Establishment and molecular characteristics of PC-9 derived erlotinib resistant cell lines

Abstract

e22193 Background: EGFR mutation is closely associated with tumor responsiveness to EGFR tyrosine kinase inhibitors (TKIs), gefitinib or erlotinib. However, many non-small cell lung cancer (NSCLC) cases with EGFR mutation that had responded to EGFR-TKIs finally acquire resistance to EGFR-TKIs. It has been reported that T790M mutation and MET amplification caused resistance to EGFR-TKIs . Recent study suggested that overexpression of HGF was also related to resistance to EGFR-TKIs. However, the whole mechanism of acquired resistance to TKIs is still not fully known. Methods: We established 5 clones of elrotinib resistant PC-9 cell line (harboring EGFR exon 19 deletion) (designated as PC-9ER1–5) by exposing PC-9 to low-dose erlotinib. The protein expression of EGFR-related molecules was examined by Western blotting. Array comparative genomic hybridization (aCGH) or real time quantitative PCR (qPCR) assay was performed to examine copy numbers of EGFR-related genes. Results: In MTS assay, the IC50 value for the parental PC-9 cells was 0.02 μmol/L. By contrast, those for PC-9ER1–5 were more than 33μmol/L. All 5 resistant cell lines retained exon19 deletions and did not obtain T790M mutation. The qPCR assay and aCGH showed no MET amplification in PC-9 or PC-9ER1–5. HGF protein was not overexpressed in PC-9ER1–5 by ELISA. The protein expressions of several molecules in the EGFR-Akt signaling pathway were examined by western blotting after erlotinib treatment. Although phospho-EGFR was suppressed in both PC-9 and PC-9ER1–5 with 2-μmol/L erlotinib, phospho-Akt was not suppressed in PC-9ER1–5. PTEN expression was not down-regulated in PC-9ER1–5. The combination of erlotinib and PHA-665752, MET tyrosine kinase inhibitor, did not suppress phospho-Akt or cell proliferation in PC-9ER1–5. Conclusions: As common molecular features of our EGFR-TKI resistant cell lines, phospho-Akt was not suppressed with exposure to erlotinib. These cell lines did not show previously reported features of resistant cell lines including T790M mutation, MET amplification or HGF overexpression. Our results indicated that other mechanisms leading to Akt activation caused resistance to EGFR-TKIs. No significant financial relationships to disclose.