Normal Human Astrocyte Cell Research Articles

P2X4R silence suppresses glioma cell growth through BDNF/TrkB/ATF4 signaling pathway.

Purinergic receptor P2X 4 (P2X4R), a member of purinergic channels family and a subtype of ionotropic adenosine triphosphate receptors, plays a critical role in tumorigenesis. Evidence suggested that P2X4R is expressed in rat C6 glioma model, however, its role and the underlying mechanism of action are still unclear in human glioblastoma multiforme (GBM). In the current study, our aim is to examine the function and the molecular basis of P2X4R in GBM. We first observed that GBM cells, U251, T98, U87, U373, and A172 were all high expressed P2X4R, when compared with the normal human astrocytes (NHA) cells. To gain the function of P2X4R, P2X4R silence cells were constructed by transfection with P2X4R small interfering RNA (siRNA). We found that P2X4R deletion impeded T98 and U87 cell viability and proliferation, and further studies indicated that cell apoptosis and caspase-3 activity was increased in T98 and U87 cell transfected with P2X4R siRNA. Subsequently, we confirmed that P2X4R silence suppressed brain-derived neurotrophic factor (BDNF), Trk receptor tyrosine kinases (TrkB), and activating transcription factor 4 (ATF4) expression in T98 and U87 cells. And P2X4R siRNA-induced ATF4-expression inhibition dependent on BDNF/TrkB signaling pathway. The impact of P2X4R silence on T98 and U87 cell growth and apoptosis was reversed by ATF4 overexpression. In summary, this study provides the first evidence that P2X4R plays important roles in GBM cell growth and apoptosis.

MiR‑454‑3p suppresses cell migration and invasion by targeting CPEB1 in human glioblastoma.

MicroRNAs (miRNA/miRs) serve crucial roles in the progression of human glioblastoma (GBM); however, the exact regulatory mechanisms of miRNAs in human GBM remain unclear. The present study aimed to investigate the roles of miR‑454‑3p in human GBM. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis was performed to examine the expression of miR‑454‑3p in glioma tissues and adjacent tissues. Human GBM cell lines (LN‑229, A172 and GL15) and a normal human astrocyte cells (HA1800) were used for analysis. In addition, RT‑qPCR and western blotting were applied for mRNA and protein expression analysis, respectively. The cell proliferation was measured using a Cell Counting kit‑8 assay. Furthermore, scratch and Transwell assays were employed for the analysis of cell migration and invasion. A luciferase reporter assay was used to verify the target of miR‑454‑3p. The results revealed that miR‑454‑3p was downregulated in the glioma tissues and GBM cell lines, including LN‑229, A172 and GL15. Additionally, the overexpression of miR‑454‑3p significantly suppressed the proliferation, migration and invasion of LN‑229 cells. Furthermore, cytoplasmic polyadenylation element‑binding protein 1 (CPEB1) was confirmed as a direct target of miR‑454‑3p. These findings indicated that the overexpression of miR‑454‑3p inhibited cell proliferation, migration and invasion by downregulating CPEB1. Therefore, miR‑454‑3p may act as a tumor suppressor and represent an effective therapeutic strategy in GBM.

Disulfiram, a Re-positioned Aldehyde Dehydrogenase Inhibitor, Enhances Radiosensitivity of Human Glioblastoma Cells In Vitro

PurposeGlioblastoma, the most common brain tumor in adults, has poor prognosis. The purpose of this study was to determine the effect of disulfiram (DSF), an aldehyde dehydrogenase inhibitor, on in vitro radiosensitivity of glioblastoma cells with different methylation status of O6-methylguanine-DNA methyltransferase (MGMT) promoter and the underlying mechanism of such effect.Materials and MethodsFive human glioblastoma cells (U138MG, T98G, U251MG, U87MG, and U373MG) and one normal human astrocyte (NHA) cell were cultured and treated with DSF or 6MV X-rays (0, 2, 4, 6, and 8 Gy). For combined treatment, cells were treated with DSF before irradiation. Surviving fractions fit from cell survival based on colony forming ability. Apoptosis, DNA damage repair, and cell cycle distributionwere assayed bywestern blot for cleaved caspase-3, γH2AX staining, and flow cytometry, respectively.ResultsDSF induced radiosensitization in most of the glioblastoma cells, especially, in the cells with radioresistance as wildtype unmethylated promoter (MGMT-wt), but did not in normal NHA cell. DSF augmented or induced cleavage of caspase-3 in all cells after irradiation. DSF inhibited repair of radiation-induced DNA damage in MGMT-wt cells, but not in cells with methylated MGMT promoter. DSF abrogated radiation-induced G2/M arrest in T98G and U251MG cells.ConclusionRadiosensitivity of glioblastoma cells were preferentially enhanced by pre-irradiation DSF treatment compared to normal cell, especially radioresistant cells such as MGMT-wt cells. Induction of apoptosis or inhibition of DNA damage repair may underlie DSF-induced radiosensitization. Clinical benefit of combining DSF with radiotherapy should be investigated in the future.

