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MALIGNANT MELANOMA

Melanocortin Receptors, Red Hair, and Skin Cancer

Jonathan L Rees and Eugene Healy Department of Dennatology, University of Newcastle upon Tyne, U.K.

Cutaneous pigmentation is a major determinant of clase, are associated with dift"erent coat colors. In the cutaneous response to ultraviolet radiation, and humans, 1 receptor variants are associ­ consequently of the risk of developing skin cancer. ated with red hair and fair skin, and work in progress Over the past 10 years, several involved in from our laboratory suggests that certain melanocor­ melanogenesis have been identified, including the tin 1 receptor variants may preferentially be associ­ . Recent work on the ated with hair color rather than skin type. In addi­ melanocortin 1 receptor suggests that it is a key tion, melanocortin 1 receptor variants are a risk player in determining whether eumelanin or factor, possibly independent of skin type, for mela­ pheomelanin is predominantly produced both in vitro noma susceptibility. Key worth: G--coupled reap­ and in vivo. In the mouse, variants of this receptor, torlpigmentationlmelanomalgenetiu.joumal of Investigative which dift"er in their ability to activate adenylyl cy- Dermatology Symposium Proceedings 2:94-98. 1997

, , n account of the bequest of the late 1996). This actual relative risk is likely to be a considerable Ezekiah Hopkins, of Lebanon, Penn., underestimate for two reasons. First, accurate skin typing is prob­ U.S.A., there is now another vacancy lematic (Rampen et ai, 1988), and second, individuals who are O open which entides a member of the sensitive to the sun will tend to spend less time in the sun, resulting League to a salary of four pounds a in an underestimation of the risks for those with sun sensitivity. week for purely nominal services. All red-headed men who are Genetic approaches to the understanding of skin type, i.e., the sound in body and mind, and above the age of twenty-one years, degree to which individuals are liable to tan or burn in response to are eligible." (from 'The Red-headed League,' in 'The Adventures ultraviolet radiation, may seem hopeless. Whereas differences of Sherlock Holmes' by Arthur Conan Doyle.) between the majority of Northern Europeans and Southern Euro­ Although it makes for good rhetoric, dissociating genetic from peans are easily detected, skin typing as used clinically is at best a environmental causes of disease, such that one forgets that genes crude instrument, although up to the present there has been litde only exert an effect in a defined environment and vice versa, makes choice. Notwithstanding these difficulties, understanding the ge­ for bad science. Understanding the causes of skin cancer provides a netics of pigmentation and the response to ultraviolet radiation are pertinent example. One can view the causes of melanoma or of major importance, not only because of their immediate relevance non-melanoma skin cancer as being predominandy environmental, to melanoma and non-melanoma skin cancer but also because or alternatively as being predominandy genetic. From an environ­ pigmentary differences between individuals are perhaps the most mental perspective, the major determinant world wide of skin striking traits that distinguish individuals of our species. That cancer appears to be ultraviolet radiation: skin cancers arise more changes in human pigmentation have occurred independendy in frequendy on sun-exposed areas; rates vary with latitude; and populations widely dispersed suggests that there is strong evolu­ migrants from Anglo-Saxon communities to AustraIia show an tionary pressure to fit particular populations to a particular envi­ elevated risk of both melanoma and non-melanoma skin cancer ronmental niche (Bodmer and Cavalli-Sforza, 1976; Kingdon, (Armstrong and Kricker, 1996). On the other hand, given that 1993). In evolutionary terms it appears likely that, with the loss of these large-scale population migrations, and increased leisure and body hair, the importance of interfollicular pigmentation as a travel, are recent phenomena, the major determinant world wide of method to protect the individual from the harmful effects of skin cancer has been genetic, namely the pigmentary status of the ultraviolet radiation assumed importance. Although, as observed in individual. Rates of both melanoma and non-melanoma skin cancer African albinos, the hazards of ultraviolet radiation are amply are up to 50 times lower in Negroes, and 4 to 12 times lower in demonstrated by the development of skin tumors at an early age Japanese, than in Caucasians (Chuang et ai, 1995; Halder and (and at an age that would influence biologic reproductive fitness), Bridgeman-Shah, 1995). Even within Caucasian populations, how­ it seems likely that the major evolutionary drive toward the ever, possession of a collection of phenotypic features including red hair, tendency to freckle, and the usually associated inability to tan development of pigment relied more on the adverse biologic effects is associated with an up to 5-fold risk in melanoma and non­ of sunburn with the consequent increased propensity to infection melanoma skin cancer (Bliss et ai, 1995; Armstrong and Kricker, and acute illness (Luande et ai, 1985; Kingdon, 1993). Nevertheless the subsequent reduction or change of pigment in populations such as those currendy inhabiting Northern Europe needs to be ac­ Reprint requests to: Jonathan L. Rees, Department of Dennatology, counted for by more than the aforementioned gifts of Mrs Ezekiah Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, United Kingdom. Hopkins. Current evolutionary theory suggests that the possession Abbreviations: MCR, ; MSH, melanocyte-stimu­ of dark skin in populations where ambient ultraviolet radiation is lating honnone. low leads to a propensity to rickets due to impaired vitamin D

