Photochemistry and Photobiology, 2017, 93: 245–258
Invited Review
Hormonal Regulation of the Repair of UV Photoproducts in Melanocytesby the Melanocortin Signaling Axis†
Stuart G. Jarrett1 and John A. D’Orazio*1,2,3,4,51Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY2Department of Toxicology and Cancer Biology, University of Kentucky College of Medicine, Lexington, KY3Department of Physiology, University of Kentucky College of Medicine, Lexington, KY4Department of Pharmacology and Nutritional Sciences, University of Kentucky College of Medicine, Lexington, KY5Department of Pediatrics, University of Kentucky College of Medicine, Lexington, KYReceived 21 July 2016, accepted 31 August 2016, DOI: 10.1111/php.12640
ABSTRACT
Melanoma is the deadliest form of skin cancer because of itspropensity to spread beyond the primary site of disease andbecause it resists many forms of treatment. Incidence of mela-noma has been increasing for decades. Although ultravioletradiation (UV) has been identified as the most important envi-ronmental causative factor for melanoma development, UV-protective strategies have had limited efficacy in melanomaprevention. UV mutational burden correlates with melanomadevelopment and tumor progression, underscoring the impor-tance of UV in melanomagenesis. However, besides amount ofUV exposure, melanocyte UV mutational load is influenced bythe robustness of nucleotide excision repair, the genome main-tenance pathway charged with removing UV photoproductsbefore they cause permanent mutations in the genome. In thisreview, we highlight the importance of the melanocortinhormonal signaling axis on regulating efficiency of nucleotideexcision repair in melanocytes. By understanding the molecu-lar mechanisms by which nucleotide excision repair can beincreased, it may be possible to prevent many cases of mela-noma by reducing UV mutational burden over time.
MELANOMA—A CANCER OF MELANOCYTESAlthough melanoma accounts for less than a tenth of all skincancers, it is responsible for approximately three quarters ofskin cancer deaths because of its aggressive nature, tendency tometastasize and to resist anticancer therapies (1). Whereas themore common skin malignancies—basal cell carcinoma andsquamous cell carcinoma—derive from epidermal keratinocytes,melanoma arises from melanocytes, which are neural crest-derived cells characterized by their ability to produce melaninpigments (2). In the skin, melanocytes can be found in the der-mis within hair follicles where they impart pigmentation to hairas it grows and more superficially in the interfollicular epider-mis where they produce melanin that accumulates in the
epidermis (Fig. 1). Epidermal melanin functions to absorb UVradiation in the skin; therefore, the more melanin that is foundin the skin, the darker the complexion and the more resistantthe skin is to UV damage (3). When epidermal or dermal mela-nocytes undergo malignant degeneration to become melanoma,they tend to retain their melanin-producing ability, which iswhy the majority of melanomas are darkly pigmented lesions.Furthermore, many melanomas arise from benign nevi, under-scoring the importance of early detection efforts to catch thedisease as early as possible based on the A-B-C-D-E model ofcarefully assessing moles for asymmetric shape, border irregu-larities, color changes, growth in diameter or evolution ofappearance over time. When detected early—before metastaticspread—melanoma can usually be cured by surgical excisionalone. Survival rates plummet, however, once the disease hasspread to regional lymph nodes or distant organs. Fortunately,the great majority of melanomas are detected and excisedbefore systemic spread, and overall 5-year survival rates cur-rently exceed ninety percent (4).
MELANOMA—EPIDEMIOLOGYU.S. melanoma incidence has risen faster than any other cancerin the last several decades, growing at an estimated 3% yearly(Fig. 2). The most recent data from the National Cancer Insti-tute’s Surveillance, Epidemiology and End Results (SEER) pro-gram suggest that an estimated 76 380 Americans will bediagnosed with melanoma and the disease will kill 10 130 in2016 alone. Roughly one in every fifty Americans will now bediagnosed with melanoma at some point in his/her life (5). Theunderlying reasons for the rising incidence of melanoma arelikely to be multifactorial (increasing age of the population, bet-ter detection methods and awareness, more recreational UVexposure, etc.) and represent a critical research question in needof clarification. Although melanoma can happen at any age, itmost commonly occurs after adolescence with incidence peakingin the fifth decade of life. Nonetheless, melanoma often affectspeople in their prime and represents the second most commoncancer in people aged 15–29. Currently, it is estimated that overone million Americans have been diagnosed with melanoma atsome point in their lives (6).
MELANOMA—RISK DETERMINANTSInherited and environmental factors heavily influence melanomarisk (Table 1; please see (7) for a comprehensive review).Genetic risk factors include number and nature of nevi as well asprior diagnosis of melanoma in the family. Some genes that areknown to influence melanoma risk include CDKN2A, CDK4,MITF and variants in pigmentation loci or DNA repair genes (8–11). Other inherited traits that are controlled by multiple loci arenumber and nature of nevi (12) and immunocompetence of T-cell responses (13).
