Melanomas, primary tumors of melanocytes, account for 15% of equine skin tumors and are classified into 4 groups according to clinical and histopathologic features. Melanocytic nevi and anaplastic malignant melanoma affect horses of any age, breed, or coat color, while dermal melanoma and dermal melanomatosis occur in gray horses.[3, 4] Dermal melanoma and melanomatosis are distinguished by the number of masses and the presence of metastasis, but are not distinguishable histopathologically.[2, 3] Up to 80% of gray horses older than 15 years develop melanomas[1, 2] and 14–66% of dermal melanomas eventually metastasize.[3, 4]
Graying and melanoma have been linked to a 4.6 kilobase duplication in intron 6 of the syntaxin-17 (STX17) gene. This duplication harbors a regulatory element containing binding sites for microphthalmia-associated transcription factor (MITF) and NR4A3, key components in the regulation of melanocyte gene expression and cell function. The expression of both STX17 and its neighboring gene, NR4A3, is up-regulated in gray horse melanomas, suggesting that the duplicated region is a melanocyte-specific, cis-acting regulatory element. MITF and NR4A3 mediated up-regulation of melanin production and proliferation of hair follicle and dermal/epidermal melanocytes is the hypothesized link between the STX17 mutation, gray coat color and melanoma formation (see Supplemental Material). Horses that are homozygous for the STX17 duplication have 2 additional copies of the regulatory element and have a higher melanoma incidence and higher mean melanoma grade than heterozygous gray horses, which have only a single extra copy of the regulatory element. NR4A3 and MITF transcription are mediated by signaling through the melanocortin-1-receptor (MC1R) pathway that results in increase in cellular cAMP concentration (Fig 1). MC1R signaling is antagonized by the agouti-signaling protein (ASIP) resulting in decreased cAMP, MITF, and NR4A3 expression (Fig 1). Horses carrying a deletion in exon 2 of the ASIP gene that results in a loss of ASIP antagonistic function, have increased signaling through the MC1R pathway and homozygotes are black in color. The relative increase in MC1R signaling in horses with the ASIP mutation has been linked to increased melanoma severity.
Figure 1. Role of STX17 and MC1R signaling in melanoma formation. Normal MC1R signaling (black boxes/arrows): MC1R signaling is activated by the binding of the agonists α-MSH or ACTH to the MC1R receptor, resulting in increased cellular cyclic-AMP (cAMP) levels (a) and increased MITF expression (b) leading to increased melanin synthesis (c); stimulation of tyrosinase leading to eumelanin (black/brown) pigment production (d); and increased NR4A3 transcription (e) which mediates cell cycle and proliferation through the action of CCND2 (f). Normal ASIP (brown boxes/arrows) antagonizes the MC1R receptor resulting in a decrease in MC1R signaling and decreased eumelanin production (g) leading to the wild-type bay/brown coat color (in horses with a wild-type MC1R). Alterations in horses with STX17 duplication (gray boxes/arrows): horses with the duplication have additional binding sites for both MITF and NR4A3 (h). It is hypothesized that binding to these additional regulatory elements results in increased NR4A3 and STX17 expression (i), leading to altered cell cycle regulation and carcinogenesis (j). Increased binding of MITF also results in excessive melanocyte proliferation (k) leading to exhaustion of hair follicle melanocytes, gray coat color (l) and abnormal proliferation, (k) regulation of apoptosis, or both in dermal/epidermal melanocytes and melanoma (m). Altered MC1R signaling in horses with the ASIP deletion (blue boxes and arrows): With the ASIP deletion, the antagonistic effect of ASIP is lost, resulting in a relative increase in MC1R signaling and increased cAMP levels compared with wild-type (n). Horses homozygous for this mutation (ASIPa/a) produce more eumelanin, resulting in black coat color (o). In horses with the STX17 duplication and the ASIP deletion, the relative increase in MC1R signaling from the ASIP deletion, results in higher levels of MITF and NR4A3, exacerbating the abnormal cellular regulation with the gray genotype (h–m). Horses with the MC1R mutation (red boxes/arrows): MC1R signaling is altered and cellular cAMP levels are decreased (p). This causes a shift from eumelanin to pheomelanin production (q). Pheomelanin is a red/orange pigment leading to the chestnut coat color in MC1Re/e horses (r). The impact of decreased MC1R signaling and thus decreased MITF and NR4A3 expression in melanoma susceptibility and severity in gray horses is unknown. (See Supplemental Materials for details).
