Great interest persists in useful prognostic and therapeutic targets in glioblastoma (GBM). In this study, we report the definition of miR-148a as a novel prognostic oncomiR in GBM. miR-148a expression was elevated in human GBM specimens, cell lines and stem cells (GSCs) compared to normal human brain and astrocytes. High levels were a risk indicator for GBM patient survival. Functionally, miR-148a expression increased cell growth, survival, migration, and invasion in GBM cells and GSCs and promoted GSC neurosphere formation. Two direct targets of miR-148a were identified, the EGFR regulator MIG6 and the apoptosis regulator BIM, which rescue experiments showed were essential to mediate the oncogenic activity of miR-148a. By inhibiting MIG6 expression, miR-148a reduced EGFR trafficking to Rab7-expressing compartments which includes late endosomes and lysosomes. This process coincided with reduced degradation and elevated expression and activation of EGFR. Lastly, inhibition of miR-148a strongly suppressed GSC and GBM xenograft growth in vivo. Taken together, our findings provide a comprehensive analysis of the prognostic value and oncogenic function of miR-148a in GBM, and further defining it as a potential target for GBM therapy.

evidence that SNAREs contribute to trafficking of MMPs. Syntaxin-4 (Miyata et al., 2004) and Ti-VAMP (tetanus-insensitive vesicleassociated membrane protein, also known as VAMP7) (Steffen et al., 2008) have been shown to be involved in cellular invasion and trafficking of MT1-MMP. SNAP25 (synaptosome-associated protein of 25 kDa) has been found to partly colocalize with MMP7 in epithelial cells (Gorodeski, 2007). There is also recent evidence that directly links SNARE-mediated intracellular membrane traffic to cell-ECM interactions (Al-Awar et al., 2000;Roberts et al., 2001). We (Gonon et al., 2005;Skalski and Coppolino, 2005;Tayeb et al., 2005), along with others (Proux-Gillardeaux et al., 2005), have reported that SNARE-mediated trafficking, including that mediated by the plasma membrane SNARE SNAP23 and the endosomal SNARE VAMP3, is required for cell adhesion and migration in cultured mammalian cell systems.In the present study, we examined the roles that SNAP23, VAMP3 and syntaxin-13 play in the secretion of MMPs and the degradation of ECM by HT-1080 fibrosarcoma cells. We report that SNAP23 and VAMP3 are required for secretion of MMP2 and MMP9, and that these SNAREs and syntaxin-13 are involved in trafficking of the membrane-bound MT1-MMP to the cell surface. Inhibiting the function of SNAP23, VAMP3 or syntaxin-13 using dominantnegative mutants of the SNAREs, RNA interference (RNAi) or tetanus toxin impaired the in situ degradation of gelatin by the cells. We present data that are the first to show that VAMP3, syntaxin-13 and SNAP23 are required for efficient invasion by HT-1080 cells in vitro. The impaired ECM degradation and reduced invasive capacity observed in cells as a consequence of inhibiting SNARE function suggests an important role for SNARE-mediated traffic in tumor progression. Results GFP-tagged SNAREs partly overlap with endogenous MMP2 and MMP9 in HT-1080 cellsMMP2 and MMP9 are secreted proteinases that have been shown to contribute to tumor-cell invasion and metastasis. Having identified roles for SNAP23 (Gonon et al., 2005) and VAMP3 (Proux-Gillardeaux et al., 2005;Skalski and Coppolino, 2005;Tayeb et al., 2005) in the non-invasive motility of cells, we sought to investigate the involvement of these SNAREs in the trafficking and secretion of MMPs during cell invasion. SNAP23 is a plasma membrane SNARE found in many cell types, and VAMP3 is a known binding partner of SNAP23 (Hepp et al., 1999). To study MMP activity during cell invasion, we used the human cell line HT-1080, which is derived from a highly invasive fibrosarcoma and is known to express MMP2 and MMP9 along with MT1-MMP (Ginestra et al., 1997;Stanton et al., 1998). The subcellular distributions of endogenous MMP2 and MMP9 were compared with those of GFP-SNAP23 and GFP-VAMP3, and it was observed that SNAP23 ( Fig. 1A) and VAMP3 (Fig. 1B) partly colocalized with both MMP2 and MMP9 at the periphery of HT-1080 cells in response to treatment of the cells with the tumor promoter PMA (phorbol 12-myristate 13-acetate). PMA was added to...

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