A wealth of epidemiologic and molecular data points to ultra-violet radiation (UV) as the most important environmental riskfactor for melanoma (14). Many melanomas arise from nevi onsun-exposed anatomic areas of the body (15–17), melanoma inci-dence increases with geographic proximity to the equator where
the sun’s rays are strongest (18), and tanning bed use clearlyincreases melanoma risk (19,20). Moreover, at least two land-mark studies have documented that melanoma has more somatic(nongermline) mutations than any other cancer and that the greatmajority of mutations are “UV-signature” base changes in adja-cent pyrimidines (21,22). Recently, the Bastian group docu-mented that somatic mutational burden increases with melanomatumor progression and that at least 80% of mutations are UV-sig-nature mutations (23). Therefore, the amount of exposure of amelanocyte to UV and its ability to resist UV-induced DNAmutations should have a direct impact on the likelihood of trans-formation into malignant melanoma.
ULTRAVIOLET RADIATION AND ITSEFFECTS ON CELLSThe International Agency for Research on Cancer (IACR) classi-fies UV as a “Group 1″ carcinogen, meaning that there is irrefu-table evidence that UV causes cancer in humans (24). Unlikemany other carcinogens that are chemical in nature, UV consistsof photons whose wavelength and energy fall between ionizingradiation (X-rays) and visible light (Fig. 3). The sun is the natu-ral source of UV, although significant UV exposure can beobtained by artificial UV sources in the form of tanning beds(25). As water vapor and particles in the atmosphere can absorbor deflect UV photons, UV in sunlight becomes attenuated whenit travels through the atmosphere. Thus, solar UV energy isstrongest in those places on Earth at the equator where the sunhits the Earth directly and at high altitudes where the landreaches highest into the sky. UV can be subdivided by wave-length and energy into UV-A (315–400 nm; 3.10–3.94 eV pho-ton�1), UV-B (280–315 nm; 3.94–4.43 eV photon�1) and UV-C(200–280 nm; 4.43–12.4 eV photon�1). In most places on Earth,ambient sunlight consists of >90% UV-A with the remainderUV-B because atmospheric ozone effectively absorbs shorter-wavelength UV-C energy.
UV energy is highly bioactive, exerting physicochemicalchanges that damage and/or cross-link a variety of cellularmacromolecules such as lipids, RNA, DNA and protein (26).Using a murine model with “humanized skin,” Noonan et al.showed that melanomas could be initiated by UV-A or UV-Bradiation (27); therefore, melanocyte carcinogenesis can be pro-moted across the UV spectrum. Longer wavelength UV promotes
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Figure 2. U.S. melanoma incidence. Melanoma incidence has increaseddramatically in the last several decades. This increase is most likelycaused by a variety of inherited and environmental factors.
Table 1. Melanoma risk factors. Various genetic and environmental riskfactors have been linked with increased melanoma incidence (recentlyreviewed in (7)).
Inherited risk factors Environmental risk factors
Family or personal history ofmelanoma and nonmelanomaskin cancer
UV exposure, especially blisteringsunburns early in life
Large number of nevi UV-rich geographic locationsNevi of large size Indoor artificial UV exposureDysplastic nevi Psoralen, UV light therapyMelanocortin 1 receptor(MC1R) function
Pharmacologic immunosuppression
T-cell immunodeficiency Various chemicals, heavy metalspossible
Defective DNA repair(e.g. xeroderma pigmentosum)
Fair skin complexion
Basal layer
Basal lamina
Spinous layer
Granular layer
Cornified layer
Melanocytes
(external surface)
(dermis)
melanin
Kera�nocytes
Figure 1. Epidermal structure. Melanocytes are neural crest-derived cellsthat produce pigment in the basal layer. Melanin is transferred to matur-ing keratinocytes where it accumulates to block incoming UV energy.
246 Stuart G. Jarrett and John A. D’Orazio
the formation of singlet oxygen (1O2) which oxidizes moleculesin lipids, amino acids as well as nucleotide bases in RNA andDNA (28). In general, these changes interfere with the functionof macromolecules and in many cases lead to their prematuredestruction (29). While lipids, RNA and protein are renewablemolecules that can be replaced when damaged, DNA cannot sim-ply be replaced and must be repaired (30). Indeed, the repair ofDNA must happen in a timely manner because many of the UVchanges interfere with the function of polymerases and/or causeerrant base pairing which yields RNAs and proteins of inappro-priate sequence and function. More serious, however, is errantbase pairing during cell division. In this manner, unrepaired UV-induced DNA changes introduce mutations into the genome ofdaughter cells. Oxidation-mediated changes to nucleotide bases,for the most part, are dealt with by the base excision repair(BER) pathway wherein specific oxidatively generated lesions
are recognized and removed from the DNA by lesion-specificglycosylases without breaking the sugar-phosphate backbone togenerate an apurinic site which is then filled in using the undam-aged sister strand as a template.