1087-0024/971$10.50 • Copyright © 1997 by The Society for Investigative Dermatology, Inc.

94 VOL 2, NO. 1 AUGUST 1997 MEIANOCORTlN RECEPTORS 9S

metabolism with the resulting biologic consequences (Kingdon, mRNA has not been detected so far in any of the other organs or 1993). As one moves progressively further north through Europe, tissues investigated (Mountjoy et aI, 1992; Chhajlani and Wikberg, skin color lightens in populations whose diets are cereal based. By 1992; De Luca et ai, 1993; Siegrist et ai, 1994; Suzuki et ai, 1996). contrast, in the Eskimos skin pigmentation is darker, an exception Expression of the ACTH receptor occurs in the adrenal gland and that some argue may be accounted for by their reliance not on a adipose tissue; MC3R is expressed in brain, placenta, and gastro­ cereal-based diet but by a diet dependent on meat with a greater intestinal tissues; MC4R is present in brain; and MC5R appears in supply of fat-soluble vitamins (including vitamin D) (Bodmer and various tissues including brain, skin, adipose tissue, adrenal gland, Cavalli-Sforza, 1976; Kingdon, 1993). skeletal muscle, bone marrow, spleen, thymus, gonads, and uterus; Over the last 10 y, genetic approaches to disease and human at present it is unclear whether the MC5R expression observed in physiology have proved extraordinarily powerful. The major intel­ murine skin is due to the presence of subcutaneous fat or to its lectual difficulty remains the framing of clinically relevant questions transcription in other cell types (Mountjoy et ai, 1992; Chhajlani et in such a way that genetic approaches can be undertaken: for aI, 1993; Gantz et ai, 1993a, 1993b; Labbe et ai, 1994; Boston and pigmentation, as for so many other areas of research, murine Cone, 1996). Interestingly, radioligand-binding assays on immor­ genetics has been especially informative Oackson 1994). The talized human epidermal keratinocytes have demonstrated that identification of the murine extension as being the gene for these cells show binding for J3-melanocyte-stimulating hormone, the melanocortin 1 receptor (Mc1r) (Robbins et ai, 1993) has but Suzuki et al were unable to detect specific mRNA transcript for opened up opportunities to obtain a genetic handle on the study of MC1R in human epidermal keratinocytes and therefore suggested the cutaneous response to ultraviolet radiation within human that keratinocytes may express another melanocortin receptor populations. In the present article we will review briefly the rather than MC1R (Chakraborty and Pawelek, 1993; Suzuki et ai, background to the cloning of the human melanocortin receptors, 1996). Immunostaining of normal skin by Xia et al (1995) has also describe recent data on cellular expression of these receptors, and failed to identify any MC1R protein in keratinocytes in vivo. then present work from our own laboratory studying the associa­ The central importance of the human MC1R gene in melano­ tion between variants of the human melanocortin 1 receptor genesis was emphasized by the recent work by Chluba-de Tapia et (MC1R) and cutaneous phenotype and associations between par­ al (1996) who induced melanin production in amelanotic mouse ticular variants of the MC1R and melanoma. melanoma cells (which do not express their own mouse Mc1r) by stable transfection of the human MC1R gene. In spite of melano­ CLONING OF A FAMILY OF MELANOCORTIN cytes having only one receptor, however, information from trans­ RECEPTORS fection experiments and from ligand assays on cultured melano­ The human MC1R gene, encoding for a seven-pass transmembrane cytes/melanoma cells suggests that other ligands, in addition to G-protein-coupled receptor of 317 amino acids, was cloned in a-melanocyte-stimulating hormone (a-MSH), including ACTH, 1992 by two separate groups (Mountjoy et ai, 1992; Chhajlani & J3-MSH, 'Y-MSH (and the synthetic analog Nle4-,D-Phe7-alpha Wikberg, 1992). Mountjoy et al (1992) used degenerate oligonu­ MSH) can bind to the MC1R (Chhajlani and Wikberg, 1992; cleotide primers from transmembrane domains 3 and 6 of earlier Mountjoy et aI, 1992; Suzuki et ai, 1996). Results from competitive cloned G-protein-coupled receptors to amplify by PCR cDNA binding assays and measurement of activityI cAMP from a human