Genetic Heterogeneity Of Epidermal Growth Factor Receptor And Hypoxia Are Important Factors In Resistance To Gefitinib In Non-Small Cell Lung Cancer

Somatic mutations in epidermal growth factor receptor (EGFR) gene, such as exon 19 deletion mutation, are important factors Rationale; to determine therapeutic response to gefitinib in non-small cell lung cancer (NSCLC). However, there are patients with activating EGFR mutations that have poor responses to gefitinib, suggesting that other factors determining responsiveness to gefitinib exist in NSCLC. Possible biological factors related to the resistance to gefitinib are genetic heterogeneity within cancer cells and microenvironment such as hypoxia. We used three NSCLC cell lines, PC9 and HCC827, which express exon 19 deletion mutation (ΔE746-A750), and A549 Methods; EGFR harboring wild-type EGFR. We examined the relative proportions of deletion mutant and wild type EGFR expressions by fragment analysis. We also exposed cells with hypoxia (1% O2), and determined the sensitivity to gefitinib under normoxic and hypoxic conditions. Phosphorylation of EGFR, AKT, and ERK were analyzed by western blot. We knocked down TGFa and HIF1a expressions in hypoxic PC9 cells by siRNA. We also introduced wild-type EGFR into HCC827 cells, and then evaluated sensitivity to gefitinib under normoxia and hypoxia. While HCC827 cells expressed only deletion mutant EGFR, PC9 cells expressed not only deletion mutant EGFR but also wild-type Results; EGFR. A549 cells expressed only wild-type EGFR. HCC827 cells were sensitive to gefitinib under both normoxic and hypoxic conditions. However, hypoxic PC9 and A549 cells were more resistant to gefitinib as compared to those in normoxia. Phosphorylation of EGFR and ERK with gefitinib was suppressed to lesser extent under hypoxic condition than those under normoxic condition in PC9 and A549 cells. Expression of TGFa which can activate wild-type EGFR was dramatically upregulated by the exposure of hypoxia, and knockdown of TGFa or HIF1a reversed the resistance to gefitinib in hypoxic PC9 cells. Finally, transfection of wild-type EGFR gene into HCC827 cells caused resistance to gefitinib in hypoxia. Our results indicate that hypoxia causes gefitinib resistance in NSCLC through the activation of wild-type EGFR mediated by Conclusion; TGFa. Genetic heterogeneity of EGFR, especially containing wild-type and mutant EGFR, within lung cancer cells and hypoxia would be important factors to be considered when treating patients with NSCLC with gefitinib.