Study on resistance of lung cancer cells to inhibitor of EGF receptor tyrosine kinase

尽管吉非替尼和厄洛替尼在EGFR突变的非小细胞肺癌（NSCLC）患者中具有显著疗效，但所有患者最终都会对EGFR酪氨酸激酶抑制剂（EGFR-TKI）产生耐药性。EGFR（T790 M）的继发突变和MET扩增已被确定为获得性EGFR-TKI耐药的主要机制。然而，在NSCLC患者中确定其他耐药机制并克服对EGFR-TKI的获得性耐药仍然很重要。我们先前报道了EGFR-TKI AG 1478诱导PC-9细胞凋亡，PC-9细胞是一种吉非替尼敏感的人NSCLC细胞系，其EGFR酪氨酸激酶结构域存在突变（delE 746-A750）。在这项研究中，我们反复处理PC-9细胞与高浓度的AG 1478（500 nM）和分离的AG 1478耐药细胞系。与PC-9细胞相比，ErbB 3在这些抗性细胞中的表达水平极低。此外，当PC-9细胞的Erb ...更多信息 siRNA下调B3表达。因此，AG 1478的促凋亡作用与ErbB 3的表达水平相关。这些结果表明ErbB 3在PC-9细胞中起到将EGFR偶联到细胞存活通路的作用，并且抗性细胞可以找到一种独立于ErbB 3有效激活存活信号传导的方法。我们接下来用较低浓度的AG 1478（50 nM）处理PC-9细胞，并分离出另一系列的AG 1478抗性细胞系。在PC-9细胞中，AG 1478降低MAPK磷酸酶-1（MKP-1）的表达，并强烈刺激JNK的磷酸化。此外，AG 1478诱导线粒体中促凋亡Bcl-2家族蛋白Bim的积累。然而，在耐药细胞系中，MKP-1的表达水平在AG 1478处理后仍然很高，JNK磷酸化和Bim的积累都没有增加。这些结果表明，Bim通过MKP-1/JNK途径易位至线粒体对于PC-9细胞中EGFR-TKI诱导的凋亡至关重要，并且MKP-1的持续高水平表达导致对EGFR-TKI的抗性。少

Despite the dramatic efficacy of gefitinib and erlotinib in non-small cell lung cancer (NSCLC) patients with EGFR mutations, all patients ultimately develop resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). A secondary mutation in EGFR (T790M) and MET amplification have been identified as major mechanisms of acquired resistance to EGFR-TKIs. However, it is still important to identify additional mechanisms of resistance and to overcome acquired resistance to EGFR-TKIs in NSCLC patients. We previously reported that EGFR-TKI AG1478 induces apoptosis in PC-9 cells, a gefitinib-sensitive human NSCLC cell line with a mutation (delE746-A750) in tyrosine kinase domain of their EGFR. In this study, we repeatedly treated PC-9 cells with a high concentration of AG1478 (500 nM) and isolated AG1478-resistant cell lines. Expression level of ErbB3 in these resistant cells was extremely low as compared with that of PC-9 cells. Furthermore, PC-9 cells became resistant to AG1478, when their Erb … More B3 expression was down-regulated by siRNA. Therefore, the apoptosis-inducing action of AG1478 was correlated with the expression level of ErbB3. These results indicate that ErbB3 served to couple EGFR to the cell survival pathway in PC-9 cells and that the resistant cells could find a way to effectively activate survival signaling independent of.We next treated PC-9 cells with a lower concentration of AG1478 (50 nM) and isolated another series of AG1478-resistant cell lines. In PC-9 cells, AG1478 decreased the expression of the MAPK phosphatase-1(MKP-1) and intensively stimulated phosphorylation of JNK. Further, AG1478 induced the accumulation of proapoptotic Bcl-2 family protein Bim in mitochondria. However, in the resistant cell lines, expression level of MKP-1 was still high after AG1478 treatment, and neither JNK phosphorylation nor accumulation of Bim was increased. These results indicate that translocation of Bim to mitochondria through the MKP-1/JNK pathway is critical for EGFR-TKI-induced apoptosis in PC-9 cells and that continuous high-level expression of MKP-1 leads to resistance to EGFR-TKIs. Less