胞质DNA-STING-IFN通路调控非小细胞肺癌脑转移放疗联合EGFR-TKI治疗敏感性的机制研究

Radiotherapy combined with EGFR-TKI improved survival in patients with brain metastasis from EGFR mutant NSCLC patients, however, relapse occurred inevitable. Studies reported that Sting signaling induce the release of IFN-I and partially contribute to radio-resistance. Our previous study showed radiation combined with EGFR-TKI significantly increase the production of cytoplasmic DNA and micronucleus, both of which induce the transcription of interferon through Sting signaling. Besides, we have exploited the RNA-sequencing data of NSCLC from the TCGA, and the results showed that the expression of Sting gene TMEM173 and interferon-stimulated genes (ISGs) is significantly elevated in EGFRm NSCLC compared with that in EGFR wild type NSCLC. A similar trends is observed in NSCLC cell lines from CCLE. Based on these results, we proposed that radiotherapy and EGFR-TKI active the cytoplasmic DNA - Sting - interferon signaling, IFN-I influence the treatment sensitivity by stimulating the immunosuppressive microenvironment and enhancing the cell intrinsic radio-resistance. By establishing syngenetic brain metastasis tumor model, and with the following methods, such as immunofluorescence, flow cytometry, qRT-PCR and WB, we will detect whether cytoplasmic DNA - Sting signaling contribute to treatment resistance in radiotherapy and EGFR-TKI both in in vivo and in vitro, the influence of interferon on the immunosuppressive microenvironment and cell intrinsic radio-sensitivity will also be detected. The results of this study might provide a new target in the treatment of brain metastasis from NSCLC.

放疗联合EGFR-TKI显著改善EGFRm NSCLC脑转移患者预后，然而仍会复发。文献报道Sting介导的IFN-I参与放疗抗拒。我们发现EGFRm NSCLC中Sting、ISGs的mRNA水平升高。放疗联合EGFR-TKI诱导胞质DNA产生。由此提出本课题假说：放疗联合EGFR-TKI激活胞质DNA-Sting-IFN通路，IFN-I激活STAT1诱导细胞内源性抗拒，诱导PD-L1、IDO表达及MDSC聚集促进抑制性免疫，从而诱导放疗及EGFR-TKI治疗抗拒。研究通过建立脑转移动物模型，采用免疫荧光、流式、qRT-PCR、WB等技术，在体内、体外探讨胞质DNA-Sting-IFN通路是否参与放疗联合EGFR-TKI治疗抗拒，以及该通路是否通过诱导细胞内源性抗拒、免疫抑制性抗拒导致治疗抵抗的。研究结果有望为改善EGFRm NSCLC脑转移预后提供新靶点。

本研究目的是明确EGFR对放疗诱导的cGAS-Sting-TBK1-IRF3通路活性及其介导的抗肿瘤免疫反应的影响；探讨EGFR调控cGAS-Sting-TBK1-IRF3通路活性的潜在机制。核质分离检测胞浆γH2AX水平、PicoGreen染色统计胞浆DNA实验表明，放疗剂量依赖性的诱导胞浆DNA水平增加，EGFR-TKI诱导胞浆DNA水平增加，放疗与EGFR-TKI联合应用时进一步增加胞浆DNA水平。western blot实验表明，放疗诱导cGAS-Sting-IRF3通路相关蛋白及其磷酸化水平增加；EGFR-TKI单独应用以及与放疗联合应用时，均可抑制cGAS-Sting-IRF3通路相关蛋白及其磷酸化水平。免疫荧光实验得出类似结果，放疗诱导胞核p IRF3水平增加；EGFR-TKI抑制细胞基础表达的及放疗诱导的p IRF3水平。ELISA实验表明，放疗诱导IFNβ水平升高；EGFR-TKI单独应用以及与放疗联合应用时，均可抑制IFNβ水平。RT-PCR结果表明，放疗诱导CCL5、CXCL10水平升高；EGFR-TKI单独应用以及与放疗联合应用时，均可抑制CCL5、CXCL10水平。在PC9和KLN205细胞中，通过siRNA干扰EGFR表达后，cGAS-Sting-IRF3通路相关蛋白及其磷酸化水平均显著下降；通过EGF诱导EGFR激活，cGAS-Sting-IRF3通路相关蛋白及其磷酸化水平均显著升高。此外，cGAMP类似物SR717可诱导EGFR敲减的PC9和KLN205细胞中pIRF3水平升高。在EGFR正常表达的PC9和KLN205细胞中，放疗可诱导cGAS-Sting-IRF3通路相关蛋白及其磷酸化水平升高；而在EGFR敲减的PC9和KLN205细胞中，放疗对cGAS-Sting-IRF3通路相关蛋白及其磷酸化表达的诱导作用显著下降。分析TCGA-LUAD数据表明，与EGFR野生组或EGFR低表达组相比，EGFR突变组及EGFR高表达组NSCLC患者中Sting、IFNAR1、IFNAR2以及肿瘤浸润DC细胞均显著升高；而CD8+ T细胞水平显著减少 。综上所述，EGFR通过影响cGAS调控Sting-IRF3通路活性。EGFR在放疗诱导的cGAS-Sting-IRF3通路活化中发挥重要作用。EGFR突变以及EGFR过表达导致的EGFR过度激活，