Mutant epidermal growth factor receptor enhances induction of vascular endothelial growth factor by hypoxia and insulin-like growth factor-1 via a PI3 kinase dependent pathway

Over-expression of truncated epidermal growth factor receptor (EGFR) occurs in a variety of malignancies including glioblastoma multiforme, breast and lung cancer. The truncation deletes an extracellular domain and results in constitutive activation of the receptor. NIH3T3 cells were transfected wit h full length or truncated human EGFR and differences in growth rates in vi vo and in vitro analysed. A growth advantage was seen for cells expressing mutant receptor compared to full length EGFR in vivo only. Administration o f an anti-mutant EGFR antibody to mice transiently reduced the growth rates of mutant tumours, confirming that the mutant receptor itself was importan t in this enhanced tumorigenicity. This showed that stimuli present in vivo and not in vitro may be contributing to growth. We therefore analysed the regulation of the angiogenic factor vascular endothelial growth factor (VEG F). Although levels of secreted VEGF did not differ significantly between w ild-type and mutant EGFR cell lines when grown in vitro under normoxic cond itions, following exposure to 0.1% hypoxia levels of VEGF produced by mutan t cells increased 3.5-6.6 fold compared to 2 or less for full length EGFR c ells. The fold induction was influenced by experimental conditions, includi ng cell confluence and percentage of fetal bovine serum, but was consistent ly higher for mutant cell lines. The increase in VEGF under hypoxic conditi ons was blocked by the addition of PI3 kinase inhibitors. indicating that t he latter pathway is important in the hypoxic stress response. Basal levels were not affected. Addition of insulin-like growth factor-1 also increased levels of VEGF under normoxic conditions in the mutant cells and no furthe r increase was seen when added to cells exposed to 0.1% oxygen, indicating that levels of VEGF were already maximally stimulated. These results show t hat the mutant EGFR interacts with other growth factors and hypoxia to regu late VEGF via a PI3 kinase pathway, and suggests a specific role for anti-m utant EGFR antibodies and PI3 kinase inhibitors as therapy of this specific tumour target. (C) 2001 Cancer Research Campaign.