melanoma that had been previously shown to production in cells transfected with human MC1R, and in cultured contain a large number of [125I]iodo-Nle4-,D-Phe7-a-melanocyte­ human melanocytes, are generally consistent, with a-MSH = stimulating hormone (a-MSH)-binding sites. Chhajlani and Wik­ ACTH > {3-MSH > -y-MSH in the order of potency (Chhajlani and berg (1992) similarly employed degenerate primers from these Wikberg, 1992; Mountjoy et ai, 1992; Suzuki et aI, 1996). The transmembrane domains but amplified from genomic DNA (this physiologic relevance of these in vitro binding studies, however, is being possible because many G-protein-coupled receptors were at present unclear; although the precursor known to be intronless), and subsequendy used the amplified is present in human skin (and is upregulated by ultraviolet product as a probe to detect an RNA band (only) in a melanoma radiation), the relative contribution of the proopiomelanocortin cell line during a Northern blot hybridization with RNA from breakdown products such as a-MSH and ACTH (which have also several tissue types. At the same time Mountjoy et al (1992) cloned been detected in mammalian skin) to melanogenesis in vivo is the murine Mc1r and human adrenocorticotrophic hormone unknown (Thody et ai, 1983; Farooqui et aI, 1993; Wintzen et ai, (ACTH) receptor (MC2R) genes and, on the basis of Southern 1996). The recent detection in human skin of the two prohormone hybridization of human genomic DNA at low stringency to a convertases PCl and PC2, which are involved in the formation of human MC1R probe, suggested that as many as five or six members ACTH and its cleavage to a-MSH, respectively, with higher of the melanocortin receptor gene family existed. To date, another immunoreactivity for pe2 in melanocytes suggests that the pattern three human melanocortin receptor genes (designated MC3R, of processing of proopiomelanocortin breakdown products in me­ MC4R, MC5R) have been identified (Chhajlani et ai, 1993; Gantz lanocytes may diJfer from that in keratinocytes (A.J. Thody, et ai, 1993a, 1993b). The MC1R has been mapped to human personal communication). 16q24.3 by fluorescent in situ hybridization (FISH) MICE AND HUMANS WITH MCIR VARIANTS HAVE analysis; however, the other melanocortin receptor genes are ALTERED PIGMENTARY PHENOTYPES located elsewhere; 18p11.2 (MC2R), 20q13.2-q13.3 (MC3R), 18q21.3 (MC4R), and 18p11.2 (MC5R) (Gantz et ai, 1993b, 1993c, Mammalian coloring, including murine coat color, is in large part 1994a; Chowdhary et ai, 1995). Investigations on MC1R mRNA dependent on the relative amounts of eumelanin and pheomelanin expression by northern hybridization have shown it to be present at that are produced (Burchill et aI, 1993; Jackson, 1994; Ozeki et ai, relatively low levels in cultured melanocytes, but expression in 1995; Prota et aI, 1995). Similar diJferences are also seen in human melanoma cells varies from undetectable to highly abundant hair, and both eumelanin and pheomelanin are found in interfol­ (Mountjoy et ai, 1992; Chhajlani and Wikberg, 1992; Cone et aI, licular human epidermis (Thody et aI, 1991). Murine genetics has 1993; Chakraborty et ai, 1995; Suzuki et ai, 1996). Immunohisto­ shown that various alleles of the extension locus are important in chemistry using a polyclonal antibody against the MC1R protein determining murine coat color (Robbins et ai, 1993). For instance, has enabled detection of the antigen in COS-7 cells transfected with loss of function mutations of this locus result in recessive yellow mice. the MC1R gene and in melanoma tissue, but melanocytes in normal Conversely, dominant mutations increase the amount of black skin are not visualized with this antibody (Xia et aI, 1995). pigment (or eumelanin) and diminish the amount of yellow pig­ ment Oackson, 1994). Although the phenotype of yellow mice is MC1R IS A KEY DETERMINANT OF PIGMENTATION different from that in red hair and may be thought to resemble the MC1R seems to be the only melanocortin receptor expressed in light colored hair found in Northern Europe, analysis of eumelanin cultured melanocytes and melanoma cells, and conversely MC1R and pheomelanin show that yellow mouse hair is rich in pheomela- 96 REES AND HEALY JID SYMPOSIUM PROCEEDINGS

