Establishment of a Human Non-Small Cell Lung Cancer Cell Line Resistant to Gefitinib

Int J Cancer. 2005 Aug 10;116(1):36-44. doi: 10.1002/ijc.20985.

Abstract

The epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitor gefitinib (Iressa, ZD1839) has shown promising activity preclinically and clinically. Because comparative investigations of drug-resistant sublines with their parental cells are useful approaches to identifying the mechanism of gefitinib resistance and select factors that determine sensitivity to gefitinib, we established a human non-small cell lung carcinoma subline (PC-9/ZD) that is resistant to gefitinib. PC-9/ZD cells are approximately 180-fold more resistant to gefitinib than their parental PC-9 cells and PC-9/ZD cells do not exhibit cross-resistance to conventional anticancer agents or other tyrosine kinase inhibitors, except AG-1478, a specific inhibitor of EGFR. PC-9/ZD cells also display significant resistance to gefitinib in a tumor-bearing animal model. To elucidate the mechanism of resistance, we characterized PC-9/ZD cells. The basal level of EGFR in PC-9 and PC-9/ZD cells was comparable. A deletion mutation was identified within the kinase domain of EGFR in both PC-9 and PC-9/ZD, but no difference in the sequence of EGFR cDNA was detected in either cell line. Increased EGFR/HER2 (and EGFR/HER3) heterodimer formations were demonstrated in PC-9/ZD cells by chemical cross-linking and immunoprecipitation analysis in cells unexposed to gefitinib. Exposure to gefitinib increased heterodimer formation in PC-9 cells, but not in PC-9/ZD cells. Gefitinib inhibits EGFR autophosphorylation in a dose-dependent manner in PC-9 cells but not in PC-9/ZD cells. A marked difference in inhibition of site-specific phosphorylation of EGFR was observed at Tyr1068 compared to other tyrosine residues (Tyr845, 992 and 1045). To elucidate the downstream signaling in the PC9/ZD cellular machinery, complex formation between EGFR and its adaptor proteins GRB2, SOS, and Shc was examined. A marked reduction in the GRB2-EGFR complex and absence of SOS-EGFR were observed in PC-9/ZD cells, even though the protein levels of GRB2 and SOS in PC-9 and PC-9/ZD cells were comparable. Expression of phosphorylated AKT was increased in PC-9 cells and inhibited by 0.02 microM gefitinib. But the inhibition was not significant in PC-9/ZD cells. These results suggest that alterations of adaptor-protein-mediated signal transduction from EGFR to AKT is a possible mechanism of the resistance to gefitinib in PC-9/ZD cells. These phenotypes including EGFR-SOS complex and heterodimer formation of HER family members are potential biomarkers for predicting resistance to gefitinib.

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism
  • Carcinoma, Non-Small-Cell Lung / drug therapy*
  • Cell Line, Tumor*
  • Drug Resistance, Neoplasm*
  • GRB2 Adaptor Protein
  • Gefitinib
  • Genes, erbB-1
  • Humans
  • Intracellular Signaling Peptides and Proteins / pharmacology*
  • Lung Neoplasms / drug therapy*
  • Mutation
  • Phosphorylation
  • Quinazolines / pharmacology*
  • Son of Sevenless Protein, Drosophila / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • GRB2 Adaptor Protein
  • GRB2 protein, human
  • Intracellular Signaling Peptides and Proteins
  • Quinazolines
  • Son of Sevenless Protein, Drosophila
  • protein kinase modulator
  • Gefitinib