Epidermal growth factor stimulates vascular endothelial growth factor production by human malignant glioma cells: a model of glioblastoma multiforme pathophysiology.

Abstract

Hypervascularity, focal necrosis, persistent cerebral edema, and rapid cellular proliferation are key histopathologic features of glioblastoma multiforme (GBM), the most common and malignant of human brain tumors. By immunoperoxidase and immunofluorescence, we definitively have demonstrated the presence of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFr) in five out of five human glioma cell lines (U-251MG, U-105MG, D-65MG, D-54MG, and CH-235MG) and in eight human GBM tumor surgical specimens. In vitro experiments with glioma cell lines revealed a consistent and reliable relation between EGFr activation and VEGF production; namely, EGF (1-20 ng/ml) stimulation of glioma cells resulted in a 25-125% increase in secretion of bioactive VEGF. Conditioned media (CM) prepared from EGF-stimulated glioma cell lines produced significant increases in cytosolic free intracellular concentrations of Ca2+ ([Ca2+]i) in human umbilical vein endothelial cells (HUVECs). Neither EGF alone or CM from glioma cultures prepared in the absence of EGF induced [Ca2+]i increases in HUVECs. Preincubation of glioma CM with A4.6.1, a monoclonal antibody to VEGF, completely abolished VEGF-mediated [Ca2+]i transients in HUVECs. Likewise, induction by glioma-derived CM of von Willebrand factor release from HUVECs was completely blocked by A4.6.1 pretreatment. These observations provide a key link in understanding the basic cellular pathophysiology of GBM tumor angiogenesis, increased vascular permeability, and cellular proliferation. Specifically, EGF activation of EGFr expressed on glioma cells leads to enhanced secretion of VEGF by glioma cells. VEGF released by glioma cells in situ most likely accounts for pathognomonic histopathologic and clinical features of GBM tumors in patients, including striking tumor angiogenesis, increased cerebral edema and hypercoagulability manifesting as focal tumor necrosis, deep vein thrombosis, or pulmonary embolism.

Overexpression of vascular endothelial growth factor induces cell transformation in cooperation with fibroblast growth factor 2

“…This Interactions between VEGF and other growth factors have already been described. EGF stimulates VEGF production in glioblastoma cells (Goldman et al, 1993), whereas TGF-b induces VEGF mRNA and protein in ®broblastic and epithelial cells (Pertovaara et al, 1994). VEGF synthesis is also increased by FGF-2 in rat vascular smooth muscle cells .…”

Oncogenes and tumor angiogenesis: the HPV-16 E6 oncoprotein activates the vascular endothelial growth factor (VEGF) gene promoter in a p53 independent manner

“…Moreover, HeLa cells have been shown to posses high expression of EGFR, which appears to be driven by their intrinsic expression of the HPV proteins E6 and E7 (Hu et al, 1997). In addition, activation of the EGFR (growth factor/receptor) system has been shown to upregulate VEGF expression (Viloria-Petit et al, 1997;Detmar et al, 1994;Goldman et al, 1993;Gille et al, 1997). Therefore we hypothesized that in HPV positive cells upregulation of VEGF may be EGFR-mediated.…”

“…Other genetic changes might include additional virus-encoded products (such as E7) as well as other cellular oncoproteins. Regarding HeLa cells, we examined the possible contribution of the TGFa/EGF receptor signaling system due to its demonstrated over-activity in HeLa cells (Derynck et al, 1987;Hu et al, 1997) and also because EGFR activation can trigger VEGF expression (Viloria-Petit et al, 1997;Detmar et al, 1994;Goldman et al, 1993;Gille et al, 1997). However, using speci®c blocking antibodies to both TGFa and EGF receptor we showed that this receptorligand system is not involved in VEGF expression in HeLa cells.…”

A Phase I Dose-Escalation Study of ZD6474 in Japanese Patients with Solid, Malignant Tumors

“…13,14 One consequence of upregulated EGFR tyrosine kinase activity is increased expression of proangiogenic factors, including VEGF, 15,16 which may lead to possible paracrine and autocrine stimulation of angiogenesis.…”