Stage-dependent dual effects of folic acid in esophageal squamous cell carcinogenesis: precancerous lesions as a potential transition point

Folic acid (FA) exerts dual effects during carcinogenesis, yet the stage at which this functional shift occurs remains unclear. This study aimed to investigate whether esophageal precancerous lesions (EPLs) may serve as a critical transition point for FA action, and to explore the potential metabolic mechanisms involved. Precancerous lesion models were established in HEEC and HET-1A cells by long-term NMBzA exposure and validated by xenograft assays. Stage-specific FA interventions (5, 10, and 20 μg/mL) were administered either before or after lesion formation for 10 passages. Findings were validated in an NMBzA-induced rat model using stage-matched FA supplementation (0.75, 1.5, and 3 mg/kg·bw) for 8 weeks. Malignant phenotypes, lesion severity, and one‑carbon (1C) metabolism–related enzymes were assessed. Bioinformatic analyses and MTHFD2 knockdown experiments were performed to investigate mitochondrial 1C metabolism and purine synthesis. Folic acid exhibited stage-dependent effects in both cellular and animal models. Supplementation before precancerous lesion formation inhibited malignant transformation and delayed lesion development, whereas supplementation after lesion establishment promoted lesion progression. Cytosolic 1C metabolism appeared to be involved throughout disease development. We further investigated whether MTHFD2-mediated mitochondrial 1C metabolism also participates in esophageal carcinogenesis. Bioinformatic analyses revealed progressive upregulation of MTHFD2 during esophageal squamous cell carcinoma development and its association with enhanced purine synthesis. Functional experiments indicated that MTHFD2 knockdown was associated with reduced malignant phenotypes and altered purine synthesis. These findings suggest that the effects of FA may differ before and after the establishment of EPLs, supporting the potential role of EPLs as a transition point for further investigation. They further suggest that MTHFD2-mediated mitochondrial 1C metabolism may be involved in esophageal carcinogenesis, with a possible link to purine biosynthesis that warrants further investigation.