MMP7激活核仁素介导EGFR-TKI原发耐药及机制研究

Our previous study found that persistent KRAS activation independent of KRAS mutations may be a new mode of EGFR - TKI primary drug resistance, and its mechanism is not clear, increased expression of MMP7 was found in EGFR-mutant lung cancer cell line with EGFR TKI resistance，exogenous MMP7 induced EGFR - TKI resistance. Recent studies indicate that MMP7 cleaved nucleolin, endogenous nucleolin interacts with EGFR and all 3 typical Ras isoforms ( H-, K and N-Ras ) in cancer cells. It is tempting to speculate that nucleolin-mediated KRAS activation may reduce or prevent the inhibitory effect of the EGFR-TKI. We examined whether or not MMP7 overexpression, nucleolin activation and its interaction promote resistance to EGFR-TKI in adenocarcinomas of the lung and effect on key enzyme in the RAS-RAF-MEK-ERK pathway through gene transfer , knockdown and immunoprecipitation..We study whether it will be prevented or overcome of gefitinib drug resistance by drug blocking test in vitro and in vivo. By the analysis of the tumor tissue samples ,we seek new targets or biomarkers of primary drug resistance to EGFR-TKI, which may provide a strong rationale to consider a strategy of treatment of lung cancer patients

我们前期研究发现不依赖KRAS突变的持续性KRAS激活可能是EGFR-TKI原发耐药的新模式，其机制并不清楚，而MMP7与核仁素（Nucleolin）在EGFR-TKI 耐药细胞株中的表达量高于敏感细胞株，外源性MMP7诱导EGFR-TKI 敏感细胞株发生耐药，近期文献报道MMP7可裂解激活核仁素，并持续激活野生型KRAS，我们推测此途径可能参与除T790M突变外肺腺癌细胞EGFR-TKI原发耐药。本研究通过基因过表达、沉默技术及免疫共沉淀，探讨MMP7与核仁素是否相互作用并影响KRAS活性参与肺腺癌细胞EGFR-TKI原发耐药，体内及体外试验明确药物阻断MMP7、活性核仁素表达与KRAS活性是否逆转吉非替尼原发耐药。肿瘤组织标本检测MMP7、核仁素表达水平了解其可否预测肺腺癌患者耐药，项目的实施有望为克服EGFR-TKI原发耐药提供可能的靶点及标志物。

我们前期研究发现不依赖KRAS突变的持续性KRAS激活可能是EGFR-TKI原发耐药的新模式，其机制并不清楚，而MMP7与核仁素（Nucleolin）在EGFR-TKI 耐药细胞株中的表达量高于敏感细胞株，外源性MMP7诱导EGFR-TKI 敏感细胞株发生耐药，文献报道MMP7可裂解激活核仁素，并持续激活野生型KRAS，我们推测此途径可能参与除T790M突变外肺腺癌细胞EGFR-TKI原发耐药。本研究通过基因过表达、沉默技术及免疫共沉淀，探讨MMP7与核仁素是否相互作用并影响KRAS活性参与肺腺癌细胞EGFR-TKI原发耐药，体内及体外试验明确药物阻断MMP7、活性核仁素表达与KRAS活性是否逆转吉非替尼原发耐药。肿瘤组织标本检测MMP7、核仁素表达水平了解其可否预测肺腺癌患者耐药。EGFR-TKI 治疗前或后肺腺癌患者恶性心包积液及胸腔积液，组织标本免疫组化检测表明活性核仁素蛋白表达增高，可能成为耐药的标志物。通过Nucleolin基因表达敲减技术，证明NSCLC细胞表面核仁素表达下调可抑制癌细胞生长。但是干扰Nucleolin表达后的肺癌细胞的MMP7表达改变不明显，KRAS下游基因蛋白表达明显减低,提示Nucleolin影响KRAS通路的激活.但不直接调控MMP7表达; 通过检测Nucleolin-ShRNA H1975 及对照细胞吉非替尼耐药程度及AS1411 （nucleolin拮抗剂）药物敏感性差异，提示AS1411发挥作用是核仁素表达依赖性的; AS1411联合Gefitinib后加剧Gefitinib对NCI-H1975的作用：阻断Nucleolin 可以增加对EGFR-TKI耐药的细胞株吉非替尼的敏感性；数据显示AS1411对核仁素mRNA表达无显著影响，而显著抑制核仁素蛋白表达，提示核仁素蛋白水平调控可能影响非小细胞肺癌细胞对吉非替尼的敏感性。体内实验中发现适配体 AS1411和Marimastat（MMP 抑制剂）均促进吉非替尼体内抗肿瘤能力。免疫共沉淀技术发现MMP7 蛋白可以直接结合核仁素参与肺腺癌细胞靶向药物耐药。