Effect of the integrin inhibitor cilengitide on TGF-beta signaling.

Abstract

2055 Background: Glioblastoma is the most malignant form of intrinsic brain tumors. One of its characteristic features, angiogenesis, is mediated by the expression of integrins αvβ3 and αvβ5 on tumor-associated vasculature. Cilengitide is a synthetic peptide which inhibits ligand binding to the αvβ3 and αvβ5 integrins. The addition of cilengitide to the standard treatment regimen of radiochemotherapy in patients with newly diagnosed glioblastoma demonstrated promising results without significant toxicity which led to the initiation of a randomized phase registration III trial. Besides other functions, av-integrins can be involved in the activation of the cytokine transforming growth factor (TGF)-β which contributes to the malignant phenotype of glioma cells. Methods: Integrin expression was examined by immunohistochemistry and flow cytometry. The effects of cilengitide on TGF-β signaling in glioma cells were investigated by PCR, immunoblot and ELISA. Further assessments included reporter assays and glioma xenografts in nude mice. Results: The target integrins of cilengitide are expressed in glioblastoma blood vessels and tumor cells. Exposure to cilengitide results in detachment and reduced phosphorylation of Smad2 in most glioma cell lines, including stem-like glioma cells. Furthermore, exposure of glioma cells to cilengitide is associated with reduced TGF-β-mediated reporter gene activity. Smad2 phosphorylation, but not attachment, can be rescued by co-exposure to recombinant TGF-β. Cilengitide results in decreased TGF-β1 and TGF-β2 mRNA and protein expression and loss of transcriptional activity of the TGF-β promoter. Blocking antibodies to integrins αv, αvβ3 or αvβ5, or silencing of integrins αv, β3, β5 or β8 by RNA interference mimics the effects of cilengitide on TGF-β expression. Intracranial LN-308 glioma xenografts in nude mice display reduced pSmad2 levels in response to cilengitide. Conclusions: This study demonstrate a novel mechanism which may in part mediate anti-glioblastoma activity of cilengitide. These anti-TGF-β properties of cilengitide might be exploited in future clinical trials.