Overcoming acquired resistance of gefitinib in lung cancer cells without T790M by AZD9291 or Twist1 knockdown in vitro and in vivo

The T790M mutation is recognized as a typical mechanism of acquired resistance to first generation of epithermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) such as gefitinib in non-small cell lung cancer (NSCLC) patients who are commonly treated by third generation of EGFR-TKI AZD9291 (osimertinib). However, the therapeutic strategy for overcoming acquired resistance to EGFR-TKIs in NSCLC patients without T790M remains to be definitively determined. In the present study, gefitinib-resistant H1650 (H1650GR) or AZD9291-resistant H1975 (H1975AR) was generated by exposing NSCLC cell line H1650 or H1975 to progressively increased concentrations of gefitinib or AZD9291 over 11months. The cytotoxic effects of gefitinib or AZD9291 in vitro were evaluated via the half maximal inhibitory concentrations (IC50s) determined by the MTT assay. IC50 of gefitinib in H1650GR (50.0 +/- 3.0 mu M) significantly increased compared with H1650 (31.0 +/- 1.0 mu M) (p<0.05). Similarly, the IC50 of AZD9291 in H1975AR (10.3 +/- 0.9 mu M) significantly increased compared with H1975 (5.5 +/- 0.6 mu M) (p<0.05). However, IC50 of AZD9291 on H1650GR (8.5 +/- 0.5 mu M) did not increase compared with H1650 (9.7 +/- 0.7 mu M). On the other hand, IC50 of AZD9291 on gefitinib-resistant A549 (A549GR established in our previous study) (12.7 +/- 0.8 mu M) was significantly increased compared with A549 (7.0 +/- 1.0 mu M) (p<0.05). AZD9291 induced caspase 3/7 activation in A549, H1650, and H1650GR, but not in A549GR. Western blot analyses showed that p-Akt played a key role in determining the sensitivities of A549, A549GR, H1650, and H1650GR to gefitinib or AZD9291. Additionally, increased expression of Twist1 was observed in all cells with acquired EGFR-TKI resistance and knockdown of Twist1 by shRNA was found to significantly enhance the sensitivity of A549GR to gefitinib or AZD9291 via reversing epithelial-mesenchymal transition and downregulating p-Akt, but not of H1975AR to AZD9291. The enhanced cytotoxic effect of AZD9291 on A549GR by Twist1 knockdown in vitro was further validated by in vivo studies which showed that Twist1 knockdown could lead to significantly delayed tumor growth of A549GR xenograft with increased sensitivity to AZD9291 treatment in nude mice without any observed side toxic effects. In summary, our study demonstrated that the mechanisms of acquired resistance in different NSCLC cell lines treated by even the same EGFR-TKI might be quite different, which provide a rationale for adopting different therapeutic strategies for those NSCLC patients with acquired EGFR-TKI resistance based on different status of heterogeneous mutations.