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Background The antiepileptic drug valproic acid (VPA) inhibits histone deacetylase in glioblastoma cells in vitro, which influences several oncogenic pathways and decreases glioma cell proliferation. The clinical relevance of these observations remains unclear, as VPA does not seem to affect glioblastoma patient survival. In this study, we analyzed whether the in vitro effects of VPA treatment on histone acetylation are also observed in tumor tissues of glioblastoma patients. Methods The in vitro effects of VPA treatment on histone acetylation were assessed with immunofluorescence and western blotting. On tissue microarrays and in fresh-frozen glioblastoma tissues we investigated the histone acetylation patterns of patients who were either treated with VPA or did not receive antiepileptic drugs at the time of their surgery. We also performed mRNA expression-based and gene set enrichment analyses on these tissues. Results VPA increased the expression levels of acetylated histones H3 and H4 in vitro, in agreement with previous reports. In tumor samples obtained from glioblastoma patients, however, VPA treatment affected neither gene (set) expression nor histone acetylation. Conclusions The in vitro effects of VPA on histone acetylation status in glioblastoma cells could not be confirmed in clinical tumor samples of glioblastoma patients using antiepileptic doses of VPA, which reflects the lack of effect of VPA on the clinical outcome of glioblastoma patients.

Osteopontin (OPN) is a secreted protein involved in most aspects of tumor progression and metastasis development. Elevated OPN expression has been reported in multiple types of cancer including glioblastoma (GBM), the highest grade and most aggressive brain tumor. GBMs contain a subpopulation of glioma-initiating cells (GICs) implicated in progression, therapeutic resistance and recurrence. We have previously demonstrated that OPN silencing inhibited GBM cell growth in vitro and in vivo. Moreover, activation of CD44 signaling upon OPN ligation has been recently implicated in the acquisition of a stem cell phenotype by GBM cells. The present study is aimed to explore OPN autocrine function using shRNA silencing strategy in GICs enriched from GBM cell lines and a human primary GBM grown in EGF and bFGF defined medium. The removal of these growth factors and addition of serum induced a significant loss of OPN expression in GICs. We showed that OPN-silenced GICs were unable to grow as spheres and this capacity was restored by exogenous OPN. Importantly, the expression of Sox2, Oct3/4 and Nanog, key stemness transcription factors, was significantly decreased in GICs upon OPN targeting. We identified Akt/mTOR/p70S6K as the main signaling pathway triggered following OPN-mediated EGFR activation in GICs. Finally, in an orthotopic xenograft mouse model, the tumorigenic potential of U87-MG sphere cells was completely abrogated upon OPN silencing. Our demonstration of endogenous OPN major regulatory effects on GICs stemness phenotype and tumorigenicity implies a greater role than anticipated for OPN in GBM pathogenesis from initiation and progression to probable recurrence.

Glioblastoma multiforme (GBM) is the most common primary brain tumor and is without exception lethal. GBMs modify the immune system, which contributes to the aggressive nature of the disease. Particularly, cells of the monocytic lineage, including monocytes, macrophages and microglia, are affected. We investigated the influence of GBM-derived extracellular vesicles (EVs) on the phenotype of monocytic cells. Proteomic profiling showed GBM EVs to be enriched with proteins functioning in extracellular matrix interaction and leukocyte migration. GBM EVs appeared to skew the differentiation of peripheral blood-derived monocytes to alternatively activated/M2-type macrophages. This was observed for EVs from an established cell line, as well as for EVs from primary cultures of GBM stem-like cells (GSCs). Unlike EVs of non-GBM origin, GBM EVs induced modified expression of cell surface proteins, modified cytokine secretion (e.g., an increase in vascular endothelial growth factor and IL-6) and increased phagocytic capacity of the macrophages. Most pronounced effects were observed upon incubation with EVs from mesenchymal GSCs. GSC EVs also affected primary human microglia, resulting in increased expression of Membrane type 1-matrix metalloproteinase, a marker for GBM microglia and functioning as tumor-supportive factor. In conclusion, GBM-derived EVs can modify cells of the monocytic lineage, which acquire characteristics that resemble the tumor-supportive phenotypes observed in patients.

