High-grade Glioma Cell Research Articles

Mutational profiling of kinases in glioblastoma.

BackgroundGlioblastoma is a highly malignant brain tumor for which no cure is available. To identify new therapeutic targets, we performed a mutation analysis of kinase genes in glioblastoma.MethodsDatabase mining and a literature search identified 76 kinases that have been found to be mutated at least twice in multiple cancer types before. Among those we selected 34 kinase genes for mutation analysis. We also included IDH1, IDH2, PTEN, TP53 and NRAS, genes that are known to be mutated at considerable frequencies in glioblastoma. In total, 174 exons of 39 genes in 113 glioblastoma samples from 109 patients and 16 high-grade glioma (HGG) cell lines were sequenced.ResultsOur mutation analysis led to the identification of 148 non-synonymous somatic mutations, of which 25 have not been reported before in glioblastoma. Somatic mutations were found in TP53, PTEN, IDH1, PIK3CA, EGFR, BRAF, EPHA3, NRAS, TGFBR2, FLT3 and RPS6KC1. Mapping the mutated genes into known signaling pathways revealed that the large majority of them plays a central role in the PI3K-AKT pathway.ConclusionsThe knowledge that at least 50% of glioblastoma tumors display mutational activation of the PI3K-AKT pathway should offer new opportunities for the rational development of therapeutic approaches for glioblastomas. However, due to the development of resistance mechanisms, kinase inhibition studies targeting the PI3K-AKT pathway for relapsing glioblastoma have mostly failed thus far. Other therapies should be investigated, targeting early events in gliomagenesis that involve both kinases and non-kinases.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-718) contains supplementary material, which is available to authorized users.

P17.07 * FENOFIBRATE AS A NOVEL THERAPEUTIC ADJUVANT FOR TARGETING GLIOMA STEM CELLS

Glioma stem cells (GSC) are being increasingly recognized as the culprits behind the resistance of glioblastoma to standard therapy, including surgery, temozolomide and radiation therapy. Intensive research is currently underway to uncover novel and effective GSC targeting strategies Fenofibrate is an FDA-approved hypolipidemic agent in widespread clinical use. Recent evidence has supported a possible role for fenofibrate as an nti-cancer agent in various cancer pathologies, including glioblastoma. Our own previous work has demonstrated that fenofibrate reduces proliferation and induces apoptosis in human high-grade glioma cells. Here, we present our results on the effects of fenofibrate on GSC in the in vitro setting. We chose to examine invasion because this property is known as a hallmark obstacle in the successful treatment of glioblastoma and recent data has implicated GSC. We found that fenofibrate treatment at a dose of 25 microM significantly decreased GSC invasion in a standard Matrigel invasion assay. We then examined the effect of fenofibrate on stem-cell marker expression and chose CD133 and Oct4. CD133 remains one of the most commonly used GSC markers to date, while Oct4 is an embryonic stem cell marker linked to glioma grade. Results demonstrated that fenofibrate treatment at 25 microM significantly diminished expression of both CD133 and Oct4 as measured by flow cytometry and/or immunofluorescence staining. Finally, we investigated possible mechanisms and identified NF-kappaB and CyclinD1 as candidates. Inhibition of NF-kappaB reportedly decreases invasion in GSC and is a well-established property of fenofibrate in the inflammatory setting. Downregulation of Cyclin D1 has been found to correlate with attenuated invasion in human glioblastoma cells and may be observed with fenofibrate treatment of lymphoma cells. Using a panel of established human glioma cell lines, we found that fenofibrate treatment robustly diminished the expression of both NF-kappaB and Cyclin D1 in a dose-dependent and p53-independent manner. In conclusion, our data show that fenofibrate significantly attenuates both GSC invasion and expression of the CD133 and Oct4 stem-cell markers, in association with diminished NF-kappaB and Cyclin D1 signaling in glioma cells. Based on these results, we propose that fenofibrate should be considered a novel and direct GSC targeting agent. Its relatively low cost, minimal toxicity and availability make it an attractive agent for rapid translation to the clinical setting as an adjuvant treatment for glioblastoma.

Activity of the polyamine-vectorized anti-cancer drug F14512 against pediatric glioma and neuroblastoma cell lines.

