Gefitinib (IRESSA), an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor, has antitumour activity in the advanced non-small-cell lung cancer (NSCLC) setting. However, in chemotherapy-naïve patients with advanced NSCLC, the addition of gefitinib to standard chemotherapy regimens failed to increase survival. These results suggest the need for improved patient selection and combination rationales for targeted therapies. We have identified subpopulations of an adenocarcinoma cell line that are naturally resistant to gefitinib, and have analysed the cDNA expression profiles, genomic status of EGFR gene and the effect of gefitinib on signalling pathways in these cell lines in order to identify key mechanisms for naturally acquired resistance to gefitinib. Gefitinib-resistant subpopulations demonstrated increased Akt phosphorylation (not inhibited by gefitinib), reduced PTEN protein expression and loss of the EGFR gene mutation when compared with parental cell lines. These differences in Akt and PTEN protein expression were not evident from the cDNA array profiles. These data suggests that (1) the EGFR gene mutation may be possibly lost in some cancer cells with other additional mechanisms for activating Akt, (2) reintroduction of PTEN or pharmacological downregulation of the constitutive PI3K -Akt-pathway activity may be an attractive therapeutic strategy in cancers with gefitinib resistance.
BackgroundPhase III trials evaluating the efficacy of gefitinib (IRESSA) in non-small cell lung cancer (NSCLC) lend support to the need for improved patient selection in terms of gefitinib use. Mutation of the epidermal growth factor receptor (EGFR) gene is reported to be associated with clinical responsiveness to gefitinib. However, gefitinib-sensitive and prolonged stable-disease-defined tumors without EGFR gene mutation have also been reported.MethodsTo identify other key factors involved in gefitinib sensitivity, we analyzed the protein expression of molecules within the EGFR family, PI3K-Akt and Ras/MEK/Erk pathways and examined the sensitivity to gefitinib using the MTT cell proliferation assay in 23 lung cancer cell lines.ResultsWe identified one highly sensitive cell line (PC9), eight cell lines displaying intermediate-sensitivity, and 14 resistant cell lines. Only PC9 and PC14 (intermediate-sensitivity) displayed an EGFR gene mutation including amplification. Eight out of the nine cell lines showing sensitivity had Akt phosphorylation without ligand stimulation, while only three out of the 14 resistant lines displayed this characteristic (P = 0.0059). Furthermore, the ratio of phosphor-Akt/total Akt in sensitive cells was higher than that observed in resistant cells (P = 0.0016). Akt phosphorylation was partially inhibited by gefitinib in all sensitive cell lines.ConclusionThese results suggest that Akt phosphorylation without ligand stimulation may play a key signaling role in gefitinib sensitivity, especially intermediate-sensitivity. In addition, expression analyses of the EGFR family, EGFR gene mutation, and FISH (fluorescence in situ hybridization) analyses showed that the phosphorylated state of EGFR and Akt might be a useful clinical marker of Akt activation without ligand stimulation, in addition to EGFR gene mutation and amplification, particularly in adenocarcinomas.
The ING1 gene is involved in the regulation of the cell cycle, senescence, and apoptosis and is a novel candidate tumor suppressor gene. ING2, another gene in the ING family, was identified and cloned. The functions of ING1 and ING2 largely depend on the activity of p53. To determine whether an alteration in these genes plays a role in carcinogenesis and tumor progression in lung cancer, we screened 30 human lung cancer cell lines and 31 primary lung cancer tumors for mutations in these genes using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing. Our findings failed to uncover any mutations in these genes. We also examined the expression of ING1 and ING2 in lung cancer cell lines that either had or lacked a p53 mutation, and in a control bronchial epithelium cell line, using quantitative real-time reverse transcriptionpolymerase chain reaction (RT-PCR). ING1 expression was up-regulated in all 7 lung cancer cell lines that had a p53 mutation, while the expression of ING2 was down-regulated in 6 of 7 lung cancer cell lines that had a p53 mutation. These results suggest that the ING1 and ING2 genes have different roles in lung carcinogenesis and progression, and the ING2 gene may be an independent tumor suppressor candidate on p53.
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