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PDF file - 25K, MIG6 mediates the effects of miR-148a on cell proliferation.

PDF file - 72K

PDF file - 247K, Transfection efficiency of U87 cells infected with lentiviruses encoding anti-miR-148, mCherry, and hygromycin resistance gene.

PDF file - 17K, BIM mediates the effects of miR-148a on aopotosis.

PDF file - 23K, miR-148a inhibits MIG6 and BIM mRNA expression.

PDF file - 30K, miR-148a levels after transfection with pre-miR-148a.

PDF file - 20K, Transfection efficiency of U87 cells infected with lentiviruses encoding anti-miR-148, mCherry, and hygromycin resistance gene.

PDF file - 72K

Great interest persists in useful prognostic and therapeutic targets in glioblastoma. In this study, we report the definition of miRNA (miR)-148a as a novel prognostic oncomiR in glioblastoma. miR-148a expression was elevated in human glioblastoma specimens, cell lines, and stem cells (GSC) compared with normal human brain and astrocytes. High levels were a risk indicator for glioblastoma patient survival. Functionally, miR-148a expression increased cell growth, survival, migration, and invasion in glioblastoma cells and GSCs and promoted GSC neurosphere formation. Two direct targets of miR-148a were identified, the EGF receptor (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. Finally, inhibition of miR-148a strongly suppressed GSC and glioblastoma xenograft growth in vivo. Taken together, our findings provide a comprehensive analysis of the prognostic value and oncogenic function of miR-148a in glioblastoma, further defining it as a potential target for glioblastoma therapy. Cancer Res; 74(5); 1541–53. ©2014 AACR.

In the present study, we examined the role of soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic in the formation of focal adhesions during cell spreading. CHO-K1 cells expressing a dominant-negative form of N-ethylmaleimide-sensitive factor (E329Q-NSF) were unable to spread as well as control cells and they formed focal adhesions (FAs) that were larger than those in control cells. FA formation was impaired in cells transfected with a dominant-negative form of RhoA, but, significantly, not in cells simultaneously expressing dominant-negative NSF. Treatment of E329Q-NSF-expressing cells with the ROCK inhibitor Y-27632 did inhibit FA formation. The results are consistent with a model of cell adhesion in which SNARE-mediated membrane traffic is required for both the elaboration of lamellipodia and the modulation of biochemical signals that control RhoA-mediated FA assembly.

In this study, the role of SNARE-mediated membrane traffic in regulating integrin localization was examined and the requirement for SNARE function in cellular spreading was quantitatively assessed. Membrane traffic was inhibited with the VAMP-specific catalytic light chain from tetanus toxin (TeTx-LC), a dominant-negative form (E329Q) of N-ethylmaleimide-sensitive fusion protein (NSF), and brefeldin A (BfA). Inhibition of membrane traffic with either E329Q-NSF or TeTx-LC, but not BfA, significantly inhibited spreading of CHO cells on fibronectin. Spreading was rescued in TeTx-LC-expressing cells by co-transfection with a TeTx-resistant cellubrevin/VAMP3. E329Q-NSF, a general inhibitor of SNARE function, was a more potent inhibitor of cell spreading than TeTx-LC, suggesting that tetanus toxin-insensitive SNAREs contribute to adhesion. It was found that E329Q-NSF prevented trafficking of alpha5beta1 integrins from a central Rab11-containing compartment to sites of protrusion during cell adhesion, while TeTx-LC delayed this trafficking. These results are consistent with a model of cellular adhesion that implicates SNARE function as an important component of integrin trafficking during the process of cell spreading.

Cell migration occurs as a highly-regulated cycle of cell polarization, membrane extension at the leading edge, adhesion, contraction of the cell body, and release from the extracellular matrix at the trailing edge. In this study, we investigated the involvement of SNARE-mediated membrane trafficking in cell migration. Using a dominant-negative form of the enzyme N-ethylmaleimide-sensitive factor as a general inhibitor of SNARE-mediated membrane traffic and tetanus toxin as a specific inhibitor of VAMP3/cellubrevin, we conducted transwell migration assays and determined that serum-induced migration of CHO-K1 cells is dependant upon SNARE function. Both VAMP3-mediated and VAMP3-independent traffic were involved in regulating this cell migration. Inhibition of SNARE-mediated membrane traffic led to a decrease in the protrusion of lamellipodia at the leading edge of migrating cells. Additionally, the reduction in cell migration resulting from the inhibition of SNARE function was accompanied by perturbation of a Rab11-containing alpha(5)beta(1) integrin compartment and a decrease in cell surface alpha(5)beta(1) without alteration to total cellular integrin levels. Together, these observations suggest that inhibition of SNARE-mediated traffic interferes with the intracellular distribution of integrins and with the membrane remodeling that contributes to lamellipodial extension during cell migration.