Table I. The Spectrwn of MC1R Variants Identmed in 222 Individualsd According to Skin Type

Skin Type Ala64Ser Phe76Tyr Asp84Glu Asn91Asp Val92Met Thr95Met Va197ne AlalO3Val Leu106Gln Asp294His

1 1 4 16 2 1 13 I-II 4 5 II 1 8 18 II-III 1 III 1 4 1 III-IV 1 IV 2

• Integen in table refer to numbers of individuals with the MC1R variant. Infurmation on skin type not available for two cases with Alp84Glu variant. and for one case with Val92Met variant. Some individuals contained more than one MC1R variant; please refer to Valverde et at (1995) and Valverde et al (1996) for further details on homozygous. heterozygous. and compound heterozygous variants.

nin and appears to be the murine counterpart of the red hair found identified with a similar phenotype some of whom are compound in humans (prota, 1992; Prota et ai, 1995). Because of this and heterozygotes and others are simple heterozygotes. It remains because of the known effects of MSH and ACTH on both murine possible that some of the heterozygotes may have abnormalities and human pigmentation, we examined the MC1R as a candidate outside the coding region, perhaps affecting , or, gene for red hair andlor skin type 1 in humans (Valverde et ai, alternatively, that the results are in keeping with what we already 1995). Because of the difficulties of skin typing we initially exam­ know about red hair, i.e., that it does not appear to segregate as a ined individuals showing the extremes of hair and skin pigmenta­ simple Mendelian recessive trait. Interestingly, Barsh (1996) has tion of phenotype. The MC1R gene from 30 unrelated individuals reviewed the results in terms of relative risks rather than in classical with different shades of red hair and a poor tanning response were Mendelian terms and points out that individuals who harbor a sequenced. Controls with brown or black hair and a good tanning variant receptor have a relative risk for red hair of 1S-fold, but this response were similarly investigated. Variants of the MC1R gene risk rises to 170-fold among those individuals carrying two variant were found in 21 of the 30 red-headed fair-skinned individuals allele. Because the population studied was not a random one, examined, but none were found in the 30 controls. A large number however, this method of analysis may tend to overemphasize the of variants were identified (see Table I). importance of the putative red hair alleles. The majority of variants clustered in a region between the first MC1R VARIANTS ARE A RISK FACTOR FOR cytoplasmic loop and the first extracelluar loop spanning the second CUTANEOUS MELANOMA transmembrane domain. The most common change, however, was an aspartate to histidine at codon 294 in the seventh transmembrane As well as being important in understanding the biology of domain. Interestingly, whereas 13 individuals had only one change cutaneous pigmentation, the identification of variants of the MC1R in the coding region, eight had two or more changes. PCR cloning gene in humans, although not entirely analogous to the situation in of these established that seven of these eight individuals were mice, may have more immediate medical consequences. Melanoma compound heterozygotes. In order to extend these results to a more incidence is apparendy increasing rapidly in many Caucasian pop­ representative population, we went on to examine a further 7 S ulations (MacLennan et ai, 1992; Elder, 1995; Rees, 1996). Whereas individuals who were classified according to their natural hair color several genetic events that are important in the development and at the age of 20 y and their skin type using the Fitzpatrick (1988) progression of sporadic melanoma have been identified, little is clinical skin typing, examining only those regions of the MC1R that known about the genetics of susceptibility to