UV photons also promote the formation of covalent basechanges in DNA at sites of neighboring pyrimidines by breakingexisting C5/C6 bonds in cytosines or thymines. Wavelengths inthe UV-B range generate cyclobutane pyrimidine dimers (CPDs)and to a lesser extent pyrimidine (6-4) pyrimidone photoproducts(6-4PPs), whereas those in the UV-A range promote CPD forma-tion rather than 6-4PPs. UV-A photons also efficiently promotephotoisomerization of 6-4PPs into Dewar valence isomers (Fig. 4)(31). All of these photolesions block transcription and DNA repli-cation, activate cell cycle checkpoints, stimulate mutagenesis andchromosomal rearrangements and can promote apoptosis. If leftunrepaired, photolesions cause transition mutations (e.g. CC-to-
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Dewar photoproductisoforms
chemiexcitedmelanin
Figure 3. UV radiation and the skin. UV can be subdivided into UV-A, UV-B and UV-C components based on photon length and energy; however,because of atmospheric ozone that absorbs UV-C, ambient sunlight is predominantly UV-A (90–95%) and UV-B (5–10%). Longer wavelength UV-Apenetrates deeply into the skin, reaching well into the dermis. In contrast, UV-B affects the epidermis with comparatively little reaching the dermis. UV-A is efficient at generating reactive oxygen species that damage DNA through oxidative changes. UV-B is directly absorbed by pyrimidine bases inDNA to produce photoproducts. Mutations and cancer result from unrepaired UV changes to DNA.
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Figure 4. UV photoproducts. UV photons are capable of breaking the C5/C6 bond in cytosines and thymines. When this happens between adjacentbases, abnormal covalent bonds can form between them to form either a four-member cyclobutane ring to create a cyclopyrimidine dimer (CPD) or a6,4 bond to produce a [6,4]-photoproduct (6-4PPs). Both lesions distort the double helix and promote base mispairing and mutagenesis.
Photochemistry and Photobiology, 2017, 93 247
TT) known as “UV-signature mutations” which are found in abun-dance in melanoma isolates (32). Indeed, the high degree of UV-signature mutations in melanoma is among the strongest pieces ofmolecular evidence linking UV with the disease (21–23).
Recently, a paradigm-shifting insight was made regardinghow photoproducts form after UV exposure. It had long beenobserved that although UV photoproducts can be identified incells immediately following UV exposure, their levels increasefor some time after the UV exposure ends. Brash et al. termedthese lesions “dark CPDs” and reported that they arise whenUV-induced reactive oxygen and nitrogen species combine toexcite an electron in fragments of melanin that creates a quantumtriplet state that has the energy of a UV photon. Triplet-statemelanin induces CPDs by energy transfer to DNA in a radiation-
independent manner up to several hours after UV exposure ends(33). Indeed, their data suggest that the majority of all UV-mediated CPDs in melanocytes might occur through this processof melanin chemiexcitation and dark CPD formation (Fig. 5).This new insight into UV-mediated DNA lesion formation hasmany implications regarding mechanisms of UV resistance andgenome maintenance. Indeed, innate UV-activated systems thatenhance the efficiency of DNA repair, such as upregulation ofthe melanocortin signaling axis (discussed below), may be mostrelevant for the repair of dark CPDs.
MELANOCORTIN 1 RECEPTOR (MC1R) ANDCAMP SIGNALINGMelanocytes are long-lived cells that must, as a result of theiranatomic location in the skin, cope with environmental insultssuch as UV radiation. They must not only survive following UVexposure, but they must also respond physiologically to rampup melanin production to protect the skin against further UVdamage. Much of their ability to respond appropriately to UV isregulated by the melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor located in the melanocyte extracellular mem-brane (34). This transmembrane protein spans the extracellularmembrane seven times and transmits signals from ligands thatinteract with MC1R’s extracellular domains to the interior of thecell (Fig. 6) (35). The MC1R is centrally placed in a broadermelanocortin signaling axis that regulates a variety of melanocyteUV responses (36) and melanoma risk (37). When appropriately
UV
oxygen andnitrogen species
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(triplet energy state)
energy transfer
adjacentpyrimidines dark CPDs
Figure 5. Dark CPD formation. UV can induce delayed formation ofCPDs by the transfer of triplet-state energy from oxidized melanin thatgives rise to a chemically excited intermediate. This process, known as“dark CPD” formation, can occur for several hours after UV exposureand may account for the majority of all UV photoproducts.
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Figure 6. Melanocortin 1 receptor (MC1R) structure. The MC1R is a Gs protein-coupled receptor with an amino terminal extracellular domain, seventransmembrane domains, three extracellular loops, three intracellular loops and a carboxy terminal cytoplasmic tail. The MC1R gene is highly polymor-phic; five “red hair color” (RHC) mutations associated with melanoma risk (D84E, R142H, R151C, R160W and D294H) are indicated.
