A SEMA3 Signaling Pathway-Based Multi-Biomarker for Prediction of Glioma Patient Survival.

Glioma is a lethal central nervous system tumor with poor patient survival prognosis. Because of the molecular heterogeneity, it is a challenge to precisely determine the type of the tumor and to choose the most effective treatment. Therefore, novel biomarkers are essential to improve the diagnosis and prognosis of glioma tumors. Class 3 semaphorin proteins (SEMA3) play an important role in tumor biology. SEMA3 transduce their signals by using neuropilin and plexin receptors, which functionally interact with the vascular endothelial growth factor-mediated signaling pathways. Therefore, the aim of this study was to explore the potential of SEMA3 signaling molecules for prognosis of glioma patient survival. The quantitative real-time PCR method was used to evaluate mRNA expression of SEMA3(A-G), neuropilins (NRP1 and NRP2), plexins (PLXNA2 and PLXND1), cadherins (CDH1 and CDH2), integrins (ITGB1, ITGB3, ITGA5, and ITGAV), VEGFA and KDR genes in 59 II-IV grade glioma tissues. Seven genes significantly associated with patient overall survival were used for multi-biomarker construction, which showed 64%, 75%, and 68% of accuracy of predicting the survival of 1-, 2-, and 3-year glioma patients, respectively. The results suggest that the seven-gene signature could serve as a novel multi-biomarker for more accurate prognosis of a glioma patient's outcome.

Isocytosine deaminase Vcz as a novel tool for the prodrug cancer therapy.

BACKGROUND: The cytosine deaminase (CD)/5-fluorocytosine (5-FC) system is among the best explored enzyme/prodrug systems in the field of the suicide gene therapy. Recently, by the screening of the environmental metagenomic libraries we identified a novel isocytosine deaminase (ICD), termed Vcz, which is able of specifically converting a prodrug 5-fluoroisocytosine (5-FIC) into toxic drug 5-fluorouracil (5-FU). The aim of this study is to test the applicability of the ICD Vcz / 5-FIC pair as a potential suicide gene therapy tool. METHODS: Vcz-expressing human glioblastoma U87 and epithelial colorectal adenocarcinoma Caco-2 cells were treated with 5-FIC, and the Vcz-mediated cytotoxicity was evaluated by performing an MTT assay. In order to examine anti-tumor effects of the Vcz/5-FIC system in vivo, murine bone marrow-derived mesenchymal stem cells (MSC) were transduced with the Vcz-coding lentivirus and co-injected with 5-FIC or control reagents into subcutaneous GL261 tumors evoked in C57/BL6 mice. RESULTS: 5-FIC alone showed no significant toxic effects on U87 and Caco-2 cells at 100 µM concentration, whereas the number of cells of both cell lines that express Vcz cytosine deaminase gene decreased by approximately 60% in the presence of 5-FIC. The cytotoxic effects on cells were also induced by media collected from Vcz-expressing cells pre-treated with 5-FIC. The co-injection of the Vcz-transduced mesenchymal stem cells and 5-FIC have been shown to augment tumor necrosis and increase longevity of tumorized mice by 50% in comparison with control group animals. CONCLUSIONS: We have confirmed that the novel ICD Vcz together with the non-toxic prodrug 5-FIC has a potential of being a new enzyme/prodrug system for suicide gene therapy.

The Anti-Tumorigenic Activity of Sema3C in the Chick Embryo Chorioallantoic Membrane Model.

Sema3C protein, a member of the class 3 family of secreted semaphorins, play an important role in tumor development by regulating cell proliferation, migration, invasion, and angiogenesis processes. Depending on the type and malignancy grade of the tumor, Sema3C function remains controversial. In this study, we constructed a stably overexpressing Sema3C glioblastoma cell line U87 MG and tested it on the chicken embryo chorioallantoic membrane (CAM) model with the aim to reveal Sema3C protein function on angiogenesis process in ovo. Our experiments showed that Sema3C not only affects angiogenesis of CAM by inhibiting neovascularization but also acts as an anti-tumorigenic molecule by hampering U87 MG cell invasion into mesenchyme. The effects of Sema3C on CAM were similar to the effects of anti-epileptic drug sodium valproate (NaVP). Both, anti-angiogenic and anti-tumorigenic activities of Sema3C were enhanced by the treatment of NaVP and, importantly, were not attributed to the cytotoxic effects. Our studies suggest that Sema3C could be a promising target for glioblastoma treatment.