Aims: Human cytomegalovirus (HCMV) is a ubiquitous beta human herpesvirus able to influence infected cell survival and proliferation and to modulate the host immune response. As there is accumulating evidence that HCMV is detected in primary intracranial astrocytic tumours, in this study we looked for the presence of HCMV in intracranial tumours and tried to correlate this eventual presence with the anti-HCMV systemic immunoreactivity and with the detection of HCMV in peripheral blood. Methods: In this study, we analysed 43 glioblastomas (GBM), 14 oligodendrogliomas (OL) and 20 meningiomas (MG) by immunofluorescence (IF) targeting HCMV immediate early antigen (IE1) and by nested PCR (nPCR) amplifying HCMV glycoprotein B (gB). Results: Detection of IE1 by IF showed the presence of HCMV in 70% of GBM, 57% of OL and 85% of MG, in contrast to gB nPCR, which detected HCMV in only 50% of GBM, 38% of OL and 46% of MG. Unexpectedly, HCMV DNA and antigens were detected within GBM, OL and MG of patients that exhibit negative viral serology. More surprisingly, PCR on the peripheral blood did not detect HCMV in patients with a HCMV-positive tumour. Conclusions: Our results are in agreement with previous observations demonstrating HCMV in glial tumours and highlight the presence of HCMV in meningiomas. We also showed that anti-HCMV specific systemic immunoreactivity and detection of HCMV in peripheral blood are not predictive of HCMV presence in primary intracranial tumours.

Primary brain tumors are among the most refractory of malignancies. Their most aggressive form, glioblastoma multiform (GBM, WHO grade IV), is also the most common and lethal subtype.1 Although multimodal therapies have been developed, the overall median survival of GBM patients hardly reaches 15 months from the time of diagnosis.2 This poor survival rate is the consequence of tumor recurrence that systematically occurs despite classical therapeutic strategies. Trying to understand the origin of GBM relapses seems mandatory, in this context, for a better understanding of the tumor's biology and improving the patients' quality of life. The infiltrative patterns of GBM make tumor cells hard to target. Furthermore, recent studies using orthotopic xenografts have demonstrated that GBM cells are able to escape the tumor mass and specifically invade the subventricular zones (SVZs) of the adult brain.3,4 In that environment, GBM cells were first shown to be highly tumorigenic and were later characterized as glioblastoma stem cells (GSCs). The SVZ is known to be the major source of neural stem cells and progenitors in adults and functions as a supportive niche promoting self-renewal and inhibits differentiation.5,6 This “seed-and-soil” relationship has also been adapted to cancer stem cell research because GSCs also rely on a specific environment or niche to maintain their stem cell properties and their ability to drive tumor growth.7,8 In this work, we questioned the role of the CXCL12/CXCR4 signaling in the GSC-oriented invasion of the SVZ. CXCR4 is known to be expressed by highly malignant gliomas,9,10 be involved in tumor cell proliferation,11 and be associated with a poor survival.12 Recent studies have shown that CXCL12 and CXCR4 enhance tumorigenesis through increased proliferation of tumor cells13 and that CXCL12 boosts the release of vascular endothelial growth factor (VEGF) from GSC, leading to tumor growth-induced angiogenesis.14 When taken altogether, those facts led to investigating the eventual role of the CXCR4 signaling in the specific invasion of the SVZ by human GSC. Counteracting with the invasion abilities of GSC would make those cells more easily targeted by classical therapeutic strategies and would definitely improve the survival rate for GBM patients.

