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对EGFR突变的非小细胞肺癌(NSCLC)患者有显著疗效，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(T790M)的继发性突变和MET扩增被认为是对EGFR-TKIs产生获得性耐药性的主要机制。然而，在非小细胞肺癌患者中，确定额外的耐药机制和克服对EGFR-TKIs的获得性耐药仍然是重要的。我们之前报道了EGFR-TKI AG1478诱导PC-9细胞凋亡，PC-9细胞是一种对吉非替尼敏感的人非小细胞肺癌细胞系，其EGFR酪氨酸激酶结构域发生突变(delE746-A750)。在这项研究中，我们反复用高浓度的AG1478(500 NM)处理PC-9细胞，并分离出对AG1478耐药的细胞系。与PC-9细胞相比，这些耐药细胞中ErbB3的表达水平极低。此外，PC-9细胞对AG1478产生抗药性，当它们的Erb…更多的B3表达被siRNA下调。因此，AG1478的诱导凋亡作用与ErbB3的表达水平有关。这些结果表明，ErbB3可以将EGFR偶联到PC-9细胞的生存途径上，耐药细胞可以找到一种有效地激活独立于生存信号的方法。接下来，我们用较低浓度的AG1478(50 NM)处理PC-9细胞，并分离出另一系列AG1478耐药细胞系。在PC-9细胞中，AG1478降低MAPK磷酸酶-1(MKP-1)的表达，并强烈刺激JNK的磷酸化。此外，AG1478还可诱导线粒体中促凋亡蛋白Bim的积聚。而在耐药细胞系中，经AG1478处理后，MKP-1的表达水平仍然较高，JNK的磷酸化水平和Bim的积聚都没有增加。这些结果表明，Bim通过MKP-1/JNK途径转位到线粒体是EGFR-TKI诱导PC-9细胞凋亡的关键，MKP-1的持续高表达导致对EGFR-TKI的抵抗。较少

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