Integrative Phosphoproteomic Profiling Reveals Stage‐Specific Signalling and Metabolism in Equine Melanocytic Neoplasm

Equine melanocytic neoplasms (EMN) are aggressive tumours characterised by high metastatic potential and limited therapeutic options available. However, the molecular mechanisms underlying their progression remain poorly understood. This study therefore presents the integrative phosphoproteomic analysis of EMN tissue, with the aim of elucidating stage-specific alterations in signalling pathways and metabolism. Nineteen tissue samples from grey horses were categorised as normal-stage (n = 6), early-stage EMN (n = 7), and severe-stage EMN (n = 6) and subjected to in-depth analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 2035 phosphoproteins were identified, of which 219 were differentially expressed across the disease stages. Interestingly, early-stage EMN showed dysregulation of inositol phosphate metabolism and activation of the PI3K-Akt pathway which involved INPP5F and PKN2. In severe-stage EMN, upregulation of SYNJ1, STRN4 and VIM indicated enhanced membrane trafficking, cytoskeletal remodelling, and MAPK signalling. Additionally, ASPM and GNAO1 upregulation reflected heightened proliferation and altered Rap1 signalling, while UBR5 dysregulation suggested aberrant protein homeostasis. Metabolic reprogramming was also noticed, with elevated TKT and GAPDH expression supporting glycolysis and NADPH production. Observably, the severe-stage EMN exhibited a higher expression of Dickkopf-3 (DKK3) which suggests a role in aberrant Wnt/β-catenin activation and tumour progression. These findings reveal stage-specific molecular mechanisms in EMN pathogenesis and highlight potential biomarkers and therapeutic targets for equine melanoma.