CRN2 binds to TIMP4 and MMP14 and promotes perivascular invasion of glioblastoma cells

CRN2 is an actin filament binding protein involved in the regulation of various cellular processes including cell migration and invasion. CRN2 has been implicated in the malignant progression of different types of human cancer. We used CRN2 knock-out mice for analyses as well as for crossbreeding with a Tp53/Pten knock-out glioblastoma mouse model. CRN2 knock-out mice were subjected to a phenotyping screen at the German Mouse Clinic. Murine glioblastoma tissue specimens as well as cultured murine brain slices and glioblastoma cell lines were investigated by immunohistochemistry, immunofluorescence, and cell biological experiments. Protein interactions were studied by immunoprecipitation, pull-down, and enzyme activity assays. CRN2 knock-out mice displayed neurological and behavioural alterations, e.g. reduced hearing sensitivity, reduced acoustic startle response, hypoactivity, and less frequent urination. While glioblastoma mice with or without the additional CRN2 knock-out allele exhibited no significant difference in their survival rates, the increased levels of CRN2 in transplanted glioblastoma cells caused a higher tumour cell encasement of murine brain slice capillaries. We identified two important factors of the tumour microenvironment, the tissue inhibitor of matrix metalloproteinase 4 (TIMP4) and the matrix metalloproteinase 14 (MMP14, synonym: MT1-MMP), as novel binding partners of CRN2. All three proteins mutually interacted and co-localised at the front of lamellipodia, and CRN2 was newly detected in exosomes. On the functional level, we demonstrate that CRN2 increased the secretion of TIMP4 as well as the catalytic activity of MMP14. Our results imply that CRN2 represents a pro-invasive effector within the tumour cell microenvironment of glioblastoma multiforme.     

Cripto-1 localizes to dynamic and shed filopodia associated with cellular migration in glioblastoma cells

Cripto-1 is a protein participating in tissue orientation during embryogenesis but has also been implicated in a wide variety of cancers, such as colon, lung and breast cancer. Cripto-1 plays a role in the regulation of different pathways, including TGF-β/Smad and Wnt/β-catenin, which are highly associated with cell migration both during embryonal development and cancer progression. Little is known about the detailed subcellular localization of cripto-1 and how it participates in the directional movement of cells. In this study, the subcellular localization of cripto-1 in glioblastoma cells was investigated in vitro with high-resolution microscopy techniques. Cripto-1 was found to be localized to dynamic and shed filopodia and transported between cells through tunneling nanotubes. Our results connect the refined subcellular localization of cripto-1 to its functions in cellular orientation and migration.     

Arginine-rich,cell penetrating peptide–anti-microRNA complexes decrease glioblastoma migration potential

MicroRNAs (miRNAs) are a class of gene regulators originating from non-coding endogenous RNAs. Altered expression, both up- and down-regulation, of miRNAs plays important roles in many human diseases. Correcting miRNA dysregulation by either inhibiting or restoring miRNA function may provide therapeutic benefit. However, efficient, nontoxic miRNA delivery systems are in need. Cell penetrating peptides (CPPs) have been widely exploited for protein, DNA, and RNA delivery. Few have examined CPP transfection efficiency with single stranded anti-miRNA. The R₈ peptide condensed both siRNA and anti-miRNA. Greater than 50% of cells had anti-miRNA/R₈ complexes associated and in these cells 68% of anti-miRNA escapes the endosome/lysosome. Single-stranded antisense miR-21 inhibitor (anti-miR-21) administered using the R₈ peptide elicited efficient downstream gene upregulation. Glioblastoma cell migration was inhibited by 25% compared to the negative control group. To our knowledge, this is the first demonstration of miRNA modulation with anti-miR-21/R₈ complexes, which has laid the groundwork for further exploring octaarginine as intracellular anti-miRNAs carrier.     

The HIF-2alpha dependent induction of PAP and adenosine synthesis regulates glioblastoma stem cell function through the A2B adenosine receptor

Glioblastomas are lethal tumors characterized by malignant proliferation and recurrence promoted partly by glioblastoma stem cells (GSCs). GSCs are known to be regulated by hypoxia, but the mechanisms involved in this regulation are not fully understood. We now demonstrate that hypoxia-inducible factor HIF2α and prostatic acid phosphatase (PAP) are preferentially expressed in hypoxic GSCs in comparison with non-stem tumor cells and normal neural stem cells and that PAP is regulated by HIF2α. Targeting PAP in hypoxic GSCs inhibits self-renewal and proliferation in vitro and attenuates tumor initiation potential of GSCs in vivo. Using specific adenosine receptor antagonists, we further find that the pro-proliferative role of PAP is stemmed from stimulated A2B adenosine receptors. Moreover, selective blockage of A2B receptor or knockdown of PAP or A2B on hypoxic GSCs results in significant reduction of phosphorylation of Akt and Erk-1/2. Our results demonstrate that PAP may play a pro-proliferative role in hypoxic GSCs with a HIF2α-induction pattern, which may be ascribed to stimulated A2B receptors and activated Akt and Erk-1/2 pathways. Therefore, we propose that these identified molecular regulators of GSCs in the hypoxic niche might represent promising targets for antiglioblastoma therapies.     

