Glioma Lines Research Articles

Glioma IL13Rα2 Is Associated with Mesenchymal Signature Gene Expression and Poor Patient Prognosis

A major challenge for successful immunotherapy against glioma is the identification and characterization of validated targets. We have taken a bioinformatics approach towards understanding the biological context of IL-13 receptor α2 (IL13Rα2) expression in brain tumors, and its functional significance for patient survival. Querying multiple gene expression databases, we show that IL13Rα2 expression increases with glioma malignancy grade, and expression for high-grade tumors is bimodal, with approximately 58% of WHO grade IV gliomas over-expressing this receptor. By several measures, IL13Rα2 expression in patient samples and low-passage primary glioma lines most consistently correlates with the expression of signature genes defining mesenchymal subclass tumors and negatively correlates with proneural signature genes as defined by two studies. Positive associations were also noted with proliferative signature genes, whereas no consistent associations were found with either classical or neural signature genes. Probing the potential functional consequences of this mesenchymal association through IPA analysis suggests that IL13Rα2 expression is associated with activation of proinflammatory and immune pathways characteristic of mesenchymal subclass tumors. In addition, survival analyses indicate that IL13Rα2 over-expression is associated with poor patient prognosis, a single gene correlation ranking IL13Rα2 in the top ~1% of total gene expression probes with regard to survival association with WHO IV gliomas. This study better defines the functional consequences of IL13Rα2 expression by demonstrating association with mesenchymal signature gene expression and poor patient prognosis. It thus highlights the utility of IL13Rα2 as a therapeutic target, and helps define patient populations most likely to respond to immunotherapy in present and future clinical trials.

Anticancer Effects of Niclosamide in Human Glioblastoma

Glioblastoma is a highly malignant, invariably fatal brain tumor for which effective pharmacotherapy remains an unmet medical need. Screening of a compound library of 160 synthetic and natural toxic substances identified the antihelmintic niclosamide as a previously unrecognized candidate for clinical development. Considering the cellular and interindividual heterogeneity of glioblastoma, a portfolio of short-term expanded primary human glioblastoma cells (pGBM; n = 21), common glioma lines (n = 5), and noncancer human control cells (n = 3) was applied as a discovery platform and for preclinical validation. Pharmacodynamic analysis, study of cell-cycle progression, apoptosis, cell migration, proliferation, and on the frequency of multipotent/self-renewing pGBM cells were conducted in vitro, and orthotopic xenotransplantation was used to confirm anticancer effects in vivo. Niclosamide led to cytostatic, cytotoxic, and antimigratory effects, strongly reduced the frequencies of multipotent/self-renewing cells in vitro, and after exposure significantly diminished the pGBMs' malignant potential in vivo. Mechanism of action analysis revealed that niclosamide simultaneously inhibited intracellular WNT/CTNNB1-, NOTCH-, mTOR-, and NF-κB signaling cascades. Furthermore, combinatorial drug testing established that a heterozygous deletion of the NFKBIA locus in glioblastoma samples could serve as a genomic biomarker for predicting a synergistic activity of niclosamide with temozolomide, the current standard in glioblastoma therapy. Together, our data advocate the use of pGBMs for exploration of compound libraries to reveal unexpected leads, for example, niclosamide that might be suited for further development toward personalized clinical application.

Resistance to Oncolytic Myxoma Virus Therapy in Nf1−/−/Trp53−/− Syngeneic Mouse Glioma Models Is Independent of Anti-Viral Type-I Interferon

Despite promising preclinical studies, oncolytic viral therapy for malignant gliomas has resulted in variable, but underwhelming results in clinical evaluations. Of concern are the low levels of tumour infection and viral replication within the tumour. This discrepancy between the laboratory and the clinic could result from the disparity of xenograft versus syngeneic models in determining in vivo viral infection, replication and treatment efficacy. Here we describe a panel of primary mouse glioma lines derived from Nf1 +/− Trp53 +/− mice in the C57Bl/6J background for use in the preclinical testing of the oncolytic virus Myxoma (MYXV). These lines show a range of susceptibility to MYXV replication in vitro, but all succumb to viral-mediated cell death. Two of these lines orthotopically grafted produced aggressive gliomas. Intracranial injection of MYXV failed to result in sustained viral replication or treatment efficacy, with minimal tumour infection that was completely resolved by 7 days post-infection. We hypothesized that the stromal production of Type-I interferons (IFNα/β) could explain the resistance seen in these models; however, we found that neither the cell lines in vitro nor the tumours in vivo produce any IFNα/β in response to MYXV infection. To confirm IFNα/β did not play a role in this resistance, we ablated the ability of tumours to respond to IFNα/β via IRF9 knockdown, and generated identical results. Our studies demonstrate that these syngeneic cell lines are relevant preclinical models for testing experimental glioma treatments, and show that IFNα/β is not responsible for the MYXV treatment resistance seen in syngeneic glioma models.

