Gray-Horse Melanoma-A Wolf in Sheep's Clothing

Abstract

Malignant melanoma (MM) affects not only humans but also animals, with gray horses being particularly predisposed to acquiring the disease. Multiomics have greatly advanced the understanding of human MM. In contrasty little is known regarding the pathogenesis of gray-horse melanoma and the unique phenomenon of melanoma "dormancy" in some animals. To help close this gap in knowledge, melanoma tissue and intact skin collected from gray horses were subjected to transcriptome analysis using RNAseq. In the next step, cultured primary tumor cells and normal skin fibroblasts were established from gray horses, and their protein expression profiles were determined. The obtained data unambiguously identified gray-horse melanoma (ghM) as a malignant tumor, as reflected by the overrepresentation of pathways typically activated in human melanoma and other human cancers. These included the RAS/RAF/MAPK, the IRS/IGF1R, and the PI3K/AKT signaling networks. In addition, the obtained data suggest that the key molecules RAC1, RAS, and BRAF, which are frequently mutated in human melanoma, may also contain activating mutations in ghM, whilst PTEN may harbor loss-of-function mutations. This issue will be subject to downstream analyses determining the mutational status in ghM to further advance the understanding of this frequent disease in gray horses.

Keywords: RNAseq; gray-horse melanoma; melanoma; primary melanoma cells; proteomics; tumor tissue.

Figure 1

Clinical presentation of ghM. (A) GhMs typically develop under the tail-root and present as firm, encapsulated nodules (green arrow), as observed in a Shagya–Arabian mare (GYN). (B) Surgical therapy bears the risk of tumor recurrence. Excision of anogenital lesions affecting a gray Icelandic horse (DOL) resulted in the reoccurrence of ghM at the same site (black nodular mass indicated by the green arrow). (C) Metastatic disease in a Trakehner cross-breed (ELL). The mare presented with a large perianal lesion (black nodular mass; green arrow) and was euthanized due to acute hemorrhage from the ruptured spleen. Necropsy revealed multiple organ metastases, involving, e.g., the interventricular sulcus of the heart (black region; green arrow), the liver, and the entire spleen (bottom).

Figure 2

Venn diagram showing the distribution of differentially expressed proteins in gray-horse melanoma (ghM) cell lines compared to normal fibroblasts. The diagram highlights both the shared (n = 155) and unique sets of deregulated proteins across the four tumor cell lines. Notably, a high number of proteins were specifically deregulated in the liver-metastasis-derived cells (ELLmtc), indicating a distinct proteomic signature associated with metastatic disease. DOLmc: cells from a primary melanoma affecting horse DOL; DOLrmc: cells from the melanoma of horse DOL that recurred following excision of the primary lesion; TIMmc: melanoma cells from horse TIM; ELLmtc: tumor cells from the liver metastasis of horse ELL.

Figure 3

PCA of differential expression data. The biplot reflects the consistency of the data obtained for the three replicates per cell line (note the co-localization of respective dots) and a clear segregation of the different cell cultures with respect to their individual protein expression profiles. This segregation was particularly pronounced in the case of ELLmtc, i.e., ghM cells derived from a liver metastasis. PC1: Principal Component 1; PC2: Principal Component 2.