248 Stuart G. Jarrett and John A. D’Orazio
activated, the MC1R engages adenylyl cyclase and intracellularlevels of the second messenger cAMP increase (38). In melano-cytes, higher cytoplasmic cAMP levels drive melanin production(39,40), increase melanocyte resistance to UV-mediated apopto-sis (41,42) and enhance genomic stability to prevent UV mutage-nesis (Fig. 7) (43–48).
Although there is evidence for ligand-independent MC1R sig-naling (49), its ability to promote cAMP signaling increases fol-lowing interaction between MC1R and either of two high-affinityagonistic ligands: alpha-melanocyte stimulating hormone (a-MSH) or adrenocorticotropic hormone (ACTH) (50). Both arederived from pro-opiomelanocortin (POMC), a hormone propep-tide expressed in the epidermis and pituitary (51,52). Interactionbetween MC1R and a-MSH or ACTH leads to adenylyl cyclaseactivation and an accumulation of cAMP (53). Higher cAMPlevels promote the activity of cAMP-activated protein kinase(protein kinase A; PKA) and upregulate levels and activity oftwo key melanocyte transcription factors: cAMP response ele-ment-binding protein (CREB) and microphthalmia transcriptionfactor (MITF) (54). Together, CREB and MITF increase tran-scription of melanin biosynthetic enzymes such as tyrosinase anddopachrome tautomerase, ultimately resulting in upregulation ofmelanin synthesis (50). Melanin produced by melanocytes istransferred to keratinocytes where it accumulates in the epidermisto protect deep layers of the skin from UV damage. In this way,MC1R directly controls the adaptive tanning response which iscritical for cutaneous UV protection (40,55). MC1R activationalso enhances melanocytes’ ability to resist UV damage, as willbe discussed in detail below.
Not only can MC1R signaling be enhanced by ligand–receptorinteractions with either a-MSH or ACTH, it can also be inhibitedby physical interactions with either of two physiologic antagonists:human beta-defensin 3 (bD3) or agouti signaling protein (ASIP)(48,56). bD3 acts as a neutral antagonist, interfering with melano-cortin binding to MC1R and preventing cAMP increases in thepresence of a-MSH or ACTH (57). In contrast, ASIP is a negativeagonist, not only inhibiting melanocortin-mediated MC1R activa-tion but also downregulating ligand-independent MC1R signaling(58). Whereas ASIP is not thought to be physiologically relevantin human skin (59), bD3 is robustly expressed in a variety of cir-cumstances including inflammation and infection (60–63). Thus,regulation of melanocyte cAMP levels and the functional down-stream consequences of melanin production and DNA repair maybe dynamic and multifaceted, being regulated by ligand-indepen-dent signaling as well as positive and negative ligands.
NUCLEOTIDE EXCISION REPAIRDNA repair is essential for maintaining the integrity of the gen-ome, which when faulty contributes to mutagenesis, geneticinstability and carcinogenesis. Nucleotide excision repair is theprimary system for removing UV-induced damage such as CPDsand 6-4PPs. The xeroderma pigmentosum complementationgroup proteins (XPs) play a critical role in coordinating and pro-moting nucleotide excision repair (reviewed in (64–66)). In ele-gant in vitro experiments, the Wood and Sancar laboratoriesindependently reported the molecular requirements for reconstitu-tion of nucleotide excision repair in vitro (67,68). In global
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Figure 7. Melanocortin–MC1R signaling axis. Melanocortins such as a-MSH and ACTH, produced in the pituitary and the epidermis, act as high-affi-nity agonistic ligands to the MC1R, a Gs protein-coupled receptor on the surface of melanocytes. As a result, adenylyl cyclase is activated and cytoplas-mic levels of the second messenger cAMP increase. This leads to activation of cAMP-dependent protein kinase (protein kinase A; PKA) andupregulation of the cAMP responsive binding element (CREB) and microphthalmia (Mitf) transcription factors. Together, CREB and Mitf stimulate mel-anin production through increased expression of melanin biosynthetic enzymes such as tyrosinase. Through increased production and accumulation ofUV-blocking melanin pigments, the skin is better protected against UV insults. In addition to stimulating melanin production, MC1R/cAMP signalingalso enhances the ability of melanocytes to resist and recover from UV damage by boosting nucleotide excision repair (nucleotide excision repair), thegenome maintenance pathway charged with the removal of mutagenic UV photolesions.
