【作者】 熊辉；

【导师】 付向宁； 李乐群；

【作者基本信息】 华中科技大学 ， 外科学（胸外科）， 2022， 博士

【摘要】 背景:肺癌是我国目前发病率和死亡率占首位的恶性肿瘤。单独使用免疫治疗或联合化疗等使用免疫治疗具有显著的抗肿瘤作用,但是大约70%的肺癌患者对免疫治疗无应答。肿瘤的弱免疫原性是导致肺癌患者对免疫治疗无响应的主要原因之一。因此,寻找有效安全地诱导和增强肿瘤细胞免疫原性的治疗策略对提高肺癌免疫治疗的应答率至关重要。近年来研究显示,干扰素基因刺激蛋白(Stimulator of interferon genes,STING)通路的激活可以增强肿瘤免疫原性,其下游靶分子Ⅰ型干扰素和相关炎症因子在抗肿瘤免疫应答中发挥重要作用。目前大量临床研究指出STING通路相关基因的表达、干扰素-γ(Interferon-gamma,IFN-γ)的表达水平与免疫治疗疗效有着密切关联,我们前期研究发现IFN-γ抑制肿瘤细胞增殖和促进肿瘤细胞凋亡,从而发挥抗肿瘤作用。但是IFN-γ是否有可能通过激活STING通路参与抗肿瘤作用,目前尚不清楚。目的:通过揭示IFN-γ对肺腺癌细胞STING信号通路的影响及其作用机制,能够进一步的认识IFN-γ抗肿瘤作用的分子机制,并且为当前靶向STING提供新的联合治疗策略。材料和方法:通过蛋白印迹(Western Blot,WB)、免疫荧光(Immunofluorescence,IF)以及彗星实验等,明确IFN-γ诱导肺腺癌细胞DNA损伤。本文还通过WB、IF、流式细胞术、实时荧光定量聚合酶链式反应(Real-Time Quantitative Polymerase Chain Reaction,RT-qPCR)以及酶联免疫吸附实验(Enzyme Linked Immunosorbent assay,ELISA)等,研究了 IFN-γ对STING信号通路的影响和作用机制。另外还探讨了依托泊苷和IFN-γ联用对STING信号强度的影响。最后采用人外周血单核细胞(Human Peripheral Blood Monocytes,PBMC)以及肿瘤组织离体培养技术验证了体外实验的结果。结果:研究发现,IFN-γ诱导DNA损伤和胞质双链DNA(double-strand DNA,dsDNA)的产生。机制研究发现,IFN-γ上调诱导型一氧化氮合成酶(Inducible Nitric Oxide Synthase,iNOS)的表达、促进一氧化氮(Nitric oxidate,NO)的产生。iNOS抑制剂和NO清除剂阻碍IFN-γ诱导的DNA损伤。研究表明,IFN-γ通过上调iNOS-NO诱导DNA损伤。进一步研究发现,IFN-γ是通过激活JAK1/2-STAT1上调肺癌中STING的表达,促进IRF3的磷酸化和转移入核,进而增加IFN-β和CCL5的表达。iNOS抑制剂和NO清除剂阻碍IFN-γ介导的STING通路活化,并且胞质DNA感受器cGAS和IFI16都参与IFN-γ介导的STING通路激活。另外依托泊苷和IFN-γ联合使用能够增强IFN-γ诱导的STING信号强度,进一步上调IFN-β的表达。抗CD3抗体激活PBMC中的T淋巴细胞同样能够诱导DNA损伤,激活STING通路,促进IFN-β和CCL5的表达,这些效应能够被IFN-γ中和抗体所消除。本文还采用肿瘤组织短期培养技术,利用抗CD3和PD-1抗体激活肿瘤组织浸润的T淋巴细胞,导致肿瘤组织STING通路的活化,表现为磷酸化IRF3水平显著上调以及IFN-β和CCL5表达的增多。结论:体外实验和肿瘤组织离体培养证明,IFN-γ能够诱导DNA损伤从而激活肿瘤细胞中STING通路,并且IFN-γ能够以STAT1依赖方式上调肺癌中STING的表达。依托泊苷可以进一步增强IFN-γ介导的STING信号强度。我们的研究首次揭示了IFN-γ对肿瘤细胞STING通路的作用及其机制,从新的切入点探讨肿瘤免疫治疗有效应答的分子机制。更多还原

