Cellular resistance to multiple proapoptotic stimuli and invasion of surrounding brain tissue by migrating tumor cells are main obstacles to an e ective therapy for human malignant glioma. Here, we report that the Wnt family of embryonic di erentiation genes modulate growth of malignant glioma cells in vitro and in vivo and inhibit cellular migration in vitro. sFRPs (soluble Frizzled-related proteins) are soluble proteins that bind to Wnt and interfere with Wnt signaling. We ®nd that sFRP-1 and sFRP-2 are produced by the majority of longterm and ex vivo malignant glioma cell lines. Glioma cells that ectopically express sFRPs exhibit increased clonogenicity and enhanced resistance to serum starvation. In contrast, sFRPs do not modulate glioma cell susceptibility to apoptosis induced by the cytotoxic cytokines, CD95 (Fas/APO-1) ligand (CD95L) or Apo2 ligand/tumor necrosis factor-related apoptosisinducing ligand (Apo2L/TRAIL), or various cytotoxic drugs. sFRP-2 strongly promotes the growth of intracranial glioma xenografts in nude mice. In contrast, enhanced expression of sFRPs inhibits the motility of glioma cells in vitro. sFRP-mediated e ects on glioma cells are accompanied by decreased expression and activity of matrix metalloproteinase-2 (MMP-2) and decreased tyrosine phosphorylation of b-catenin. Thus, sFRPs promote survival under non-supportive conditions and inhibit the migration of glioma cells. We suggest that the regulation of these cellular processes involves expression of MMP-2 and tyrosine phosphorylation of bcatenin. These data support a function for Wnt signaling and its modulation by sFRPs in the biology of human gliomas. Oncogene (2000) 19, 4210 ± 4220.

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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.

Cutaneous melanoma represents one of the most aggressive human tumor entities possessing a high tendency to metastasize. Cancer cells frequently exploit a highly conserved developmental program, the epithelial-to-mesenchymal transition (EMT), to gain migratory and invasive properties promoting their metastatic spread. Cytoplasmic localization of the oncogenic transcription and translation factor Y-box binding protein 1 (YB-1) is a powerful inducer of EMT in breast carcinoma cells. Interestingly, EMT-like processes have also been observed in cutaneous melanoma despite its neural crest origin. Here, increased expression of YB-1 negatively affects patient survival in malignant melanoma and promotes melanoma cell tumorigenicity both and Intriguingly, this effect seems to be mainly mediated by cytoplasmic YB-1 that does not exhibit phosphorylation at serine-102 (S102). Moreover, S102 unphosphorylated YB-1 enhances the migratory and invasive potential of human melanoma cells in two-dimensional (2D) and three-dimensional (3D) culture systems and facilitates acquisition of a mesenchymal-like invasive phenotype in the chick embryo model. Collectively, these data demonstrate that the cytoplasmic activity of YB-1 stimulates tumorigenicity and metastatic potential of melanoma cells by promoting EMT-like properties. This study reveals for the first time that YB-1 efficiently drives tumorigenicity and invasiveness of melanoma cells in its S102 unphosphorylated cytoplasmic state and that YB-1 expression represents a negative prognostic factor in primary melanoma patients. .

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