Ezrin-Dependent Promotion of Glioma Cell Clonogenicity, Motility, and Invasion Mediated by BCL-2 and Transforming Growth Factor-β₂

Abstract

Ezrin belongs to the ezrin-radixin-moesin family proteins, which cross-link actin cytoskeleton and plasma membrane. Malignant glioma cells are paradigmatic for their strong migratory and invasive properties. Here, we report that the expression of dominant-negative ezrins inhibits clonogenicity, migration, and invasiveness of human malignant glioma cells. Furthermore, dominant-negative ezrins block hepatocyte growth factor (HGF)-mediated stimulation of clonogenicity and migration, without altering HGF-induced protein kinase B/Akt and focal adhesion kinase phosphorylation. Glioma cells expressing dominant-negative ezrins exhibit a shift of the BCL-2/ BAX rheostat toward apoptosis, reduced ␣ V ␤ 3 integrin expression and reduced matrix metalloproteinase (MMP) expression and activity. These changes are associated with a dramatic loss of transforming growth factor ␤ 2 (TGF-␤ 2 ) release. Exogenous supplementation of TGF-␤ 2 overcomes the inhibitory effects of dominant-negative ezrins on migration and clonogenicity. A neutralizing TGF-␤ 2 antibody mimics the effects of dominantnegative ezrins on clonogenicity and migration. Exogenous HGF markedly induces TGF-␤ 2 protein levels, and a neutralizing TGF-␤ 2 antibody abolishes the HGF-mediated increase in glioma cell motility. Finally, TGF-␤ 2 does not modulate BCL-2 or BAX expression, but BCL-2 gene transfer increases the levels of latent and active TGF-␤ 2 . Intracranial xenografts of U87MG glioma cells transfected with the dominant-negative ezrins in athymic mice grow to significantly smaller volumes, and the median survival of these mice is 50 d compared with 28 d in the control group. These data define a novel pathway for HGFinduced glioma cell migration and invasion, which requires ezrin, changes in the BCL-2/BAX rheostat, and the induction of TGF-␤ 2 expression in vitro, and underscore the important role of HGF signaling in vivo.

BCL-xL: time-dependent dissociation between modulation of apoptosis and invasiveness in human malignant glioma cells

“…However, the sensitivity towards chemotherapeutics, death ligands or therapeutic irradiation remained unaltered. 10 Since reduction of BCL-2 expression using antisense oligonucleotides strongly reduced survival of LNT-229 cells (data not shown) and since BCL-x L is more relevant in human gliomas, we chose the alternative approach of generating conditionally BCL-x L -overexpressing cells (Figures 1 and 2). We postulated that subtle changes in the expression level of BCL-2 family proteins would affect motility, but might not be sufficient to alter resistance towards apoptosis.…”

“…Viable cell density following 12 h of incubation with CD95L (120 U/l) and CHX (10 mg/ml) was assessed by crystal violet staining. 10 …”

“…7,8 Accordingly, expression of TGF-b 2 correlates with glioma cell invasiveness 9 and inhibition of TFG-b by a neutralizing antibody or by a small molecule TGF-b receptor antagonist, SD-208, significantly reduced glioma cell migration. 10,11 Ectopic expression of the antiapoptotic BCL-2 protein conferred a more migratory and invasive phenotype to two human malignant glioma cell lines in vitro. 12 The inter-relation between proteins of the BCL-2 family and motility has also been shown for other tumor types.…”

MicroRNA-125b-2 confers human glioblastoma stem cells resistance to temozolomide through the mitochondrial pathway of apoptosis

“…Pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins are known to regulate the apoptosis of glioma cells (10). Bcl-2, which resists induction of apoptosis, constitutes one major obstacle to radiotherapy and chemotherapy in many cancer cells (11). Bax plays a major role in the apoptotic response of glioblastoma multiforme cells, the high expression of which is shown to correlate with an increased survival of GBM patients (12).…”

Is there a world beyond bevacizumab in targeting angiogenesis in glioblastoma?

“…The MET ligand HGF also binds NRP-1 and thus may be related to VEGFR-2 proangiogenic signaling [72]. However, HGF-MET signaling is also involved in processes of migration and invasion in glioma and thus represents a promising therapeutic target [73]. In a mouse glioma model the proliferation of tumor and endothelial cells was reduced, resulting in a dose-dependent inhibition of tumor growth [74].…”