Siglec‐h on activated microglia for recognition and engulfment of glioma cells

Sialic-acid-binding immunoglobulin-like lectin-h (Siglec-h) is a recently identified mouse-specific CD33-related Siglec that signals via DAP12/TYROBP. Expression of Siglec-h has been observed on plasmacytoid dendritic cells and microglia, but the ligand and the function of Siglec-h remained elusive. Here, we demonstrate gene transcription and protein expression of Siglec-h by mouse microglia after interferon-γ treatment or polarization into a M1-subtype. Microglial Siglec-h acted as phagocytosis receptor since targeting of microsphere beads to Siglec-h triggered their uptake into the microglia. The extracellular domain of Siglec-h protein bound to mouse glioma lines, but not to astrocytes or other normal mouse cells. Microglial cells stimulated to express Siglec-h engulfed intact glioma cells without prior induction of apoptosis and slightly reduced glioma cell number in culture. Phagocytosis of glioma cells by activated microglia was dependent on Siglec-h and its adapter molecule DAP12. Thus, data show that M1-polarized microglial cells can engulf glioma cells via a DAP12-mediated Siglec-h dependent mechanism.

Abstract 4476: Cytotoxicity of a novel bi-functional temozolomide analog, DP68, is independent of MGMT status in glioblastoma models.

Abstract Resistance to temozolomide (TMZ) in the majority of glioblastoma multiforme (GBM) is mediated by O6-methylguanine methyltransferase (MGMT). In this study, the efficacy of TMZ, a mono-functional methylating agent, was compared to a bi-functional TMZ analog (DP68) and a corresponding mono-functional TMZ analog (DP86) in established glioma lines (T98, U251), primary GBM xenograft lines (GBM12, GBM22, GBM6) and a TMZ-resistant model with high level MGMT expression (GBM12TMZ). Using a combination of CyQuant and neurosphere assays, the IC50 with TMZ treatment for MGMT expressing lines (U251, GBM12, GBM22) was much lower than non-expressing lines (T98, G6, G12TMZ), while all lines were sensitive to DP68 regardless of MGMT status (see Table). In the same assays, cells were less sensitive to DP86, but cytotoxicity was also independent of MGMT status. In both U251 and T98, DP68 resulted in a time-and dose-dependent induction of markers for DNA damage and checkpoint activation (P-H2AX, ATM, KAP1, Chk1 and Chk2) and a pronounced S-phase arrest within 24 hours of treatment. Based on these results and the hypothesis that DP68 induces DNA cross-links, siRNA knockdown was used to further dissect the importance of the ATM and ATR signaling pathways and the Fanconi Anemia (FA) cross-link repair pathway for DP68 cytotoxicity. Compared to no treatment, 1 microM DP68 resulted in 100% relative absorbance in siCONtrol transfected U251 cells, 41% for siATR, 35% for siFANCD2 and 90% for siATM. In contrast, only siATR and not siFAND2 nor siATM sensitized U251 cells to DP86. Similarly, inhibition of ATR (VE-821), but not ATM (KU-60019) potently sensitized cells to DP68 (see Table). Taken together, the inhibitory effects of DP68 are superior to TMZ in the GBM cell lines tested, regardless of MGMT expression. Furthermore, the siRNA and western blotting suggest that recovery from DP68 is dependent on ATR and the FA repair pathway. TMZ (IC50, microM) DP68 (IC50, microM)) DP86 (IC50, microM)) CyQuant Ctrl 1uM VE-821 1uM KU-60019 Ctrl 1uM VE-821 1uM KU-60019 Ctrl 1uM VE-821 1uM KU-60019 U251 25 20 20 2.5 0.8 2.1 70 40 65 T98 >320 200 260 9 2 7 115 60 115 Neurosphere Ctrl VE-821 KU-60019 Ctrl VE-821 KU-60019 Ctrl VE-821 KU-60019 G12 4 3.2 4.2 0.9 0.08 0.7 18 6 18 G22 40 NA NA 1 NA NA 40 NA NA G6 160 NA NA 0.5 NA NA 15 NA NA G12TMZ >300 NA NA 2 NA NA 22 NA NA Citation Format: Ann C. Mladek Tuma, Yulian Ramirez, Dimitrios Pletsas, Richard T. Wheelhouse, Roger M. Phillips, Ross H. Alonzo, Kaitlyn Knudson, Jann N. Sarkaria. Cytotoxicity of a novel bi-functional temozolomide analog, DP68, is independent of MGMT status in glioblastoma models. [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 4476. doi:10.1158/1538-7445.AM2013-4476

