PO.TB10.09 · 肿瘤生物学
NAT10-PBX1 ac4C 轴使 MHC II 类抗原提呈失能，从而促进免疫冷型胆管癌
The NAT10-PBX1 ac4C axis disables MHC Class II antigen presentation to promote immune‑cold cholangiocarcinoma
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摘要 Abstract
中文摘要
背景：肝内胆管癌（ICC）是一种预后不良的侵袭性恶性肿瘤，其部分驱动因素为固有的免疫冷型肿瘤微环境。N4-乙酰胞苷（ac4C）是一种保守的 RNA 修饰，调控 RNA 稳定性和翻译，而 NAT10 介导的 ac4C 重塑已被证实与肿瘤转移和免疫细胞浸润有关。在此，我们研究 ac4C 修饰如何促进 ICC 进展和免疫逃逸，以识别潜在的治疗易感性。
方法：我们利用公开可用队列和临床组织样本定量 ICC 中的 ac4C 修饰和 NAT10 表达，并进行单细胞转录组分析以研究 NAT10 表达对免疫细胞浸润的影响。基于 CRISPR-Cas9 对 ICC 细胞系和小鼠模型中 NAT10 的基因操作，使我们能够对其在肿瘤起始和进展中的作用进行功能探究。通过整合的多组学和分子方法获得了机制层面的见解，包括 RNA-seq、acRIP-seq、CUT&Tag、RIP、ChIP、FISH、双荧光素酶报告基因实验和 RNA 稳定性实验。
结果：我们发现，与癌旁非肿瘤组织相比，ICC 组织中的 ac4C 修饰和 NAT10 表达均显著升高（P < 0.05）。临床上，高 NAT10 表达与更短的 mOS 相关（17.3 vs 26.7 个月；log-rank P = 0.021）。在细胞模型中，NAT10 敲低降低了 ac4C 水平和 ICC 细胞增殖（EdU 阳性的 HUCCT1 和 RBE 细胞分别减少 11.7% 和 15.6%），并损害了侵袭和转移，Transwell 迁移分别减少 45.2% 和 38.7%。在体内，NAT10 敲除抑制了小鼠异种移植瘤的生长；肿瘤体积减少了 67%（P < 0.05）。在机制上，NAT10 敲低减少了 PBX1 mRNA 上的 ac4C 修饰，导致 mRNA 稳定性降低和 PBX1 蛋白下调。PBX1 被鉴定为 CIITA 的转录抑制因子：NAT10 沉默通过下调 PBX1 增加了 CIITA 表达，进而升高了 MHC II 类基因的转录本和蛋白水平。相反，PBX1 过表达逆转了这些效应。单细胞转录组分析表明，高 NAT10 表达的 ICC 肿瘤表现出 CD4⁺/CD8⁺ T 细胞浸润水平降低，这在小鼠模型中经多重免疫荧光和流式细胞术得到验证。
结论：我们的研究揭示，NAT10 通过对 PBX1 进行 ac4C 修饰以抑制 CIITA-MHC II 类通路，从而限制 T 细胞浸润，促进 ICC 进展和免疫排斥。靶向 NAT10 可能恢复抗原提呈并改善 ICC 的免疫治疗应答。
查看英文原文 English abstract
Background: Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with poor prognosis, partly driven by an inherently immune-cold tumor microenvironment. N4-acetylcytidine (ac4C) is a conserved RNA modification that regulates RNA stability and translation, and NAT10-mediated ac4C remodeling has been implicated in tumor metastasis and immune cell infiltration. Here, we investigate how ac4C modification promotes ICC progression and immune evasion to identify potential therapeutic vulnerabilities.
Methods: We quantified ac4C modification and NAT10 expression in ICC using publicly available cohorts and clinical tissue samples, and performed single-cell transcriptomic profiling to investigate the impact of NAT10 expression on immune cell infiltration. CRISPR-Cas9-based genetic manipulation of NAT10 in ICC cell lines and mouse models enabled functional interrogation of its contribution to tumor initiation and progression. Mechanistic insights were obtained through an integrated multi-omics and molecular approach, including RNA-seq, acRIP-seq, CUT&Tag, RIP, ChIP, FISH, dual-luciferase reporter assays and RNA stability assays.
Results: We found that both ac4C modification and NAT10 expression were significantly elevated in ICC tissues compared with adjacent non-tumor tissues (P < 0.05). Clinically, high NAT10 expression was associated with shorter mOS (17.3 vs 26.7 months; log-rank P = 0.021). In cellular models, NAT10 knockdown reduced ac4C levels and ICC cell proliferation (EdU-positive HUCCT1 and RBE cells decreased by 11.7% and 15.6%, respectively) and impaired invasion and metastasis, with Transwell migration reduced by 45.2% and 38.7%. In vivo, NAT10 knockout suppressed tumor growth in murine xenografts; tumor volumes were reduced by 67% (P < 0.05). Mechanistically, NAT10 knockdown diminished ac4C modification on PBX1 mRNA, leading to reduced mRNA stability and downregulation of PBX1 protein. PBX1 was identified as a transcriptional repressor of CIITA: NAT10 silencing increased CIITA expression via PBX1 downregulation, subsequently elevating transcript and protein levels of MHC class II genes. Conversely, PBX1 overexpression reversed these effects. Single-cell transcriptomic analysis demonstrated that ICC tumors with high NAT10 expression exhibited reduced infiltration levels of CD4⁺/CD8⁺ T cells, which was validated by multiplex immunofluorescence and flow cytometry in murine models.
Conclusion: Our study reveals that NAT10 promotes ICC progression and immune exclusion by ac4C-modifying PBX1 to repress the CIITA-MHC class II pathway, thereby limiting T-cell infiltration. Targeting NAT10 may restore antigen presentation and improve immunotherapy responsiveness in ICC.
利益披露 Disclosure
K. Luo, None..
Y. Fang, None..
Y. Chen, None..
Y. Zhao, None..
J. Qu, None..
D. Shang, None..
C. Xu, None..
G. Zhang, None.