PIM-1信号通路在非小细胞肺癌EGFR-TKI获得性耐药中的作用及其分子机制

Non-small cell lung cancer patients with sensitizing mutations of EGFR initially respond to epidermal growth factor receptor (EGFR)- tyrosine kinase inhibitors (TKI) invariably will develop progressive disease ultimately because of acquired resistance. The reactivation of EGFR downstream pathway is the key factor of acquired resistance. As the key kinase and effector of EGFR, PI3K/AKT, and STAT signaling pathway, PIM-1 is the intermediator to mediate cellular signals for proliferation and drug resistance, therefore, PIM-1 may play the crucial role in the lung cancer acquired resistance to EGFR-TKI. In order to further understand the mechanism of EGFR-TKI acquired resistance and the function of PIM-1, our project will establish controllable drug resistance cell lines by introducing drug resistance related genes with Tet-On lentivirus system, and to elucidate the controlling function of EGFR, PI3K, and c-met to PIM-1 in the lung cancer cell proliferation and acquird resistance to EGFR-TKI by modulating gene expression. Moreover, we will investigate the function of PIM-1 pathway in acquird resistance to EGFR-TKI by knockdown or overexpression of the key factors, including PIM-1. In addition, we will investigate the cross-talk between PIM-1 downstream factors by gene expression microarray and obtain the molecular expression profile. Moreover, by clinical detection of these important signalling factors, we will clarify the relationship between the factors and prognosis. Taken together, with this project we will further elucidate the molecular mechanism of PIM-1 in lung cancer acquired resistance to EGFR-TKI, and therefore provide the theoretical base for the finding of novel targets of acquired resistance to EGFR-TKI.

EGFR敏感性突变阳性的肺癌患者能从EGFR-TKI治疗中显著获益，但最终都会出现耐药，EGFR通路重新活化是获得性耐药的关键因素。PIM-1作为EGFR、PI3K和STAT传导途径的关键激酶和效应分子，是传递细胞增殖和耐药信号的中继站，因而可能在肺癌EGFR-TKI获得性耐药中发挥重要作用。为深入理解PIM-1在EGFR-TKI获得性耐药中的作用及机制，本研究拟运用Tet On慢病毒系统导入耐药基因建立可调控型耐药细胞系，通过调控耐药基因表达，明确在获得性耐药中EGFR、PI3K、c-MET通路对PIM-1的调控作用；通过敲除或过表达PIM-1等关键分子，探讨PIM-1通路在获得性耐药中的作用，利用基因芯片分析PIM-1下游分子交互网络，筛选分子表达谱，并利用肿瘤标本对PIM-1等分子进行临床验证，以期阐明其在EGFR-TKI获得性耐药的分子机制，为克服EGFR-TKI耐药奠定理论基础。

EGFR敏感性突变阳性的肺癌患者能从EGFR-TKI治疗中显著获益，但最终都会出现耐药，EGFR通路重新活化是获得性耐药的关键因素。PIM-1作为EGFR、PI3K和STAT传导途径的关键激酶和效应分子，是传递细胞增殖和耐药信号的中继站，因而可能在肺癌EGFR-TKI获得性耐药中发挥重要作用。本课题通过对PIM-1在肺癌细胞增殖和耐药中的作用进行研究：（1）采用实时定量RT-PCR分析了NSCLC细胞系中PIM-1 mRNA的表达水平，Western blotting方法检测了NSCLC细胞系中PIM-1蛋白的表达水平。用免疫荧光的方法检测了PIM-1在细胞中的定位。采用免疫组化的方法检测了194例肺癌组织标本及其中31例相对应的正常肺组织标本中PIM1、 p-STAT3以及 c-MYC的表达；并对上述基因的表达以及临床病理因素及总生存间的关系进行了统计学分析。（2）回顾性分析2008年1月-2012年10月1016例手术切除的I-IIIA期非小细胞肺癌患者，分析血清肿瘤标志物与EGFR突变以及生存指标的关系、明确相关预后因素。（3）运用免疫组化的方法检测肺癌组织标本PIM-1、SET的表达；对二者的表达以及临床病理因素及总生存间的关系进行了统计学分析。通过特异性抑制药物干扰EGFR、SET、PIM-1的表达，通过细胞增殖、细胞周期、细胞侵袭等细胞功能实验，观察细胞生物功能的改变。通过Western Blot检测SET、PIM-1、EGFR等表达，进一步探索SET、PIM-1在NSCLC增殖、耐药中的作用机制。（4）运用免疫组化的方法检测215例肺腺癌患者PIM-1及c-MET表达，并分析其与临床病理因素、预后、生存的关系；逐步增加靶向药物厄罗替尼浓度诱导HCC827细胞耐药、建立靶向治疗耐药细胞株，通过Western Blot检测PIM1、c-MET表达。通过PIM-1抑制剂SGI-1776、17-DMAG以及PIM-1 siRNA抑制PIM1表达，通过细胞增殖、细胞周期、细胞侵袭等细胞功能实验，观察细胞生物功能的改变。通过Western Blot检测c-MET表达情况。结果证明PIM-1通过调控MET信号通路参与肺癌细胞增殖和EGFR-TKI厄罗替尼靶向药物耐药，为遏制肿瘤生长和逆转肿瘤耐药提供了新的理论依据。