Photochemistry and Photobiology, 2017, 93 249
genomic repair (GG-nucleotide excision repair), the recognitionof helical distorting lesions is achieved by XPC-RAD23B (69),and in some cases UV-DDB (70). Initial events in lesion recog-nition remain somewhat unclear with several studies supporting amodel of initial recognition by XPC followed by binding ofTFIIH, XPA and RPA (71–74), but it is generally accepted thatthe XPC/HR23B complex is recruited to photodamage by inter-actions with the undamaged complementary strand (64). In tran-scription-coupled repair (TC-nucleotide excision repair), ablocked RNA polymerase acts as the damage recognition signal.TC-nucleotide excision repair and GG-nucleotide excision repairdiffer in damage recognition, but subsequent steps converge intoone repair pathway (Fig. 8). After damage recognition, there isrecruitment of transcription factor II H (containing XPB andXPD) leading to strand separation to facilitate binding of othernucleotide excision repair factors including XPA, replication pro-tein A (RPA), XPG and excision repair cross-complementation
group 1 (ERCC1)-XPF (75,76). Once ERCC1-XPF is positionedon DNA via its interaction with XPA (77), it cuts the damagedstrand 50 to the lesion (77), followed by XPG incising 30 to thedamage (78). DNA is restored to its original form (with preserva-tion of original sequence) by the action of DNA polymerases(e.g. Pold and Polj) and associated factors using the undamagedcomplementary strand as a template (79). Failure to fill the gapleads to activation of cellular damage responses (80).
XERODERMA PIGMENTOSUMThe importance of nucleotide excision repair in cancer resistanceis best illustrated by considering the natural history of patientswith XP, a rare UV hypersensitivity syndrome caused byhomozygous defects in certain components of the pathway:XPA, ERCC1, ERCC3 (XP-B), XPC, ERCC2 (XPD), DDB2(XPE), ERCC4 (XPF), ERCC5 (XPG) and POLH (81). Patients
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Figure 8. A simplified overview of nucleotide excision repair. UV photodamage is repaired by the nucleotide excision repair genome maintenance path-way. In TC-nucleotide excision repair, a blocked RNA polymerase acts as the damage recognition signal, whereas in GG-nucleotide excision repair,XPC initiates repair by locating the damage and recruiting the subunits of a large complex called transcription factor II H (containing XPB and XPD).These proteins initiate strand separation, enabling other nucleotide excision repair factors to bind, including XPA, replication protein A (RPA), XPG andexcision repair cross-complementation group 1 (ERCC1)-XPF. Once ERCC1-XPF is positioned on DNA via its interaction with XPA, it incises the dam-aged strand 50 to the lesion, followed by XPG performing the 30 incision. Repair is completed by polymerases in combination with proliferating cellnuclear antigen that replace the gap using the undamaged complementary strand for fidelity. Please see the following excellent recent reviews for moredetail about nucleotide excision repair (46, 65, 66).
250 Stuart G. Jarrett and John A. D’Orazio
with XP demonstrate hyper UV sensitivity and develop charac-teristic skin changes including pigmentary abnormalities, capil-lary telangiectasias and atrophy on UV-exposed anatomic sites atvery early ages (82). Premalignant lesions and skin cancersdevelop at high frequency and much sooner than in unaffectedpersons. Melanomas, basal cell carcinomas and squamous cellcarcinomas typically develop before the second decade of life,many years before the general population (83). In addition, XP-associated skin cancers frequently demonstrate “UV-signaturemutations,” clearly indicating the importance of nucleotide exci-sion repair in resisting cancers caused by UV (84).
MELANOCORTIN 1 RECEPTOR ANDNUCLEOTIDE EXCISION REPAIRThe melanocortin signaling axis controls not only skin pigmen-tary responses but also directly regulates melanocyte DNA repair(46,47,85,86). When activated and functional, MC1R/cAMP sig-naling boosts the ability of melanocytes to rid their DNA of UVphotoproducts. MC1R-mediated signaling influences nucleotideexcision repair at the level of gene transcription as well asthrough the post-translational modifications of repair factors.Much of the downstream transcriptional effects of MC1R-cAMP
stimulation in melanocytes are controlled by MITF. By combin-ing high-throughput sequencing (ChIP-seq) and RNA sequencinganalyses, MITF was identified to target several nucleotide exci-sion repair genes; loss of Mitf resulted in downregulation of thecrucial nucleotide excision repair components XPA, RPA, DNAligase I and DNA polymerase delta (Pol d) (87). In anotherlarge-scale gene expression study, Mc1r loss of function inC57BL/6J mice was further associated with reduced XPAB1gene expression in neonatal whole skin (88). Although transcrip-tional activation of the expression of repair factors may positionmelanocytes in the skin to cope with future UV insults, the mela-nocortin signaling cascade also facilitates more immediate repairresponses through post-translational modifications of key proteinsin the UV damage response pathway. Our data establish an inter-section between MC1R/cAMP signaling and the ataxia telangiec-tasia mutated and Rad3-related (ATR) protein. ATR function iscritical to UV DNA damage signaling (89), cell survival (90)and is linked with nucleotide excision repair (91).
