title: Establishment of a human non-small cell lung cancer cell line resistant to gefitinib.
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.
authors.affiliation: Shien-Lab, National Cancer Center Hospital, Tokyo, Japan.
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Koizumi
authors.forename: Tatsu
authors.initials: T
authors.lastname: Shimoyama
authors.forename: Fumiko
authors.initials: F
authors.lastname: Taguchi
authors.forename: Nagahiro
authors.initials: N
authors.lastname: Saijo
authors.forename: Kazuto
authors.initials: K
authors.lastname: Nishio
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy*; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D051380:GRB2 Adaptor Protein; D000077156:Gefitinib; D018773:Genes, erbB-1; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology*; D008175:Lung Neoplasms / Q000188:drug therapy*; D009154:Mutation; D010766:Phosphorylation; D011799:Quinazolines / Q000494:pharmacology*; D020839:Son of Sevenless Protein, Drosophila / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D011799:Quinazolines; D020839:Son of Sevenless Protein, Drosophila; C015482:protein kinase modulator; D000077156:Gefitinib