NAT10-mediated ac 4 C RNA modification promotes colorectal cancer stemness and 5-FU resistance via ATG5-dependent autophagy

Background: Colorectal cancer (CRC) is the third most common malignancy and the second leading cause of cancer-related death globally. Although 5-fluorouracil (5-FU)-based chemotherapy remains a mainstay, acquired resistance severely limits its clinical benefit. The objective response rate to first-line combination therapy remains only 40-50%, and the 5-year survival for metastatic CRC hovers around 12%. Autophagy enables tumor cells to survive chemotherapy-induced stress, and targeting protective autophagy has emerged as a promising approach to reverse chemoresistance. RNA modifications can influence this process. N-acetyltransferase 10 (NAT10), the sole RNA N⁴-acetylcytidine (ac⁴C) acetyltransferase, has been implicated in cancer progression, but its role in autophagy-driven 5-FU resistance remains undefined. This study explores how NAT10 regulates autophagic activity and contributes to 5-FU resistance in CRC. Methods: Integrative clinical and bioinformatics analyses were conducted to examine the association between NAT10 expression and 5-FU resistance in CRC. Complementary in vitro assays assessed the functional role of NAT10 in CRC progression and chemoresistance. Mechanistic studies, including pathway enrichment analyses, autophagic flux assays, and genetic epistasis experiments, were conducted to elucidate the signaling pathways downstream of NAT10. RNA immunoprecipitation (RIP) and ac⁴C-specific RIP were used to identify direct molecular targets, and pharmacologic inhibition was applied to evaluate NAT10 as a therapeutic target. Results: NAT10 was found to play a key oncogenic role in CRC by promoting cell proliferation, migration, and stemness. NAT10 expression was significantly higher in 5-FU-resistant CRC cells ( p <0.001). NAT10 depletion markedly suppressed proliferation ( p <0.001) and restored sensitivity to 5-FU. Mechanistically, NAT10 stabilized ATG5 mRNA through ac⁴C modification (t 1/2 = 12.2 h vs. 20.1 h), thereby enhancing autophagic flux, which sustains cancer stemness and supports resistance to 5-FU. Loss of NAT10 resulted in reduced LC3-II accumulation and increased p62 expression, indicating impaired autophagy, and significantly decreased sphere-forming ability ( p <0.001), along with downregulation of stemness markers, ultimately sensitizing resistant cells to 5-FU. Similarly, pharmacological inhibition of NAT10 repressed autophagy and significantly lowered the 5-FU IC50 values (~2.2-fold), thereby restoring 5-FU sensitivity in CRC models. Conclusion: The NAT10-ATG5-autophagy axis represents a critical mechanism underlying chemoresistance in CRC. Targeting NAT10-mediated RNA acetylation disrupts autophagy-driven survival and restores 5-FU responsiveness, offering a promising therapeutic strategy for overcoming treatment resistance in colorectal cancer.