The poor prognosis of children with high-grade glioma (HGG) and high-risk neuroblastoma, despite multidisciplinary therapeutic approaches, demands new treatments for these indications. F14512 is a topoisomerase II inhibitor containing a spermine moiety that facilitates selective uptake by tumor cells via the Polyamine Transport System (PTS) and increases topoisomerase II poisoning. Here, F14512 was evaluated in pediatric HGG and neuroblastoma cell lines. PTS activity and specificity were evaluated using a fluorescent spermine-coupled probe. The cytotoxicity of F14512, alone or in combination with ionizing radiation and chemotherapeutic agents, was investigated in vitro. The antitumor activity of F14512 was assessed in vivo using a liver-metastatic model of neuroblastoma. An active PTS was evidenced in all tested cell lines, providing a specific and rapid transfer of spermine-coupled compounds into cell nuclei. Competition experiments confirmed the essential role of PTS in the cell uptake and cytotoxicity of F14512. This cytotoxicity appeared greater in neuroblastoma cells compared with HGG cells but appeared independent of PTS activity levels. In vivo evaluation confirmed a marked and prolonged antitumoral effect in neuroblastoma cells. The combinations of F14512 with cisplatin and carboplatin were often found to be synergistic, and we demonstrated the significant radiosensitizing potential of F14512 in the MYCN-amplified Kelly cell line. Thus, F14512 appears more effective than etoposide in pediatric tumor cell lines, with greater efficacy in neuroblastoma cells compared with HGG cells. The synergistic effects observed with platinum compounds and the radiosensitizing effect could lead to a clinical development of the drug in pediatric oncology.

Piperlongumine treatment inactivates peroxiredoxin 4, exacerbates endoplasmic reticulum stress, and preferentially kills high-grade glioma cells.

Piperlongumine, a natural plant product, kills multiple cancer types with little effect on normal cells. Piperlongumine raises intracellular levels of reactive oxygen species (ROS), a phenomenon that may underlie the cancer-cell killing. Although these findings suggest that piperlongumine could be useful for treating cancers, the mechanism by which the drug selectively kills cancer cells remains unknown. We treated multiple high-grade glioma (HGG) sphere cultures with piperlongumine and assessed its effects on ROS and cell-growth levels as well as changes in downstream signaling. We also examined the levels of putative piperlongumine targets and their roles in HGG cell growth. Piperlongumine treatment increased ROS levels and preferentially killed HGG cells with little effect in normal brain cells. Piperlongumine reportedly increases ROS levels after interactions with several redox regulators. We found that HGG cells expressed higher levels of the putative piperlongumine targets than did normal neural stem cells (NSCs). Furthermore, piperlongumine treatment in HGG cells, but not in normal NSCs, increased oxidative inactivation of peroxiredoxin 4 (PRDX4), an ROS-reducing enzyme that is overexpressed in HGGs and facilitates proper protein folding in the endoplasmic reticulum (ER). Moreover, piperlongumine exacerbated intracellular ER stress, an effect that was mimicked by suppressing PRDX4 expression. Our results reveal that the mechanism by which piperlongumine preferentially kills HGG cells involves PRDX4 inactivation, thereby inducing ER stress. Therefore, piperlongumine treatment could be considered as a novel therapeutic option for HGG treatment.

Assembly and interrogation of regulatory networks elucidates synergistic dependencies in human malignancies (475.3)

The recent onslaught of molecular data, across multiple human malignancies, is producing an unprecedented repertoire of genetic and epigenetic alterations contributing to tumorigenesis and progression. Yet, the direct impact of this knowledge on tumor treatment and prevention is still largely unproven. Loss of tumor suppressor function is difficult to target pharmacologically and, with a handful of exceptions, alterations providing potential drug targets are relatively infrequent in cancer patients and are thus unlikely to support clinical development.By reconstructing and interrogating the in vivo regulatory logic of distinct cancer cells, which integrates multiple aberrant signals resulting from genetic and epigenetic alterations, systems biology is starting to elucidate and mechanistically validate both oncogene and non‐oncogene addiction mechanisms. These mechanisms are exquisitely dependent on the molecular landscape of cancer subtypes, can be targeted pharmacologically, and are frequently synergistic, thus providing uniquely specific entry points for combination therapy.In this presentation, we will discuss recent result in the discovery of synergistic, non‐oncogene addiction mechanisms and their application to the stratification and treatment of prostate cancer, high‐grade glioma, and non‐small cell lung cancer. This approach is highly extensible and has been applied to a variety of additional tumor subtypes, to the study of stem cell differentiation, reprogramming, and pluripotency control, as well as to the study of neurodegenerative diseases, such as ALS.

