NAT10-mediated ac4C modification and CEBPB-mediated transcriptional activation converge on CBS to promote malignant progression in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignant tumor, and its development mechanism remains unclear. Ferroptosis, as a novel regulatory mode of cell death, plays an important role in tumor regulation. However, the function of cystathionine-β-synthase (CBS) in ESCC and its upstream regulatory mechanism have not been fully elucidated. The differentially expressed genes were screened by GEO database (GSE157804 and GSE17351) and PathCards ferroptosis-related protein data. The functions of CBS and its upstream regulatory molecules N-acetyltransferase 10 (NAT10) and CCAAT/enhancer binding protein beta (CEBPB) in ESCC cells were evaluated by quantitative real-time PCR, western blot, Transwell assay, wound healing assay, CCK8 assay, sphere formation assay and detection of ferroptosis-related indexes (Fe 2+ , GSH, and lipid peroxidation). The intermolecular regulatory relationship was verified by dual-luciferase reporter assay, N4-acetylcytosine (ac4C)-RIP assay, RIP assay, actinomycin D treatment assay, and ChIP assay. A subcutaneous xenograft model in nude mice was established to verify the effect in vivo. By GEO database and PathCards screening, CBS was identified as a important gene that was upregulated in ESCC and was associated with ferroptosis. CBS was highly expressed in ESCC cells ( P < 0.05), and its silencing could inhibit ESCC cell invasion, migration, viability and stemness, while promoting ferroptosis ( P < 0.05). Mechanistically, NAT10 enhanced CBS mRNA stability and upregulated its expression through ac4C modification ( P < 0.05). Transcription factor CEBPB directly bound to the promoter region of CBS to activate its transcription. The recovery of function experiment confirmed that CBS overexpression reversed the inhibitory effect of NAT10 or CEBPB knockdown on ESCC cell malignant phenotype and the promotion effect of ferroptosis ( P < 0.05). Furthermore, interference of NAT10 inhibited the growth of ESCC xenografts, while co-overexpression of CBS partially reversed the above effects ( P < 0.05). Collectively, our findings identify NAT10-mediated ac4C modification and CEBPB-mediated transcriptional activation as two distinct upstream layers that converge on CBS to promote ESCC malignancy and ferroptosis resistance in vitro, providing a mechanistic basis for functional investigation of this axis in ESCC models. In the future, the contribution of ferroptosis to the observed in vivo tumor growth inhibition by NAT10 knockdown remains to be directly demonstrated.