Combination of Droplet Extraction and Pico-ESI-MS Allows the Identification of Metabolites from Single Cancer Cells.

We have combined droplet extraction and a pulsed direct current electrospray ionization mass spectrometry method (Pico-ESI-MS) to obtain information-rich metabolite profiling from single cells. We studied normal human astrocyte cells and glioblastoma cancer cells. Over 600 tandem mass spectra (MS2) of metabolites from a single cell were recorded, allowing the successful identification of more than 300 phospholipids. We found the ratios of unsaturated phosphatidylcholines (PCs) to saturated PCs were significantly higher in glioblastoma cells compared to normal cells. In addition, both isomeric PC (17:1) and (phosphatidylethanolamine) PE (20:1) were found in glioblastoma cells, whereas only PC (17:1) was observed in astrocyte cells. Our method paves the way to characterize the chemical contents of single cells, providing rich metabolome information. We suggest that this technique is general and can be applied to other life science studies such as differentiation and drug resistance of individual cells.

MicroRNA-599 suppresses glioma progression by targeting RAB27B.

MicroRNAs (miRNAs/miRs) serve tumor promoting or suppressive roles in different human cancer types, including glioma; however, the regulatory underlying mechanism by which miR-599 affects glioma progression remains largely unknown. The aim of the present study was to investigate the expression of miR-599 in glioma, as well as the underlying regulatory mechanism. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to examine mRNA and protein expression, respectively. MTT, wound healing and transwell assays were conducted to study cell proliferation, migration and invasion, respectively. A dual-luciferase reporter gene assay was used to confirm the targeting association between miR-599 and Ras-related protein Rab-27B (hereafter RAB27B). In the present study, miR-599 expression was observed to be significantly downregulated in human glioma tissues and cell lines, when compared with normal brain tissues and normal human astrocyte cells, respectively. Low miR-599 expression was significantly associated with glioma progression. Ectopic expression of miR-599 caused a significant reduction in the proliferation, migration and invasion of U-87MG Uppsala and U251 cells. Bioinformatics analysis and dual-luciferase reporter gene assay data identified that RAB27B was a direct target gene of miR-599. The mRNA and protein expression of RAB27B was significantly downregulated following miR-599 overexpression in U-87MG Uppsala and U251 cells. Rescue experiments demonstrated that RAB27B rescued the miR-599-induced inhibition of glioma cell growth, migration and invasion. In addition, RAB27B expression was significantly upregulated in glioma tissues and cell lines, and the expression levels of RAB27B were inversely correlated with miR-599 levels in glioma tissues; therefore, the present study demonstrated that miR-599 exerts a tumor-suppressive role in glioma progression by targeting RAB27B, indicating that miR-599 may be used as a potential candidate for glioma treatment.

Knockdown of RWD domain containing 3 inhibits the malignant phenotypes of glioblastoma cells via inhibition of phosphoinositide 3-kinase/protein kinase B signaling.

Glioblastoma is the most common and malignant primary brain tumor. RWD domain containing 3 (RWDD3) has been previously reported to serve a promoting role in pituitary tumors. However, the exact role of RWDD3 in glioblastoma remains unclear. Therefore, the present study aimed to investigate the expression levels of RWDD3 in human glioblastoma tissues and cell lines, as well as to examine the regulatory mechanism of RWDD3 underlying glioblastoma growth and metastasis. The results revealed that RWDD3 was significantly upregulated in glioblastoma tissues compared with normal brain tissues, while high expression of RWDD3 was associated with a shorter survival time of glioblastoma patients. The expression levels of RWDD3 were also higher in the glioblastoma cell lines compared with the normal human astrocyte cell line. Subsequent to knockdown of RWDD3, the proliferation of glioblastoma U87 and U251 cells was significantly decreased, possibly due to the cell cycle arrest at G1 phase, as well as the increased cell apoptosis. Furthermore, downregulation of RWDD3 also suppressed U87 and U251 cell invasion by inhibiting the expression levels of matrix metalloproteinase 2 (MMP2) and MMP9. Molecular mechanism investigation demonstrated that knockdown of RWDD3 significantly downregulated the activity of the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway. Activation of PI3K/AKT signaling prevented the suppressive effects of RWDD3 downregulation on glioblastoma cell proliferation and migration, concurrent with increased protein levels of MMP2 and MMP9. In conclusion, the current study demonstrated for the first time that inhibition of RWDD3 expression inhibited glioblastoma progression, at least partly, via suppressing the PI3K/AKT signaling activity, and thus RWDD3 may be a novel potential therapeutic target for glioblastoma.