Therapeutic Potential of an Anti-diabetic Drug, Metformin: Alteration of miRNA expression in Prostate Cancer Cells

Prostate cancer is the most commonly diagnosed cancer in males in many populations. Metformin is the most widely used anti-diabetic drug in the world, and there is increasing evidence of a potential efficacy of this agent as an anti-cancer drug. Metformin inhibits the proliferation of a range of cancer cells including prostate, colon, breast, ovarian, and glioma lines. MicroRNAs (miRNAs) are a class of small, non- coding, single-stranded RNAs that downregulate gene expression. We aimed to evaluate the effects of metformin treatment on changes in miRNA expression in PC-3 cells, and possible associations with biological behaviour. Average cell viability and cytotoxic effects of metformin were investigated at 24 hour intervals for three days using the xCELLigence system. The IC50 dose of metformin in the PC-3 cells was found to be 5 mM. RNA samples were used for analysis using custom multi-species microarrays containing 1209 probes covering 1221 human mature microRNAs present in miRBase 16.0 database. Among the human miRNAs investigated by the arrays, 10 miRNAs were up-regulated and 12 miRNAs were down-regulated in the metformin-treated group as compared to the control group. In conclusion, expression changes in miRNAs of miR-146a, miR-100, miR-425, miR-193a-3p and, miR-106b in metformin-treated cells may be important. This study may emphasize a new role of metformin on the regulation of miRNAs in prostate cancer.

Targeting and killing of glioblastoma with activated T cells armed with bispecific antibodies

BackgroundSince most glioblastomas express both wild-type EGFR and EGFRvIII as well as HER2/neu, they are excellent targets for activated T cells (ATC) armed with bispecific antibodies (BiAbs) that target EGFR and HER2.MethodsATC were generated from PBMC activated for 14 days with anti-CD3 monoclonal antibody in the presence of interleukin-2 and armed with chemically heteroconjugated anti-CD3×anti-HER2/neu (HER2Bi) and/or anti-CD3×anti-EGFR (EGFRBi). HER2Bi- and/or EGFRBi-armed ATC were examined for in vitro cytotoxicity using MTT and 51Cr-release assays against malignant glioma lines (U87MG, U118MG, and U251MG) and primary glioblastoma lines.ResultsEGFRBi-armed ATC killed up to 85% of U87, U118, and U251 targets at effector:target ratios (E:T) ranging from 1:1 to 25:1. Engagement of tumor by EGFRBi-armed ATC induced Th1 and Th2 cytokine secretion by armed ATC. HER2Bi-armed ATC exhibited comparable cytotoxicity against U118 and U251, but did not kill HER2-negative U87 cells. HER2Bi- or EGFRBi-armed ATC exhibited 50—80% cytotoxicity against four primary glioblastoma lines as well as a temozolomide (TMZ)-resistant variant of U251. Both CD133– and CD133+ subpopulations were killed by armed ATC. Targeting both HER2Bi and EGFRBi simultaneously showed enhanced efficacy than arming with a single BiAb. Armed ATC maintained effectiveness after irradiation and in the presence of TMZ at a therapeutic concentration and were capable of killing multiple targets.ConclusionHigh-grade gliomas are suitable for specific targeting by armed ATC. These data, together with additional animal studies, may provide the preclinical support for the use of armed ATC as a valuable addition to current treatment regimens.

