Abstract #4216: A role for c-Src in EGFR-induced upregulation of HIF-1\#945;

Non-small cell lung cancer (NSCLC) is the leading cause of cancer related deaths in the United States and worldwide. Receptor tyrosine kinase (RTK) signaling through Epidermal Growth Factor Receptor (EGFR) has been shown to play a key role in the development and progression of NSCLC. Two activating mutations within EGFR, L858R and del19, have been demonstrated to lead to oncogene addiction and have also been shown to confer sensitivity to targeted EGFR inhibitors (e.g. erlotinib and gefitinib). Earlier work has demonstrated a crucial role for the EGFR downstream effector c-Src in the upregulation of Vascular Endothelial Growth Factor (VEGF), a HIF-1\#945; regulated gene. We have identified a correlation between mutant EGFR and elevated HIF-1\#945; protein levels. Thus, we hypothesized that c-Src may be a critical downstream effector of mutant EGFR and may have a central role in the upregulation of HIF-1\#945; signaling by activated EGFR. NSCLC cell lines were treated with erlotinib to demonstrate a role for mutant EGFR in the upregulation of HIF-1\#945; and subsequently VEGF protein levels. HCC827 NSCLC cells, expressing the EGFR sensitizing mutant, had significantly reduced HIF-1\#945; protein levels and VEGF secretion when treated with the EGFR inhibitor, whereas, A549 cells, expressing the wild-type EGFR, were minimally effected by erlotinib treatment, compared their respective untreated controls. Furthermore, transient transfection with del19 EGFR led to increased VEGF promoter activity as demonstrated by a luciferase assay. These data demonstrate a central role for mutant EGFR signaling in the upregulation of HIF-1\#945; protein levels and VEGF secretion. Hence, signaling through mutant EGFR uncouples the normal regulation of HIF-1\#945; from hypoxia leading to aberrant expression of the angiogenic growth factor, VEGF. To identify critical signaling pathways potentially involved in the EGFR-induced up-regulation of HIF-1\#945;, multiple inhibitors were utilized. Inhibition of Mek did not affect HIF-1\#945; protein levels compared to controls, whereas inhibition of phosphotidylinositol-3-kinase (PI3K) led to a 50% reduction in HIF-1\#945; protein levels versus untreated cells. The Src family inhibitor PP2 led to a complete reduction of HIF-1\#945; protein, and similar results were seen with erlotinib. Moreover, erlotinib and PP2 led to a partial reduction of HIF-2\#945; protein levels. Collectively, our data demonstrate that NSCLC cells expressing activating mutations in EGFR have elevated HIF-1\#945; protein levels and that the c-Src signaling pathway may play a central role in the uncoupling of HIF-1\#945; signaling from hypoxia-induced regulation. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4216.