We recently reported that a critical molecular event linkingMC1R signaling to DNA repair is a cAMP-dependent phospho-rylation event on ATR (46). When cAMP levels are inducedeither by MSH-MC1R interactions or pharmacologically withadenylyl cyclase activation by forskolin, PKA becomes activated
Figure 9. Summary figure of PKA-ATR-XPA model. In melanocytes, enhancement of nucleotide excision repair by cAMP is dependent on a post-translational modification of ATR. MC1R activation by melanocortins results in PKA-induced phosphorylation of ATR at the S435 residue. This eventpromotes binding between XPA and ATR in the nucleus, and together, ATR and XPA localize to UV photodamage in an accelerated and enhancedmanner. MC1R agonists a-MSH or ACTH activate PKA-mediated ATR phosphorylation and nucleotide excision repair, whereas MC1R antagonistsASIP or bD3 inhibit this repair-enhancing pathway. In this way, genomic stability and susceptibility to UV mutagenesis in melanocytes may be hormon-ally influenced.
Photochemistry and Photobiology, 2017, 93 251
and phosphorylates ATR on its serine 435 (S435) residue. Thispost-translational modification causes ATR to associate with thekey nucleotide excision repair factor XPA (46). Together, XPAand ATR-pS435 co-localize to sites of UV-induced DNA dam-age in an accelerated and enhanced manner (Fig. 9). Loss ofS435 within ATR prevents PKA-mediated ATR phosphorylation,disrupts ATR–XPA binding, delays recruitment of XPA to UV-damaged DNA and elevates UV-induced mutagenesis in melano-cytes. Our data suggest that PKA modification promotes nuclearentry of an ATR–XPA complex to “prime” DNA damageresponses in melanocytes immediately upon UV exposure (46).Indeed, S435 is part of a PKA target sequence within ATR’spredicted nuclear localization sequence (425-DGISPKRRRLSSSLNPSKRAP), suggesting that its phosphorylation might impactATR’s nuclear localization, possibly through interactions withnuclear importins (92). Interestingly, PKA-directed nuclear local-ization of DNA-PK, another PIKK family member, has also beenreported (93). Our data suggest that PKA may be an importantregulator of nuclear import of key nucleotide excision repair fac-tor(s) during DNA repair. Alternatively, PKA-mediated phospho-rylation of ATR at S435 may optimize nucleotide excision repairthrough enhanced intranuclear interactions with XPA to facilitatetransport and/or assembly of nucleotide excision repair factors atsites of UV damage. By a variety of possible mechanisms, it isclear that PKA-mediated modification of ATR may “prime” mel-anocytes to better cope with UV injury.
Melanocortin signaling also impacts nucleotide excision repairthrough other mechanisms. For example, the nucleotide excisionrepair proteins, DDB1, DDB2 and XPC have all been shown tobe upregulated following cAMP activation (94). Of note, itappears that MC1R wild-type melanocytes regulate DDB2 pro-tein levels through a p38 MAPK-associated signaling. Recently,melanocortin-enhanced DNA repair was shown to be influencedby increased levels of XPC and H2AX, potentially promotingthe formation of DNA repair complexes (47). Other componentsof the UV DNA damage repair response also are impacted byMC1R and include the NR4A superfamily of nuclear receptors(86,95,96). NR4A is recruited to sites of nuclear DNA damagetogether with XPC and XPE. Taken together, these studies pro-vide a mechanistic view of MC1R as a master orchestrator ofnucleotide excision repair in melanocytes.
HORMONAL REGULATION OF NUCLEOTIDEEXCISION REPAIR BY MC1R AGONISTS ANDANTAGONISTSHaving previously established that MC1R-enhanced nucleotideexcision repair is dependent on PKA-mediated phosphorylationof ATR on S435, recruitment of XPA and accelerated localiza-tion to nuclear UV photodamage (46), we sought to determinethe influence of physiologic MC1R ligands on ATR-pS435 accu-mulation and nucleotide excision repair function (48). To studythe kinase activity of the MC1R–cAMP–PKA axis, high-through-put methodologies were developed. ATR-pS435 was detectedin vitro using a 14-mer peptide (consisting of residues 428–441of ATR) using a phospho-specific (ATR-pS435) antibody. Inmelanocytes with intact MC1R signaling, the ATR–pS435–XPAaxis is heavily influenced by agonists and antagonists of MC1R,with melanocyte genomic stability regulated by MC1R signalingstatus and concentrations of MC1R ligands in the local milieu.Enzyme kinetic studies revealed higher Vmax and lower Km
values for forskolin-mediated ATR-pS435 compared with a-MSH-mediated ATR-pS435 (48). These different kinetic proper-ties suggest an increased “cAMP load” may enhance the capabil-ity of PKA to recognize ATR-S435 and/or impact how stronglyPKA binds with the S435 substrate.
In primary human melanocytes and MC1R-transfectedHEK293 cells, either a-MSH or ACTH enhanced ATR-pS435,XPA’s association with UV-damaged DNA and optimizednucleotide excision repair. In contrast, either ASIP or bD3 inter-fered with ATR-pS435 generation, impaired XPA–DNA interac-tion and impeded DNA repair. Interestingly, ASIPdownregulated basal levels of ATR-pS435, consistent with itbeing an MC1R inverse agonist capable of downregulatingligand-independent MC1R signaling. bD3, however, had noimpact on constitutive levels of ATR-pS435, supporting its func-tion as a neutral MC1R antagonist instead (48).