Large-Scale Screening of Preferred Interactions of Human Src Homology-3 (SH3) Domains Using Native Target Proteins as Affinity Ligands.

The Src Homology-3 (SH3) domains are ubiquitous protein modules that mediate important intracellular protein interactions via binding to short proline-rich consensus motifs in their target proteins. The affinity and specificity of such core SH3 - ligand contacts are typically modest, but additional binding interfaces can give rise to stronger and more specific SH3-mediated interactions. To understand how commonly such robust SH3 interactions occur in the human protein interactome, and to identify these in an unbiased manner we have expressed 324 predicted human SH3 ligands as full-length proteins in mammalian cells, and screened for their preferred SH3 partners using a phage display-based approach. This discovery platform contains an essentially complete repertoire of the ∼300 human SH3 domains, and involves an inherent binding threshold that ensures selective identification of only SH3 interactions with relatively high affinity. Such strong and selective SH3 partners could be identified for only 19 of these 324 predicted ligand proteins, suggesting that the majority of human SH3 interactions are relatively weak, and thereby have capacity for only modest inherent selectivity. The panel of exceptionally robust SH3 interactions identified here provides a rich source of leads and hypotheses for further studies. However, a truly comprehensive characterization of the human SH3 interactome will require novel high-throughput methods based on function instead of absolute binding affinity.

Hepatitis C virus NS5A protein blocks epidermal growth factor receptor degradation via a proline motif- dependent interaction.

Hepatitis C virus (HCV) establishes a persistent infection that in many cases leads to cirrhosis and hepatocellular carcinoma. The non-structural 5A protein (NS5A) has been implicated in this process as it contains a C-terminal polyproline motif (termed P2) that binds to Src homology 3 (SH3) domains to regulate cellular signalling and trafficking pathways. We have shown previously that NS5A impaired epidermal growth factor (EGF) receptor (EGFR) endocytosis, thereby inhibiting EGF-stimulated EGFR degradation by a mechanism that remained unclear. As EGFR has been implicated in HCV cell entry and trafficking of the receptor involves several SH3-domain containing proteins, we investigated in more detail the mechanisms by which NS5A perturbs EGFR trafficking. We demonstrated that the P2 motif was required for the NS5A-mediated disruption to EGFR trafficking. We further demonstrated that the P2 motif was required for an interaction between NS5A and CMS, a homologue of CIN85 that has previously been implicated in EGFR endocytosis. We provided evidence that CMS was involved in the NS5A-mediated perturbation of EGFR trafficking. We also showed that NS5A effected a loss of EGFR ubiquitination in a P2-motif-dependent fashion. These data provide clues to the mechanism by which NS5A regulates the trafficking of a key cellular receptor and demonstrate for the first time the ability of NS5A to regulate host cell ubiquitination pathways.

Hepatitis C virus NS5A inhibits mixed lineage kinase 3 to block apoptosis.

Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K(+) channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis, which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication.

Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV), Sindbis (SINV), and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication.

Recognition of tandem PxxP motifs as a unique Src homology 3-binding mode triggers pathogen-driven actin assembly.

Src homology 3 (SH3) domains are globular protein interaction modules that regulate cell behavior. The classic SH3 ligand-binding site accommodates a hydrophobic PxxP motif and a positively charged specificity-determining residue. We have determined the NMR structure of insulin receptor tyrosine kinase substrate (IRTKS) SH3 domain in complex with a repeat from Escherichia coli-secreted protein F-like protein encoded on prophage U (EspF(U)), a translocated effector of enterohemorrhagic E. coli that commandeers the mammalian actin assembly machinery. EspF(U)-IRTKS interaction is among the highest affinity natural SH3 ligands. Our complex structure reveals a unique type of SH3 interaction based on recognition of tandem PxxP motifs in the ligand. Strikingly, the specificity pocket of IRTKS SH3 has evolved to accommodate a polyproline type II helical peptide analogously to docking of the canonical PxxP by the conserved IRTKS SH3 proline-binding pockets. This cooperative binding explains the high-affinity SH3 interaction and is required for EspF(U)-IRTKS interaction in mammalian cells as well as the formation of localized actin "pedestals" beneath bound bacteria. Importantly, tandem PxxP motifs are also found in mammalian ligands and have been shown to contribute to IRTKS SH3 recognition similarly.