Characterization of fenofibrate-mediated anti-proliferative pro-apoptotic effects on high-grade gliomas and anti-invasive effects on glioma stem cells

Glioblastoma is the most common, and at the same time, most aggressive type of high-grade glioma (HGG). The prognosis of glioblastoma patients treated with standard therapy including surgery, temozolomide and radiation therapy remains poor. Peroxisome proliferator-activated receptor-α (PPARα) agonists are in widespread clinical use for the treatment of hyperlipidemia. Recent evidence has suggested a potential role in various cancers including glioblastoma. In this study, we characterized the effects of PPARα agonist, fenofibrate, directly on HGG cells and glioma stem cells (GSC). Fenofibrate exhibited dose-dependent p53-independent anti-proliferative effects on HGG starting at 25μM and pro-apoptotic effects starting at 50μM, suggesting that the anti-proliferative actions are present only at 25μM. PPARα was expressed in all HGG cell lines. Inhibition of PPARα with specific inhibitor GW6471 did not affect either proliferation or apoptosis suggesting that these are PPARα-independent effects. Fenofibrate treatment of HGG cells robustly diminished the expression of key signaling pathways, including NF-κB and cyclin D1. Phosphorylation of Akt was also diminished, with no change in total Akt. Effects on apoptotic signaling molecules, Bax and Bcl-xL, had a trend towards pro-apoptotic effects. With respect to GSC, fenofibrate treatment at 25μM significantly decreased invasion in association with a decrease in CD133 and Oct4 expression. Overall, results support consideration of fenofibrate as an anti-glioma agent and establish its potential as an adjunct treatment strategy for HGG. Translation to the clinical setting could be rapid given its current use as a clinical agent and its low toxicity profile.

RHPS4 G-Quadruplex Ligand Induces Anti-Proliferative Effects in Brain Tumor Cells

BackgroundTelomeric 3′ overhangs can fold into a four-stranded DNA structure termed G-quadruplex (G4), a formation which inhibits telomerase. As telomerase activation is crucial for telomere maintenance in most cancer cells, several classes of G4 ligands have been designed to directly disrupt telomeric structure.MethodsWe exposed brain tumor cells to the G4 ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) and investigated proliferation, cell cycle dynamics, telomere length, telomerase activity and activated c-Myc levels.ResultsAlthough all cell lines tested were sensitive to RHPS4, PFSK-1 central nervous system primitive neuroectodermal cells, DAOY medulloblastoma cells and U87 glioblastoma cells exhibited up to 30-fold increased sensitivity compared to KNS42 glioblastoma, C6 glioma and Res196 ependymoma cells. An increased proportion of S-phase cells were observed in medulloblastoma and high grade glioma cells whilst CNS PNET cells showed an increased proportion of G1-phase cells. RHPS4-induced phenotypes were concomitant with telomerase inhibition, manifested in a telomere length-independent manner and not associated with activated c-Myc levels. However, anti-proliferative effects were also observed in normal neural/endothelial cells in vitro and ex vivo.ConclusionThis study warrants in vivo validation of RHPS4 and alternative G4 ligands as potential anti-cancer agents for brain tumors but highlights the consideration of dose-limiting tissue toxicities.

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor. Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Targeting Wee1 for the treatment of pediatric high-grade gliomas