To elucidate the functional effect of MC1R ligands on DNArepair, the oligonucleotide retrieval assay that quantifies repair byPCR-based amplification was adapted (97). In this assay, thepresence of photoproduct(s) interferes with the DNA polymerase;therefore, the amount of amplification across the oligonucleotideis proportional to clearance of photolesions by nucleotide exci-sion repair. We adapted this method by directly UV-irradiatingthe oligonucleotide (instead of a chemically generated singleCPD lesion) which resulted in more CPDs and 6-4PPs (48). Weobserved that nucleotide excision repair responses were regulatedby MC1R status and ligand interactions and that they correlatedwith ATR-pS435 accumulation and XPA–DNA binding (48).Thus, a-MSH promoted nucleotide excision repair, while ASIPand bD3 blocked a-MSH-mediated enhancement of repair. ASIPblunted repair of UV-induced DNA damage to a greater extentthan bD3, which is explained by the fact that ASIP has a greaterability to inhibit ATR-pS435 generation than bD3. Together,these findings support the hypothesis that MC1R/cAMP signalingcontrols melanocytic nucleotide excision repair through down-stream PKA-mediated ATR phosphorylation on S435. Further-more, these data raise the possibility that dysregulated expressionof ASIP or bD3 in the skin may impair DNA repair responses inmelanocytes to heighten UV mutagenesis and melanoma risk.
MC1R AS A MELANOMA RISKDETERMINANTIt has been known for many years that inherited signaling defectsin MC1R correlate with melanoma risk (37,98–100). WhileMC1R loss certainly yields a UV-sensitive fair complexion phe-notype, it is clear that melanocyte genome stability is reduced asa result of MC1R loss. Indeed, a recent study established a rolefor germline loss-of-function MC1R variants in increasing thesomatic mutational landscape of melanoma (101). Because ofMC1R’s role in regulating melanocyte UV responses (102,103),it is not surprising that inherited MC1R signaling defects impactmelanoma susceptibility (Fig. 10).
The human MC1R gene is highly polymorphic with over100 variants reported (104). Although many mutants affect itssignaling ability, five in particular—the so-called “red hairvariants” (or “RHC variants”)—D84E, R142H, R151C,R160W and D294H—are especially associated with increasedmelanoma risk (105,106). When MC1R signaling is blunted,melanocytes are less able to respond to a-MSH or ACTH andcytoplasmic cAMP levels are lower. Instead of robust
252 Stuart G. Jarrett and John A. D’Orazio
production of a brown/black UV-blocking pigment known aseumelanin, there is production of a pigment variant known aspheomelanin, which is a red/blonde melanin bioaggregate thatis less able to block UV radiation and actually promotes freeradical formation (107). Indeed, pheomelanin is an independentmelanoma risk factor (108); therefore, MC1R deficiency influ-ences mutagenic risk not only because of diminished eume-lanin formation, but also because of increased pheomelaninproduction. MC1R deficiency also results in less effectivenucleotide excision repair (43,46–48); therefore, MC1R defectscontribute to melanoma development not only by permitting
more UV damage in melanocytes but also by blunting theability of melanocytes to repair that damage.
TRANSLATIONAL IMPLICATIONSThe MSH–MC1R signaling axis is an innate melanocyte protec-tive pathway recruited after UV exposure to help melanocytesrecover from damage and protect the skin against further UVdamage. Persons with inherited defects in MC1R signaling func-tion suffer higher melanoma risk because of suboptimalmelanization and defective DNA repair, each of which leads to
MC1R Signaling GoodPoor
Melanocyte cAMP Levels HighLow
Melanoma Risk LowHigh
Pigmentation Dark Skin ComplexionFair Skin Complexion
Skin Color
Melanocyte NER OptimalDiminished
Tanning response Robust Impaired
UV mutagenesis LowIncreased
Epidermal melanin EumelaninPheomelanin
MED 90-150 mJ/cm215-30 mJ/cm2
Sunburn Risk LowHigh
pS435-ATR AdequateMinimal
Figure 10. MC1R function and UV physiologic responses. Strength of MC1R signaling correlates with melanization, UV sensitivity, robustness of mel-anocyte DNA repair. Individuals with inherited loss-of-function MC1R polymorphisms have heightened melanoma risk, in part, because their melano-cytes accrue more UV-signature mutations over time.
