EphA2 and EFNA1 are transcriptional targets of EGFR in gefitinib-sensitive non-small cell lung cancer cell lines Free

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EGF receptor (EGFR) mutations are observed in the tumors of a subset of non-small cell lung cancer (NSCLC) patients, and several clinical trials with EGFR tyrosine kinase inhibitors, Gefitinib and Erlotinib, showed significant response rates in these patients. However, the mechanisms by which these drugs induce tumor shrinkage are poorly understood. To address this question, microarray gene expression profiling was performed on 5 gefitinib-resistant NSCLC (3 with wild-type and 2 with mutant-type EGFR) and 3 gefitinib-sensitive NSCLC (all with mutant type EGFR) cell lines to uncover the immediate responses associated with sensitivity to gefitinib. The data for gene expression profiling was analyzed in two ways; first, by comparing gefitinib-resistant versus -sensitive (including only EGFR-mutant cell lines); second, by comparing gefitinib-resistant versus -sensitive (including both EGFR-wild-type and mutant cell lines). Using strict criteria, we have identified 130 genes from the first and 135 genes from the second comparisons to be significantly modulated by gefitinib. To validate the data, 50 genes were selected for SYBR-Green based quantitative Real-time PCR, and 46 genes among the 50 were confirmed to be similarly regulated. The Ingenuity Pathway Analysis software was used to sort those genes by their biological functions, which revealed genes involved in the regulation of cell cycle, apoptosis, cell morphology, cellular growth and proliferation, gene transcription, and cell signaling. EphA2 and EFNA1 were two genes found to be regulated only in gefitinib-sensitive cell lines. Gefitinib increased the expression of EFNA1 3-fold and decreased the expression of EphA2 7 to 20-fold. These changes were also observed at the protein level by western blotting. The expression of other EFNA1 receptors did not change. Treatment with 5ug/ml of EphA2 antagonist, recombinant EphA2/Fc, decreased cell viability approximately 30% in sensitive but not in resistant cell lines. Additional experiments are underway to examine the role of EphA2 and EFNA1 in gefitinib response.