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Veterinary and comparative oncology2026; doi: 10.1111/vco.70070

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

Abstract: 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.
© 2026 The Author(s). Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.
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Publication Date: 2026-04-16 PubMed ID: 41992374DOI: 10.1111/vco.70070Google Scholar: Lookup
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Summary

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Overview

  • This study investigates the molecular changes that occur at different stages of equine melanocytic neoplasm (EMN), an aggressive horse tumor, by analyzing protein phosphorylation patterns.
  • The research identifies specific signaling pathways and metabolic processes that are altered as the disease progresses, providing insight into potential biomarkers and therapeutic targets.

Background and Significance

  • Equine melanocytic neoplasms (EMN) are aggressive skin tumors found primarily in grey horses.
  • These tumors have a high tendency to metastasize and limited treatment options, making understanding their molecular basis critical.
  • Despite their clinical impact, the detailed molecular mechanisms driving EMN progression remain largely unknown.
  • Phosphoproteomics, which studies protein phosphorylation (a key regulatory modification), can reveal active signaling pathways and metabolic alterations relevant to tumor development.

Methodology

  • Tissue samples were collected from grey horses and classified into three categories based on disease severity:
    • Normal-stage (healthy tissue, n=6)
    • Early-stage EMN (tumor initiation, n=7)
    • Severe-stage EMN (advanced tumor, n=6)
  • Samples underwent liquid chromatography-tandem mass spectrometry (LC-MS/MS), a high-sensitivity technique to detect and quantify phosphorylated proteins.
  • A total of 2035 phosphoproteins were identified across all samples.
  • Differential expression analysis detected 219 phosphoproteins showing significant changes between disease stages.

Key Findings: Early-Stage EMN

  • Alterations were observed in inositol phosphate metabolism, a pathway involved in cellular signaling and membrane dynamics.
  • Activation of the PI3K-Akt signaling pathway was detected, which is known to regulate cell growth, survival, and metabolism.
    • Phosphoproteins INPP5F and PKN2 were implicated in this activation.
  • These changes suggest early EMN favors signaling that promotes cell proliferation and survival.

Key Findings: Severe-Stage EMN

  • Upregulation of SYNJ1, STRN4, and VIM indicated enhanced:
    • Membrane trafficking, important for vesicle movement and signaling.
    • Cytoskeletal remodeling, which can facilitate cell motility and invasion.
    • MAPK signaling pathway activation, a key route regulating proliferation and stress responses.
  • Increased levels of ASPM and GNAO1 suggested:
    • Heightened cellular proliferation.
    • Altered Rap1 signaling, involved in cell adhesion and movement.
  • Dysregulation of UBR5, a protein involved in protein degradation and homeostasis, suggested disruptions in cellular protein quality control.
  • Metabolic reprogramming was noted:
    • Elevated TKT (transketolase) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) indicated increased glycolysis and NADPH production, supporting tumor growth and antioxidant defenses.
  • Notably, Dickkopf-3 (DKK3) showed higher expression, implicating aberrant activation of Wnt/β-catenin signaling, a pathway well-known for its role in cancer progression and metastasis.

Implications and Conclusions

  • The study demonstrates that EMN progression involves distinct stage-specific signaling and metabolic changes.
  • Early-stage EMN is characterized by activation of survival and growth pathways (PI3K-Akt), while severe-stage involves enhanced cell motility, altered protein homeostasis, and metabolic remodeling.
  • Wnt/β-catenin pathway activation via DKK3 in late-stage EMN suggests a potential target for therapeutic intervention.
  • The identified phosphoproteins offer potential biomarkers for disease staging and progression monitoring.
  • These molecular insights provide a foundation for developing novel treatments targeting the specific pathways dysregulated in EMN.

Cite This Article

APA
Srimontri P, Kingkaw A, Prapaiwan N, Sujittosakul R, Iamkaewprasert N, Piputwat J, Isama-Al P, Munkongdee T, Chotikaprakal T, Yanyongsirikarn P, Phaonakrop N, Roytrakul S, Vongsangnak W, Tesena P. (2026). Integrative Phosphoproteomic Profiling Reveals Stage-Specific Signalling and Metabolism in Equine Melanocytic Neoplasm. Vet Comp Oncol. https://doi.org/10.1111/vco.70070

Publication

Veterinary and comparative oncology
ISSN: 1476-5829
NlmUniqueID: 101185242
Country: England
Language: English

Researcher Affiliations

Srimontri, Paitoon
  • Department of Clinical Science and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand.
Kingkaw, Amornthep
  • Kasetsart University International College (KUIC), Kasetsart University, Bangkok, Thailand.
Prapaiwan, Nawarus
  • Department of Clinical Science and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand.
Sujittosakul, Rangsima
  • Department of Clinical Science and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand.
Iamkaewprasert, Nichapat
  • Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand.
Piputwat, Jiraschaya
  • Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand.
Isama-Al, Puetta
  • Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand.
Munkongdee, Thanutchanok
  • Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand.
Chotikaprakal, Thanapon
  • Biochemistry Unit, Department of Physiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
Yanyongsirikarn, Petchpailin
  • Equine Clinic, Prasuarthon Small Animal Hospital, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand.
Phaonakrop, Narumon
  • Functional Proteomics Technology Laboratory, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, Thailand.
Roytrakul, Sittiruk
  • Functional Proteomics Technology Laboratory, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, Thailand.
Vongsangnak, Wanwipa
  • Department of Zoology, Faculty of Science, Kasetsart University, Bangkok, Thailand.
  • Omics Center for Agriculture, Bioresources, Food, and Health, Kasetsart University (OmiKU), Bangkok, Thailand.
Tesena, Parichart
  • Department of Clinical Science and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand.

Grant Funding

  • MU-SRF-RS-14A/66 / Mahidol University's Strategic Research Fund: 2023
  • Faculty of Veterinary Science, Mahidol University.

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