<br>构建CD74-ROS1 D2033N和CD74-ROS1 S1986F点突变的克唑替尼耐药模型，探讨耐药机制和治疗策略,Cellular Signalling

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构建CD74-ROS1 D2033N和CD74-ROS1 S1986F点突变的克唑替尼耐药模型，探讨耐药机制和治疗策略
Cellular Signalling ( IF 4.4 ) Pub Date : 2022-10-17 , DOI: 10.1016/j.cellsig.2022.110497
Rui Sun ₁ , Yuting Meng ₁ , Rui Xu ₁ , Ye Li ₁ , Xiaobo Xu ₁ , Zengqiang Li ₁ , Daiying Zuo ₁

Affiliation

靶向治疗是非小细胞肺癌（NSCLC）的重要治疗手段，且与耐药性密切相关。 -ros癌基因1（ROS1）基因点突变是引起耐药性的主导因素之一。然而，克唑替尼的点突变细胞模型难以获得，导致其耐药机制和治疗策略的报道较少。采用融合PCR技术构建CD74-ROS1 D2033N和CD74-ROS1 S1986F点突变质粒并转染A549细胞。 Western blot和MTT实验证明耐药细胞系转染成功。 Transwell实验证实，与野生型组相比，突变细胞的运动能力显着增强。此外，突变细胞中粘着斑激酶（FAK）显着增加。此外，通过激活FAK/磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（AKT）途径，突变细胞中出现克唑替尼耐药。接下来，克唑替尼与FAK抑制剂德法替尼联合，进一步探讨其治疗效果。结果表明，该组合能够显着抑制突变细胞的增殖、侵袭和迁移。总之，我们证明CD74-ROS1 D2033N和CD74-ROS1 S1986F点突变NSCLC细胞通过激活FAK/PI3K/AKT信号通路对克唑替尼产生耐药，并且通过defactinib抑制FAK/PI3K/AKT信号通路激活可以克服药物突变细胞的耐药性。

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Construction of crizotinib resistant models with CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutations to explore resistance mechanism and treatment strategy

Targeted therapy is an essential treatment for non-small cell lung cancer (NSCLC) that is always associated with the drug resistance. -ros oncogene 1 (ROS1) gene point mutation is one of the leading factors causing drug resistance. However, the point mutation cell models of crizotinib are challenging to obtain, causing few reports on the drug resistance mechanism and the treatment strategy. We constructed CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutant plasmids by fusion PCR technology and transfected them into A549 cells. Western blot and MTT assay proved that the drug-resistant cell lines were successfully transfected. The transwell assay confirmed that the mutant cells' motor abilities were significantly increased compared with the wild-type group. In addition, focal adhesion kinase (FAK) was significantly increased in mutant cells. Moreover, crizotinib resistance occurred in the mutant cells through the activation of FAK / phosphatidylinositol 3-kinase (PI3K) / protein kinase B (AKT) pathway. Next, crizotinib was combined with defactinib, a FAK inhibitor, to further explore its therapeutic effect. The results showed that the combination could significantly inhibit the proliferation, invasion and migration of mutant cells. In conclusion, we proved that CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutant NSCLC cells were resistant to crizotinib through the activation of FAK/PI3K/AKT signaling pathway, and inhibiting FAK/PI3K/AKT signaling pathway activation by defactinib could overcome drug resistance in mutant cells.

更新日期：2022-10-17

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