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Detroit, MI, United States

Lee H.K.,Davidson Laboratory of Cell Signaling and Tumorigenesis | Lee H.K.,Hermelin Brain Tumor Center | Finniss S.,Davidson Laboratory of Cell Signaling and Tumorigenesis | Finniss S.,Hermelin Brain Tumor Center | And 10 more authors.
Oncotarget | Year: 2015

Glioblastoma (GBM), the most aggressive primary brain tumors, are highly infiltrative. Although GBM express high Ras activity and Ras proteins have been implicated in gliomagenesis, Ras-activating mutations are not frequent in these tumors. RasGRP3, an important signaling protein responsive to diacylglycerol (DAG), increases Ras activation. Here, we examined the expression and functions of RasGRP3 in GBM and glioma cells. RasGRP3 expression was upregulated in GBM specimens and glioma stem cells compared with normal brains and neural stem cells, respectively. RasGRP3 activated Ras and Rap1 in glioma cells and increased cell migration and invasion partially via Ras activation. Using pull-down assay and mass spectroscopy we identified the actin-related protein, Arp3, as a novel interacting protein of RasGRP3. The interaction of RasGRP3 and Arp3 was validated by immunofluorescence staining and co-immunoprecipitation, and PMA, which activates RasGRP3 and induces its translocation to the peri-nuclear region, increased the association of Arp3 and RasGRP3. Arp3 was upregulated in GBM, regulated cell spreading and migration and its silencing partially decreased these effects of RasGRP3 in glioma cells. In summary, RasGRP3 acts as an important integrating signaling protein of the DAG and Ras signaling pathways and actin polymerization and represents an important therapeutic target in GBM. Source


Ziv-Av A.,Bar - Ilan University | Giladi N.D.,Bar - Ilan University | Lee H.K.,Hermelin Brain Tumor Center | Cazacu S.,Hermelin Brain Tumor Center | And 7 more authors.
Oncotarget | Year: 2015

Glioblastoma (GBM) are characterized by increased invasion into the surrounding normal brain tissue. RTVP-1 is highly expressed in GBM and regulates the migration and invasion of glioma cells. To further study RTVP-1 effects we performed a pull-down assay using His-tagged RTVP-1 followed by mass spectrometry and found that RTVP-1 was associated with the actin polymerization regulator, N-WASP. This association was further validated by co-immunoprecipitation and FRET analysis. We found that RTVP-1 increased cell spreading, migration and invasion and these effects were at least partly mediated by N-WASP. Another protein which was found by the pull-down assay to interact with RTVP-1 is hnRNPK. This protein has been recently reported to associate with and to inhibit the effect of N-WASP on cell spreading. hnRNPK decreased cell migration, spreading and invasion in glioma cells. Using co-immunoprecipitation we validated the interactions of hnRNPK with N-WASP and RTVP-1 in glioma cells. In addition, we found that overexpression of RTVP-1 decreased the association of N-WASP and hnRNPK. In summary, we report that RTVP-1 regulates glioma cell spreading, migration and invasion and that these effects are mediated via interaction with N-WASP and by interfering with the inhibitory effect of hnRNPK on the function of this protein. Source


Lee H.K.,Hermelin Brain Tumor Center | Finniss S.,Hermelin Brain Tumor Center | Cazacu S.,Hermelin Brain Tumor Center | Xiang C.,Hermelin Brain Tumor Center | And 2 more authors.
Stem Cells and Development | Year: 2014

MicroRNAs (miRNAs) are potential therapeutic targets in a variety of pathological conditions in the brain; however, their clinical application is hampered by lack of efficient delivery modes. Mesenchymal stromal stem cells (MSCs) migrate to sites of injury and inflammation and exert therapeutic effects in various neurological disorders. Here, we examined the ability of MSCs to deliver exogenous miRNA mimics and pre-miRNAs to human neural progenitor cells (NPCs) and astrocytes and characterized the functional impact of this delivery. We found that MSCs efficiently delivered fluorescent-labeled miR-124 and miR-145 mimics to cocultured NPCs and astrocytes. We further demonstrated the delivery of the miRNAs using novel reporter plasmids that contain a sequence complementary to miR-124 or miR-145 downstream of luciferase or mCherry. Binding of the specific miRNAs to these sequences results in decreased luciferase activity or mCherry fluorescence and therefore enable analysis of miRNA delivery in living cells. The delivered exogenous miR-124 significantly decreased the expression of the target gene Sox9 by targeting its 3′-UTR, and increased the neuronal differentiation of the NPCs. In addition, the delivered miR-124 increased the expression of the glutamate transporters, EAAT1 in NPCs and EAAT2 in both NPCs and astrocytes. Similar results were obtained with MSCs transfected with pre-miR-124. The miRNA delivery was mediated by MSC-derived exosomes and was cell contact independent. These results suggest that MSCs can functionally deliver exogenous miRNAs to neural cells and provide an efficient route of therapeutic miRNA delivery to the brain in pathological conditions with clinical implications for regenerative medicine. © 2014 Mary Ann Liebert, Inc. Source