Aims Synaptic vesicle proteins 2 (SV2) are neuronal vesicles membrane glycoproteins that appear as important targets in the treatment of partial and generalized epilepsies. Therefore, we analysed the expression of SV2 isoforms in the hippocampus of patients with temporal lobe epilepsy (TLE). Methods SV2A, SV2B and SV2C immunostaining and QuantiGene branched DNA assay were performed on biopsies from 31 consecutive TLE patients with mesial temporal sclerosis (MTS) and compared with 10 autopsy controls. SV2 expression was further compared with Timm's staining, and synaptophysin, Zinc transporter 3 (ZnT3), dynorphin, vesicular glutamate transporter 1 (VGLUT1) and vesicular GABA transporter (VGAT) expression. Results In TLE patients, SV2A and SV2B expression was decreased in areas of synaptic loss. SV2C, which is weakly expressed or absent in the hippocampus of controls, was overexpressed in 10/11 cases with classical MTS1A and mossy fibre sprouting but not in cases with other types of MTS. SV2C staining was located in the inner molecular layer of the dentate gyrus and colocalized with dynorphin, ZnT3 and VGLUT1, suggesting selective expression in presynaptic glutamatergic Zn2+-rich terminals of abnormal sprouting fibres. SV2 expression patterns correlated with histological subtypes of MTS, but not with clinical features or therapeutic regimens in this patient cohort. Conclusion In classical MTS1A, the expression of SV2 isoforms is altered with a marked decrease of SV2A and SV2B paralleling synaptic loss and a selective increase of SV2C in sprouting mossy fibres. These findings suggest a different physiology of sprouting synapses and the possibility to target them with SV2C-specific strategies.

Glioblastoma multiforme (GBM, WHO grade IV) is the most common and lethal subtype of primary brain tumor with a median overall survival of 15 months from the time of diagnosis. The presence in GBM of a cancer population displaying neural stem cell (NSC) properties as well as tumor-initiating abilities and resistance to current therapies suggests that these glioblastoma-initiating cells (GICs) play a central role in tumor development and are closely related to NSCs. However, it is nowadays still unclear whether GICs derive from NSCs, neural progenitor cells or differentiated cells such as astrocytes or oligodendrocytes. On the other hand, NSCs are located in specific regions of the adult brain called neurogenic niches that have been shown to control critical stem cell properties, to nourish NSCs and to support their self-renewal. This “seed-and-soil” relationship has also been adapted to cancer stem cell research as GICs also require a specific micro-environment to maintain their “stem cell” properties. In this review, we will discuss the controversies surrounding the origin and the identification of GBM stem cells and highlight the micro-environment impact on their biology.

Background. Patients with glioblastoma (GBM) have an overall median survival of 15 months despite multimodal therapy. These catastrophic survival rates are to be correlated to systematic relapses that might arise from remaining glioblastoma stem cells (GSCs) left behind after surgery. In this line, it has recently been demonstrated that GSCs are able to escape the tumor mass and preferentially colonize the adult subventricular zone (SVZ). At a distance from the initial tumor site, these GSCs might therefore represent a high-quality model of clinical resilience to therapy and cancer relapses as they specifically retain tumor-initiating abilities. Method. While relying on recent findings that have validated the existence of GSCs in the human SVZ, we questioned the role of the SVZ niche as a potential GSC reservoir involved in therapeutic failure. Results. Our results demonstrate that (i) GSCs located in the SVZ are specifically resistant to radiation in vivo, (ii) these cells display enhanced mesenchymal roots that are known to be associated with cancer radioresistance, (iii) these mesenchymal traits are specifically upregulated by CXCL12 (stromal cell-derived factor-1) both in vitro and in the SVZ environment, (iv) the amount of SVZ-released CXCL12 mediates GBM resistance to radiation in vitro, and (v) interferes with the CXCL12/CXCR4 signalling system, allowing weakening of the tumor mesenchymal roots and radiosensitizing SVZ-nested GBM cells. Conclusion. Together, these data provide evidence on how the adult SVZ environment, through the release of CXCL12, supports GBM therapeutic failure and potential tumor relapse.

Casein kinase II contributes to the growth and survival of malignant gliomas and attracts increasing attention as a therapeutic target in these tumors. Several reports have suggested that this strategy might be most relevant for specific subgroups of patients, namely Verhaak's classical and TP53 wild-type tumors. Using kinase assays and microarray genetic profiling in a series of 27 proprietary fresh frozen surgical glioma samples, we showed that constitutive CK2 kinase activation is not restricted to tumors that present increased copy numbers or mRNA expression of its catalytic or regulatory subunits, and can result from a functional activation by various cytokines from the glioma microenvironment. Using corresponding primary tumor and human astrocyte cell cultures as well as glioma cell lines, we confirmed that CK2 inhibition is selectively toxic to malignant glial tumors, without any restriction to tumor class or to TP53 status. We finally showed that while the contribution of CK2 to the constitutive NF-κB hyperactivation in malignant gliomas is at best moderate, a delayed activation of NF-κB may associate with the therapeutic resistance of glioma cells to CK2 inhibition.