this neoplasm (Rees had harbored abnormalities in the earlier study. Eighty two percent and Healy, 1996). Red hair and the inability to tan in response to of individuals with light redldeep red hair had changes in one or ultraviolet radiation are known risk factors for the development of both alleles compared to fewer than a quarter of the auburns, one melanoma (Bliss et ai, 1995). It is to be expected, therefore, that third of those who were fair or blonde, and fewer than 20% of the there may be a preponderance of individuals with variant alleles brown or blacks. Changes in both alleles (29% of the total) were among individuals who develop melanoma. If this were just the only found in those individuals who had light or deep red hair. case, then genotypic examination may be a useful predictor of Interestingly, when the individuals were classified according to melanoma but one would question whether it would impart their skin type, the association was even more striking: all the information not obtainable from the phenotype. On the other hand, black- or brown-haired individuals who had changes in the MC1R the situation may be more complex than this initial interpretation. were classified as skin types I or ll. We didn't detect any alterations First, although associations between variant alleles and melanoma in individuals with skin type IV, and only two of the skin type III might be expected because of the association between variant subjects in this study contained MC1R variants. alleles and skin type, the majority of individuals with red hair or These data have several enigmatic and puzzling features and skin type I do not develop melanoma. It seems possible that the suggest important differences from that seen in the mouse. First, a possession of particular variants may not be equal in their biologic large number of variants have been detected. Whereas some of effect, raising the possibility that the presence of particular alleles these changes might be expected to be functionally significant, e.g., may render certain individuals particularly at risk. Second, the the replacement of an acidic residue with the basic one at codon 294 majority of patients with melanoma do not have skin type I. Based and others in the second transmembrane region that might be on present evidence, the genetics of the MC1R in humans are expected to alter the a-helical structure, some of the variants may complex, and it is possible that even in the absence of the features prove to be simple polymorphisms in linkage with other changes that render a person at increased risk, such as red hair or freckling, (perhaps in the promoter region), which alter the expression of the some individuals with dark hair who tan intermediately or well may gene. Second, abnormalities were not found in all individuals with also harbor variant alleles; there may be differences, therefore, red hair. This is perhaps not surprising given the complex nature of between genotypic examination and phenotypic examination. Fi­ human pigmentation and, as discussed below, given the possibility nally, there is some evidence that MSH may be important in that the physiologic antagonist of the MC1R receptor, agouti, may controlling melanocyte growth in the context of melanoma pro­ also be important in determining human hair color. These results, gression (Lunec et ai, 1990; Lunec et ai, 1992; De Luca et ai, 1993; however, do show that, unlike inbred mouse strains in which loss Suzuki et ai, 1996). If this is indeed the case, then variant receptor of function mutations are recessive (Robbins et ai, 1993), in humans alleles could be associated with melanoma independent of the the situation appears more complex: many individuals have been effects on cutaneous pigmentation. VOL. 2. NO. 1 AUGUST 1997 MELANOCORTIN RECEPTORS 97

