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Interactions between cancer cells and their microenvironment are critical for the development and progression of solid tumors. This study is the first to examine the role of all members of the ErbB tyrosine kinase receptors (epidermal growth factor receptor [EGFR], ErbB-2, ErbB-3, or ErbB-4), expressed singly or as paired receptor combinations, in the regulation of angiogenesis both in vitro and in vivo. Comparison of all receptor combinations reveals that EGFR/ErbB-2 and ErbB-2/ErbB-3 heterodimers are the most potent inducers of vascular endothelial growth factor (VEGF) mRNA expression compared with EGFR/ErbB-3, EGFR/ErbB-4, ErbB-2/ErbB-4, and ErbB-3/ErbB-4. Immunohistochemistry of tumor xenografts overexpressing these heterodimers shows increased VEGF expression and remarkably enhanced vascularity. Enhanced VEGF expression is associated with increased VEGF transcription. Deletional analysis reveals that ErbB-mediated transcriptional up-regulation of VEGF involves a hypoxia-inducible factor 1-independent responsive region located between nucleotides −88 to −66 of the VEGF promoter. Mutational analysis reveals that the Sp-1 and AP-2 transcription factor binding elements within this region are required for up-regulation of VEGF by heregulin β1 and that this up-regulation is dependent on the activity of extracellular signal-related protein kinases. These results emphasize the biological implications of cell signaling diversity among members of the ErbB receptor family in regulation of the tumor microenvironment.

The vascular endothelial growth factor (VEGF) is implicated in the progression of cancers. Its expression is well correlated with tumor growth and metastases. The availability of a rapid and sensitive method to detect the amounts of VEGF mRNA in biological samples of limited size, very small biopsies, or samples containing relatively few cells could provide an interesting prognostic tool for clinicians. We have developed an RT-PCR method that allows us to detect the VEGF mRNA from as little as 3 mg total mRNA. We have also shown that this protocol can be generalized to all cell lines tested. This method constitutes a very potent tool for the analysis of VEGF mRNA expression in different contexts.

Vascular endothelial growth factor (VEGF), a potent agonist secreted by virtually all cells, controls migration and division of vascular endothelial cells. Disruption of one VEGF allele in mice has revealed a dramatic lethal effect in early embryogenesis, suggesting a key role in vasculogenesis. We analyzed the regulation of VEGF mRNA in normal and transformed CCL39 fibroblasts and then dissected the VEGF promoter to identify the signaling pathway(s) controlling the activation of this promoter in response to growth factors, oncogenes, and hypoxic stress. We demonstrated that the p42p44 MAP kinase signaling cascade controls VEGF expression at least at two levels. In normoxic conditions, MAPKs activate the VEGF promoter at the proximal (?88?66) region where Sp-1AP-2 factors bind. Activation of p42p44 MAPKs is sufficient to turn on VEGF mRNA. At low O2tension, hypoxia inducible factor-1? (HIF-1?), a limiting factor rapidly stabilized and phosphorylated, plays a key role in the expression of several genes including VEGF. We demonstrated that p42p44MAPKs stoichiometrically phosphorylate HIF-1?in vitroand that HIF-1-dependent VEGF gene expression is strongly enhanced by the exclusive activation of p42p44MAPKs. Finally, we demonstrated that the regulation of p42p44MAPK activity is critical for controlling proliferation and growth arrest of vascular endothelial cells at confluency. These results point to at least three major targets of angiogenesis where p42p44 MAP kinases exert a determinant action.