Inhibitors of casein kinase 2 (CK2), a regulator of cell proliferation and mediator of the DNA damage response, are being evaluated in clinical trials for the treatment of cancers. Apigenin was capable of inhibiting the activation of CK2 following γ irradiation in LN18 and U87 malignant glioma cells. Apigenin and siRNA-mediated CK2 protein depletion further inhibited NF-κB activation and altered the Tyr68 phosphorylation of Chk2 kinase, a DNA damage response checkpoint kinase, following irradiation. However, CK2 inhibition did not decrease the ability of these glioma cells to repair double-strand DNA breaks, as assessed by COMET assays and γ-H2Ax staining. Likewise, apigenin and siRNA-induced depletion of CK2 failed to sensitize glioma cells to the cytotoxic effect of 2 to 10 G-rays of γ irradiation, as assessed by clonogenic assays. These results contrast with those found in other cancer types, and urge to prudence regarding the inclusion of malignant glioma patients in clinical trials that assess the radiosensitizing role of CK2 inhibitors in solid cancers.

In patients with glioblastoma multiforme, recurrence is the rule despite continuous advances in surgery, radiotherapy and chemotherapy. Within these malignant gliomas, glioblastoma stem cells or initiating cells have been recently described, and they were shown to be specifically involved in experimental tumorigenesis. In this study, we show that some human glioblastoma cells injected into the striatum of immunodeficient nude mice exhibit a tropism for the subventricular zones. There and similarily to neurogenic stem cells, these subventricular glioblastoma cells were then able to migrate toward the olfactory bulbs. Finally, the glioblastoma cells isolated from the adult mouse subventricular zones and olfactory bulbs display high tumorigenicity when secondary injected in a new mouse brain. Together, these data suggest that neurogenic zones could be a reservoir for particular cancer-initiating cells.

International audience; Osteopontin (OPN) is a secreted protein involved in most aspects of tumor progression and metastasis development. Elevated OPN expression has been reported in multiple types of cancer including glioblastoma (GBM), the highest grade and most aggressive brain tumor. GBMs contain a subpopulation of glioma-initiating cells (GICs) implicated in progression, therapeutic resistance and recurrence. We have previously demonstrated that OPN silencing inhibited GBM cell growth in vitro and in vivo. Moreover, activation of CD44 signaling upon OPN ligation has been recently implicated in the acquisition of a stem cell phenotype by GBM cells. The present study is aimed to explore OPN autocrine function using shRNA silencing strategy in GICs enriched from GBM cell lines and a human primary GBM grown in EGF and bFGF defined medium. The removal of these growth factors and addition of serum induced a significant loss of OPN expression in GICs. We showed that OPN-silenced GICs were unable to grow as spheres and this capacity was restored by exogenous OPN. Importantly, the expression of Sox2, Oct3/4 and Nanog, key stemness transcription factors, was significantly decreased in GICs upon OPN targeting. We identified Akt/mTOR/p70S6K as the main signaling pathway triggered following OPN-mediated EGFR activation in GICs. Finally, in an orthotopic xenograft mouse model, the tumorigenic potential of U87-MG sphere cells was completely abrogated upon OPN silencing. Our demonstration of endogenous OPN major regulatory effects on GICs stemness phenotype and tumorigenicity implies a greater role than anticipated for OPN in GBM pathogenesis from initiation and progression to probable recurrence.

