MTHFD2 promotes esophageal squamous cell carcinoma progression via m6A modification‑mediated upregulation and modulation of the PEBP1‑RAF1 interaction.

One‑carbon metabolism plays an important role in cancer progression. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a mitochondrial enzyme in one‑carbon metabolism, is dysregulated in several cancer types. However, the precise role and mechanisms of MTHFD2 in esophageal squamous cell carcinoma (ESCC) remain unclear. The present study unravels the multifaceted mechanisms by which MTHFD2 contributes to ESCC pathogenesis. Bioinformatics analyses revealed significant upregulation of MTHFD2 in ESCC tumor tissues, which was associated with advanced disease stage and poor patient prognosis. Validating these findings in clinical samples, MTHFD2 overexpression was confirmed through immunohistochemistry, Reverse transcription‑quantitative PCR and western blotting. Knockdown of MTHFD2 inhibited ESCC cell viability, colony formation, invasion, and tumor growth in vivo, indicating its oncogenic potential. Mechanistically, the present study elucidated a novel regulatory axis involving N6‑methyladenosine modification and MTHFD2 mRNA stability. Specifically, methyltransferase‑like 3 (METTL3) and insulin‑like growth factor 2 mRNA binding protein 2 (IGF2BP2) were identified as key mediators of m6A‑dependent stabilization of MTHFD2 mRNA, contributing to its elevated expression in ESCC. Furthermore, MTHFD2 was found to activate PI3K/AKT and ERK signaling pathways by modulating interaction between phosphatidylethanolamine‑binding protein 1 (PEBP1) and raf‑1 proto‑oncogene (RAF1). This modulation was achieved through direct binding of MTHFD2 to PEBP1, disrupting the inhibitory effect of PEBP1 on RAF1 and promoting downstream pathway activation. The oncogenic functions of MTHFD2 were attenuated upon PEBP1 knockdown, underscoring the role of the MTHFD2‑PEBP1 axis in ESCC progression. In summary, the present study uncovers a novel regulatory mechanism involving m6A modification and the MTHFD2‑PEBP1 axis, unveiling potential therapeutic avenues for targeting MTHFD2 in ESCC.