PO.ET03.07 · 实验与分子治疗
NAT10介导的ac4C RNA修饰通过ATG5依赖性自噬促进结直肠癌干性和5-FU耐药
NAT10-mediated ac 4 C RNA modification promotes colorectal cancer stemness and 5-FU resistance via ATG5-dependent autophagy
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：结直肠癌（CRC）是全球第三大常见恶性肿瘤，也是癌症相关死亡的第二大原因。尽管以5-氟尿嘧啶（5-FU）为基础的化疗仍是主要治疗手段，但获得性耐药严重限制了其临床获益。一线联合治疗的客观缓解率仍仅为40-50%，转移性CRC的5年生存率徘徊在12%左右。自噬使肿瘤细胞得以在化疗诱导的应激下存活，靶向保护性自噬已成为逆转化疗耐药的一种有前景的策略。RNA修饰可影响这一过程。N-乙酰转移酶10（NAT10）是唯一的RNA N4-乙酰胞苷（ac4C）乙酰转移酶，已被证实与癌症进展相关，但其在自噬驱动的5-FU耐药中的作用仍不明确。本研究探讨NAT10如何调控自噬活性并参与CRC的5-FU耐药。
方法：开展整合性临床与生物信息学分析，以考察NAT10表达与CRC中5-FU耐药之间的关联。辅以体外实验评估NAT10在CRC进展和化疗耐药中的功能作用。开展机制研究，包括通路富集分析、自噬流检测和遗传上位性实验，以阐明NAT10下游的信号通路。采用RNA免疫沉淀（RIP）和ac4C特异性RIP鉴定直接分子靶点，并应用药理学抑制评估NAT10作为治疗靶点的价值。
结果：研究发现NAT10通过促进细胞增殖、迁移和干性，在CRC中发挥关键的致癌作用。NAT10表达在5-FU耐药的CRC细胞中显著升高（p<0.001）。敲低NAT10显著抑制增殖（p<0.001）并恢复对5-FU的敏感性。机制上，NAT10通过ac4C修饰稳定ATG5 mRNA（t1/2=12.2 h对比20.1 h），从而增强自噬流，维持肿瘤干性并支持对5-FU的耐药。NAT10缺失导致LC3-II积累减少和p62表达增加，提示自噬受损，并显著降低成球能力（p<0.001），同时下调干性标志物，最终使耐药细胞对5-FU重新敏感。类似地，对NAT10的药理学抑制抑制了自噬并显著降低5-FU的IC50值（约2.2倍），从而在CRC模型中恢复5-FU敏感性。
结论：NAT10-ATG5-自噬轴是CRC化疗耐药的关键机制。靶向NAT10介导的RNA乙酰化可破坏自噬驱动的存活并恢复5-FU反应性，为克服结直肠癌治疗耐药提供了一种有前景的治疗策略。
查看英文原文 English abstract
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.
利益披露 Disclosure
X. Kong, None..
J. Weng, None..
Z. Wang, None..
A. Goel, None.