CHFR drives gastric cancer stemness and hepatic metastasis by suppressing ITCH-mediated ubiquitination and degradation of OCT4

Abstract

Gastric cancer (GC) is one of the most lethal malignancies worldwide, and hepatic metastasis is a major cause of its poor clinical prognosis. The stemness regulator octamer-binding transcription factor 4 (OCT4) drives the migration, invasion and metastasis of GC cells. As a RING-type E3 ubiquitin ligase, Checkpoint with Forkhead-associated and RING finger domains (CHFR) has been identified as a key promoter of metastasis in advanced GC. However, the precise molecular mechanisms underlying CHFR-mediated modulation of GC metastasis remain unclear. This study systematically explored the biological functions and molecular mechanism of CHFR in GC via public data analysis, in vitro cell experiments and in vivo hepatic metastasis models. Functional assays revealed that CHFR overexpression enhanced the stemness and metastatic capacity of GC cells but inhibited cell proliferation. Mechanistically, CHFR stabilized OCT4 protein by blocking itchy E3 ubiquitin-protein ligase (ITCH)-mediated ubiquitination and proteasomal degradation of OCT4. Molecular docking predicted a direct interaction between CHFR and ITCH, and site mutation studies showed that their binding interface was essential for CHFR-induced OCT4 stabilization. DynaMut2 and FoldX mutagenesis confirmed Tyr530 (CHFR) and Thr222 (ITCH) as critical interfacial residues, whose alanine substitution weakened complex binding. Biochemical Co-IP ubiquitination assays verified CHFR targets ITCH for proteasomal degradation to suppress ITCH-dependent OCT4 breakdown. OCT4 knockdown rescue experiments further validated OCT4 as the essential downstream mediator of CHFR-induced glycolytic reprogramming, stemness and invasion in GC cells. Importantly, in vivo xenograft assays demonstrated that CHFR overexpression significantly promoted GC liver metastasis, whereas simultaneous overexpression of CHFR and ITCH reversed this metastatic phenotype. CHFR enhances the stemness and metastatic progression of advanced GC cells by antagonizing ITCH-dependent OCT4 degradation, highlighting the CHFR/ITCH/OCT4 axis as a therapeutic target for GC treatment.