α-MSH, ACTH analogues
Melanocortin 1 Receptor
cAMP
adenylyl cyclase activators(e.g. forskolin)
adenylylcyclase
phosphodiesterase inhibitors(e.g. rolipram)
melanin production(eumelanin)
genome stability(nucleotide excision repair)
βD3, ASIP inhibitors
Figure 11. Pharmacologic strategies to mimic MC1R signaling in melanocytes. Raising cAMP levels in melanocytes enhances UV protection throughenhanced melanin production and improved melanocyte genome stability. MC1R-specific approaches include melanocortin analogs or, theoretically, inhi-bitors of MC1R antagonists such as bD3, but would be expected to be of benefit only for individuals with intact MC1R signaling. Pharmacologic induc-tion of cAMP could be achieved by activation of adenylyl cyclase or through inhibition of phosphodiesterases and would be independent of MC1Rfunction.
Photochemistry and Photobiology, 2017, 93 253
accumulation of UV-induced mutations known to be causativefor melanoma. Because MC1R-mediated melanization andenhanced genome maintenance are dependent on cellular cAMPlevels, it is possible to pharmacologically stimulate MC1Rresponses to protect MC1R-impaired individuals from UV-induced mutagenesis by manipulating melanocyte cAMP levels.There are two fundamental approaches to achieve this: (1) thosethat specifically target melanocytes by taking advantage of thefact that MC1R expression is limited to melanocytes in the skinand (2) those that raise cAMP through MC1R-independentmechanisms (Fig. 11). Targeting approaches that limit cAMPinduction in melanocytes is important because of the potentialfor off-target effects of cAMP stimulation on other cells (e.g.keratinocytes) and systems (immune system, circulatory system,etc.). Therefore, cutaneous application of agents that mimic mela-nocortin agonists or that inhibit MC1R antagonists would beexpected to increase cAMP levels only in melanocytes becauseof selective action on MC1R (44). Although these approachesoffer melanocyte specificity, neither melanocortin analogs norinhibitors of MC1R antagonists would be expected to offer bene-fit to individuals with inherited MC1R signaling defects. Asthese persons are among the most melanoma-prone in the generalpopulation, MC1R-independent mechanisms of raising melano-cyte cAMP levels seem more appropriate. Our group has usedforskolin, a powerful activator of adenylyl cyclase, to show thattopical induction of cAMP rescues melanization (40,109,110)and optimizes nucleotide excision repair (46) in a humanizedmouse model of the MC1R-defective human. Similarly, othershave rescued melanization in this model by interfering withphosphodiesterases (111), the enzymes responsible for degradingcAMP. Global induction of cAMP in the skin would overcomeMC1R defects and enhance melanocyte UV resistance, but thisapproach lacks melanocyte specificity. While more research isrequired to understand the risks and benefits of different thera-peutic approaches aimed at augmenting MC1R signaling, it isnow clear that it is possible to reduce short- and long-term UVinjury to the skin by rationally targeting the innate melanocortinsignaling axis UV-protective pathway.
Acknowledgements—We are grateful for support from the NationalCancer Institute (R01 CA131075), the Melanoma Research Alliance(MRA) and the Regina Drury Endowment for Pediatric Research. Wealso acknowledge the technical support of the Markey ResearchCommunications Office for their help in preparing figures for thismanuscript and NCI Cancer Center Support Grant (P30 CA177558) ofthe Markey Cancer Center at the University of Kentucky.
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Stuart G. Jarrett’sresearch interests arefocused on the biochemi-cal mechanisms of DNArepair active against ultra-violet radiation. Recently,he delineated a pathwayby which melanocytesregulate nucleotide exci-sion repair, involving acAMP-mediated phospho-rylation event at Serine435 on the ATM andRad3-related protein. Stu-art completed his PhD atthe University of Cardiff,where his researchfocused on mitochondrialbase excision repair,oxidative stress and aging.Stuart’s long-term career
goal is to discover the molecular events that control melanocyte DNArepair to inform development of targeted melanoma-preventive strategies.
John A. D’Orazio, MD,PhD, is an associate pro-fessor of pediatrics and aboard-certified pediatrichematologist/oncologist atthe University of Ken-tucky who actively treatspediatric patients withcancer and blood dis-eases. His particular inter-est is in inherited cancersyndromes that affectchildren and youngadults. He earned MDand PhD degrees fromthe University of Miamibefore joining the pedi-atric residency program atMassachusetts GeneralHospital. After a year as
Pediatric Chief Resident, Dr. D’Orazio joined the Hematology and Oncol-ogy Fellowship Program at Boston Children’s Hospital and the Dana-Far-ber Cancer Institute. He joined the University of Kentucky in 2004 wherehe directs an NIH-funded laboratory studying melanoma inherited risk fac-tors. His laboratory focuses on the melanocortin 1 receptor (MC1R),which is a major determinant of lifetime melanoma risk. By understandingthe molecular events that cause cancer, Dr. D’Orazio and his team striveto develop effective antimelanoma therapies. Dr. D’Orazio holds theDrury Pediatric Research Endowed Chair and is co-leader of the MarkeyCancer Center’s Genetic Instability, Epigenetics and Metabolism ResearchProgram. He has authored numerous scientific and clinical manuscripts aswell as reviews and book chapters and has presented data from his labo-ratory at regional, national and international scientific meetings.
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