GRB2 Promotes Sorafenib Resistance in Hepatocellular Carcinoma Cells Under Hypoxia by Activating the PI3K/AKT Signaling Pathway

Purpose: Sorafenib resistance remains a major therapeutic challenge in advanced hepatocellular carcinoma (HCC). This study aimed to investigate the role of growth factor receptor-bound protein 2 (GRB2) in sorafenib resistance of HCC cells under hypoxic conditions and to elucidate the underlying molecular mechanisms involving the PI3K/AKT signaling pathway. Methods: Bioinformatics analysis was performed using TCGA-LIHC, GEO datasets, and multiple databases to evaluate GRB2 expression, its correlation with hypoxia signatures, and prognostic significance in HCC. Western blot was used to detect GRB2 expression in HCC cell lines and normal hepatocytes. Huh7 cells were cultured under normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions, treated with sorafenib alone or combined with PI3K inhibitor LY294002. GRB2 knockdown was performed using lentiviral shRNA. Cell proliferation, migration, and apoptosis were evaluated by CCK-8 assay, wound healing assay, and flow cytometry, respectively. Results: Bioinformatics analysis revealed that GRB2 was significantly upregulated in HCC tissues compared to normal tissues, positively correlated with hypoxia-related genes (HIF-1α, VEGFA) and hypoxia scores, and associated with poor patient prognosis. Western blot confirmed that GRB2 was highly expressed in HCC cell lines compared to normal hepatocytes. Under hypoxic conditions, HIF-1α expression increased in a time-dependent manner, validating the hypoxia model. Hypoxia attenuated sorafenib-induced inhibition of cell proliferation and migration while reducing apoptosis, as evidenced by increased IC50 values (from 4.934 μM to 8.676 μM). Sorafenib treatment under hypoxia upregulated GRB2, PI3K, and p-AKT protein levels. PI3K inhibition by LY294002 or GRB2 knockdown restored sorafenib sensitivity, reduced PI3K and p-AKT expression, and promoted apoptosis in hypoxic HCC cells. Conclusions: Our findings suggest that hypoxia reduces the sensitivity of HCC cells to sorafenib, and that GRB2 contributes to this process through activation of the PI3K/AKT pathway. Targeting GRB2 may represent a potential strategy to enhance sorafenib efficacy in HCC treatment under hypoxic conditions. Keywords: hepatocellular carcinoma, hypoxia, GRB2, sorafenib, drug sensitivity, PI3K/AKT pathway