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

Abstract

Transforming growth factor-β is a central mediator of the malignant phenotype of glioblastoma, the most common and malignant form of intrinsic brain tumours. Transforming growth factor-β 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-β. We report that αvβ3, αvβ5 and αvβ8 integrins are broadly expressed not only in glioblastoma blood vessels but also in tumour cells. Exposure to αv, β3 or β5 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-β-mediated reporter gene activity, coinciding with reduced transforming growth factor-β protein levels in the supernatant. Time course experiments indicated that the loss of transforming growth factor-β 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-β gene transcription as confirmed by decreased activity of the transforming growth factor-β gene promoter and decreased transforming growth factor-β(1) and transforming growth factor-β(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-β-controlled features of malignancy including invasiveness, stemness and immunosuppression in human glioblastoma.

The aryl hydrocarbon receptor links integrin signaling to the TGF-β pathway

“…These findings were further corroborated using RNA interference to specifically silence single αv-integrins. As shown before, exposure to integrin-specifc siRNA molecules resulted in reduced expression of the targeted integrin in LN-308 glioma cells (11). Integrin gene silencing was paralleled by a significant reduction of DRE reporter activity (Fig.…”

“…Based on the findings that integrin inhibition interferes with TGF-β signaling (11) and that AhR controls the TGF-β pathway in human glioma cells (8) suggesting that this effect is AhR-mediated (Fig. 4A, bottom).…”

“…4C). Based on the finding that murine hepatocytes detached and died upon integrin inhibition (data not shown) and previous work demonstrating that detachment may also affect TGF-β signaling (11,18), we extended our investigations to human HaCaT keratinocytes, with lentivirus-mediated AhR silencing, which neither detached nor died upon integrin inhibition (data not shown). Compared to control cells, AhR and CYP1A1 mRNA levels were strongly diminished in AhR-deficient keratinocytes.…”

“…TGF-β is a central mediator of the malignant phenotype of gliomas that promotes growth, invasiveness, angiogenesis as well as 4 stemness and contributes to the immune evasion of these tumors (9,10). Recently, we have shown that inhibition of αv integrins reduces TGF-β expression and down-stream signaling (11).…”

Overview of Transforming Growth Factor β Superfamily Involvement in Glioblastoma Initiation and Progression

“…When treated with integrin inhibitors, glioma cells undergo atypical cell death in combination of anoikis, apoptosis and necrosis, which can be rescued by TGFβ treatment (Silginer et al, 2014). Cilengitide is an antiangiogenic small molecule, an inhibitor of αvβ3 and αvβ5 integrin, that prevents TGFβ's tumor promotion in glioma cells by decreasing TGFβ level and dephosphorylating SMAD2 (Roth et al, 2013). After promising results in animal models (Mikkelsen et al, 2009), this molecule has been translated to clinical trials with as-yet unsatisfactory results (Stupp et al;Reardon et al, 2008;Eisele et al, 2014) Blocking the abnormal signal at its origin by inhibiting TGFβ receptor is another strategy to prevent its harmful effect.…”