Targeting NAT10 Inhibits Hepatocarcinogenesis via ac4C‐Mediated SMAD3 mRNA Stability

ABSTRACT Hepatocellular carcinoma (HCC) is characterized by high morbidity and mortality, with limited effective treatment options. N ‐acetyltransferase 10 (NAT10) is the only known acetyltransferase for mRNA ac4C modification and is recognized as a biomarker for HCC, promoting its progression. However, the critical role of NAT10 in hepatocarcinogenesis remains to be fully elucidated, and the identification of suitable small‐molecule inhibitors targeting NAT10 is of great interest. Here, we report that NAT10 promotes HCC progression by stabilizing SMAD family member 3 (SMAD3) mRNA through ac4C modification. Clinically, NAT10 is highly expressed in HCC tissues and is significantly associated with poor prognosis. Functionally, NAT10 downregulation inhibits HCC cell proliferation, invasion, and epithelial‐mesenchymal transition, while promoting anoikis in vitro. Additionally, NAT10 depletion significantly impairs tumor growth, metastasis, and hepatocarcinogenesis in vivo. Mechanistically, NAT10 enhances oncogene SMAD3 mRNA stability via ac4C modification, thereby activating TGF‐β signaling pathway. We also identify a novel small‐molecule inhibitor, NAT10‐2023, which effectively blocks NAT10 activity. Notably, NAT10‐2023 treatment significantly reduces intracellular RNA ac4C modification levels and disrupts NAT10‐RNA interactions, leading to suppressed tumor progression. Overall, NAT10 drives HCC progression via SMAD3 mRNA stability regulation, and NAT10‐2023 could be a promising therapeutic candidate for targeting NAT10 in cancer treatment.