Adipose Tissue-Derived Stem Cell Extracellular Vesicles Suppress Glioblastoma Proliferation, Invasiveness and Angiogenesis.

Extracellular vesicles (EVs) are attractive anticancer drug delivery candidates as they confer several fundamental properties, such as low immunogenicity and the ability to cross biological barriers. Mesenchymal stem cells (MSCs) are convenient producers for high EV yields, and patient-derived adipose tissue MSC-EVs could serve as personalised carriers. However, MSC-EV applications raise critical concerns as their natural cargo can affect tumour progression in both inducing and suppressing ways. In this study, we investigated the effect of adipose tissue-derived mesenchymal stem cell EVs (ASC-EVs) on several glioblastoma (GBM) cell lines to define their applicability for anticancer therapies. ASC-EVs were isolated from a cell-conditioned medium and characterised by size and specific markers. The internalisation of fluorescently labelled ASC-EVs by human GBM cells HROG36, U87 MG, and T98G was evaluated by fluorescent microscopy. Changes in GBM cell proliferation after ASC-EV application were determined by the metabolic PrestoBlue assay. Expression alterations in genes responsible for cell adhesion, proliferation, migration, and angiogenesis were evaluated by quantitative real-time PCR. ASC-EV effects on tumour invasiveness and neoangiogenesis in ovo were analysed on the chicken embryo chorioallantoic membrane model (CAM). ASC-EV treatment reduced GBM proliferation in vitro and significantly downregulated invasiveness-related genes ITGα5 (in T98G and HROG63) and ITGß3 (in HROG36) and the vascularisation-inducing gene KDR (in all GBM lines). Additionally, an approximate 65% reduction in the GBM invasion rate was observed in CAM after ASC-EV treatment. Our study indicates that ASC-EVs possess antitumour properties, reducing GBM cell proliferation and invasiveness, and can be applied as anticancer therapeutics and medicine carriers.

Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation.

Enterohemorrhagic Escherichia coli O157:H7 translocates 2 effectors to trigger localized actin assembly in mammalian cells, resulting in filamentous actin "pedestals." One effector, the translocated intimin receptor (Tir), is localized in the plasma membrane and clustered upon binding the bacterial outer membrane protein intimin. The second, the proline-rich effector EspF(U) (aka TccP) activates the actin nucleation-promoting factor WASP/N-WASP, and is recruited to sites of bacterial attachment by a mechanism dependent on an Asn-Pro-Tyr (NPY(458)) sequence in the Tir C-terminal cytoplasmic domain. Tir, EspF(U), and N-WASP form a complex, but neither EspF(U) nor N-WASP bind Tir directly, suggesting involvement of another protein in complex formation. Screening of the mammalian SH3 proteome for the ability to bind EspF(U) identified the SH3 domain of insulin receptor tyrosine kinase substrate (IRTKS), a factor known to regulate the cytoskeleton. Derivatives of WASP, EspF(U), and the IRTKS SH3 domain were capable of forming a ternary complex in vitro, and replacement of the C terminus of Tir with the IRTKS SH3 domain resulted in a fusion protein competent for actin assembly in vivo. A second domain of IRTKS, the IRSp53/MIM homology domain (IMD), bound to Tir in a manner dependent on the C-terminal NPY(458) sequence, thereby recruiting IRTKS to sites of bacterial attachment. Ectopic expression of either the IRTKS SH3 domain or the IMD, or genetic depletion of IRTKS, blocked pedestal formation. Thus, enterohemorrhagic E. coli translocates 2 effectors that bind to distinct domains of a common host factor to promote the formation of a complex that triggers robust actin assembly at the plasma membrane.