Aflatoxin B1 Impairs TACE Efficacy Through Downregulated Carbonic Anhydrase 2: A Bioinformatics Analysis

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide, and transcatheter arterial chemoembolization (TACE) remains the core locoregional therapeutic modality for patients with intermediate to advanced disease. However, a considerable proportion of patients exhibit nonresponse to this treatment, which highlights an urgent need to identify reliable predictive biomarkers and elucidate the underlying mechanisms. Environmental carcinogens such as aflatoxin B1 (AFB1) are closely associated with the initiation and progression of liver cancer, yet their impact on the therapeutic efficacy of TACE has not been systematically elucidated. In this study, we integrated transcriptomic profiling, single-cell RNA sequencing (scRNA-seq), immune infiltration analysis, and molecular docking techniques to investigate the molecular mechanisms underlying AFB1-associated TACE nonresponse in HCC. We performed differential expression analysis using public cohorts to screen for genes associated with TACE response status (278 differentially expressed genes [DEGs] identified in the GSE104580 dataset) and tumorigenesis and progression (1365 DEGs identified in the TCGA-LIHC database), as well as AFB1 exposure. Intersection analysis with the predicted targets of AFB1 was subsequently conducted to identify key regulatory molecules. Functional enrichment analysis was applied to clarify the potential biological pathways involved in TACE nonresponse, and molecular docking was used to evaluate the binding potential between AFB1 and the core target. Our findings revealed that carbonic anhydrase 2 (CA2) could bind stably to AFB1 (Vina score = -7.8 kcal/mol) and possessed diagnostic potential for distinguishing tumor samples from normal ones (AUC = 0.819). Meanwhile, CA2 was identified as the core molecule linking AFB1 exposure to TACE nonresponse, with its expression consistently downregulated in HCC tumor tissues, TACE nonresponders (log₂FC = -1.063, P.adj = 0.0001), and AFB1-treated liver cells (log₂FC = -0.978, P.adj = 0.005). Immune infiltration analysis demonstrated that CA2 expression was significantly correlated with multiple immune cell subsets, suggesting its role in shaping the tumor immune microenvironment. Furthermore, scRNA-seq and pseudotime trajectory analyses revealed heterogeneous and dynamically downregulated CA2 expression across hepatocyte subpopulations, indicating a potential "gene-switch" pattern of this gene during the state transition of tumor cells. Collectively, our results suggest that AFB1 may contribute to the development of TACE nonresponse by downregulating CA2, which in turn mediates the regulation of tumor heterogeneity and immune remodeling. This study provides a novel candidate biomarker and a mechanistic framework for optimizing individualized treatment strategies for patients with AFB1-related HCC.