NAT10 drives head and neck squamous cell carcinoma proliferation via ac4C modification of DDIT4 mRNA

N-acetyltransferase 10 (NAT10) is the principal enzyme responsible for N4-acetylcytidine (ac4C) modification in mRNA, a process that regulates target gene expression and contributes to the development of various diseases. Emerging evidence indicates that NAT10 plays a role in tumor progression across multiple cancer types; however, its specific function in head and neck squamous cell carcinoma (HNSCC) remains unclear. This study aimed to investigate the role of NAT10-mediated ac4C modification in HNSCC cell proliferation. NAT10 expression was analyzed at the RNA and protein levels by quantitative real-time PCR and western blot, respectively. Dot blot was used to assess global ac4C levels. Cell proliferation was evaluated through CCK-8 and colony formation assays. ac4C RNA immunoprecipitation quantitative PCR (acRIP-qPCR) was employed to detect ac4C modification on DDIT4 mRNA, and RNA immunoprecipitation (RIP) was used to examine the interaction between NAT10 and DDIT4 mRNA. Results showed that NAT10 was significantly overexpressed in HNSCC tissues and cell lines, and high NAT10 expression was associated with poor overall survival. Knockdown of NAT10 markedly inhibited HNSCC cell proliferation. Furthermore, DDIT4, which was also upregulated in HNSCC samples, was identified as a downstream target of NAT10. NAT10 directly bound to DDIT4 mRNA and enhanced its stability. Restoration of DDIT4 expression reversed the anti-proliferative effect induced by NAT10 silencing. In summary, NAT10 promotes HNSCC proliferation by mediating ac4C modification of DDIT4 mRNA, underscoring the NAT10/ac4C/DDIT4 axis as a potential therapeutic target in HNSCC.