We investigated the efficacy of the Wee1 inhibitor MK-1775 in combination with radiation for the treatment of pediatric high-grade gliomas (HGGs), including diffuse intrinsic pontine gliomas (DIPGs). Gene expression analysis was performed for 38 primary pediatric gliomas (3 grade I, 10 grade II, 11 grade III, 14 grade IV) and 8 normal brain samples using the Agilent 4 × 44 K array. Clonogenic survival assays were carried out in pediatric and adult HGG cell lines (n = 6) to assess radiosensitizing effects of MK-1775. DNA repair capacity was evaluated by measuring protein levels of γ-H2AX, a marker of double strand DNA breaks. In vivo activity of MK-1775 with radiation was assessed in 2 distinct orthotopic engraftment models of pediatric HGG, including 1 derived from a genetically engineered mouse carrying a BRAF(V600E) mutation, and 1 xenograft model in which tumor cells were derived from a patient's DIPG. Wee1 is overexpressed in pediatric HGGs, with increasing expression positively correlated with malignancy (P = .007 for grade III + IV vs I + II) and markedly high expression in DIPG. Combination treatment of MK-1775 and radiation reduced clonogenic survival and increased expression of γ-H2AX to a greater extent than achieved by radiation alone. Finally, combined MK-1775 and radiation conferred greater survival benefit to mice bearing engrafted, orthotopic HGG and DIPG tumors, compared with treatment with radiation alone (BRAF(V600E) model P = .0061 and DIPG brainstem model P = .0163). Our results highlight MK-1775 as a promising new therapeutic agent for use in combination with radiation for the treatment of pediatric HGGs, including DIPG.

Tumor-Specific Chromosome Mis-Segregation Controls Cancer Plasticity by Maintaining Tumor Heterogeneity

Aneuploidy with chromosome instability is a cancer hallmark. We studied chromosome 7 (Chr7) copy number variation (CNV) in gliomas and in primary cultures derived from them. We found tumor heterogeneity with cells having Chr7-CNV commonly occurs in gliomas, with a higher percentage of cells in high-grade gliomas carrying more than 2 copies of Chr7, as compared to low-grade gliomas. Interestingly, all Chr7-aneuploid cell types in the parental culture of established glioma cell lines reappeared in single-cell-derived subcultures. We then characterized the biology of three syngeneic glioma cultures dominated by different Chr7-aneuploid cell types. We found phenotypic divergence for cells following Chr7 mis-segregation, which benefited overall tumor growth in vitro and in vivo. Mathematical modeling suggested the involvement of chromosome instability and interactions among cell subpopulations in restoring the optimal equilibrium of tumor cell types. Both our experimental data and mathematical modeling demonstrated that the complexity of tumor heterogeneity could be enhanced by the existence of chromosomes with structural abnormality, in addition to their mis-segregations. Overall, our findings show, for the first time, the involvement of chromosome instability in maintaining tumor heterogeneity, which underlies the enhanced growth, persistence and treatment resistance of cancers.

Comparative evaluation of transport mechanisms of trans-1-amino-3-[18F]fluorocyclobutanecarboxylic acid and l-[methyl-11C]methionine in human glioma cell lines

Positron emission tomography (PET) with amino acid tracers is useful for the visualization and assessment of therapeutic effects on gliomas. Our purpose is to elucidate the transport mechanisms of trans-1-amino-3-[18F]fluorocyclobutanecarboxylic acid (anti-[18F]FACBC) and l-[methyl-11C]methionine ([11C]Met) in normal human astrocytes (NHA), low-grade (Hs683, SW1088), and high-grade (U87MG, T98G) human glioma cell lines. Because the short half-lives of fluorine-18 and carbon-11 are inconvenient for in vitro experiments, trans-1-amino-3-fluoro[1-14C]cyclobutanecarboxylic acid (anti-[14C]FACBC) and l-[methyl-14C]methionine ([14C]Met) were used instead of the PET tracers. Time-course uptake experiments showed that uptake of anti-[14C]FACBC was 1.4–2.6 times higher than that of [14C]Met in NHA and low-grade glioma cells, and was almost equal to that of [14C]Met in high-grade glioma cells. To identify the amino acid transporters (AATs) involved in the transport of anti-[14C]FACBC and [14C]Met, we carried out competitive inhibition experiments using synthetic/naturally-occurring amino acids as inhibitors. We found that anti-[14C]FACBC uptake in the presence of Na+ was strongly inhibited by l-glutamine and l-serine (the substrates for ASC system AATs), whereas l-phenylalanine and 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH, the substrates for L system AATs) robustly inhibited Na+-independent anti-[14C]FACBC uptake. Regardless of Na+, [14C]Met uptake was inhibited strongly by l-phenylalanine and BCH. Moreover, the exchange transport activity of l-glutamine for anti-[14C]FACBC was stronger than that of BCH in the presence of Na+, whereas that for [14C]Met was almost equal to BCH. These results demonstrate that ASC and L are important transport systems for anti-[18F]FACBC uptake, while system L is predominantly involved in [11C]Met transport in human astrocytes and glioma cells.