OASIS/CREB3L1 Is Induced by Endoplasmic Reticulum Stress in Human Glioma Cell Lines and Contributes to the Unfolded Protein Response, Extracellular Matrix Production and Cell Migration

OASIS is a transcription factor similar to ATF6 that is activated by endoplasmic reticulum stress. In this study we investigated the expression of OASIS in human glioma cell lines and the effect of OASIS knock-down on the ER stress response and cell migration. OASIS mRNA was detected in three distinct glioma cell lines (U373, A172 and U87) and expression levels were increased upon treatment with ER stress-inducing compounds in the U373 and U87 lines. OASIS protein, which is glycosylated on Asn-513, was detected in the U373 and U87 glioma lines at low levels in control cells and protein expression was induced by ER stress. Knock-down of OASIS in human glioma cell lines resulted in an attenuated unfolded protein response to ER stress (reduced GRP78/BiP and GRP94 induction) and decreased expression of chondroitin sulfate proteoglycan extracellular matrix proteins, but induction of the collagen gene Col1a1 was unaffected. Cells in which OASIS was knocked-down exhibited altered cell morphology and reduced cell migration. These results suggest that OASIS is important for the ER stress response and maintenance of some extracellular matrix proteins in human glioma cells.

Serum Amyloid A impacts the growth of melanoma cells and may be a link between inflammation and cancer outcome.

Event Abstract Back to Event Serum Amyloid A impacts the growth of melanoma cells and may be a link between inflammation and cancer outcome. Luziane P. Bellé1*, Franciele H. Knebel1, Renata Albuquerque1, Silvya S. Maria-Engler1 and Ana Campa1 1 University of Sao Paulo, Brazil Serum amyloid A (SAA), an inflammatory protein, has been called attention due its increased concentrations in plasma of cancer patients. Recently, it was suggested an immune modulation mediated by SAA in melanoma patients (Nat Immunol., 11:1039-46, 2010). Previous study from our group showed increased proliferation of human glioma lines in the presence of SAA. Furthermore SAA also affected migration and invasion (Mediators of Inflammation, 2013, in press). To verify if these findings could be extended to other tumor we evaluate the effects of SAA (5 or 10 µg/mL) on proliferation, migration, invasion and cytokine release on melanoma cells (SK-Mel 19, 28, 103 and 147). We used [3H]-thymidine incorporation and clonogenic assay to evaluate proliferation, flow cytometry for necrosis and apoptosis, scratch test and Boyden chamber assay to evaluate mobility and ELISA for cytokine release. We observed that SAA inhibited proliferation without inducing cells death, however, by optical microscopy, we observed that SAA could cause morphologic alterations similar to cell senescence. Some of the cell lines seemed to be more sensitive to SAA in relation to cellular motility. In these cases it was observed decreased migration and/or invasion. Furthermore, SAA induced the release of IL-6 and IL-8 cytokines on melanoma cells. Our finding demonstrated that SAA might, in some cases, impact melanoma growth and emphasizes the possibility to be one of the factors that links inflammation with cancer outcome. Acknowledgements This work was supported by Grants from support: Sao Paulo Research Foundation (FAPESP, Sao Paulo); National Counsel of Technological and Scientific Development (CNPq, Brasília); and Coordination of Improvement of Higher Education Personnel (CAPES, Brasília) References De Santo, C., Arscott, R., Booth, S., Karydis, I., Jones, M., Asher, R., Salio, M., Middleton, M., Cerundolo, V. (2010). Invariant NKT cells modulate the suppressive activity of IL-10-secreting neutrophils differentiated with serum amyloid A. Nat Immunol..11, 1039-46. Knebel, F.H., Albuquerque, R.C., Massaro, R.R., Maria-Engler, S.S., Campa, A. (2013). Dual effect of serum amyloid A on the invasiveness of glioma cells. Mediators of inflammation. 2013. Keywords: Melanoma, Serum Amyloid A Protein, proliferation, Inflammation, invasiveness Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune receptors and signaling Citation: Bellé LP, Knebel FH, Albuquerque R, Maria-Engler SS and Campa A (2013). Serum Amyloid A impacts the growth of melanoma cells and may be a link between inflammation and cancer outcome.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00912 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Ms. Luziane P Bellé, University of Sao Paulo, São Paulo, Brazil, luzibelle@usp.br Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Luziane P Bellé Franciele H Knebel Renata Albuquerque Silvya S Maria-Engler Ana Campa Google Luziane P Bellé Franciele H Knebel Renata Albuquerque Silvya S Maria-Engler Ana Campa Google Scholar Luziane P Bellé Franciele H Knebel Renata Albuquerque Silvya S Maria-Engler Ana Campa PubMed Luziane P Bellé Franciele H Knebel Renata Albuquerque Silvya S Maria-Engler Ana Campa Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Cell death and neuronal differentiation of glioblastoma stem‐like cells induced by neurogenic transcription factors

