Porcine recombinant NK-lysin inhibits canine mammary tumor progression by targeting elongation factor 1-alpha and T-complex protein 1 subunit gamma to disrupt the organization of cytoskeletal architecture.

Canine mammary tumor (CMT) is the most prevalent neoplasms in female dogs and frequently undergo malignant progression and metastasis, leading to poor clinical outcomes and highlighting a crucial need for effective therapies beyond surgery. The Porcine Recombinant NK-lysin (prNK-lysin), a recombinant peptide produced from our lab, has previously shown inhibitory effects on orthotopic murine breast cancer growth and lung metastasis. However, its efficacy against CMT remains unclear. In this study, the CMT cell lines (CHMp and CHMm) and Orthotopic CMT model with CHMp in BALB/c nude mice were employed to investigate the anticancer effects of prNK-lysin. Mechanistic studies were performed using quantitative proteomics and pull-down assays, followed by PPI network analysis, molecular docking, CETSA, SPR, and gene overexpression. Our results revealed that prNK-lysin inhibits the proliferation, migration, adhesion, and invasion of both CHMp and CHMm cell lines and suppresses the tumor growth of CMT model. Mechanistically, Elongation Factor 1-Alpha (EEF1A1) and T-Complex Protein 1 Subunit Gamma (CCT3) were identified as high-affinity direct targets of prNK-lysin. The direct engagement of prNK-lysin with these two targets resulted in the perturbation of the MEK/ERK/ROCK2 signaling axis, the subsequent downregulation of a core set of actin-binding regulatory proteins, ultimately leading to cytoskeletal collapse and inhibition of CMT progression. Our findings strongly indicate that prNK-lysin is a promising therapeutic candidate for CMT by dismantling the cytoskeleton.