Abstract 724: Role of ERK reactivation mediated by Src in acquired resistance to gefitinib in non-small cell lung cancer with EGFR mutation
Authors: Ochi Nobuaki , Takigawa Nagio , Ohashi Kadoaki , Takeda Hiromasa , Fujii Masanori , Kubo Toshio , Ichihara Eiki , Hotta Katsuyuki , Tanimoto Mitsune , Kiura Katsuyuki 
Published: June 2012

Abstract
Introduction: Non-small cell lung cancer (NSCLC) harboring some mutations in epidermal growth factor receptor (EGFR) often shows dramatic responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs). Although the problem of acquired resistance to EGFR-TKI is widely recognized and has grown in clinical situation, some mechanisms have been elucidated. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was known as tobacco specific nitrosamine. To clarify the resistant mechanisms to EGFR-TKIs associated with smoking, we established novel gefitinib-resistant NSCLC cells using gefitinib and NNK.
Materials and Methods: The EGFR mutant (frame deletion in exon 19) NSCLC cell line, PC-9, was treated with gefitinib and NNK. The drug concentration required to inhibit the growth of tumor cells by 50% (IC50) was evaluated by MTT assay. Protein expression was determined by immunoblotting using specific antibodies. Extracted mRNA and DNA were analyzed by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and by sequencing with specific primers, respectively, to determine gene amplification and mutation. Some specific small interfering RNA (siRNA) was used to down-regulate the target gene expression.
Result: IC50s of the established resistant cells (PC9-GR) to gefitinib ranged from 2 to 4 µM, which was a 100- to 200-fold decrease in gefitinib sensitivity compared with parental PC-9 cells. The resistant mechanisms in PC9-GR cells were different from T790M mutation, RAS mutation, MET amplification, phosphatase and tensin homolog (PTEN) loss or the insulin-like growth factor-1 receptor (IGF-1R) signaling. Although gefitinib promptly suppressed phosphorylations of EGFR, Akt and the mitogen-activated protein/extracellular signal-regulated kinase (ERK), only ERK was reactivated 6 hours after treatment with gefitinib in PC9-GR cells but not in parental PC-9 cells. the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor (U0126) significantly suppressed ERK reactivation and also inhibited cell growth. These results suggest ERK reactivation play a key role in this resistant mechanism. Furthermore, we found the combination of gefitinib with Src inhibitor (dasatinib) suppressed ERK reactivation in PC9-GR cells and significantly inhibited cell growth. In addition, the combination of gefitinib with Src-specific siRNA brought improvement of gefitinib sensitivity in PC9-GR cells.
Conclusion: ERK reactivation mediated by Src participated in the novel gefitinib-resistant mechanism. Src inhibition might be a new therapeutic strategy to overcome an acquired gefitinib-resistant NSCLC with EGFR mutation.
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 724. doi:10.1158/1538-7445.AM2011-724