Radiation-induced upregulation of telomerase activity escapes PI3-kinase inhibition in two malignant glioma cell lines

Tumor relapse after radiotherapy is a great concern in the treatment of high-grade gliomas. Inhibition of the PI3-kinase/AKT pathway is known to radiosensitize cancer cells and to delay their DNA repair after irradiation. In this study, we show that the radiosensitization of CB193 and T98G, two high-grade glioma cell lines, by the PI3K inhibitor LY294002, correlates with the induction of G1 and G2/M arrest, but is inconsistently linked to a delayed DNA double-strand break (DSBs) repair. The PI3K/AKT pathway has been shown to activate radioprotective factors such as telomerase, whose inhibition may contribute to the radiosensitization of cancer cells. However, we show that radiation upregulates telomerase activity in LY-294002-treated glioma cells as well as untreated controls, demonstrating a PI3K/AKT-independent pathway of telomerase activation. Our study suggests that radiosensitizing strategies based on PI3-kinase inhibition in high-grade gliomas may be optimized by additional treatments targeting either telomerase activity or telomere maintenance.

A phase I dose-escalation study of intratumoral herpes simplex virus-1 mutant HSV1716 in pediatric/young adult patients with refractory non-central nervous system solid tumors.

10047 Background: HSV1716 is a mutant herpes simplex virus type 1 deleted in the RL1 gene which encodes the protein ICP34.5, a specific determinant of virulence. Mutants lacking the RL1 gene are capable of replication in actively dividing cells but not in terminally differentiated cells – a phenotype exploited to selectively kill tumor cells. Studies in adult patients with high grade glioma, melanoma and squamous cell carcinoma report that HSV1716 is safe when administered by intra-tumoral injection. This is the first study of HSV1716 in pediatric/young adult patients. Methods: The primary endpoint of the study is to assess safety of intratumoral HSV1716 in pediatric/young adult patients, and to determine any dose-limiting toxicities (DLT) at the doses given. Patients undergo long-term follow up in accordance with FDA guidelines for viral vectors. The secondary endpoints of the study are to measure: (i) antiviral immune response; (ii) systemic viremia and viral shedding and (iii) the antitumor activity of HSV1716 by radiological response within the confines of a Phase I study. Patients aged 7 to 30 with solid non-CNS tumors refractory to standard therapy (or for which no therapy exists) are eligible for the study provided the target lesion is accessible to image-guided injection. Eligible patients receive a single dose of HSV1716 (Virttu Biologics Limited) at either 105 or 2x106 plaque forming units (pfu) HSV1716 administered directly into the tumor via ultrasound or CT-guided injection by an interventional radiologist. Tumor response between baseline and day 28 is assessed by modified RECIST criteria. Patients showing at least stable disease may receive up to a 3 additional doses of HSV1716. Results: Recruitment of the first cohort of 3 patients has been completed without DLT or procedure related severe adverse events. In the second cohort, 2 patients have been treated without DLT or procedure related SAE. Conclusions: The trial is in progress and the study is open to recruitment. Clinical trial information: NCT00931931.

An Integrative Systems Analysis of High-grade Glioma Cell Lines: TLRs, Wnt, BRCA1, Netrins, STXBP1 and MDH1 Provide Putative Molecular Targets for Therapeutic Intervention

Gliomas are the most common form of brain tumor, originate from glial cells and characterized by rapidly proliferating cells frequently associated with an invasive phenotype. Several focused studies have successfully demonstrated the genetic origin of these tumors; however, a systems level study to provide comprehensive overview of complex molecular alterations associated with glioma pathogenesis is essential. In this study the main aim was to identify molecular targets associated with high-grade gliomas using systems based investigation, and to identify pathways, molecular networks and functional modules associated with curated genes/ proteins that have altered expression in highgrade glioma cell lines. DAVID and PANTHER analysis tools were used to identify molecular functions, and functional annotation clusters associated with curated dataset. IPA, MetaCore and GeneSpring were used to study the complex interaction of curated molecules and identification of associated signaling pathways. The signaling pathways mediated by TLRs, IL-8, STATs and Wnt, which regulate malignant behavior of high-grade gliomas, were identified as candidates for therapeutic targets. STXBP1, PDK1, MAP3K8 and HIF1A/ARNT appeared as most significant modules in GeneSpring functional analysis. GLIPR1 and MDH1 were identified as potential candidates for further study in gliomas. This study represents a systems level approach to extract biologically relevant information from existing resources and provide new contexts for future research in gliomas.

