Sex-determining region Y-Box 4 promotes the progression of advanced hepatocellular carcinoma and enhances regulatory T-cell infiltration and immune suppression.

OBJECTIVE: This study examined the role of sex-determining region Y-box 4 (SOX4) in sorafenib-resistant hepatocellular carcinoma (HCC) cells and its potential therapeutic relevance by focusing on the effects of SOX4 knockdown on tumor growth, apoptosis, and immune infiltration. MATERIAL AND METHODS: A sorafenib-resistant HCC cell line (sorafenib-resistant HepG2 [SR-HepG2]) was established by gradually increasing the sorafenib dose (1-7 μM) over 12 months. The messenger RNA and protein expression levels of SOX4 in HepG2 and SR-HepG2 cells were analyzed by a quantitative reverse transcription-polymerase chain reaction and Western blot. Small interfering RNA (SOX4) or SOX4 overexpression plasmids were introduced into SR-HepG2 cells through transfection, and the effects on cell proliferation, colony formation, and apoptosis were evaluated using 5-ethynyl-2'-deoxyuridine staining, colony formation assays, and terminal deoxynucleotidyl transferase dUTP nick end labeling assays. For in vivo experiments, HepG2 or SR-HepG2 cells were subcutaneously injected into BALB/c nude mice to monitor tumor growth. In the sorafenib-resistant HCC mouse model, SOX4 knockdown (small-interfering RNA SOX4 [si-SOX4]) was delivered through lentiviral vectors to assess its effect on tumor growth. Immune cell infiltration was assessed by immunofluorescence staining, and the influences on immune escape markers were evaluated by Western blot. RESULTS: Compared with those in the parental HepG2 cells, the transcriptional and translational expression levels of SOX4 were significantly elevated in the SR-HepG2 cells (P < 0.001). Si-SOX4 markedly suppressed the proliferation and colony formation of SR-HepG2 cells and increased their cell apoptosis (P < 0.001). In vivo experiments revealed that si-SOX4 inhibited tumor growth in the sorafenib-resistant HCC model, accompanied by a significant reduction in tumor volume and weight (P < 0.001). Histological analysis showed that si-SOX4 disrupted the tumor structure, characterized by increased necrosis and reduced collagen fibers. In addition, si-SOX4 decreased the infiltration of Forkhead box P3+regulatory T cells and cluster of differentiation 11b + myeloid-derived suppressor cells while increasing the number of cluster of differentiation 8 (CD8)+ T cells and granzyme B + CD8+ cytotoxic T cells (P < 0.001). SOX4 knockdown also reduced the expression of two immune escape markers, programmed cell death ligand 1 and C-C motif chemokine ligand 12 (P < 0.001). CONCLUSIONS: SOX4 overexpression drives sorafenib resistance in HCC cells by promoting cellular growth, inhibiting apoptosis, and enhancing immune evasion. Conversely, SOX4 knockdown inhibits tumor growth, alters immune cell infiltration, and reduces immune escape. Hence, targeting SOX4 is a promising therapeutic approach to overcome sorafenib resistance in HCC.