Cucurbitacin I Induces Protective Autophagy in Glioblastoma in Vitro and in Vivo

There is an urgent need for new therapeutic avenues to improve the outcome of patients with glioblastoma multiforme (GBM). Current studies have suggested that cucurbitacin I, a natural selective inhibitor of JAK2/STAT3, has a potent anticancer effect on a variety of cancer cell types. This study showed that autophagy and apoptosis were induced by cucurbitacin I. Exposure of GBM cells to cucurbitacin I resulted in pronounced apoptotic cell death through activating bcl-2 family proteins. Cells treatment with cucurbitacin I up-regulated Beclin 1 and triggered autophagosome formation and accumulation as well as conversion of LC3I to LC3II. Activation of the AMP-activated protein kinase/mammalian target of rapamycin/p70S6K pathway, but not the PI3K/AKT pathway, occurred in autophagy induced by cucurbitacin I, which was accompanied by decreased hypoxia-inducible factor 1α. Stable overexpression of hypoxia-inducible factor 1α induced by FG-4497 prevented cucurbitacin I-induced autophagy and down-regulation of bcl-2. Knockdown of beclin 1 or treatment with the autophagy inhibitor 3-methyladenine also inhibited autophagy induced by cucurbitacin I. A coimmunoprecipitation assay showed that the interaction of Bcl-2 and Beclin 1/hVps34 decreased markedly in cells treated with cucurbitacin I. Furthermore, knockdown of beclin 1 or treatment with the lysosome inhibitor chloroquine sensitized cancer cells to cucurbitacin I-induced apoptosis. Finally, a xenograft model provided additional evidence for the occurrence of cucurbitacin I-induced apoptosis and autophagy in vitro. Our findings provide new insights into the molecular mechanisms underlying cucurbitacin I-mediated GBM cell death and may provide an efficacious therapy for patients harboring GBM.     

The Src Homology 3 Domain-containing Guanine Nucleotide Exchange Factor Is Overexpressed in High-grade Gliomas and Promotes Tumor Necrosis Factor-like Weak Inducer of Apoptosis-Fibroblast Growth Factor-inducible 14-induced Cell Migration and Invasion via Tumor Necrosis Factor Receptor-associated Factor 2

Glioblastoma (GB) is the highest grade of primary adult brain tumors, characterized by a poorly defined and highly invasive cell population. Importantly, these invading cells are attributed with having a decreased sensitivity to radiation and chemotherapy. TNF-like weak inducer of apoptosis (TWEAK)-Fn14 ligand-receptor signaling is one mechanism in GB that promotes cell invasiveness and survival and is dependent upon the activity of multiple Rho GTPases, including Rac1. Here we report that Src homology 3 domain-containing guanine nucleotide exchange factor (SGEF), a RhoG-specific guanine nucleotide exchange factor, is overexpressed in GB tumors and promotes TWEAK-Fn14-mediated glioma invasion. Importantly, levels of SGEF expression in GB tumors inversely correlate with patient survival. SGEF mRNA expression is increased in GB cells at the invasive rim relative to those in the tumor core, and knockdown of SGEF expression by shRNA decreases glioma cell migration in vitro and invasion ex vivo. Furthermore, we showed that, upon TWEAK stimulation, SGEF is recruited to the Fn14 cytoplasmic tail via TRAF2. Mutation of the Fn14-TRAF domain site or depletion of TNF receptor-associated factor 2 (TRAF2) expression by siRNA oligonucleotides blocked SGEF recruitment to Fn14 and inhibited SGEF activity and subsequent GB cell migration. We also showed that knockdown of either SGEF or RhoG diminished TWEAK activation of Rac1 and subsequent lamellipodia formation. Together, these results indicate that SGEF-RhoG is an important downstream regulator of TWEAK-Fn14-driven GB cell migration and invasion.     

