UBE2S promotes gastric cancer progression through RACGAP1 stabilization associated with K292-dependent K63-linked ubiquitination

OBJECTIVE: Gastric cancer (GC) remains highly lethal, and effective molecular targets are limited. This study investigated the expression, exploratory survival associations, and mechanistic role of UBE2S in GC. METHODS: UBE2S expression and survival associations were analyzed using public datasets, whereas UBE2S protein expression was assessed in seven paired GC tissues and GC cell lines. Loss- and gain-of-function assays, xenograft models, quantitative proteomics, genetic rescue, JNK inhibition, co-immunoprecipitation, cycloheximide chase, proteasome inhibition, and linkage-specific ubiquitination assays were performed. RESULTS: UBE2S was upregulated in GC and showed apparent within-dataset discriminatory ability at the tissue-transcript level (AUC = 0.909); higher expression showed an exploratory association with unfavorable survival in public datasets. Protein assessment in seven paired tissues was consistent with this expression pattern. UBE2S depletion suppressed GC cell viability, clonogenicity, migration, and xenograft growth. Proteomics prioritized RACGAP1 as a candidate downstream effector, and subsequent rescue experiments supported its functional relevance. UBE2S associated with RACGAP1 and increased RACGAP1 protein stability without altering its mRNA expression. UBE2S depletion decreased K63-linked and increased K48-linked ubiquitination of RACGAP1, whereas MG132 partially restored RACGAP1 abundance. K292 mutation impaired UBE2S-dependent K63-linked ubiquitination, RACGAP1 accumulation, JNK/c-Jun activation, and malignant phenotypes, although RACGAP1-K292R retained partial activity. Pharmacological JNK inhibition with SP600125 suppressed RACGAP1-mediated JNK/c-Jun reactivation and clonogenic rescue. CONCLUSION: Our findings support a model in which UBE2S promotes GC progression by stabilizing RACGAP1 in association with K292-dependent K63-linked ubiquitination, providing a mechanistic basis for further preclinical investigation of the UBE2S/RACGAP1/JNK/c-Jun axis.