Abstract 719: Erlotinib-resistant non-small cell lung cancer cells exhibit enhanced cell motility via activation of TGFβ/Smad signaling pathway

Abstract

Abstract Background: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib have been shown to be effective in non-small cell lung cancer (NSCLC) patients with EGFR-activating mutations. However, the majority of responders ultimately develop acquired resistance to EGFR-TKIs mainly due to the EGFR T790M mutation or the MET gene amplification which accounts for about 50 % and 20 % of the cases with acquired resistance, respectively. It is urgently needed to further investigate other mechanisms of acquired resistance than those reported previously and on top of that, little has been elucidated about the biological behavior of EGFR-TKI-resistant tumors. Materials and Methods: Erlotinib-resistant PC-9/ER cells were generated after continuous exposure of PC-9 NSCLC cells which harbor the EGFR-activating mutation (exon 19 deletion) to increasing concentrations of erlotinib. Growth-inhibitory effect of inhibitors was evaluated by MTT assay. EGFR mutations and MET amplification were examined by PNA-LNA clamping method and fluorescent in situ hybridization, respectively. Immunoblot analysis was performed to investigate the modulation of molecules relevant to EGFR signaling pathways. Wound closure assay and transwell assay were performed to evaluate the cell migration. Expression level of receptor ligands was detected by ELISA. Transcriptional regulatory activity of Smad was measured by luciferase assay. Results: PC-9/ER cells were 100-150-fold more resistant to erlotinib than parental PC-9 cells. Neither the EGFR T790M mutation nor the MET gene amplification was detected in the resistant cells suggesting novel mechanisms responsible for acquired resistance to erlotinib. PC-9/ER cells showed cross-resistance to gefitinib and were insensitive to irreversible EGFR-TKI and MET inhibitor as well. Most notably, cell motility of PC-9/ER cells was significantly enhanced than that of PC-9 cells. Phosphorylation of Smad2 turned out to be markedly induced and the subsequent transcriptional activity was elevated in PC-9/ER cells and TGFβ2 overexpression was suggested to be responsible for its activation. TGFβ receptor I inhibitor LY364947 attenuated the cell motility of PC-9/ER cells, while it had no effect in PC-9 parental cells. Combined treatment of erlotinib and LY364947 effectively suppressed the motility of PC-9/ER cells though they either alone or in combination showed little growth-inhibitory effect, suggesting that cell motility and cell growth are driven by different signaling pathways in PC-9/ER cells. Conclusion: Activated TGFβ/Smad pathway is suggested to confer enhanced cell motility in PC-9/ER cells while it is unlikely to drive cell growth. Blockade of TGFβ signaling pathway with continuative EGFR-TKIs treatment seems to be beneficial in preventing metastasis after the failure of EGFR-TKIs monotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 719. doi:10.1158/1538-7445.AM2011-719