Background: A third generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib is effective against gefitinib/erlotinib-resistant non-small-cell lung cancer (NSCLC) harboring EGFR T790M mutation. Although the majority of these patients initially responds to osimertinib, they eventually develop resistance. Acquired mutations such as EGFR C797S mutation have been reported to confer resistance to osimertinib, but mechanisms of resistance to osimertinib or biological behaviors of osimertinib-resistant NSCLC have not been fully elucidated.
Materials and methods: Osimertinib-resistant PC-9/ZD/OR1 and PC-9/ZD/OR3 cells were generated after continuous exposure of gefitinib-resistant PC-9/ZD cells harboring both EGFR exon 19 deletion and T790M mutations to increasing concentrations of osimertinib. Growth-inhibitory effect of inhibitors gefitinib and osimertinib was evaluated by MTT assay. Common EGFR mutational status was examined by RainDrop Digital PCR System (RainDance Technologies). Immunoblot analysis was performed to investigate the modulation of molecules relevant to EGFR signaling pathways. Cell-adhesion assay, wound closure assay and transwell assay were performed to evaluate the ability of cell adhesion and migration.
Results: Both PC-9/ZD/OR1 and PC-9/ZD/OR3 cells showed 50- to 120-fold more resistant to osimertinib than parental PC-9/ZD cells, and maintained resistance to gefitinib as well. PC-9/ZD/OR cells maintain EGFR exon 19 deletion and T790M mutations and C797S mutation was not detected. Although phosphorylation of EGFR and ERK1/2 were inhibited by osimertinib treatment in PC-9/ZD/OR cells, phosphorylation of Akt was not inhibited. PC-9/ZD/OR cells express the elevated level of c-MET and its phosphorylated form, suggesting that bypass signaling is activated to evade EGFR pathway blockade. PC-9/ZD/OR cells also express marked level of E-cadherin which parental PC-9/ZD cells do not express. PC-9/ZD/OR cells exhibited enhanced adherent activity, cell motility and migration ability compared to PC-9/ZD cells. Notably, osimertinib treatment significantly reduced the number of migrated cells not only in PC-9/ZD cells but also in PC-9/ZD/OR cells. These results suggest that osimertinib-resistant PC-9/ZD/OR cells acquired enhanced migration ability through c-MET pathway activation and/or induction of E-cadherin. Osimertinib treatment may be still effective to inhibit cell migration, suggesting that cell migration and cell growth are driven by different signaling pathways in PC-9/ZD/OR cells.
Conclusion: c-MET overexpression was suggested to confer resistance to osimertinib and the resistant cells exhibited enhanced cell migration. Continuative osimertinib treatment may be beneficial in preventing metastasis even after the failure and involvement of c-MET and E-cadherin in osimertinib resistance should be further investigated.
Citation Format: Satoshi Kambayashi, Yasuhiro Koh, Maiha Ohyama, Ayaka Tanaka, Hiroaki Akamatsu, Nahomi Tokudome, Hiroki Ueda, Nobuyuki Yamamoto. Increased migration ability of osimertinib-resistant EGFR-T790M mutant non-small-cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4149. doi:10.1158/1538-7445.AM2017-4149