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Research Articles: Therapeutics, Targets, and Development

Dual targeting of Raf and VEGF receptor 2 reduces growth and metastasis of pancreatic cancer through direct effects on tumor cells, endothelial cells, and pericytes

¹ Departments of Surgery and Surgical Oncology and ² Institute of Pathology, University of Regensburg Medical Center, Regensburg, Germany; and ³ Novartis Institutes for Biomedical Research, Cambridge, Massachusetts

Requests for reprints: Oliver Stoeltzing, Departments of Surgery and Surgical Oncology, University of Regensburg Medical Center, Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany. Phone: 49-941-944-6801; Fax: 49-941-944-6802. E-mail: oliver.stoeltzing{at}arcor.de

Abstract

The Ras/Raf/MEK pathway represents an important oncogenic signaling pathway in gastrointestinal malignancies, including pancreatic cancer. Although activating B-Raf mutations are infrequent in pancreatic cancer, we hypothesized that targeting Raf could be valuable for therapy of this cancer entity. Moreover, as vascular endothelial growth factor receptor 2 (VEGFR2) is involved in tumor angiogenesis, we sought to investigate the effects of dual inhibition of Raf and VEGFR2 on pancreatic tumor growth, vascularization, and metastasis. Effects of a Raf/VEGFR2 inhibitor (NVP-AAL881) on pancreatic cancer cells, endothelial cells, and vascular smooth muscle cells were determined by Western blotting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, and migration assays, respectively. Changes in the expression of VEGF-A or survivin were investigated by ELISA and/or real-time PCR. The growth-inhibitory effects of Raf/VEGFR2 inhibition were additionally evaluated in orthotopic tumor models. Results showed that various Raf isoforms were activated in pancreatic cancer cells and NVP-AAL881 diminished the activation of MEK, Akt, Erk, and also STAT3. Moreover, dual inhibition of Raf/VEGFR2 significantly reduced VEGF expression and impaired cancer cell migration. Importantly, besides blocking VEGF-induced Erk and SAPK phosphorylation in endothelial cells, the Raf inhibitor diminished STAT3 phosphorylation, independent of a VEGFR2 blockade, and reduced the expression of survivin. In addition, cell proliferation and migration of both endothelial cells and vascular smooth muscle cells were significantly reduced. In vivo, blocking Raf/VEGFR2 significantly inhibited orthotopic tumor growth and vascularization and reduced cancer metastasis. In conclusion, blocking Raf exerts growth-inhibitory effects on pancreatic tumor cells, endothelial cells, and pericytes and elicits antiangiogenic properties. Dual targeting of Raf and VEGFR2 appears to be a valid strategy for therapy of pancreatic cancer. [Mol Cancer Ther 2008;7(11):3509–18]

Grant support: German Cancer Aid (Deutsche Krebshilfe, Max-Eder Program; O. Stoeltzing) and University of Regensburg, Medical Faculty (ReForM; O. Stoeltzing and S.A. Lang).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: S.A. Lang and P. Schachtschneider contributed equally to this work.

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⁵ Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

Received 4/21/08; revised 7/23/08; accepted 9/ 1/08.