EGFR-TKIs耐药通过上调肺癌PD-L1的表达促进肿瘤免疫逃逸的研究

发布时间：2018-06-20 19:28

本文选题：PD-L1 + 免疫治疗　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究背景及目的肺癌是最常见的肿瘤,同时也是国内外癌症死因之首。表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKIs)能有效的延长部分肺癌患者的生存期。然而EGFR-T790M 突变、MET 扩增、HGF 分泌、HER2 扩增、IGF-1R 的活化、EMT、PTEN缺失等机制使患者对EGFR-TKIs产生耐药。因此,寻找有效的治疗方法对于肺癌患者尤其是EGFR-TKIs耐药的患者而言意义重大。靶向PD-1/PD-L1通路的免疫检查点治疗已在多种恶性肿瘤的治疗中取得重大突破,2017年NCCN指南推荐抗PD-1/PD-L1疗法用于高表达PD-L1的肺癌患者。既往研究报道EGFR-TKIs可降低EGFR突变肺癌细胞PD-L1的表达,然而肺癌患者发生EGFR-TKIs耐药后肿瘤PD-L1的表达情况却鲜有报道,EGFR-TKIs耐药患者能否从抗PD-L1疗法中获益也有待明确。本研究通过TCGA数据库明确EGFR-TKIs敏感及耐药肿瘤及肿瘤微环境的差异,通过体内外实验证明EGFR-TKIs耐药可上调肺癌PD-L1的表达,并探究内在调控机制,同时通过体内外实验评估了抗PD-L1疗法对EGFR-TKIs耐药肺癌的疗效,为EGFR-TKI耐药患者提供新的治疗方法。方法使用吉非替尼耐药MET扩增的肺癌细胞PC-9R、肝细胞生长因子HGF刺激后的肺癌细胞、转染EGFR-19Del和/或EGFR-T790M突变质粒的293FT细胞,分别建立MMET扩增、HGF和EGFR-T790M突变的EGFR-TKIs耐药细胞模型。用Western Blot、流式细胞技术和RT-qPCR等技术比较前述细胞PD-L1的表达,并探究调控机制。将前述模型与T细胞共培养并用LDH细胞毒性实验比较肿瘤的免疫逃逸能力。PD-L1基因敲除的肺癌细胞及其阴性对照建立皮下瘤模型并用T细胞治疗比较肿瘤的免疫逃逸能力。分析TCGA数据库中肺腺癌相关的数据,比较EGFR-TKIs敏感及耐药肺癌的肿瘤突变负荷、PD-L1等免疫抑制分子及的表达、肿瘤微环境中肿瘤浸润淋巴细胞、细胞毒活性的改变。结果体外实验结果表明前述三种机制均可上调肿瘤PD-L1的表达,PI3K/Akt、MAPK、NF-kappa B信号通路参与EGFR-T790M突变诱导的PD-L1的表达;PI3K/Akt信号通路、MAPK信号通路而非NF-kappa B信号通路参与HGF和MET扩增诱导的PD-L1的表达;体内外实验结果表明MET扩增、HGF、EGFR-T790M突变上调肿瘤细胞PD-L1的表达降低T淋巴细胞的杀伤效果,敲除PD-L1基因降低PD-L1表达或抗PD-L1单克隆抗体可恢复人T淋巴细胞的杀伤能力。TCGA数据库中肺腺癌相关的数据提示:较EGFR-TKIs敏感的肿瘤而言,EGFR-TKIs耐药肺癌的肿瘤突变负荷高,PD-L1、PD-1等免疫抑制分子的表达增加。结论(1)相比EGFR-TKIs敏感肺癌而言,EGFR-TKIs耐药肺癌肿瘤突变负荷更高,肿瘤免疫抑制作用更强。(2)MMET扩增通过P13K/Akt和MAPK信号通路上调肿瘤细胞PD-L1的表达促进非小细胞肺癌的免疫逃逸(3)HGF通过PI3K/Akt和MAPK信号通路诱导PD-L1的表达促进非小细胞肺癌免疫逃逸(4)EGFR-T790M 突变通过 PI3K/Akt、MAPK 和 NF-kappa B信号通路诱导PD-L1的表达促进肺癌的免疫逃逸(5)EGFR-TKIs耐药细胞及皮下瘤通过上调PD-L1降低T细胞的杀伤能力,抗PD-L1疗法可恢复T细胞的杀伤能力。
[Abstract]:Background and objective lung cancer is the most common tumor, and it is also the leading cause of cancer death at home and abroad. Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKIs) can effectively prolong the survival period of some lung cancer patients. However, EGFR-T790M mutation, MET amplification, HGF secretion, HER2 amplification, IGF-1R activation, EMT, PTEN deletion and other mechanisms make Patients are resistant to EGFR-TKIs. Therefore, finding effective treatments is of great significance for patients with lung cancer, especially for EGFR-TKIs resistant patients. Immunologic checkpoints targeting PD-1/PD-L1 pathway have made significant breakthroughs in the treatment of multiple malignant tumors. In 2017, NCCN refers to the recommendation of anti PD-1/PD-L1 therapy for high expression of PD-L1. EGFR-TKIs can reduce the expression of PD-L1 in EGFR mutant lung cancer cells. However, the expression of PD-L1 is rarely reported in patients with lung cancer after EGFR-TKIs resistance, and it is also unclear whether EGFR-TKIs resistant patients can benefit from the anti PD-L1 therapy. This study uses a TCGA database to clarify EGFR-TKIs sensitivity and tolerance. The difference between drug tumor and tumor microenvironment shows that EGFR-TKIs resistance can up regulate the expression of PD-L1 in lung cancer and explore the internal regulation mechanism, and evaluate the efficacy of anti PD-L1 therapy on EGFR-TKIs resistant lung cancer in vivo and in vitro, and provide a new treatment for EGFR-TKI resistant patients. Methods use gefitinib resistance. MET expanded lung cancer cells PC-9R, lung cancer cells stimulated by hepatocyte growth factor HGF, transfected 293FT cells with EGFR-19Del and / or EGFR-T790M mutant plasmids, respectively, to establish MMET amplification, HGF and EGFR-T790M mutant EGFR-TKIs resistant cell models, compared with Western Blot, flow cytometry, and other techniques. The previous model was co cultured with T cells and used LDH cytotoxicity test to compare the tumor immune escape ability.PD-L1 knockout lung cancer cells and their negative controls to establish a subcutaneous tumor model and compare the immune escape ability of the tumor with T cells. Analysis of the data related to lung adenocarcinoma in the TCGA database and compared E GFR-TKIs sensitive and drug-resistant lung cancer mutation load, PD-L1 and other immunosuppressive molecules and expression, tumor infiltrating lymphocytes, cytotoxic activity changes in tumor microenvironment. Results in vitro results show that the above three mechanisms can up-regulate the expression of tumor PD-L1, PI3K/Akt, MAPK, NF-kappa B signaling pathway involved in EGFR-T790M mutation The expression of induced PD-L1; PI3K/Akt signaling pathway, MAPK signaling pathway and not NF-kappa B signaling pathway involved in HGF and MET induced PD-L1 expression; in vitro and in vivo experimental results showed that MET amplification, HGF, EGFR-T790M mutation up regulation of tumor cell PD-L1 expression reduced the killing effect of lymphocyte. D-L1 monoclonal antibodies can restore human T lymphocyte killing ability in.TCGA database associated with lung adenocarcinoma data: compared with EGFR-TKIs sensitive tumors, EGFR-TKIs resistant lung cancer has high mutation load, and the expression of PD-L1, PD-1 and other immunosuppressive molecules increases. Conclusion (1) compared with EGFR-TKIs sensitive lung cancer, EGFR-TKIs resistant lung cancer The tumor mutation load is higher and the tumor immune inhibition is stronger. (2) MMET amplification through P13K/Akt and MAPK signaling pathway up-regulated the expression of PD-L1 in tumor cells promoting the immune escape of non small cell lung cancer (3) HGF induced PD-L1 expression through PI3K/Akt and MAPK signaling pathway to promote non small cell lung cancer immune escape (4) EGFR-T790M mutation through PI3K. /Akt, MAPK and NF-kappa B expression induced by PD-L1 signaling pathway to promote lung cancer immune escape (5) and EGFR-TKIs resistant cell subcutaneous tumor decreased T cell killing ability through upregulation of PD-L1, anti PD-L1 therapy can restore T cell killing ability.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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