BACKGROUND: The objective was to analyze the role of IkappaB-zeta in the cell death resistance and growth of glioblastomas. METHODS: The CNV, mRNA and protein expression of NFKBIZ/IKB-zeta were assessed in low grade and high grade glioma samples from the TCGA, the Rembrandt and proprietary cohorts of tumors, totaling 1195 samples. Data were correlated to histological grade and patient survival. IKBzeta expression was knocked down using inducible sh-RNA technology in GBM cultures, and clonogenic assays, MTT counts, caspase activation and LDH release assays, Western blotting, FACS and DNA damage assays and in situ xenograft models of malignant gliomas in nude mice were analyzed in these conditions. IKB-zeta interacting proteins were defined using immunoprecipitation, mass spectroscopy and proximity ligation assays, and protein localization was identified using selective compartmental protein extractions methods. RESULTS: NFKBIZ was amplified in 2.3% of LGG and 10% of GBM (P < 0.0001, Chi-Sqare), and its mRNA and protein expression increased with grade. NFKBIZ mRNA expression significantly associated with poor survival in a multivariate continuous Cox model (465 tumors) that also included patient age and molecular subclass (classical, mesenchymal, neural, proneural) : p = 0.030, HR: 1.12, 95%-CI: 1.01-1.24. IKB-zeta knock-down allows the translocation of STAT proteins to the mitochondria, induces a massive necroptotic cell death through a transcription-independent, TNF-related mechanism, and prevents tumor growth in xenograft models of human GBM. CONCLUSIONS: NFKBIZ is overexpressed in malignant gliomas, protects them from necroptotic cell death via a transcription-independent mechanism and constitutes a promising therapeutic target for these tumors.

BACKGROUND: In 30-40% of glioblastoma patients, the disease present with an epileptic seizure. Some studies suggest that tumor-associated epilepsy influences the survival of glioblastoma patients, possibly due to early diagnosis or the tumors' biology. However, the in vivo mechanisms underlying the suggested prognostic effect of epilepsy in glioblastoma patients are still unclear. METHODS: In a retrospective cohort study, we investigated the effect of epilepsy on survival of 230 glioblastoma patients using multivariate Weibull analysis. Gene set enrichment analyses defined the transcriptional effects of epilepsy and anti-epileptic drug treatments using gene expression microarrays of 50 fresh frozen surgical specimens. Protein expression was analyzed using immunohistochemistry on tissue microarrays containing surgical specimens of 230 patients. In vitro reporter gene assays, western blotting, kinase assays and immunocytochemistry experiments were performed to confirm our findings. RESULTS: Epilepsy is an independent prognostic factor in glioblastoma patients (HR 0.71, 95%CI: 0.53 - 0.96, p = 0.024), irrespective of other clinical characteristics. The epilepsy subgroup displayed a distinct gene expression pattern, with a significant downregulation of, notably, the oncologically relevant NF-κB, C/EBPβ and STAT3, -5A and -5B transcription factor associated genes. At a protein level, a significant downregulation of phosphorylated STAT3 and C/EBPβ expression, but not of phosphorylated STAT5b or NF-κB p65, was confirmed in the surgical samples. In addition, a significant repression of NF-κB was observed in the patients who were treated with the anti-epileptic drug valproic acid (VPA) prior to their surgery. In vitro, VPA inhibited NF-κB transcriptional activity and was shown to increase acetylated histone H3 and H4 expression. CONCLUSION: Glioblastoma patients with tumor-associated epilepsy form a distinct subgroup with a favorable prognosis and a distinct gene expression pattern involving downregulation of oncologically relevant pathways. The anti-epileptic drug VPA downregulates NF-κB signaling, which might, as suggested in recent literature, contribute to a longer survival of glioblastoma patients.

BACKGROUND Multiple choroid plexus papillomas (CPPs) are rare. Usually, they correspond to villous hypertrophy or metastasis occurring during cerebrospinal dissemination. Multiple CPPs have rarely been reported as synchronous tumors. CASE REPORT Three synchronous CPPs were resected in a 59-year-old female 6 years after their first imaging description. Pathology showed mucus-producing CPP in all 3, 1 of the 3 presenting some signs of atypia. No p53 or hSNF5/INI1 mutation, or signs of polyoma viruses infection were found. CONCLUSION Although no clear cause for the multifocality was found, the simultaneous presence of the three tumors and their benign histology suggest that they were synchronous and not metastatic. The issue of differentiating synchronous CPPs from metastatic CPP is discussed.