Glioblastoma multiform (GBM) are devastating brain tumors containing a fraction of multipotent stem-like cells which are highly tumorigenic. These cells are resistant to treatments and are likely to be responsible for tumor recurrence. One approach to eliminate GBM stem-like cells would be to force their terminal differentiation. During development, neurons formation is controlled by neurogenic transcription factors such as Ngn1/2 and NeuroD1. We found that in comparison with oligodendrogenic genes, the expression of these neurogenic genes is low or absent in GBM tumors and derived cultures. We thus explored the effect of overexpressing these neurogenic genes in three CD133(+) Sox2(+) GBM stem-like cell cultures and the U87 glioma line. Introduction of Ngn2 in CD133(+) cultures induced massive cell death, proliferation arrest and a drastic reduction of neurosphere formation. Similar effects were observed with NeuroD1. Importantly, Ngn2 effects were accompanied by the downregulation of Olig2, Myc, Shh and upregulation of Dcx and NeuroD1 expression. The few surviving cells adopted a typical neuronal morphology and some of them generated action potentials. These cells appeared to be produced at the expense of GFAP(+) cells which were radically reduced after differentiation with Ngn2. In vivo, Ngn2-expressing cells were unable to form orthotopic tumors. In the U87 glioma line, Ngn2 could not induce neuronal differentiation although proliferation in vitro and tumoral growth in vivo were strongly reduced. By inducing cell death, cell cycle arrest or differentiation, this work supports further exploration of neurogenic proteins to oppose GBM stem-like and non-stem-like cell growth.

ATM inhibitor KU-55933 increases the TMZ responsiveness of only inherently TMZ sensitive GBM cells

Ataxia telangiectasia mutated (ATM) kinase is critical in sensing and repairing DNA double-stranded breaks (DSBs) such as those induced by temozolomide (TMZ). ATM deficiency increases TMZ sensitivity, which suggests that ATM inhibitors may be effective TMZ sensitizing agents. In this study, the TMZ sensitizing effects of 2 ATM specific inhibitors were studied in established and xenograft-derived glioblastoma (GBM) lines that are inherently sensitive to TMZ and derivative TMZ-resistant lines. In parental U251 and U87 glioma lines, the addition of KU-55933 to TMZ significantly increased cell killing compared to TMZ alone [U251 survival: 0.004 ± 0.0015 vs. 0.08 ± 0.01 (p < 0.001), respectively, and U87 survival: 0.02 ± 0.005 vs. 0.04 ± 0.002 (p < 0.001), respectively] and also elevated the fraction of cells arrested in G2/M [U251 G2/M fraction: 61.8 ± 1.1 % vs. 35 ± 0.8 % (p < 0.001), respectively, and U87 G2/M fraction 25 ± 0.2 % vs.18.6 ± 0.4 % (p < 0.001), respectively]. In contrast, KU-55933 did not sensitize the resistant lines to TMZ, and neither TMZ alone or combined with KU-55933 induced a G2/M arrest. While KU-55933 did not enhance TMZ induced Chk1/Chk2 activation, it increased TMZ-induced residual γ-H2AX foci in the parental cells but not in the TMZ resistant cells. Similar sensitization was observed with either KU-55933 or CP-466722 combined with TMZ in GBM12 xenograft line but not in GBM12TMZ, which is resistant to TMZ due to MGMT overexpression. These findings are consistent with a model where ATM inhibition suppresses the repair of TMZ-induced DSBs in inherently TMZ-sensitive tumor lines, which suggests an ATM inhibitor potentially could be deployed with an improvement in the therapeutic window when combined with TMZ.

Stem-like Tumor-Initiating Cells Isolated from IL13Rα2 Expressing Gliomas Are Targeted and Killed by IL13-Zetakine–Redirected T Cells