In order to examine further these possibilities, we screened 43 only changes in MC1R, but also differences in expression or, unrelated patients with sporadic cutaneous melanoma and 44 conceivably, mutations [although as Barsh (1996) has argued this unrelated control patients (Valverde et ai, 1996). The patient's skin would be unusual in a ligand] might also account for the red­ type was assessed according to the Fitzpatrick scale and the history headed and skin type 1 phenotype. of freckling and hair color was taken. As expected, a weak It is not clear at present whether the reported variations in the association between melanoma and skin type was observed ifskin MC1R gene are associated with red hair per se or with pale skin type was treated as a quantitative variable (p = 0.046). In keeping (Valverde et ai, 1995). The two characteristics do not always go with the previous work 20 of 43 melanoma patients harbored hand in hand. For instance, in Irish populations there are many variant alleles compared with only eight of the 44 controls. The individuals with dark hair who are skin type I. Results on these are association between variant alleles and melanoma was significant incomplete but preliminary observations from our laboratory sug­ with the relative risk of 3.91 (95% confidence intervals, 1.48 - gest that certain MC1R variants may preferentially be associated 10.35). The population risk attributable to carriers, which provides with hair color rather than skin type, and that these variants are a measure of what the disease incidence would be reduced by ifthe found at different frequencies in groups of individuals with similar risk in carriers was reduced to the same level as the non-carriers, phenotypes from different European populations. was 35%: more than one third of melanomas could be attributed to Finally, the association of a particular alteration at codon 84 in the presence of variant alleles in the population; for comparison, the melanomas in the UK population needs to be confirmed or refuted INK4 relevant figure for melanoma cases due to germline p16 in other populations. The current data suggest that genotype may mutation is 3 to 5%. What was perhaps unexpected, at least in predict tumor risk better than phenotype. This may relate to the degree, however, was that the majority of variant alleles among difficulties of skin typing using the Fitzpatrick classification individuals with melanoma comprised one particular variant, the (Rampen et aI, 1988), but at least raises the possibility that in Asp84Glu variant. Two individuals were homozygous for this melanoma the influence of the MC1R signaling pathway may be variant and eight were heterozygous. Although at the biochemical additional to any effects on the pigmentary phenotype. It would be level the functional significance of this change is as yet unknown, of interest to see whether similar MC1R variants are present in the importance of these results is highlighted by the finding that this non-melanoma skin cancer; if no association exists it may be that variant was found in none of the control subjects studied as part of the effect of MC1R variants in melanoma are independent of the the melanoma study and was only found in two of 135 subjects in skin type phenotype and that their influence on the growth and a previous study that included 47 subjects with red or auburn hair development of melanocytes and melanoma cells is via the MSH­ and 77 individuals who were skin type I or II (Valverde et ai, 1995). signaling pathway. Thus, the Asp84Glu is rare even in a population enriched for those with marked sun sensitivity and red hair who are potentially at risk of melanoma development. Although the change from aspartate to E.H. is a Medical Research Council (MRC) Training Fellow. Work in J.L.R. 's glutamate appears minor, an aspartate at codon 84 is highly laboratOlY is supported by NECRC, the Department ofHealth, AICR, and MRC. conserved throughout not only the melanocortin receptor family but also other G-protein-coupled receptors (Chhajlani and Wik­ berg, 1992; Mountjoy et ai, 1992; Chhajlani et ai, 1993; Gantz et ai, 1993a, 1993b). As with previous studies of the variant alleles, it REFERENCES remains formally possible that this change may be in linkage Armstrong BK, Kricker A: Epidemiology of sun exposure and skin cancer. In: Leigh dis-equilibrium with other changes outside the coding region of the 1M, Newton Bishop JA, Kripke ML (eds.). Cancer Surveys Skin Cancer. Vol. 26. 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