【Abstract】 Background:Lung cancer is the most common malignancy and the leading cause of cancer death in China.Immunotherapy alone or in combination with chemotherapy has shown significant anti-tumor effects for patients with advanced non-small-cell lung cancer(NSCLC).However,a substantial fraction of patients with lung cancer do not respond to immunotherapy.Lung cancer is considered to possess poor immunogenicity.The low immunogenicity of cancer cells contributes to their unresponsiveness to immunotherapy.Recently,substantial evidence has shown that activation of the stimulator of interferon genes(STING)pathway enhances tumor immunogenicity.Activation of the STING pathway promotes the expression of type Ⅰ interferons and STING-related inflammatory factors,which play key roles in anti-tumor immune responses.Clinically related studies have shown that the expression levels of STING-associated genes in tumors correlate with the responsiveness to immunotherapy.Interestingly,the amount of IFN-γ in tumor tissues also reflects better responsiveness to immunotherapy.Previously,we showed that IFN-γinduces cell cycle arrest and apoptosis in lung cancer cells.Whether IFN-γ plays a role in STING activation in lung cancer cells remains unclarified.Objective:This study investigated the effect and mechanism of IFN-γ on the STING pathway in lung cancer cells.Methods:In this study,we mainly used western blotting(WB)and immunofluorescence(IF)to detect the phosphorylation of H2AX.The comet assay was used to determine IFN-γ-triggered double-strand DNA breaks.We used WB,IF,flow cytometry,and real-time quantitative polymerase chain reaction(RT-qPCR)to assess IFN-γ-induced iNOS expression.Nitric oxide level was assessed by enzyme-linked immunosorbent assay(ELISA).We used WB and RT-qPCR to determine expression levels of STING pathway-associated molecules.The role of IFI16 and cGAS in STING activation was determined by transfection of siRNA specific for IFI16 and cGAS,respectively.The association between cGAS and γ-H2AX was examined by immunoprecipitation assay.Results:The results showed that IFN-γ induced DNA damage and cytosolic dsDNA formation by increasing iNOS expression and NO production in lung adenocarcinoma cells.iNOS inhibitors and NO scavengers blocked IFN-γ-induced DNA damage and reduced IFN-γ-induced STING signals.Further studies showed that IFN-γ up-regulated the expression of STING in lung cancer by activating JAK1/2-STAT1 pathway.IFN-γactivated STING pathways,as evidenced by increased phosphorylation and nuclear translocation of IRF3 and induction of IFN-β and CCL5 expression in tumor cells treated with IFN-γ compared to untreated cells.iNOS inhibitors and NO scavengers could block IFN-γ-mediated STING pathway activation.Furthermore,IFN-γ-induced STING activation requires cytoplasmatic DNA receptors cGAS and IFI16.In addition,Etoposide further enhanced IFN-γ-induced STING signals and significantly up-regulated IFN-βexpression compared to cells treated with IFN-γ alone.The supernatants collected from anti-CD3 antibody-stimulated T lymphocytes co-cultured with tumor cells led to DNA damage and STING activation.These effects were eliminated by IFN-γ neutralizing antibodies.Our data suggest that stimulation of T cells can activate STING mediated by IFN-γ in tumor cells.We used the tumor tissue explant method to show that stimulation of tumor-infiltrating T lymphocytes with anti-CD3 and anti-PD-1 antibodies resulted in the activation of STING pathways in tumor tissues as manifested by significant up-regulation of IRF3 phosphorylation and an increase in the expression of IFN-β and CCL5.Conclusions:IFN-γ induces DNA damage and activates STING signaling in lung adenocarcinoma cells.IFN-γ-induced STING activation is cGAS-and IFI16 dependent.Moreover,IFN-γ increases STING expression in a STAT1-dependent manner.Etoposide further enhances IFN-γ-mediated STING signaling and the expression of IFN-β.We for the first time reveal the role of IFN-γ in the STING activation in tumor cells.Our study provides a new understanding of the mechanism underlying the responsiveness of tumors to immunotherapy.更多还原