Twist1在ROS1融合基因突变非小细胞肺癌EMT和侵袭转移中的调控机制及靶向干预研究

Although targeted therapy of ROS1 fusion gene positive non-small cell lung cancer (NSCLC) has made some progress, the secondary mutation and drug resistance leading to tumor invasion and metastasis is still a difficult problem in basic and clinical research. In our previous study, we found that CD74-ROS1 G2032R mutant NSCLC cells had obvious epithelial mesenchymal transition (EMT) phenomenon, and the invasion and migration ability of tumor cells was significantly enhanced, accompanied by high expression of Twist1. Therefore, we hypothesized that Twist1 may play important effects on the EMT, migration and invasion in NSCLC with ROS1 fusion gene mutations. Based on this hypothesis, we will build different types of ROS1 mutations in NSCLC cells through plasmid construction and transfection. Then, the regulatory mechanism of Twist1 on tumor invasion and metastasis of NSCLC with different types of ROS1 mutations will be investigated in molecular, cell and animal levels through the qRT-PCR, western blot, siRNA and nude mice xenograft methods. The main signal pathways by which Twist1 regulates invasion and metastasis of NSCLC during the process of EMT will be clarified, and the targeted intervention will be conducted. Our study will be helpful for finding new target to effectively reverse migration and metastasis and provide new ideas for individualized therapy of ROS1 mutation NSCLC.

ROS1融合基因阳性非小细胞肺癌（NSCLC）的靶向治疗虽取得了一定的进展，但继发性突变耐药导致肿瘤侵袭转移仍是基础和临床研究中的难题。本课题组前期发现CD74-ROS1 G2032R突变的NSCLC细胞发生了明显的上皮-间质转化（EMT）现象，肿瘤细胞的侵袭迁移能力明显增强，伴有Twist1高表达。由此我们提出假说：Twist1可能在ROS1突变NSCLC的EMT及侵袭转移中发挥重要作用。本课题拟采用质粒构建和转染的方式构建不同ROS1突变位点的NSCLC细胞，并通过qRT-PCR、western blot、siRNA和裸鼠移植瘤等技术，在分子、细胞和动物水平上研究Twist1在不同类型ROS1突变NSCLC中对肿瘤侵袭和转移的调控机制，明确Twist1调控NSCLC侵袭和转移的主要信号通路，并进行靶向干预治疗，为寻找有效逆转ROS1突变NSCLC侵袭转移的新靶点和个体化治疗提供新思路。

ROS1融合基因阳性非小细胞肺癌（NSCLC）患者的治疗得益于crizotinib的使用。但患者在接受治疗1年后均不可避免地出现了耐药现象，其中ROS1激酶结构域内发生的点突变为其重要的耐药原因之一。为探索ROS1点突变诱导的耐药机制，我们构建了CD74-ROS1 L2026M、CD74-ROS1 D2033N及CD74-ROS1 S1986F突变基因，将其连接至慢病毒载体并转染至NSCLC细胞内。Western blot与MTT实验表明，CD74-ROS1野生型与耐药突变型NSCLC细胞系构建成功。在形态学上，我们发现与CD74-ROS1组相比，NSCLC细胞在表达CD74-ROS1 L2026M、D2033N和S1986F突变后发生了纺锤形转变。Transwell实验结果显示，这些突变基因显著增强了NSCLC细胞的侵袭与迁移能力，且Twist1表达水平明显升高。MDC染色与western blot的结果表明，与CD74-ROS1组相比，CD74-ROS1 L2026M突变基因显著增强了H460与A549细胞的自噬水平，自噬抑制剂氯喹的预处理显著抑制了CD74-ROS1 L2026M突变NSCLC细胞的侵袭迁移能力并克服其crizotinib耐药；此外，CD74-ROS1 L2026M突变诱导H460与A549细胞内MEK/ERK通路发生过度激活，MEK抑制剂的使用可以显著抑制细胞的自噬水平与侵袭迁移能力，且该过程可被自噬激活剂rapamycin所阻断；MEK抑制剂可增强CD74-ROS1 L2026M突变细胞对crizotinib的敏感性并促进细胞凋亡，进而克服crizotinib耐药。对于CD74-ROS1 D2033N与S1986F突变，我们发现突变细胞内FAK的表达与PI3K/AKT信号通路发生显著上调，FAK抑制剂defactinib在抑制两耐药突变细胞侵袭迁移的同时对PI3K/AKT通路同样产生明显的抑制作用；此外，defactinib可增加CD74-ROS1 D2033N与S1986F突变细胞对crizotinib的敏感性，并增强crizotinib对其抑制作用。总之，我们发现了CD74-ROS1 L2026M、D2033N和S1986F突变NSCLC新的侵袭迁移和crizotinib耐药机制，并为突变耐药患者的临床用药提供了新思路。