The Rac1-USP11 feedback amplification loop: a radiation-activated engine driving radioresistance in hepatocellular carcinoma.

[BACKGROUND] Radioresistance is an objective biological factor that affects the efficacy of clinical radiotherapy in hepatocellular carcinoma (HCC). However, the mechanism involved has not yet been fully understood.

[METHODS] Integrative analysis of HCC patient data with RNA-seq and IHC. Radiosensitivity assessed by colony formation assays using HCC cell lines and xenograft models established in B-NDG mice. USP11 promoter activity measured by dual luciferase reporter. Protein interactions analyzed by Co-IP/GST pulldown; post-translational modifications by ubiquitylation assays. Rac1 activity was quantified by measuring GTP-bound levels. Rac1 structural dynamics simulated through molecular dynamics. Functional validation performed using pharmacological inhibitors, genetic depletion and site-directed mutagenesis.

[RESULTS] Elevated activated Rac1(Rac1-GTP) predicted poor radiotherapeutic response. Ionizing radiation (IR) activated Rac1 and induced its nuclear translocation. Rac1-GTP is required for the transcriptional upregulation of USP11. USP11 stabilised Rac1-GTP via deubiquitination at residues K123/K147/K183, forming a self-reinforcing Rac1-USP11 amplification loop driving radioresistance. USP11 knockdown or mutation of Rac1 deubiquitination sites (K123/K147/K183) destabilized Rac1-GTP and reversed radioresistance. Elevated Rac1-GTP and USP11 correlated with adverse clinical outcomes. Critically, combined NSC23766/Mitoxantrone treatment showed enhanced radiosensitization.

[CONCLUSION] This study identifies the Rac1-USP11 reciprocal feedback loop as a novel, self-reinforcing mechanism driving radioresistance in HCC. Targeting this loop via combined Rac1-GTP/USP11 inhibition represents a promising therapeutic strategy for radiosensitizing HCC.