Abstract 2618: MDA-9/syntenin is a key regulator of glioma pathogenesis.

Abstract It is predicted that 66,290 new cases of primary central nervous system (CNS) tumors will be diagnosed in the United States in 2012. Of these, glioblastoma multiforme (GBM) is among the most aggressive tumors. The extraordinary invasiveness of GBM contributes to treatment failure and the grim prognosis of patients diagnosed with this tumor. Consequently, it is imperative to define further the cellular and molecular mechanisms that control GBM invasion and identify novel therapeutic targets. Melanoma differentiation associated gene-9 (MDA-9/syntenin) is a highly conserved PDZ domain-containing scaffolding protein that promotes invasion and metastasis in in vitro and in vivo in human melanoma models. The ability of single high-grade glioma cells to invade the surrounding normal brain tissue is an important characteristic that makes treating these tumors so difficult. Additionally, the highly angiogenic nature of GBM tumors contributes to their malignancy. Therefore, if MDA-9/syntenin plays a role in GBM invasion and angiogenesis, this gene or its downstream-regulated molecules could represent new targets that will aid in furthering the efficacy of current and future therapeutics. The objectives of our studies were to: (1) Determine the expression of MDA-9/syntenin in astrocytic tumors, (2) Assess the role of MDA-9/syntenin on the invasive ability of astrocytomas in vitro and in vivo, (3) Elucidate important molecular signaling events associated with mda-9/syntenin, and (4) Assess the role of MDA-9/syntenin on angiogenic potential in malignant astrocytomas. We now demonstrate that in primary human patient samples, MDA-9/syntenin is overexpressed in a large proportion of high-grade astrocytomas and this expression pattern correlates with tumor grade. Forced overexpression of MDA-9/syntenin enhanced Matrigel invasion, while knockdown of MDA-9/syntenin inhibited invasion, suppressed migration as measured by rate of gap closure, and reduced anchorage-independent growth in soft agar. Moreover, overexpression of MDA-9/syntenin in cells with low endogenous levels (Sw1783 and GBM5) increased activation of c-Src, p-P38MAPK, and NF-κB, leading to elevated MMP2 expression. Conversely, the knockdown of MDA-9/syntenin in GBM cell lines T98G and U87MG resulted in reduced levels of p-Src, p-IκBα, and MMP2. GBM6 cells that stably express shmda-9 had decreased invasive ability and formed smaller tumors in a mouse xenograft model. Furthermore, knockdown of MDA-9/syntenin was found to decrease the production and secretion of IL-8, a known angiogenic cytokine. Using intracranial orthotopic xenografts, MDA-9/syntenin knockdown resulted in a less invasive tumor phenotype with decreased CD31 expression. Our findings indicate that MDA-9/syntenin is a novel and important mediator of invasion and angiogenesis, a key regulator of pathogenesis in GBM, and this gene represents a potential target for anti-invasive treatment in human astrocytomas. Citation Format: Timothy P. Kegelman, Swadesh K. Das, Bin Hu, Mitchell Menezes, Luni Emdad, Santanu Dasgupta, Albert S. Baldwin, Jeffrey N. Bruce, Paul Dent, Maurizio Pellecchia, Devanand Sarkar, Paul B. Fisher. MDA-9/syntenin is a key regulator of glioma pathogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2618. doi:10.1158/1538-7445.AM2013-2618

Abstract B2: Oligodendritic precursor mediates vascular remodeling within glioma microenvironment in PDGF-C dependent manner