Download figure to PowerPoint
A loss-of-function mutation in the MC1R gene results in the “chestnut” coat color in horses. This mutation, by decreasing melanocyte cAMP levels, also has the potential to affect melanoma formation and severity in gray horses through the downstream effect of decreased MITF and NR4A3 transcription (Fig 1). In humans, similar MC1R variants that alter MITF expression have been linked to better outcomes in melanoma patients. The impact of altered MC1R signaling associated with the MC1R mutation on melanoma development and severity in horses has not been evaluated. In this study, we evaluate melanoma prevalence and severity in a population of greater than 300 gray Quarter Horses (QH), and the effects of genotypes at the STX17, ASIP, and MC1R loci on melanoma prevalence and severity. We hypothesized that melanoma susceptibility is lower in gray QHs relative to gray horses from other breeds; and further, this lower susceptibility is because of decreased MC1R signaling resulting from a high incidence of the MC1R chestnut coat color allele in the QH population.
- Top of page
- Materials and Methods
- Conflicts of Interest
- Supporting Information
The melanoma prevalence in our QH cohort was 16%, which is lower than previously described in the Lipizzaner (50%), Camargue (31.4%), and Pura Raza Española (89.6%). Our study confirmed the effect of age on melanoma prevalence in gray QH horses as previously described.[1, 3, 11] However, the melanoma prevalence in QH older than 15 years old (52%) in our study was still much lower than the prevalence reported in Lipizzaner (75%) and Camargue (68%) horses older than 15 years,[3, 11] or Pura Raza Española horses and crosses older than 10 years (100%). Further, the mean melanoma grade in this entire cohort was 0.35 (scale 0–4), and 2.15 in cases alone (horses with melanoma), which is again lower than melanoma grade reported in a population of 296 gray Lipizzaner horses, where mean melanoma grade across the entire study population was 1.19 and 2.40 in cases (grades 0–4).
To date, studies of melanoma grade and prevalence have been limited to the Lipizzaner, Pura Raza Española (Spanish pure breed or Andalusians of Spanish origin), and Camargue breeds.[11, 14, 13, 12] In these breeds gray coat color is predominant or breed-defining, the ASIP mutation is relatively common, and the MC1R chestnut allele is absent or segregating at extremely low frequency. Thus, the potential impact of altered MC1R signaling associated with the MC1R chestnut mutation on melanoma development and grade cannot be evaluated in any of these 3 breeds. The high chestnut allele frequency and resulting high homozygous chestnut genotype frequency in our gray QH cohort made it possible to determine the effect of diminished MC1R signaling resulting from this mutation on melanoma risk.
In a population of 694 Lipizzaner horses, the number of STX17 duplications appeared to impact melanoma risk, as STX17 homozygotes had greater melanoma prevalence than STX17 heterozygotes, and the estimated mean melanoma grade (least-squares mean) for heterozygotes was 0.67 compared to 1.43 for homozygotes. In our study cohort, melanoma prevalence and mean melanoma grade were higher in horses homozygous for the gray mutation (27.27% and 0.52, respectively), compared to heterozygous gray horses (15.91% and 0.34, respectively), although this difference was not statistically significant. There were very few homozygous gray horses in the study cohort (n = 22) and only 6 of these horses had melanoma, resulting in poor statistical power to demonstrate the effect of the additional copies of the STX17 mutation on melanoma prevalence, grade, or both. The low frequency of the SXT17 homozygosity in our population is not surprising, as QH are not primarily bred for the gray coat color.
Study in the Lipizzaner breed also demonstrated an effect of the ASIP genotype on melanoma grade after accounting for the gray genotype. The effect of the ASIP deletion in that study was estimated as 1.06 (least-squares mean) for heterozygotes (ASIPA/a) and 1.22 for homozygotes (ASIPa/a). We did not detect a statistically significant effect of the ASIP mutation on melanoma grade in our study cohort. There are several possible explanations for this result. First, although no effect of the ASIP deletion was observed, a trend of increasing mean melanoma grade with increased copy of the mutate allele (ASIPa) was observed in melanoma cases (Table 1); however, after accounting for age (Table 2), these differences were not statistically significant. The confidence intervals around the estimated additive effect of the ASIP deletion on melanoma grade in our study cohort (estimate: 0.07; 95% CI −0.065 to 0.204) overlap with the additive effect reported by Pielberg et al (0.16–0.18).