Constitutive Activation of Signal Transducer and Activator of Transcription 3 (STAT3) and Nuclear Factor κB Signaling in Glioblastoma Cancer Stem Cells Regulates the Notch Pathway

Malignant gliomas are locally aggressive, highly vascular tumors that have a dismal prognosis, and present therapies provide little improvement in the disease course and outcome. Many types of malignancies, including glioblastoma, originate from a population of cancer stem cells (CSCs) that are able to initiate and maintain tumors. Although CSCs only represent a small fraction of cells within a tumor, their high tumor-initiating capacity and therapeutic resistance drives tumorigenesis. Therefore, it is imperative to identify pathways associated with CSCs to devise strategies to selectively target them. In this study, we describe a novel relationship between glioblastoma CSCs and the Notch pathway, which involves the constitutive activation of STAT3 and NF-κB signaling. Glioma CSCs were isolated and maintained in vitro using an adherent culture system, and the biological properties were compared with the traditional cultures of CSCs grown as multicellular spheres under nonadherent culture conditions. Interestingly, both adherent and spheroid glioma CSCs show constitutive activation of the STAT3/NF-κB signaling pathway and up-regulation of STAT3- and NF-κB-dependent genes. Gene expression profiling also identified components of the Notch pathway as being deregulated in glioma CSCs, and the deregulated expression of these genes was sensitive to treatment with STAT3 and NF-κB inhibitors. This finding is particularly important because Notch signaling appears to play a key role in CSCs in a variety of cancers and controls cell fate determination, survival, proliferation, and the maintenance of stem cells. The constitutive activation of STAT3 and NF-κB signaling pathways that leads to the regulation of Notch pathway genes in glioma CSCs identifies novel therapeutic targets for the treatment of glioma.     

C-Myc negatively controls the tumor suppressor PTEN by upregulating miR-26a in glioblastoma multiforme cells

The c-Myc oncogene is amplified in many tumor types. It is an important regulator of cell proliferation and has been linked to altered miRNA expression, suggesting that c-Myc-regulated miRNAs might contribute to tumor progression. Although miR-26a has been reported to be upregulated in glioblastoma multiforme (GBM), the mechanism has not been established. We have shown that ectopic expression of miR-26a influenced cell proliferation by targeting PTEN, a tumor suppressor gene that is inactivated in many common malignancies, including GBM. Our findings suggest that c-Myc modulates genes associated with oncogenesis in GBM through deregulation of miRNAs via the c-Myc–miR-26a–PTEN signaling pathway. This may be of clinical relevance.     

Cathepsin L silencing enhances arsenic trioxide mediated in vitro cytotoxicity and apoptosis in glioblastoma U87MG spheroids

Despite improved treatment options, glioblastoma multiforme (GBM) remains the most aggressive brain tumour with the shortest post-diagnostic survival. Arsenite (As₂O₃) is already being used in the treatment of acute promyelocytic leukaemia (APL), yet its effects on GBM have not been evaluated in detail. In U87MG cell monolayers, we have previously shown that arsenite cytotoxicity significantly increases upon transient inhibition of lysosomal protease Cathepsin L (CatL). As multicellular spheroids more closely represent in vivo tumours, we aimed to evaluate the impact of permanent CatL silencing on arsenite treatment in U87MG spheroids. CatL was stably silenced using shRNA expression plasmid packed lentiviruses. By using metabolic- and cell viability assays, we demonstrated that long-term CatL silencing significantly increased arsenite cytotoxicity in U87MG spheroids. Silenced CatL also increased arsenite-mediated apoptosis in spheroids via elevated p53 expression, Bax/Bcl2 ratio and caspase 3/7 activity, though with lower efficacy than in monolayers. Arsenite cytotoxicity was enhanced by lower CatL activity, since similar cytotoxicity increase was also observed using the novel CatL inhibitor AT094. The results have significant translational impact, since stable CatL silencing would enable the application of lower systemic doses of arsenite to achieve the desired cytotoxic effects on GBMs in vivo.     

Analogues of the Inhoffen–Lythgoe diol with anti-proliferative activity

The anti-proliferative activity of a series of ester- and amide-linked Inhoffen–Lythgoe side chain analogues is reported. Whereas the Inhoffen–Lythgoe diol was inactive in these studies, a number of aromatic and aliphatic ester-linked side chains demonstrated modest in vitro growth inhibition in two human cancepar cell lines, U87MG (glioblastoma) and HT-29 (colorectal adenocarcinoma). Structure–activity relationship (SAR) studies demonstrated the most active aromatic (13) and aliphatic (25 and 29) substituted analogues were approximately equipotent in U87MG and HT-29 cells. Further evaluation of 13, 25, and 29 indicated these analogues do not activate canonical vitamin D signaling nor antagonize Hedgehog (Hh) signaling. Thus, the cellular mechanism(s) that govern the anti-proliferative activity for this class of truncated vitamin D-based structures appears to be different from classical mechanisms previously identified for these scaffolds.