Abstract The platelet derived growth factor (PDGF) signaling pathway is upregulated in many glioma subtypes, plays a role in gliomagenesis and promotes brain tumor progression. In this study, we demonstrate a role for PDGF signaling in the maintenance of brain tumor microenvironment homeostasis. We detected PDGFRa-expressing stromal cells potentially derived from oligodendritic precursor cells in both human and mouse high-grade gliomas. PDGFRa+ cells are predominately located at the invasive front of gliomas, and have a unique perivascular distribution. We suggest that these stromal cells play an important role in both initiating angiogenesis and supporting tumor progression. In an orthotopic model of glioma in PDGFRa deficient mice, tumor growth was significantly inhibited and gadolinium enhancement was decreased shown by bioluminescence and MRI, respectively. Glioma-associated blood vessels in PDGFRa knockout mice have smaller lumens and relatively normal vascular morphology. Glioma-associated oligodendritic precursor cells promoted endothelial sprouting and tubule formation by abrogating the inhibitory effect of astrocytes. We also demonstrated stromal-derived PDGF-CC as a key factor in the recruitment and activation of PDGFRa+ stromal cells. Utilizing PDGF-CC deficient mice, we recapitulate the glioma phenotype demonstrated in PDGFRa; knockout mice, suggesting their joint contribution to this pathway's integrity. In conclusion, the PDGF-CC/PDGFRa signaling axis in glioma stroma appears to contribute to the remodeling of the vascular architecture in the brain and influences subsequent angiogenesis. Citation Format: Yujie Huang, David Lyden, Jeffrey Greenfield. Oligodendritic precursor mediates vascular remodeling within glioma microenvironment in PDGF-C dependent manner. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B2.

Abstract B85: MDA-9/syntenin is a key regulator of glioma pathogenesis

Abstract It is predicted that 66,290 new cases of primary central nervous system (CNS) tumors will be diagnosed in the United States in 2012. Of these, glioblastoma multiforme (GBM) is among the most aggressive tumors. The extraordinary invasiveness of GBM contributes to treatment failure and the grim prognosis of patients diagnosed with this tumor. Consequently, it is imperative to define further the cellular and molecular mechanisms that control GBM invasion and identify promising novel therapeutic targets. Melanoma differentiation associated gene-9 (MDA-9/syntenin) is a highly conserved PDZ domain-containing scaffolding protein that promotes invasion and metastasis in in vitro and in vivo in human melanoma models. The ability of single high-grade glioma cells to invade the surrounding normal brain tissue is an important characteristic that makes treating these tumors so difficult. Additionally, the highly angiogenic nature of GBM tumors contributes to their malignancy. Therefore, if MDA-9/syntenin plays a role in GBM invasion and angiogenesis, this gene or its downstream-regulated molecules could represent new targets that will aid in furthering the efficacy of current and future therapeutics. The objectives of our studies were to: (1) Determine the expression of MDA-9/syntenin in astrocytic tumors, (2) Assess the role of MDA-9/syntenin on the invasive ability of astrocytomas in vitro and in vivo, (3) Elucidate important molecular signaling events associated with mda-9/syntenin, and (4) Assess the role of MDA-9/syntenin on angiogenic potential in malignant astrocytomas. To achieve these objectives we have employed gain-of-function (GOF) and Loss-of-function (LOF) strategies that incorporate both Genetic and Pharmacological approaches. We now demonstrate that in primary human patient samples, MDA-9/syntenin is overexpressed in a large proportion of high-grade astrocytomas and this expression pattern correlates with tumor grade. Forced overexpression (GOF) of MDA-9/syntenin through an adenoviral vector enhanced Matrigel invasion, while knockdown (LOF) of MDA-9/syntenin inhibited invasion, suppressed migration as measured by rate of gap closure, and reduced anchorage-independent growth in soft agar. Moreover, overexpression of MDA-9/syntenin in cells with low endogenous levels (Sw1783, a grade III astrocytoma, and GBM5) increased activation of c-Src, p-P38MAPK, and NF-κB, leading to elevated MMP2 expression. Conversely, the knockdown of MDA-9/syntenin in GBM cell lines with high levels of MDA-9/syntenin, T98G and U87MG, resulted in reduced levels of p-Src, p-IκBα, and MMP2. GBM6 cells that stably express shmda-9 had decreased invasive ability and formed smaller tumors in a mouse xenograft model. Furthermore, knockdown of MDA-9/syntenin was found to decrease the production and secretion of IL-8, a known angiogenic cytokine. Using intracranial orthotopic xenografts, MDA-9/syntenin knockdown resulted in a less invasive tumor phenotype with decreased CD31 expression. Our findings indicate that MDA-9/syntenin is a novel and important mediator of invasion and angiogenesis, key regulators of pathogenesis, in GBM and this gene represents a potential target for anti-invasive treatment in human astrocytomas. Citation Format: Timothy P. Kegelman, Swadesh K. Das, Bin Hu, Mitchell Menezes, Luni Emdad, Santanu Dasgupta, Albert S. Baldwin, Jr., Jeffery N. Bruce, Paul Dent, Maurizio Pellecchia, Devanand Sarkar, Paul B. Fisher. MDA-9/syntenin is a key regulator of glioma pathogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B85.

