H3K18乳酸化通过转录激活POM121促进胰腺癌免疫逃逸的机制研究

H3K18 lactylation promotes immune escape in pancreatic cancer through transcriptional activation of POM121

摘要:

背景与目的 胰腺癌具有高度侵袭性及显著免疫抑制特征，其免疫逃逸机制尚未完全阐明。肿瘤糖酵解增强导致乳酸大量积累，可通过组蛋白乳酸化参与基因转录调控，但其在胰腺癌免疫微环境重塑中的作用及机制尚不清楚。本研究旨在探讨组蛋白H3第18位赖氨酸乳酸化（H3K18la）调控核孔膜蛋白121（POM121）表达促进胰腺癌免疫逃逸的作用及分子机制。方法 利用TCGA数据库分析乳酸脱氢酶A（LDHA）和POM121在胰腺癌中的表达及预后价值。采用Western blot检测胰腺癌细胞中泛赖氨酸乳酸化（Pan-Kla）、H3K18la及POM121表达水平；通过LDH抑制剂oxamate处理或siRNA沉默LDHA抑制乳酸生成，采用乳酸检测试剂盒、CCK-8实验和平板克隆形成实验评价细胞乳酸水平及增殖能力。构建胰腺癌细胞与CD8⁺ T细胞共培养体系，采用流式细胞术检测GZMB、TNF-α和IFN-γ表达。应用ChIP-qPCR和双荧光素酶报告基因实验验证H3K18la对POM121转录活性的调控作用；通过POM121敲低及过表达实验明确其在乳酸化介导免疫调控中的作用。建立PANC02荷瘤小鼠模型验证体内效应。结果 LDHA和POM121在胰腺癌组织中均呈高表达，并与患者较差预后相关。胰腺癌细胞中Pan-Kla及H3K18la水平明显升高。oxamate处理或LDHA沉默均可降低细胞内乳酸水平及H3K18la表达，抑制胰腺癌细胞增殖，同时促进CD8⁺ T细胞效应分子GZMB、TNF-α和IFN-γ表达。POM121在胰腺癌细胞中高表达，其表达受H3K18la调控。ChIP-qPCR及双荧光素酶实验表明，H3K18la富集于POM121启动子区域并增强其转录活性。敲低POM121可抑制肿瘤细胞增殖并增强CD8⁺ T细胞功能；过表达POM121则可部分逆转oxamate诱导的抗肿瘤免疫效应。动物实验进一步证实，抑制乳酸生成可降低POM121表达并促进CD8⁺ T细胞浸润，而POM121过表达能够削弱上述作用。结论 胰腺癌中糖酵解产生的乳酸通过促进H3K18乳酸化修饰激活POM121转录，进而抑制CD8⁺ T细胞抗肿瘤免疫功能并促进免疫逃逸。乳酸-H3K18la-POM121信号轴有望成为胰腺癌免疫治疗的新靶点。

Abstract:

Background and Aims Pancreatic cancer is characterized by high aggressiveness and a profoundly immunosuppressive microenvironment, while the mechanisms underlying its immune evasion remain incompletely understood. Enhanced glycolysis in tumor cells leads to excessive lactate accumulation, which can regulate gene transcription through histone lactylation. However, the role of histone lactylation in remodeling the immune microenvironment of pancreatic cancer and its underlying molecular mechanisms remains unclear. This study aimed to investigate the role and molecular mechanism of histone H3 lysine 18 lactylation (H3K18la) in regulating nuclear pore membrane protein 121 (POM121)-mediated immune escape in pancreatic cancer.Methods The expression patterns and prognostic significance of lactate dehydrogenase A (LDHA) and POM121 in pancreatic cancer were analyzed using The Cancer Genome Atlas (TCGA) database. Western blotting was performed to determine the levels of pan-lysine lactylation (Pan-Kla), H3K18la, and POM121 in pancreatic cancer cells. Lactate production was inhibited by treatment with the LDH inhibitor oxamate or by siRNA-mediated LDHA silencing, followed by assessment of intracellular lactate levels, cell proliferation, and colony-forming ability using lactate assays, CCK-8 assays, and colony formation assays. A co-culture system of pancreatic cancer cells and CD8⁺ T cells was established, and the expression of GZMB, TNF-α, and IFN-γ was analyzed by flow cytometry. ChIP-qPCR and dual-luciferase reporter assays were used to verify the transcriptional regulation of POM121 by H3K18la. Gain- and loss-of-function experiments of POM121 were performed to determine its role in lactylation-mediated immune regulation. A PANC02 tumor-bearing mouse model was established to validate the in vivo effects.Results LDHA and POM121 were significantly upregulated in pancreatic cancer tissues and were associated with poor patient prognosis. Levels of Pan-Kla and H3K18la were markedly increased in pancreatic cancer cells. Oxamate treatment or LDHA silencing reduced intracellular lactate production and H3K18la levels, suppressed pancreatic cancer cell proliferation, and enhanced CD8⁺ T-cell effector functions, as evidenced by increased expression of GZMB, TNF-α, and IFN-γ. POM121 was highly expressed in pancreatic cancer cells and positively regulated by H3K18la. ChIP-qPCR and dual-luciferase assays demonstrated that H3K18la was enriched at the POM121 promoter region and enhanced its transcriptional activity. Knockdown of POM121 inhibited tumor cell proliferation and enhanced CD8⁺ T-cell function, whereas POM121 overexpression partially reversed the antitumor immune effects induced by oxamate. In vivo experiments further confirmed that inhibition of lactate production reduced POM121 expression and promoted CD8⁺ T-cell infiltration, while POM121 overexpression attenuated these effects.Conclusion Lactate generated by glycolytic reprogramming promotes H3K18 lactylation, which transcriptionally activates POM121, thereby suppressing CD8⁺ T-cell-mediated antitumor immunity and facilitating immune escape in pancreatic cancer. The lactate-H3K18la-POM121 signaling axis may represent a promising therapeutic target for pancreatic cancer immunotherapy.