanoma survival factors include MITF regulators such as histone deacetylase inhibitors and candidates from ongoing high throughput screens for MITF regulators. Strategies may also target MITF posttranscriptional modification processes such as SUMO modification, dimerization, and ubiquitination/deubiquitination. Future mechanism-based studies of UVR are needed to help completely elucidate molecular pathways responsible for the carcinogenic effects of UVR on the melanocyte lineage. We hope to develop better strategies to regulate pigmentation and in doing so, identify further opportunities for prevention, early detection, and treatment of melanoma.

      ACKNOWLEDGMENTS

      We thank members of the Fisher lab, particularly E. Roydon Price, for invaluable discussions and advice. We apologize to the authors of many papers that have significantly contributed toward the current state of the melanoma and pigmentation fields but could not be acknowledged here due to space constraints. This work was supported by grants to DEF from the National Institutes of Health (1P01 CA163222-01; R01 AR043369-17; R01CA150226; R21CA175907) and The Miriam and Sheldon G. Adelson Medical Research Foundation.

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