To evaluate IL13Rα2 as an immunotherapeutic target for eliminating glioma stem-like cancer initiating cells (GSC) of high-grade gliomas, with particular focus on the potential of genetically engineered IL13Rα2-specific primary human CD8(+) CTLs (IL13-zetakine(+) CTL) to target this therapeutically resistant glioma subpopulation. A panel of low-passage GSC tumor sphere (TS) and serum-differentiated glioma lines were expanded from patient glioblastoma specimens. These glioblastoma lines were evaluated for expression of IL13Rα2 and for susceptibility to IL13-zetakine(+) CTL-mediated killing in vitro and in vivo. We observed that although glioma IL13Rα2 expression varies between patients, for IL13Rα2(pos) cases this antigen was detected on both GSCs and more differentiated tumor cell populations. IL13-zetakine(+) CTL were capable of efficient recognition and killing of both IL13Rα2(pos) GSCs and IL13Rα2(pos) differentiated cells in vitro, as well as eliminating glioma-initiating activity in an orthotopic mouse tumor model. Furthermore, intracranial administration of IL13-zetakine(+) CTL displayed robust antitumor activity against established IL13Rα2(pos) GSC TS-initiated orthotopic tumors in mice. Within IL13Rα2 expressing high-grade gliomas, this receptor is expressed by GSCs and differentiated tumor populations, rendering both targetable by IL13-zetakine(+) CTLs. Thus, our results support the potential usefullness of IL13Rα2-directed immunotherapeutic approaches for eradicating therapeutically resistant GSC populations.

Effective Radiovirotherapy for Malignant Gliomas by Using Oncolytic Measles Virus Strains Encoding the Sodium Iodide Symporter (MV-NIS)

Engineered measles virus (MV) strains deriving from the vaccine lineage represent a promising oncolytic platform and are currently being tested in phase I trials. In this study, we have demonstrated that MV strains genetically engineered to express the human sodium iodide symporter (NIS) have significant antitumor activity against glioma lines and orthotopic xenografts; this compares favorably with the MV strain expressing the human carcinoembryonic antigen, which is currently in clinical testing. Expression of NIS protein in infected cells results in effective concentration of radioactive iodine, which allows for in vivo monitoring of localization of MV-NIS infection by measuring uptake of (123)I or (99m)Tc. In addition, radiovirotherapy with MV-NIS followed by (131)I administration resulted in significant increase of MV-NIS antitumor activity as compared with virus alone in both subcutaneous (p=0.0003) and orthotopic (p=0.004) glioblastoma models. In conclusion, MV-NIS-based radiovirotherapy has significant antitumor activity against glioblastoma multiforme and represents a promising candidate for clinical translation.

Equine Herpesvirus Type 1-Mediated Oncolysis of Human Glioblastoma Multiforme Cells

The cytolytic animal virus equine herpesvirus type 1 (EHV-1) was evaluated for its oncolytic potential against five human glioblastoma cell lines. EHV-1 productively infected four of these cell lines, and the degree of infection was positively correlated with glioma cell death. No human major histocompatibility complex class 1 (MHC-I) was detected in the resistant glioma line, while infection of the susceptible glioma cell lines, which expressed human MHC-I, were blocked with antibody to MHC-I, indicating that human MHC-I acts as an EHV-1 entry receptor on glioma cells.

A dialog between glioma and microglia that promotes tumor invasiveness through the CCL2/CCR2/interleukin-6 axis

Glioma cells in situ are surrounded by microglia, suggesting the potential of glioma-microglia interactions to produce various outcomes. As chemokines are important mediators of cell-cell communication, we sought first to identify commonly expressed chemokines in 16 human glioma lines. We found CCL2 (macrophage chemoattractant protein-1) messenger RNA to be expressed by the majority of glioma lines. However, these lines did not express the CCL2 receptor, CCR2, which was found on microglia. Next, we overexpressed CCL2 in the U87 glioma line, which has low basal level of CCL2, to investigate the hypothesis that glioma-secreted CCL2 interacts with microglia to affect glioma growth. Stable clones with 10- to 12-fold elevation of CCL2 have similar growth rate and invasive capacity as vector controls when cultured in isolation. However, in coculture with microglia in a three-dimensional collagen gel matrix, the invasiveness of CCL2-overexpressing clones was increased. Gene array analyses were then undertaken and they revealed that interleukin (IL)-6 was consistently increased in the coculture. Recombinant IL-6 enhanced the invasiveness of glioma cells when these were cultured alone, whereas a neutralizing antibody to IL-6 attenuated the microglia-stimulated glioma invasiveness. Finally, we found that human glioma specimens in situ contained IL-6 immunoreactivity that was expressed on CD68+ cells. This study has uncovered a mechanism by which glioma cells exploit microglia for increased invasiveness. Specifically, glioma-derived CCL2 acts upon CCR2-bearing microglia, which then produces IL-6 to stimulate gliomas. The CCL2/CCR2/IL-6 loop is a potential therapeutic target for the currently incurable malignant gliomas.