GDNF mediates glioblastoma-induced microglia attraction but not astrogliosis

High-grade gliomas are the most common primary brain tumors. Their malignancy is promoted by the complex crosstalk between different cell types in the central nervous system. Microglia/brain macrophages infiltrate high-grade gliomas and contribute to their progression. To identify factors that mediate the attraction of microglia/macrophages to malignant brain tumors, we established a glioma cell encapsulation model that was applied in vivo. Mouse GL261 glioma cell line and human high-grade glioma cells were seeded into hollow fibers (HF) that allow the passage of soluble molecules but not cells. The glioma cell containing HF were implanted into one brain hemisphere and simultaneously HF with non-transformed fibroblasts (controls) were introduced into the contralateral hemisphere. Implanted mouse and human glioma- but not fibroblast-containing HF attracted microglia and up-regulated immunoreactivity for GFAP, which is a marker of astrogliosis. In this study, we identified GDNF as an important factor for microglial attraction: (1) GL261 and human glioma cells secret GDNF, (2) reduced GDNF production by siRNA in GL261 in mouse glioma cells diminished attraction of microglia, (3) over-expression of GDNF in fibroblasts promoted microglia attraction in our HF assay. In vitro migration assays also showed that GDNF is a strong chemoattractant for microglia. While GDNF release from human or mouse glioma had a profound effect on microglial attraction, the glioma-induced astrogliosis was not affected. Finally, we could show that injection of GL261 mouse glioma cells with GDNF knockdown by shRNA into mouse brains resulted in reduced tumor expansion and improved survival as compared to injection of control cells.

Cytotoxicity and Radiosensitization of High Grade Glioma Cells by CI-1033, an Irreversible Pan-Erbb Inhibitor

Background: High-grade gliomas (HGG) are highly infiltrative malignancies, causing considerable mortality in child- and adulthood, necessitating new therapies. Novel therapies directed against multiple epidermal growth factor family (ErbB) members are potentially effective in HGG. Aim: To assess ErbB family expression in normal brain and pediatric and adult HGG in silico and to determine radiosensitizing property of the pan-ErbB inhibitor CI-1033, in HGG cells in vitro. Material and methods: In silico mRNA array expression analysis was performed to assess EGFR, ERBB2, ERBB3, ERBB4 gene expression in normal brain, adult and pediatric HGG. ErbB family protein expression was determined in HGG cell lines using Western blot. Sulforhodamine-B assay was used to assess cytotoxicity of CI- 1033 andclonogenic assays to determine radiosensitization. The effect on cell cycle distribution and PI3K-Akt/Ras- MAPK signalling of CI-1033 ± radiation was measured using flow cytometry. Results: EGFR and ERBB2 were significantly overexpressed in datasets of pediatric and adult HGG. Heterogeneous protein expression of EGFR, ErbB2, 3 and 4 was observed in HGG cell lines. CI-1033 IC50 values of 1.0 μM, 2.5 μM and 4.3 μMwere found in D384MG, U-251 MG and Gli-6 cells, respectively. CI-1033 significantly sensitized Gli-6 and D384MGcells to radiation, with 24 and 48 hrs pre-treatment respectively. Conclusion: EGFR and ErbB2 are overexpressed in adult and childhood HGG. Irreversible pan-ErbB inhibition by CI-1033 is cytotoxic and radiosensitizes HGG cell lines in vitro, warranting further in vivo studies.