Activated Neu/ErbB-2 induces expression of the vascular endothelial growth factor gene by functional activation of the transcription factor Sp 1 Gu¨nter Finkenzeller 1,4 , Karin Weindel 1 , Wolfgang Zimmermann 2 , Gunnar Westin 3 & Dieter Marme´ 1 1 Institute of Molecular Oncology, Tumor Biology Center, Freiburg, Germany; 2 Tumor Immunology Group, Department of Urology, University Clinic Grosshadern, Ludwig-Maximilians-University, Mu ¨nchen, Germany; 3 Department of Surgical Sciences, Endocrine Unit, Uppsala University Hospital, Uppsala, Sweden; 4 Current address: Department of Plastic Surgery, University Hospital Freiburg, Freiburg, Germany Received 19 October 2003; accepted in revised form 9 January 2004 Key words: Neu, promoter, Sp 1, tumor angiogenesis, VEGF Abstract The neu (c-erbB-2 or HER2) proto-oncogene which encodes a receptor protein homologous to the epidermal growth factor receptor is overexpressed in 20%–30% of human breast and ovarian cancers. Oncogenic activation of Neu can also occur through multiple molecular mechanisms, including a point mutation in the transmembrane domain, deletion of the extracellular domain and short in-frame deletions of 7–12 amino acids in the extracellular region proximal to the transmembrane domain. Because of the highly vascularized phenotype of breast and ovarian cancers and the contribution of the Neu receptor to the development and progression of these tumors, we investigated the effect of Neu on the expression of the tumor angiogenesis factor VEGF. Expression of various activated Neu receptors but not wild-type Neu in Rat-1 cells, leads to increased VEGF expression on mRNA as well as on protein level. This effect is mediated by transcriptional activation of the VEGF promoter via a cluster of Sp 1 binding sites. Molecular analysis of the activation mechanism of Sp 1 revealed that neither the VEGF promoter binding activity of Sp 1 nor the expression of Sp 1 is affected by Neu transformation of the cells. Instead, functional Neu-induced transactivation of Sp 1 was observed by using a GAL4-based transactivation assay. These results demonstrate that functional changes of the transcription factor Sp 1 mediates a Neu-signaling cascade leading to VEGF promoter activation. Abbreviations: ECD – extracellular domain; EGFR – epidermal growth factor receptor; PDGF – platelet-derived growth factor; TK – thymidine kinase; TNF – tumor necrosis factor; VEGF – vascular endothelial growth factor; VHL – von Hippel Lindau Introduction The Neu (ErbB-2 or HER2) receptor protein belongs to the subclass I of receptor tyrosine kinases (for review, see [1, 2]). Currently, this family of receptors consist of the epidermal growth factor receptor (EGFR), Neu (ErbB-2/HER2), ErbB-3 (HER3) and ErbB-4 (HER4) [3–7]. Neu plays an important role in mitogenic stimu- lation of breast cancer cells and overexpression of Neu is directly correlated with a poor clinical outcome for breast cancer patients [8, 9]. The clinical relevance of Neu/ErbB-2 in human cancer was further established with the observation that approximately 20%–30% of human breast and ovarian cancer show increased expression of Neu/ErbB-2 [10, 11]. Furthermore, onco- genic activation of rat Neu can occur as a result of a point mutation in the transmembrane domain of the receptor, leading to a valine to glutamic acid substitu- tion [12]. This mutation results in a constitutive, ligand- independent activation of the tyrosine kinase [13]. Recently, it was shown that mammary tumorigenesis in transgenic mouse strains overexpressing Neu is accompanied by oncogenic activation of Neu through short in-frame deletions of 7–12 amino acids in the extracellular region proximal to the transmembrane domain [14]. Interestingly, a similar in-frame deletion in the extracellular domain (ECD) of the human ErbB-2 receptor was also detected in human breast tumor samples, suggesting that activating mutations in the ErbB-2 receptor may also contribute to the development and progression of human breast tumors [15]. Breast and ovarian cancers are characterized by a highly vascularized phenotype [16, 17]. In pathological Correspondence to: Gu¨nter Finkenzeller, Department of Plastic Surgery, University Hospital Freiburg, Hugstetterstr. 55, 79106 Freiburg, Germany. Tel: +49-761-2706367; Fax: +49-761-2706368; E-mail: finkenzeller@ch11.ukl.uni-freiburg.de Angiogenesis 7: 59–68, 2004. 59 Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands.