[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Glioblastomas (GBMs), the most aggressive primary brain tumors, exhibit increased invasiveness and resistance to anti-tumor treatments. We explored the role of RTVP-1, a glioma-associated protein that promotes glioma cell migration, in the mesenchymal transformation of GBM. Analysis of The Cancer Genome Atlas (TCGA) demonstrated that RTVP-1 expression was higher in mesenchymal GBM and predicted tumor recurrence and poor clinical outcome. ChiP analysis revealed that the RTVP-1 promoter binds STAT3 and C/EBPβ, two master transcription factors that regulate mesenchymal transformation of GBM. In addition, IL-6 induced RTVP-1 expression in a STAT3-dependent manner. RTVP-1 increased the migration and mesenchymal transformation of glioma cells. Similarly, overexpression of RTVP-1 in human neural stem cells induced mesenchymal differentiation, whereas silencing of RTVP-1 in glioma stem cells (GSCs) decreased the mesenchymal transformation and stemness of these cells. Silencing of RTVP-1 also increased the survival of mice bearing GSC-derived xenografts. Using gene array analysis of RTVP-1 silenced glioma cells we identified IL-6 as a mediator of RTVP-1 effects on the mesenchymal transformation and migration of GSCs, therefore acting in a positive feedback loop by upregulating RTVP-1 expression via the STAT3 pathway. Collectively, these results implicate RTVP-1 as a novel prognostic marker and therapeutic target in GBM.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Hepatitis C virus (HCV) enters its target cell via clathrin-mediated endocytosis. AP-2-associated protein kinase 1 (AAK1) and cyclin G-associated kinase (GAK) are host kinases that regulate clathrin adaptor protein (AP)-mediated trafficking in the endocytic and secretory pathways. We previously reported that AAK1 and GAK regulate HCV assembly by stimulating binding of the μ subunit of AP-2, AP2M1, to HCV core protein. We also discovered that AAK1 and GAK inhibitors, including the approved anticancer drugs sunitinib and erlotinib, could block HCV assembly. Here, we hypothesized that AAK1 and GAK regulate HCV entry independently of their effect on HCV assembly. Indeed, silencing AAK1 and GAK expression inhibited entry of pseudoparticles and cell culture grown-HCV and internalization of Dil-labeled HCV particles with no effect on HCV attachment or RNA replication. AAK1 or GAK depletion impaired epidermal growth factor (EGF)-mediated enhanced HCV entry and endocytosis of EGF receptor (EGFR), an HCV entry cofactor and erlotinib's cancer target. Moreover, either RNA interference-mediated depletion of AP2M1 or NUMB, each a substrate of AAK1 and/or GAK, or overexpression of either an AP2M1 or NUMB phosphorylation site mutant inhibited HCV entry. Last, in addition to affecting assembly, sunitinib and erlotinib inhibited HCV entry at a postbinding step, their combination was synergistic, and their antiviral effect was reversed by either AAK1 or GAK overexpression. Together, these results validate AAK1 and GAK as critical regulators of HCV entry that function in part by activating EGFR, AP2M1, and NUMB and as the molecular targets underlying the antiviral effect of sunitinib and erlotinib (in addition to EGFR), respectively.
Understanding the host pathways hijacked by HCV is critical for developing host-centered anti-HCV approaches. Entry represents a potential target for antiviral strategies; however, no FDA-approved HCV entry inhibitors are currently available. We reported that two host kinases, AAK1 and GAK, regulate HCV assembly. Here, we provide evidence that AAK1 and GAK regulate HCV entry independently of their role in HCV assembly and define the mechanisms underlying AAK1- and GAK-mediated HCV entry. By regulating temporally distinct steps in the HCV life cycle, AAK1 and GAK represent "master regulators" of HCV infection and potential targets for antiviral strategies. Indeed, approved anticancer drugs that potently inhibit AAK1 or GAK inhibit HCV entry in addition to assembly. These results contribute to an understanding of the mechanisms of HCV entry and reveal attractive host targets for antiviral strategies as well as approved candidate inhibitors of these targets, with potential implications for other viruses that hijack clathrin-mediated pathways.
Full-text · Article · Feb 2015 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Glioblastoma (GBM), the most aggressive primary brain tumors, are categorized into the major subgroups: proneural, neural,
classical and mesenchymal, the latter being characterized by increased invasion and poor prognosis. We recently identified
RTVP-1 as a glioma-associated protein that regulates glioma cell migration and invasion. Using ChiP analyses, we found that
the RTVP-1 promoter binds STAT3 and C/EBPbeta, the two master transcription factors that regulate mesenchymal transformation
of GBM. Analysis of TCGA tumor specimens demonstrated that the expression of RTVP-1 was higher in the mesenchymal GBM and
was inversely correlated with patient survival. We further found that RTVP-1 was expressed in glioma stem cells (GSCs) but
not in human neural stem cells (NSCs). Overexpression of RTVP-1 in NSCs induced their mesenchymal transformation, whereas
silencing of RTVP-1 in GSCs decreased their mesenchymal signature, increased their neural phenotypes and inhibited the self
renewal and stemness of these cells. Silencing of RTVP-1 also decreased tumor volume of GSC-derived xenografts and increased
animal survival. Using gene array analysis of RTVP-1 silenced cells we identified IL-6 and CXCR4 as major mediators of RTVP-1
effects on the mesenchymal transformation and self-renewal of GSCs. Using a pull down assay with His-tagged RTVP-1 and FRET
analysis, we identified HSP27, N-WASP and hnRNPK as novel interacting proteins of RTVP-1, that mediate its effects on GSC
migration and invadopodia formation. In summary, RTVP-1 expression is regulated by STAT3 and CEBPbeta and is promoting the
mesenchymal transformation of GSCs. RTVP-1 induces self-renewal and migration of GSCs by the increased expression of IL-6
and CXCR4 and via its interaction with N-WASP, hnRNPK and HSP27. The upregulation of IL-6 by RTVP-1 acts in a positive feedback
loop to further increase RTVP-1 expression by activating the STAT3 pathway. Collectively, these results implicate RTVP-1 as
a novel prognostic marker and therapeutic target in GBM.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) have emerged as potential cancer therapeutics; however, their clinical use is hindered by lack of effective delivery mechanisms to tumor sites. Mesenchymal stem cells (MSCs) have been shown to migrate to experimental glioma and to exert anti-tumor effects by delivering cytotoxic compounds. Here, we examined the ability of MSCs derived from bone marrow, adipose tissue, placenta and umbilical cord to deliver synthetic miRNA mimics to glioma cells and glioma stem cells (GSCs). We examined the delivery of miR-124 and miR-145 mimics as glioma cells and GSCs express very low levels of these miRNAs. Using fluorescently labeled miRNA mimics and in situ hybridization, we demonstrated that all the MSCs examined delivered miR-124 and miR-145 mimics to co-cultured glioma cells and GSCs via gap junction- dependent and independent processes. The delivered miR-124 and miR-145 mimics significantly decreased the luciferase activity of their respected reporter target genes, SCP-1 and Sox2, and decreased the migration of glioma cells and the self-renewal of GSCs. Moreover, MSCs delivered Cy3-miR-124 mimic to glioma xenografts when administered intracranially. These results suggest that MSCs can deliver synthetic exogenous miRNA mimics to glioma cells and GSCs and may provide an efficient route of therapeutic miRNA delivery in vivo.
[Show abstract][Hide abstract] ABSTRACT: Background: Novel therapies are urgently needed against hepatitis C virus (HCV) infection. HCV assembly is poorly understood and is currently not being targeted pharmacologically. We identified a conserved heretofore unrecognized YXXΦ motif (Φ is a bulky hydrophobic residue) within the HCV core (capsid) protein. This motif is homologous to sorting signals within host cargo proteins known to mediate binding of AP2M1, the μ subunit of clathrin adaptor protein complex 2, and intracellular trafficking. We hypothesized that core binds AP2M1, that this interaction is essential for infectious HCV production, and can be disrupted pharmacologically.
Methods: Novel microfluidics affinity analysis, protein-fragment complementation assays, and co-immunoprecipitations were used to study core-AP2M1 binding in vitro, in cells, and in the context of HCV infection, respectively. Infectivity assays were combined with RNAi, genetic, dominant interfering, and pharmacological approaches for probing functional relevance of core-AP2M1 binding to the HCV life cycle. Quantitative confocal immunofluorescence analysis was used for studying the effect of disrupting this interaction on core's subcellular localization.
Results: Core's YXXΦ motif mediates AP2M1 binding. Either YXXΦ core mutations, silencing AP2M1 expression or overepressing a dominant negative AP2M1 mutant dramatically inhibit intracellular and extracellular infectivity, consistent with a defect in HCV assembly. Core's YXXΦ motif mediates recruitment of AP2M1 to assembly sites on lipid droplets. Moreover, the observed defect in assembly following disruption of core-AP2M1 binding correlates with accumulation of core on lipid droplets and reduced core colocalization with the E2 (envelope) protein and markers of trans-Golgi network, the presumed site of HCV egress. AAK1 and GAK, serine/threonine kinases known to stimulate AP2M1 binding to host cargo proteins, regulate core-AP2M1 binding and are essential for HCV assembly. Last, approved anti-cancer drugs that inhibit AAK1 or GAK not only disrupt core-AP2M1 binding, but also significantly inhibit HCV assembly and infectious viral production.
Conclusion: These results validate viral-host interactions essential for HCV assembly and yield compounds for pharmaceutical development.
[Show abstract][Hide abstract] ABSTRACT: Novel therapies are urgently needed against hepatitis C virus infection (HCV), a major global health problem. The current model of infectious virus production suggests that HCV virions are assembled on or near the surface of lipid droplets, acquire their envelope at the ER, and egress through the secretory pathway. The mechanisms of HCV assembly and particularly the role of viral-host protein-protein interactions in mediating this process are, however, poorly understood. We identified a conserved heretofore unrecognized YXXΦ motif (Φ is a bulky hydrophobic residue) within the core protein. This motif is homologous to sorting signals within host cargo proteins known to mediate binding of AP2M1, the μ subunit of clathrin adaptor protein complex 2 (AP-2), and intracellular trafficking. Using microfluidics affinity analysis, protein-fragment complementation assays, and co-immunoprecipitations in infected cells, we show that this motif mediates core binding to AP2M1. YXXΦ mutations, silencing AP2M1 expression or overexpressing a dominant negative AP2M1 mutant had no effect on HCV RNA replication, however, they dramatically inhibited intra- and extracellular infectivity, consistent with a defect in viral assembly. Quantitative confocal immunofluorescence analysis revealed that core's YXXΦ motif mediates recruitment of AP2M1 to lipid droplets and that the observed defect in HCV assembly following disruption of core-AP2M1 binding correlates with accumulation of core on lipid droplets, reduced core colocalization with E2 and reduced core localization to trans-Golgi network (TGN), the presumed site of viral particles maturation. Furthermore, AAK1 and GAK, serine/threonine kinases known to stimulate binding of AP2M1 to host cargo proteins, regulate core-AP2M1 binding and are essential for HCV assembly. Last, approved anti-cancer drugs that inhibit AAK1 or GAK not only disrupt core-AP2M1 binding, but also significantly inhibit HCV assembly and infectious virus production. These results validate viral-host interactions essential for HCV assembly and yield compounds for pharmaceutical development.
[Show abstract][Hide abstract] ABSTRACT: Gliomas are characterized by increased infiltration into the surrounding normal brain tissue. We recently reported that RTVP-1 is highly expressed in gliomas and plays a role in the migration of these cells, however the regulation of RTVP-1 expression in these cells is not yet described. In this study we examined the role of PKC in the regulation of RTVP-1 expression and found that PMA and overexpression of PKCα and PKCε increased the expression of RTVP-1, whereas PKCδ exerted an opposite effect. Using the MatInspector software, we identified a SRF binding site on the RTVP-1 promoter. Chromatin immunoprecipitation (ChIP) assay revealed that SRF binds to the RTVP-1 promoter in U87 cells, and that this binding was significantly increased in response to serum addition. Moreover, silencing of SRF blocked the induction of RTVP-1 expression in response to serum. We found that overexpression of PKCα and PKCε increased the activity of the RTVP-1 promoter and the binding of SRF to the promoter. In contrast, overexpression of PKCδ blocked the increase in RTVP-1 expression in response to serum and the inhibitory effect of PKCδ was abrogated in cells expressing a SRFT160A mutant. SRF regulated the migration of glioma cells and its effect was partially mediated by RTVP-1. We conclude that RTVP-1 is a PKC-regulated gene and that this regulation is at least partly mediated by SRF. Moreover, RTVP-1 plays a role in the effect of SRF on glioma cell migration.
[Show abstract][Hide abstract] ABSTRACT: Here, we report the cloning and characterization of RTVP-1b, a novel splice variant of human RTVP-1, which was isolated from the U87 glioma cell line. Sequence analysis revealed that RTVP-1b contains an additional 71 base exon between exons 2 and 3 that is missing in RTVP-1, leading to a frame-shift and a different putative protein. The deduced protein was 237 amino acids in length, sharing the N-terminal 141 amino acids with RTVP-1. RT-PCR analysis demonstrated that RTVP-1b was expressed in a wide range of tissues and that its expression was different from that of RTVP-1. In contrast, RTVP-1 and RTVP-1b showed similar patterns of expression in astrocytic tumors; highly expressed in glioblastomas as compared to normal brains, low-grade astrocytomas and anaplastic oligodendrogliomas. Overexpression of RTVP-1b increased glioma cell proliferation but did not affect cell migration. Our results suggest that RTVP-1b represents a potential prognostic marker and therapeutic target in gliomas.
No preview · Article · Nov 2007 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: In this study, we examined the expression and functions of related to testes-specific, vespid, and pathogenesis protein 1 (RTVP-1) in glioma cells. RTVP-1 was expressed in high levels in glioblastomas, whereas its expression in low-grade astrocytomas and normal brains was very low. Transfection of glioma cells with small interfering RNAs targeting RTVP-1 decreased cell proliferation in all the cell lines examined and induced cell apoptosis in some of them. Overexpression of RTVP-1 increased astrocyte and glioma cell proliferation and the anchorage-independent growth of the cells. In addition, overexpression of RTVP-1 rendered glioma cells more resistant to the apoptotic effect of tumor necrosis factor-related apoptosis-inducing ligand and serum deprivation. To delineate the molecular mechanisms involved in the survival effects of RTVP-1, we examined the expression and phosphorylation of various apoptosis-related proteins. We found that overexpression of RTVP-1 decreased the phosphorylation of c-Jun-NH2-kinase and increased the expression of Bcl2 and that the protective effect of RTVP-1 was partially mediated by Bcl2. Finally, we found that RTVP-1 regulated the invasion of glioma cells as was evident by their enhanced migration through Matrigel and by their increased invasion in a spheroid confrontation assay. The increased invasive potential of the RTVP-1 overexpressors was also shown by the increased activity of matrix metalloproteinase 2 in these cells. Our results suggest that the expression of RTVP-1 is correlated with the degree of malignancy of astrocytic tumors and that RTVP-1 is involved in the regulation of the growth, survival, and invasion of glioma cells. Collectively, these findings suggest that RTVP-1 is a potential therapeutic target in gliomas.