In vitro study of normoxic epidermal growth factor receptor–induced hypoxia‐inducible factor‐1‐alpha, vascular endothelial growth factor, and BNIP3 expression in head and neck squamous cell carcinoma cell lines: Implications for anti–epidermal growth factor receptor therapy

Abstract Background We previously showed that activation of epidermal growth factor receptor (EGFR) induces hypoxia inducible factor‐1α (HIF‐1α) in head and neck squamous cell carcinoma (HNSCC) cells. In this study, we have furthered this by investigating the mechanism of HIF‐1α activation by epidermal growth factor (EGF) and its association with the sensitivity to gefitinib. Methods EGFR/HIF‐1α signaling was tested by immunoblot, polymerase chain reaction (PCR), cell proliferation, and apoptosis assays. Results HIF‐1α accumulated in cells overexpressing EGF and phosphorylated epidermal growth factor receptor (pEGFR), phosphatidylinositol‐3‐kinase (pPI3K), and mitogen‐activated protein kinase (pMAPK). EGF‐induced expression of HIF‐1α and its targets, vascular endothelial growth factor (VEGF) and BNIP3, were blocked by gefitinib and PI3K‐inhibitors and MAPK‐inhibitors. HIF‐1α‐siRNAs abrogated EGF‐induced BNIP3 but not VEGF expression. Gefitinib inhibited cell proliferation and induced apoptosis more strongly in cells with constitutively active EGFR/HIF‐1α signaling than in cells lacking activation of these pathways. HIF‐1α‐siRNA treatment reduced sensitivity to gefitinib. Conclusion The search for molecular predictors of sensitivity to gefitinib in HNSCC should be extended to the activation status of EGFR‐downstream pathways, phosphorylated protein kinase B, pMAPK, and HIF‐1α. © 2014 Wiley Periodicals, Inc. Head Neck 37: 1150–1162, 2015