Lung cancer is the leading cause of cancer related death in the world. Platinum-based combination regimen or molecular target drugs, such as EGFR tyrosine kinase inhibitors (EGFR-TKIs) and ALK inhibitors are used for first line chemotherapy of NSCLC. Somatic mutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) have been identified in NSCLC and those mutations confer sensitivity to the EGFR-TKIs such as gefitinib. Unfortunately, it is well known that the almost cases are developed to drug resistance after chemotherapy. Several EGFR-TKI resistant mechanisms have been reported, including gatekeeper mutation (T790M), c-MET amplification and ErbB3 activation. Re-biopsy is generally required for determine the resistance mechanism and second line chemotherapy. However, re-biopsy is invasive test for cancer patients. Recently, microRNAs (miRNAs) are paid attention as biomarkers for cancer diagnosis and sensitivity to chemotherapy in various cancers. In the previous study, we revealed that the miRNA biosynthesis pathway is critical for maintain the cisplatin resistant phenotype. Further, it was reported that EGFR modulated microRNA maturation through the phosphorylation of AGO2. Protein phosphorylation is one of most important post transcriptional modification mechanisms and miRNA is also associated with fine tuning of mRNA expression. Thus, we focused on the relationship between miRNA expression and kinase signaling in gefitinib resistance. In this study, we performed characterization of gefitinib resistant cell lines, PC-9/MET200 and PC-9/MET1000 established from NSCLC cell line, PC-9, which were confirmed to have a c-MET amplification using miRNA microarray and quantitative PCR. Furthermore, we analyzed miRNA activity and kinase signaling using a luciferase reporter assay system, siRNA, RTK/phosphokinase protein array and kinase inhibitors. We revealed that miR-205 was significantly elevated in both gefitinib resistant cell lines. By the computational analysis using miRNA target prediction database, we found that ErbB3 was a target of miR-205. However, ErbB3 expression levels were rather up-regulated and highly phosphorylated in both resistant cell lines compared with a parental cell line. Under ErbB3 knockdown condition, miR-205 expression levels were slightly down regulated. Furthermore, under c-MET knockdown or Foretinib treatment condition, miR-205 activity were down regulated and ErbB3 dephosphorylated in resistant cell lines. This down-regulation of miR-205 appears to be mediated by inhibition of kinase signals. Moreover, we found that some kinase inhibitors suppressed miR-205 activity in gefitinib resistant cell lines. In conclusion, our results suggest that alterations of kinase signals are more effective for miR-205 regulation than protein expression of target molecule itself, such as ErbB3.

Citation Format: Toshihiro Suzuki, Ikuko Nagasawa, Toshimitsu Yamaoka, Tohru Ohmori, Kazuto Nishio, Kiyotaka Koyama, Yuki Ogasawara. The effect of kinase signaling for miR-205 regulation in gefitinib-resistant lung cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1893.