Alam R.,Hermelin Brain Tumor Center | Schultz C.R.,Hermelin Brain Tumor Center | Golembieski W.A.,Hermelin Brain Tumor Center | Poisson L.M.,Ford Motor Company | Rempel S.A.,Hermelin Brain Tumor Center
Neuro-Oncology | Year: 2013

Background. Secreted protein acidic and rich in cysteine (SPARC) is overexpressed in astrocytomas (World Health Organization grades II-IV). We previously demonstrated that SPARC promotes glioma migration and invasion-in part, by activating the P38 mitogen-activated protein kinase (MAPK)-heat shock protein (HSP)27 signaling pathway. The commonly lost tumor suppressor phosphatase and tensin homolog (PTEN) suppresses SPARC-induced migration, which is accompanied by suppression of Shc-Ras-Raf-MEK-ERK1/2 and Akt signaling. As PTEN completely suppresses SPARC-induced migration, we proposed that PTEN must also interfere with SPARC-induced HSP27 signaling. Therefore, this study determined the effects of PTEN expression on SPARC-induced expression and phosphorylation of HSP27. Methods. Control and SPARC-expressing clones transfected with control- or PTEN-expression plasmids were plated on fibronectin-coated tissue culture plates for 3, 6, 24, and 48 h and then lysed. Equal amounts of protein were subjected to Western blot and densitometric analyses. Results. The results show that SPARC enhances phosphorylated (p)P38 MAPK, phosphorylated MAPKactivated protein kinase 2 (pMAPKAPK2), and serine (Ser)78 HSP27 phosphorylation relative to total HSP27. PTEN suppresses pAkt and pMAPKAPK2, suggesting that PTEN effects are downstream of pP38 MAPK. PTEN suppressed SPARC-induced sustained phosphorylation at Ser78 HSP27. As the level of total HSP27 differed based on the presence of SPARC or PTEN, the ratios of phosphorylation-specific to total HSP27 were examined. The data demonstrate that SPARC-induced phosphorylation at Ser78 remains elevated despite increasing levels of total HSP27. In contrast, PTEN inhibits SPARC-induced increases in Ser78 HSP27 phosphorylation relative to total HSP27. Conclusion. These data describe a novel mechanism whereby PTEN inhibits SPARC-induced migration through suppression and differential regulation of pAkt and the P38 MAPK-MAPKAPK2-HSP27 signaling pathway. © The Author(s) 2013. Source


Thomas S.L.,Hermelin Brain Tumor Center | Alam R.,Hermelin Brain Tumor Center | Lemke N.,Hermelin Brain Tumor Center | Schultz L.R.,Ford Motor Company | And 2 more authors.
Neuro-Oncology | Year: 2010

SPARC (secreted protein acidic and rich in cysteine) is expressed in all grades of astrocytoma, including glioblastoma (GBM). SPARC suppresses glioma growth but promotes migration and invasion by mediating integrin and growth factor receptor-regulated kinases and their downstream effectors. PTEN (phosphatase and tensin homolog deleted on chromosome 10), which is commonly lost in primary GBMs, negatively regulates proliferation and migration by inhibiting some of the same SPARC-mediated signaling pathways. This study determined whether PTEN reconstitution in PTEN-mutant, SPARC-expressing U87MG cells could further suppress proliferation and tumor growth but inhibit migration and invasion in SPARC-expressing cells in vitro and in vivo, and thereby prolong survival in animals with xenograft tumors. In vitro, PTEN reduced proliferation and migration in both SPARCexpressing and control cells, with a greater suppression in SPARC-expressing cells. PTEN reconstitution suppressed AKT activation in SPARC-expressing and control cells but suppressed the SHC-RAF-ERK signaling pathway only in SPARC-expressing cells. Importantly, coexpression of SPARC and PTEN resulted in the smallest, least proliferative tumors with reduced invasive capacity and longer animal survival. Furthermore, direct inhibition of the AKT and SHCRAF- ERK signaling pathways suppressed the proliferation and migration of SPARC-expressing cells in vitro. These findings demonstrate that PTEN reconstitution or inhibition of signaling pathways that are activated by the loss of PTEN provide potential therapeutic strategies to inhibit SPARC-induced invasion while enhancing the negative effect of SPARC on tumor growth. © The Author(s) 2010. Source

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