Timescale-dependent Phosphoproteomic Remodeling and Motility-associated Adaptation under Chronic Cabozantinib Exposure in Renal Cell Carcinoma.

BACKGROUND/AIM: Cabozantinib is a multi-target tyrosine kinase inhibitor used in renal cell carcinoma (RCC), yet the exposure-dependent remodeling of phosphorylation networks under short-term versus chronic treatment remains insufficiently defined. This study applied quantitative phosphoproteomics to delineate remodeling programs induced by acute and chronic cabozantinib exposure and to examine cellular features within the same signaling background. MATERIALS AND METHODS: RCC cells were subjected to acute (48 h) or chronic (>4-month) cabozantinib exposure. Dimethyl-labeling-based phosphoproteomics was used to quantify phosphosites and derive pathway- and kinase-substrate-level modules, integrated with functional enrichment, 2D-annotation and PTM-signature analyses, immunoblotting, migration, and Matrigel invasion assays. RESULTS: A total of 6,305 phosphosites were quantified. Acute cabozantinib exposure predominantly downregulated cell-cycle and CDK-associated phosphorylation, consistent with a broad cytostatic remodeling pattern, whereas chronic cabozantinib exposure produced a more selective redistribution enriched for adhesion- and stress-associated modules, including MAPK/AP-1/MAPKAPK2/HSPB1-linked signatures. Activation-loop MET phosphorylation (Y1234/1235) remained suppressed under both exposure conditions, while phosphorylation of MET at T977 increased under chronic cabozantinib treatment and was interpreted as site-specific regulation within the remodeled phosphorylation context rather than restoration of MET signaling activity. Motility features examined in the same cellular background showed pattern-specific differences: migration exhibited modest but significant increases with a larger effect size in chronically cabozantinib-exposed cells under drug treatment, whereas invasion was consistently higher in chronically cabozantinib-exposed cells than in parental cells across conditions without a marked treatment-specific change. CONCLUSION: Chronic cabozantinib exposure is characterized by sustained suppression of MET phosphorylation and a selective adhesion- and MAPK/AP-1-associated phosphorylation program, accompanied by modest, pattern-specific motility differences observed within the same signaling context. These findings provide a systems level framework for future mechanistic and in vivo evaluation.