It is also possible that the presence of the MC1R mutation mitigates an effect of the ASIP mutation on melanoma grade. Although we did not find a significant effect of the MC1R mutation itself, it is conceivable that the altered signaling in MC1R heterozygotes and homozygotes, specifically decreased levels of cAMP, balance the up-regulation of signaling in horses with the ASIP mutation (Fig 1). A high frequency of the MC1R mutation and the MC1Re/e and MC1RE/e genotypes were observed in this cohort. Only 5% of the horses were homozygous wild-type for the chestnut MC1R gene (MC1RE/E), thus the vast majority of horses in this study had decreased MC1R signaling relative to wild-type. Although, we did not identify a statistically significant interaction between these 2 genotypes, the low frequency of the MC1RE/E genotype resulted in poor statistical power for the identification of this interaction. It is possible that the ASIP mutation has little to no effect on melanoma grade in the presence of the MC1R mutation because of decreased signaling through that pathway.
Another possible explanation for the lack of association between the ASIP mutation and melanoma grade is that the ASIP mutation might not be the functional allele underlying increased melanoma risk in Lipizzaners. In humans, ASIP variants have been shown to directly alter pigmentation phenotypes; and large genome wide association studies have implicated haplotypes containing ASIP in melanoma risk.[15, 16] Yet, associations between specific variants in the ASIP gene and melanoma have not been demonstrated,[17, 18] leading to speculation that in humans, the increased risk for melanoma is attributed to other genes within the associated haplotype.[15, 16] Thus, it is possible that the genetic variant responsible for increased risk in Lipizzaner horses is simply in linkage disequilibrium with the ASIP mutation, and is not the ASIP mutation itself. Because of the differences in haplotype length between those 2 breeds, the 2 alleles might not be in linkage disequilibrium in QH, preventing detection of a positive association.
Although genetic variants in MC1R pathway, MITF regulated genes, or both frequently underlie melanoma risk in humans, the pathophysiology of melanoma appears to be very distinct from horses. An increase in MC1R signaling associated with increased melanoma risk in horses is in contrast with decreased MC1R signaling that is associated with red hair, fair skin, freckles, poor UVR-induced tanning response, and increased melanoma risk in people. Gray horses maintain dark pigmentation in the skin through the process of graying and commonly develop melanomas in areas that are not exposed to the sunlight, thus UV exposure is unlikely an important environmental component in gray horse melanoma. Although MC1R variants are associated with increased melanoma risk in people, it was recently demonstrated that melanoma patients carrying MC1R variants had a better outcome than melanoma cases with black/brown hair, suggesting that MC1R mutations have a protective effect on survival. Signaling through MC1R regulates expression of the MITF transcription factor (Fig 1) that has many target genes in addition to pigment biosynthesis enzymes, including genes that regulate DNA repair, the cell cycle, apoptosis and invasion. The hypotheses is that melanoma cells carrying MC1R variants would have less resistance to apoptosis, less sustained proliferation and poorer DNA repair, leading to better patient survival. Although we found no effect of the MC1R mutation on melanoma prevalence/grade in gray QH, it is possible that this genotype is also associated with better survival in horses.
A better understanding of the risk factors involved in melanoma susceptibility, severity, and the risk of metastasis may allow for better prediction of tumor behavior and allow early intervention in horses that are more likely to develop severe, life-threatening consequences to dermal melanomas. Classification of melanoma patients into risk categories (high and low risk groups) based on genetic predispositions would allow veterinarians to identify patients that are candidates for early intervention, before treatment options are limited, enabling owners and veterinarians to make informed decisions before embarking in expensive or prolonged treatment regimens. In humans, genetic testing is routinely used to identify individuals at high risk for melanoma formation.
Our findings suggest a lower prevalence and severity of melanoma in gray QH compared to other breeds in which melanoma prevalence and grade have been investigated, but could not conclusively tie this decrease in prevalence and severity to altered MC1R signaling. Further work is needed to determine if the decreased prevalence and severity of melanoma in gray QH is simply because of infrequent STX17 homozygosity, a potential mitigating effect of the MC1R mutation on ASIP mediated enhancement of melanoma progression, or other genes in the MC1R pathway. Addressing these possibilities would require an expanded cohort of melanoma cases and controls, investigation of other loci, or both. It is also possible, as in humans, that carrying MC1R variants do not prevent horses from getting melanomas, but rather provide better survival rates. A prolonged follow-up of this cohort would be necessary to prove this hypothesis.