Integrin control of the transforming growth factor-β pathway in glioblastoma

Transforming growth factor-beta is a central mediator of the malignant phenotype of glioblastoma, the most common and malignant form of intrinsic brain tumours. Transforming growth factor-beta promotes invasiveness and angiogenesis, maintains cancer cell stemness and induces profound immunosuppression in the host. Integrins regulate cellular adhesion and transmit signals important for cell survival, proliferation, differentiation and motility, and may be involved in the activation of transforming growth factor-beta. We report that alpha v beta 3, alpha v beta 5 and alpha v beta 8 integrins are broadly expressed not only in glioblastoma blood vessels but also in tumour cells. Exposure to alpha v, beta 3 or beta 5 neutralizing antibodies, RNA interference-mediated integrin gene silencing or pharmacological integrin inhibition using the cyclic RGD peptide EMD 121974 (cilengitide) results in reduced phosphorylation of Smad2 in most glioma cell lines, including glioma-initiating cell lines and reduced transforming growth factor-beta-mediated reporter gene activity, coinciding with reduced transforming growth factor-beta protein levels in the supernatant. Time course experiments indicated that the loss of transforming growth factor-beta bioactivity due to integrin inhibition likely results from two distinct mechanisms: an early effect on activation of preformed inactive protein, and second, major effect on transforming growth factor-beta gene transcription as confirmed by decreased activity of the transforming growth factor-beta gene promoter and decreased transforming growth factor-beta 1 and transforming growth factor-beta 2 messenger RNA expression levels. In vivo, EMD 121974 (cilengitide), which is currently in late clinical development as an antiangiogenic agent in newly diagnosed glioblastoma, was a weak antagonist of pSmad2 phosphorylation. These results validate integrin inhibition as a promising strategy not only to inhibit angiogenesis, but also to block transforming growth factor-beta-controlled features of malignancy including invasiveness, stemness and immunosuppression in human glioblastoma.