Integrin signaling is central to cell growth and differentiation, and critical for the processes of apoptosis, cell migration and wound repair. Previous research has demonstrated a requirement for SNARE-dependent membrane traffic in integrin trafficking, as well as cell adhesion and migration. The goal of the present research was to ascertain whether SNARE-dependent membrane trafficking is required specifically for integrin-mediated signaling. Membrane traffic was inhibited in Chinese hamster ovary cells by expression of dominant-negative (E329Q) N-ethylmaleimide-sensitive fusion protein (NSF) or a truncated form of the SNARE SNAP23. Integrin signaling was monitored as cells were plated on fibronectin under serum-free conditions. E329Q-NSF expression inhibited phosphorylation of focal adhesion kinase (FAK) on Tyr397 at early time points of adhesion. Phosphorylation of FAK on Tyr576, Tyr861 and Tyr925 was also impaired by expression of E329Q-NSF or truncated SNAP23, as was trafficking, localization and activation of Src and its interaction with FAK. Decreased FAK–Src interaction coincided with reduced Rac activation, decreased focal adhesion turnover, reduced Akt phosphorylation and lower phosphatidylinositol 3,4,5-trisphosphate levels in the cell periphery. Over-expression of plasma membrane-targeted Src or phosphatidylinositol 3-kinase (PI3K) rescued cell spreading and focal adhesion turnover. The results suggest that SNARE-dependent trafficking is required for integrin signaling through a FAK/Src/PI3K-dependent pathway.

Cellular remodeling of the extracellular matrix (ECM), an essential component of many physiological and pathological processes, is dependent on the trafficking and secretion of matrix metalloproteinases (MMPs). Soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic has documented roles in cell-ECM interactions and the present study specifically examines SNARE function in the trafficking of MMPs during ECM degradation. Using the invasive human fibrosarcoma cell line HT-1080, we demonstrate that a plasma membrane SNARE, SNAP23, and an endosomal v-SNARE, VAMP3 (also known as cellubrevin), partly colocalize with MMP2 and MMP9, and that inhibition of these SNAREs using dominant-negative SNARE mutants impaired secretion of the MMPs. Inhibition of VAMP3, SNAP23 or syntaxin-13 using dominant-negative SNARES, RNA interference or tetanus toxin impaired trafficking of membrane type 1 MMP to the cell surface. Consistent with these observations, we found that blocking the function of these SNAREs reduced the ability of HT-1080 cells to degrade a gelatin substrate in situ and impaired invasion of HT-1080 cells in vitro. The results reveal the importance of VAMP3, syntaxin-13 and SNAP23 in the trafficking of MMP during degradation of ECM substrates and subsequent cellular invasion.

In this study, we uncovered microRNA-148a (miR-148a) as a novel prognostic and oncogenic microRNA in glioblastoma (GBM). MiR-148a expression was significantly higher in human GBM tissues, cell lines and stem cells (GSCs) as compared to normal human brain and astrocytes. High miR-148a levels were a risk indicator for GBM patient survival. MiR-148a increased GBM cell and GSC growth, survival, migration, and invasion as well as GSC neurosphere formation. We identified the EGFR regulator, MIG6, and the apoptosis regulator, BIM as two direct targets of miR-148a and showed with rescue experiments that they mediate the oncogenic effects of miR-148a. Importantly, by inhibiting MIG6 expression, miR-148a reduced EGFR trafficking to the Rab7-expressing late endosome/lysosomal compartments. This coincided with a reduction of EGFR degradation and induction of EGFR expression and activation. Lastly, inhibition of miR-148a expression led to a strong inhibition of GSC and GBM xenograft growth in vivo. These findings represent the first comprehensive analysis of the role of miR-148a in GBM. They show that miR-148a expression levels are predictive of patient survival and that this microRNA enhances malignancy by inhibiting apoptosis and indirectly activating EGFR. These results also suggest that miR-148a is a potential target for GBM therapy. Citation Format: Jungeun Kim, Ying Zhang, Michael Skalski, Josie Hayes, Benjamin Kefas, David Schiff, Benjamin Purow, Sarah Parsons, Sean Lawler, Roger Abounader. microRNA-148a is a prognostic oncomiR that targets MIG6 and BIM to regulate EGFR and apoptosis in glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5222. doi:10.1158/1538-7445.AM2014-5222

