LARS inhibition overcomes regorafenib resistance in hepatocellular carcinoma via ferroptosis and drug metabolism.

BACKGROUND

Regorafenib improves outcomes in advanced hepatocellular carcinoma (HCC), but acquired resistance limits its durable therapeutic benefit. The metabolic and redox mechanisms driving regorafenib resistance remain incompletely defined.

METHODS

Regorafenib-resistant Huh7 and Hep3B cell (RegR-) models were generated by long-term stepwise drug exposure. Transcriptomic profiling, LC-MS/MS, Seahorse extracellular flux analysis, ferroptosis-related assays, and apoptosis assays were used to characterize resistance-associated alterations. LARS1 was modulated by shRNA knockdown, overexpression, and pharmacological inhibition using BC-LI-0186. Therapeutic efficacy was evaluated in vitro and in Regᴿ Huh7 xenografts. Clinical relevance was assessed using paired HCC specimens, clinicopathological analysis, TCGA-LIHC, and TNMplot datasets.

RESULTS

Regᴿ HCC cells showed increased regorafenib IC₅₀ values, retained migratory, invasive, clonogenic, and spheroid-forming capacity under regorafenib exposure, and exhibited extensive transcriptomic remodeling. Differential expression analysis identified 200 DEGs in Regᴿ Huh7 cells and 1,635 DEGs in Regᴿ Hep3B cells, with LARS1 and GPX4 among the upregulated candidates. Regᴿ cells displayed enhanced regorafenib glucuronidation, with approximately 2.3-fold and 2.7-fold higher glucuronide accumulation in Huh7 and Hep3B models, respectively. LARS1 expression positively correlated with the regorafenib-glucuronide/regorafenib ratio. Clinically, LARS1 and GPX4 were upregulated in HCC tissues and associated with aggressive clinicopathological features. LARS1 knockdown or BC-LI-0186 reduced regorafenib IC50, suppressed GPX4/SLC7A11 expression, increased lipid ROS and apoptosis, and disrupted the hypermetabolic phenotype of Regᴿ cells by reducing both OCR and ECAR. In xenograft models, combined regorafenib and BC-LI-0186 treatment produced the strongest tumor growth inhibition and reduced LARS1/GPX4 expression, although body-weight monitoring indicated the need for toxicity evaluation.

CONCLUSIONS

LARS1 acts as a druggable metabolic regulator that links regorafenib biotransformation, mTOR-related metabolic adaptation, and GPX4/SLC7A11-mediated ferroptosis defense in resistant HCC. Targeting LARS1 with BC-LI-0186 may restore regorafenib sensitivity and represents a potential strategy for overcoming acquired regorafenib resistance.