BACKGROUND: Several studies suggest that involvement of the subventricular zone (SVZ) by glioblastoma is a negative prognostic factor associating to poor patient survival. We confirm this relationship and explore the biological significance of this prognostic effect in a series of 647 consecutive glioblastoma patients. METHODS: We performed multivariable Cox regression analysis using a retrospective consecutive cohort of 647 primary GBM patients. SVZ involvement was determined using gadolinium-enhanced T1 pre-operative MRI scans. The prognostic effect was adjusted for age, gender, KPS, surgery type, post-operative treatment and tumor volume. Molecular classification and gene set enrichment analyses were performed using mRNA expression microarrays of a subset of these patients. Oncologically relevant protein expression patterns (CD133, NF-kB, C/EBP-b and STAT) were determined using immunohistochemistry on tissue microarrays containing a large subset of the tumors. RESULTS: Involvement of the SVZ is a significant prognostic factor in glioblastoma, and remains associated to survival after correction for age, gender, KPS, surgery type, post-operative treatment and tumor volume (adjusted HR: 1.58, 95% CI: 1.27-1.95). Gene and protein expression correlations to SVZ status to explore the biological significance of this effect will be presented at the SNO meeting. CONCLUSIONS: Involvement of the SVZ is an independent prognostic factor in glioblastoma. The biological relevance of this effect will be presented.

Glioblastoma multiforme (GBM) is the most prevalent and dismal central nervous system tumor.1 It thrives due to rapid proliferation, widespread brain invasion, and resistance to conventional treatments. While the biological events and key signaling pathway disruptions that drive its growth are being progressively deciphered,2,3 the tumor's mechanisms of resistance to conventional DNA-damaging agents and ionizing radiation—the mainstays of current therapies4—remain largely unknown.5 Connexins, the major constituents of gap junction channels, constitute a family of 21 transmembrane proteins6 that contribute to intercellular communication, help regulate cell proliferation and survival, and are frequently underexpressed in the early stages of oncogenesis.7 Astrocytes can express connexin (Cx)43, Cx30, and Cx26.8–10 Astrocytic gliomas, by contrast, frequently underexpress Cx43,11 which can act as a tumor suppressor in these tumors.12,13 Studies also suggest that astrocytomas may lack the expression of Cx30,14 and forced expression of this protein could reduce the tumorigenicity of rodent glioma cells.14 The tumor suppressive effects of connexins do not exclusively stem from the restoration of functional gap junction structures.14–16 Cx43, for instance, interacts with zona occludens (ZO)–1, ZO-2, Src, tubulin, and beta-catenin to modulate the growth control pathways of ZO-1–associated nucleic acid-binding protein, mitogen-activated protein kinase/extracellular signal-regulated kinase, and Wnt beta-catenin17–21 and to regulate the expression of cell cycle regulators like p27Kip, cyclins D1 and D3, and Skp2.15,22,23 Cx43 can also shuttle to the mitochondria and help protect cells against ischemia-reperfusion stresses.24 In addition, the carboxy terminus of Cx43 can shuttle to the nucleus, where it is believed to directly regulate gene expression and help inhibit cell growth.25,26 In contrast to Cx43, however, little is known of the mechanisms of Cx30-dependent cell growth inhibition, even though this protein can also associate with ZO-2 and alpha- and beta-catenin and can shuttle to the nucleus upon forced expression.20,27 In this study, we analyzed the tumor suppressive effects of Cx30 in malignant astrocytic gliomas. We demonstrated its growth inhibitory potential in these tumors and dissected the involved signaling cascades. In addition, however, Cx30 protected human gliomas against radiation-induced DNA damage via a heat shock protein (HSP)90–dependent translocation into the mitochondria that favors ATP production, DNA repair, and cell survival. As a consequence, Cx30 expression adversely affected patient survival in 2 independent cohorts of radiation-treated glioblastoma patients. These results demonstrate the central regulatory role of Cx30 in the biology of human gliomas. Acting as both a tumor growth suppressor and a radioprotective agent, this protein is a potential biomarker and an attractive target for specific therapeutic interventions against these tumors.