Tumor-cell TOP2A contributes to ochratoxin A-enhanced hepatocellular carcinoma progression and impaired CD8⁺ T-cell responses

Ochratoxin A (OTA) is a widespread foodborne mycotoxin with hepatotoxic and immunotoxic effects, but its role in hepatocellular carcinoma (HCC) progression and antitumor immunity remains unclear. Here, we integrated network toxicology, bulk and single-cell transcriptomics, machine learning, molecular interaction assays, and experimental validation to identify key molecular factors associated with OTA-enhanced HCC progression. DNA topoisomerase II alpha (TOP2A) was prioritized, and direct OTA–TOP2A interaction was supported by surface plasmon resonance and drug affinity-responsive target stability assays. OTA increased TOP2A expression, promoted HCC-cell proliferation, migration, and invasion, and suppressed apoptosis. TOP2A knockdown attenuated these effects, whereas shRNA-resistant TOP2A rescue substantially restored the malignant phenotypes. In HCC cell–T-cell coculture, OTA-pretreated tumor cells impaired CD8⁺ T-cell responses, as indicated by reduced CD69, granzyme B, and IFN-γ expression, increased PD-1 expression, and enhanced T-cell apoptosis. These effects were alleviated by TOP2A knockdown, and this alleviation was reversed by TOP2A rescue. CCK-8 and LDH release assays further showed that OTA pretreatment reduced CD8⁺ T-cell-associated cytotoxicity against Hep3B cells, whereas tumor-cell TOP2A knockdown attenuated this reduction and shRNA-resistant TOP2A rescue reversed the protective effect of knockdown. Direct OTA treatment of T cells alone did not produce comparable changes in the measured endpoints. Integrative transcriptomic analysis further revealed opposing metabolic programs between OTA exposure and TOP2A knockdown. Notably, glycolysis-related gene sets were positively enriched following OTA treatment but negatively enriched after TOP2A knockdown, accompanied by reciprocal regulation of glycolysis- and tumor microenvironment-associated genes, including LDHA , SLC2A1 , PGK1 , CA9 , and SERPINE1 . In an immunocompetent orthotopic HCC model, OTA promoted tumor growth and proliferative activity and reduced intratumoral CD8⁺ T-cell infiltration, whereas tumor-cell Top2a knockdown attenuated these effects. Collectively, these findings provide gene-specific evidence for a functional contribution of tumor-cell TOP2A to OTA-enhanced HCC progression and impaired CD8⁺ T-cell responses, including reduced antitumor cytotoxicity, while TOP2A-associated glycolytic alterations represent a potential downstream feature that requires further functional validation.