MiR-185 Targets the DNA Methyltransferases 1 and Regulates Global DNA Methylation in human glioma

BackgroundPerturbation of DNA methylation is frequent in cancers and has emerged as an important mechanism involved in tumorigenesis. To determine how DNA methylation is modified in the genome of primary glioma, we used Methyl-DNA immunoprecipitation (MeDIP) and Nimblegen CpG promoter microarrays to identify differentially DNA methylation sequences between primary glioma and normal brain tissue samples.MethodsMeDIP-chip technology was used to investigate the whole-genome differential methylation patterns in glioma and normal brain tissues. Subsequently, the promoter methylation status of eight candidate genes was validated in 40 glioma samples and 4 cell lines by Sequenom's MassARRAY system. Then, the epigenetically regulated expression of these genes and the potential mechanisms were examined by chromatin immunoprecipitation and quantitative real-time PCR.ResultsA total of 524 hypermethylated and 104 hypomethylated regions were identified in glioma. Among them, 216 hypermethylated and 60 hypomethylated regions were mapped to the promoters of known genes related to a variety of important cellular processes. Eight promoter-hypermethylated genes (ANKDD1A, GAD1, HIST1H3E, PCDHA8, PCDHA13, PHOX2B, SIX3, and SST) were confirmed in primary glioma and cell lines. Aberrant promoter methylation and changed histone modifications were associated with their reduced expression in glioma. In addition, we found loss of heterozygosity (LOH) at the miR-185 locus located in the 22q11.2 in glioma and induction of miR-185 over-expression reduced global DNA methylation and induced the expression of the promoter-hypermethylated genes in glioma cells by directly targeting the DNA methyltransferases 1.ConclusionThese comprehensive data may provide new insights into the epigenetic pathogenesis of human gliomas.

Taurine Biosynthesis by Neurons and Astrocytes

The physiological roles of taurine, a product of cysteine degradation and one of the most abundant amino acids in the body, remain elusive. Taurine deficiency leads to heart dysfunction, brain development abnormalities, retinal degradation, and other pathologies. The taurine synthetic pathway is proposed to be incomplete in astrocytes and neurons, and metabolic cooperation between these cell types is reportedly needed to complete the pathway. In this study, we analyzed taurine synthesis capability as reported by incorporation of radioactivity from [(35)S]cysteine into taurine, in primary murine astrocytes and neurons, and in several transformed cell lines (human (SH-SY5Y) and murine (N1E-115) neuroblastoma, human astrocytoma (U-87 MG and 1321 N1), and rat glioma (C6)). Extensive incorporation of radioactivity from [(35)S]cysteine into taurine was observed in rat glioma cells as well as in primary mouse astrocytes and neurons, establishing the presence of an intact taurine synthesis pathway in these cells. Interestingly, exposure of cells to cysteine or cysteamine resulted in elevated intracellular hypotaurine without a corresponding increase in taurine levels, suggesting that oxidation of hypotaurine limits taurine synthesis in cells. Consistent with its role as an organic osmolyte, taurine synthesis was stimulated under hypertonic conditions in neurons.

Abstract 3847: Cytoplasmic ECT2: Implications for invasion and migration and RAS-RAC cross-talk in glioma