Co-overexpression of the epidermal growth factor (EGF) receptor (EGFR) and c-Src often occurs in tumors, resulting in synergistic increases in oncogenic activity and correlation with poor patient prognosis in several cancers1. We have demonstrated that overexpression of EGFR and c-Src, and stimulation with EGF results in the transient translocation of both kinases to the mitochondria, which correlates with reduced adriamycin-induced apoptosis, and lowered O2 consumption and ATP production. Translocation requires EGFR endocytosis, as well as EGFR and c-Src catalytic activity2,3. It has been suggested that to translocate to the mitochondria, EGFR is retrograde trafficked to the ER, where it is removed from the membrane and then enters the mitochondria4. Here we show that EGFR remains associated with endosomal membranes upon translocation from the plasma membrane to mitochondria, as demonstrated by the colocalizaton of EGFR, mitochondria and extracellularly applied dextran. This translocation requires the activity of integrins, as cells plated on poly-L-lysine show significantly reduced mitochondrial-EGFR in response to EGF stimulation. Alternatively, adhesion to fibronectin in the absence of other extracellular signals was sufficient to activate EGFR and cause mitochondrial translocation. Adhesion resulted in phosphorylation on Y845, Y992, and Y1068, but not Y1045 of EGFR, while EGF stimulation, even on poly-L-lysine, resulted in the phosphorylation of all these sites. pY1045 is involved in Cbl recruitment and lysosomal degradation of EGFR5, suggesting adhesion stimulates EGFR signaling and mitochondrial translocation, while preventing lysosomal degradation. Future work will examine the role of Y1045 in mitochondrial EGFR translocation, mitochondrial function and resistance to chemotherapy. References: 1. Biscardi, J. S., Tice, D. A., and Parsons, S. J. (1999) Adv. Cancer Res. 76, 61-119. 2. Demory, M. L., et. al. (2009) J Biol Chem, 284: 36592-36604. 3. Boerner, J. L., Demory, M. L., Silva, C., and Parsons, S. J. (2004) Mol Cell Biol, 24: 7059-7071. 4. Cao, X., Zhu, H., Ali-Osman, F., and Lo, L.W. (2010) Mol. Cancer. 10:26. 5. Groval, L.M., Stang, E., Sorkin, A., and Madshus, I.H. (2004) Exp Cell Res, 300:388-95. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1202. doi:1538-7445.AM2012-1202

Background Intracellular membrane traffic is an essential component of the membrane remodeling that supports lamellipodium extension during cell adhesion. The membrane trafficking pathways that contribute to cell adhesion have not been fully elucidated, but recent studies have implicated SNARE proteins. Here, the functions of several SNAREs (SNAP23, VAMP3, VAMP4 and syntaxin13) are characterized during the processes of cell spreading and membrane ruffling. Results We report the first description of a SNARE complex, containing SNAP23, syntaxin13 and cellubrevin/VAMP3, that is induced by cell adhesion to an extracellular matrix. Impairing the function of the SNAREs in the complex using inhibitory SNARE domains disrupted the recycling endosome, impeded delivery of integrins to the cell surface, and reduced haptotactic cell migration and spreading. Blocking SNAP23 also inhibited the formation of PMA-stimulated, F-actin-rich membrane ruffles; however, membrane ruffle formation was not significantly altered by inhibition of VAMP3 or syntaxin13. In contrast, membrane ruffling, and not cell spreading, was sensitive to inhibition of two SNAREs within the biosynthetic secretory pathway, GS15 and VAMP4. Consistent with this, formation of a complex containing VAMP4 and SNAP23 was enhanced by treatment of cells with PMA. The results reveal a requirement for the function of a SNAP23-syntaxin13-VAMP3 complex in the formation of lamellipodia during cell adhesion and of a VAMP4-SNAP23-containing complex during PMA-induced membrane ruffling. Conclusions Our findings suggest that different SNARE-mediated trafficking pathways support membrane remodeling during ECM-induced lamellipodium extension and PMA-induced ruffle formation, pointing to important mechanistic differences between these processes.