Abstract Gliomas are a highly malignant primary brain tumour characterized by their adept ability to invade normal brain parenchyma. We have previously identified the cytoplasmic and normally nuclear sequestered pro-cytokinetic, small cytoskeletal GTPase ECT2 to be involved in the invasive process of malignant glioma. ECT2 shows dysregulated spatial regulation in malignant gliomas with increased cytoplasmic expression, in particular at the leading edge of invading primary glioma cells. This aberrant ECT2 plays a role in RAC1 and CDC42 activation, as shRNA mediated loss of ECT2 leads to no effect on cell cycle, but a less invasive phenotype and diminished RAC1 and CDC42 levels. Using immunoprecipitation and mass spectrometry we identified the novel RAS-GAP, RASAL2 as a significant cytoplasmic interactor with ECT2 in gliomas. We show this interaction represents a mechanism by which RAS (which is aberrantly activated in gliomas) can cross-talk with the RAC/RHO pathway and coordinate growth and invasion of gliomas. Over-expression of ECT2 leads to mesenchymal-amoeboid transition (MAT) in glioma cells. Cells expressing GFP-ECT2 show hyperactive cortical dynamics with the formation of membrane blebs, similar to an amoeboid phenotype. ECT2 localized to the cortical blebs during retraction in a similar manner to active RHOA in amoeboid cells. It is known that MAT relies on an antagonistic relationship between RHOA (pro-amoeboid) and RAC1 (promesenchymal). We tested whether our phenotype was mediated by RHOA or RAC1 by administration of the RHOA downstream inhibitor of ROCK, Y27632 and RAC1 NSC23766. RHOA pathway inhibition by Y27632 at 25uM was able to reverse this hypercortical activity and resulted in formation of lamellipodia, as seen with RAC1 activation. NSC23766 was unable to abolish the amoeboid phenotype in concentrations of up to 100uM. A proportion of GFP-ECT2 amoeboid cells were capable of migrating at velocities of up to 10 um/sec. Interestingly, although loss of ECT2 has no phenotypic changes in cells plated in a 2D context, loss of ECT2 by shRNA in cells plated within a collagen matrix show marked reduction in amoeboid phenotype. The ability of glioma cells to undergo MAT is a relatively new concept, however well documented in other cancer subtypes such as melanoma. The molecular switches involved in MAT remain elusive, however our data reveals a potential role for ECT2 in migratory phenotype switching. Importantly, when a stably expressing ECT2 shRNA glioma line is orthotopically injected into mouse brains, these ECT2 knockdown xenografts exhibit enhanced survival and diminished tumour engraftment as compared to controls. Taken together, this suggests that ECT2 is a viable target for malignant gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3847. doi:10.1158/1538-7445.AM2011-3847

The CD133+ tumor stem-like cell-associated antigen may elicit highly intense immune responses against human malignant glioma

To explore the immunogenicity of glioma stem-like cell-associated antigens (SAAs) from sorted or unsorted glioma tumor stem-like cells (TSCs) as well as irradiated TSCs. Two primary human malignant glioma lines (SHG62, SHG66) and U87 cell line were primarily cultured in the serum-free medium (SFM) supplemented with EGF/bFGF. TSCs were identified by their self-renewal, multi-lineage differentiation and tumorigenic activity. To prepare SAAs in vitro, CD133+ TSCs were sorted either by magnetic cell sorting or with irradiation (6 Gy).The cytotoxicity induced by autogenous myeloid dendritic cell (DC)-mediated SAA-specific cytotoxic T lymphocytes (CTLs) was assessed by the Just Another Method test. SHG62, SHG66, and U87 cells contained TSCs. CD133+ SAAs-specific CTLs were significantly more cytotoxic than effector cells loaded with unsorted SAA (P < 0.05). Effector cells loaded with irradiated SAAs were more cytotoxic than those with regular SAAs (P < 0.01). SAAs from CD133+ TSCs and irradiated TSCs provide highly immunogenic antigens. TSCs might be a novel source of antigens for DC vaccination against malignant gliomas.

A Dual PI3K/mTOR Inhibitor, PI-103, Cooperates with Stem Cell–Delivered TRAIL in Experimental Glioma Models

The resistance of glioma cells to a number of antitumor agents and the highly invasive nature of glioma cells that escape the primary tumor mass are key impediments to the eradication of tumors in glioma patients. In this study, we evaluated the therapeutic efficacy of a novel PI3-kinase/mTOR inhibitor, PI-103, in established glioma lines and primary CD133(+) glioma-initiating cells and explored the potential of combining PI-103 with stem cell-delivered secretable tumor necrosis factor apoptosis-inducing ligand (S-TRAIL) both in vitro and in orthotopic mouse models of gliomas. We show that PI-103 inhibits proliferation and invasion, causes G(0)-G(1) arrest in cell cycle, and results in significant attenuation of orthotopic tumor growth in vivo. Establishing cocultures of neural stem cells (NSC) and glioma cells, we show that PI-103 augments the response of glioma cells to stem cell-delivered S-TRAIL. Using bimodal optical imaging, we show that when different regimens of systemic PI-103 delivery are combined with NSC-derived S-TRAIL, a significant reduction in tumor volumes is observed compared with PI-103 treatment alone. To our knowledge, this is the first study that reveals the antitumor effect of PI-103 in intracranial gliomas. Our findings offer a preclinical rationale for application of mechanism-based systemically delivered antiproliferative agents and novel stem cell-based proapoptotic therapies to improve treatment of malignant gliomas.