Circular RNA hsa-circ-0012129 Promotes Cell Proliferation and Invasion in 30 Cases of Human Glioma and Human Glioma Cell Lines U373, A172, and SHG44, by Targeting MicroRNA-661 (miR-661).

BackgroundCircular RNA (circRNA) is a stable non-coding RNA without 5′-3′ polarity and without a poly-A tail, that contains response elements for microRNAs (miRNAs) such as miR-661. There have previously been few reported studies on the role of circRNAs in glioma. The aim of this study was to investigate the effects of the expression of the circRNA, hsa-circ-0012129, and miR-661 in human glioma tissue and human glioma cell lines.Material/MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of hsa-circ-0012129 and miR-661 in glioma tissues from 31 patients (WHO grades I–IV), compared with adjacent normal tissue, and in human glioma cell lines, U373, A172, and SHG44, compared with the normal human astrocyte cell line, NHA. The MTT assay, colony formation assay, transwell and wound scratch assays were performed to analyze and compare cell viability, cell migration, and invasion.ResultsExpression of hsa-circ-0012129 was significantly increased in glioma tissues and cell lines; hsa-circ-0012129 knockdown significantly suppressed the proliferation, migration, and invasion abilities of U373 and SHG44 cells. A dual-luciferase reporter assay showed that hsa-circ-0012129 contained the complementary binding region with miR-661 and that hsa-circ-0012129 expression negatively regulated miR-661. Rescue experiments showed that miR-661 could reverse the effects of hsa-circ-0012129 on cell viability, cell migration and invasion of glioma cells in vitro.ConclusionsThe findings of this study indicated that, in human glioma cells, the circRNA, hsa-circ-0012129 might act as a natural miR-661 sponge, and that miR-661 could have suppressive effects on the expression of circ-0012129.

Mir-758-5p Suppresses Glioblastoma Proliferation, Migration and Invasion by Targeting ZBTB20

Background/Aims: To determine the cellular functions and clinical significance of micro-758-5p (miR-758-5p) in glioblastoma (GBM) by targeting zinc finger and BTB domain-containing protein 20 (ZBTB20). Methods: Fifty-five paired GBM tissues and adjacent normal tissues, GBM cell lines (U118, LN-299, H4, A172, U87-MG, and U251), and normal human astrocyte cell line (HEB) were used. miR-758-5p mimics, ZBTB20 siRNA, and pcDNA3.1-ZBTB20 were transiently transduced into cancer cells independently or together. qRT-PCR was conducted to analyze the expression of miR-758-5p and ZBTB20. Luciferase reporter assays were performed to determine the effect of miR-758-5p on ZBTB20. Western blot was applied to measure the expression of ZBTB20, PCNA, and cleaved caspase3. Cell Counting Kit-8 (CCK8), colony formation, FACS, and Transwell assays were carried out to detect cellular proliferation, apoptosis, migration, and invasion. Xenograft experiments were implemented to evaluate tumor growth and metastasis in vivo. Results: miR-758-5p was significantly downregulated in GBM tissues and cell lines compared with that in adjacent normal tissues and HEB cells. miR-758-5p overexpression inhibited the proliferation, migration, and invasion of GBM cells and induced apoptosis by regulating the ZBTB20 expression. Pearson correlation analysis also confirmed that miR-758-5p was inversely correlated with ZBTB20 in GBM tissues. miR-758-5p suppressed tumor growth and metastasis in vivo. The restored ZBTB20 expression partially rescued the miR-758-5p-induced inhibition of GBM cell proliferation, migration, and invasion. Kaplan–Meier curve analysis revealed that a high miR-758-5p expression indicated an enhanced prognosis of patients with GBM. Conclusion: miR-758-5p suppressed GBM progression by targeting ZBTB20. The miR-758-5p/ZBTB20 axis might be a promising therapeutic target for GBM treatment.

Abstract LB-195: Bortezomib sensitizes glioblastoma for NK cell immunotherapy

Abstract Introduction: Glioblastoma (GBM) is the most aggressive and lethal brain cancer in adults where best available therapy combining surgery and concomitant chemo radiation prolongs patient survival from 12.1 to 14.6 months. Approximately half of the patients benefit from addition of Temozolomide chemotherapy (those with methylated promoter of the DNA repair gene O6 methylguanine DNA methyltransferase). Immunotherapy has demonstrated unprecedented efficacy in solid tumours and kill targets in unique ways to the conventional therapies. Natural Killer (NK) cells are attractive effectors not only because of their ability to recognize and kill malignant and virus infected cells, but also their potential for modifying the tumor microenvironment to pro-inflammatory state. We have previously demonstrated NK-cell cytotoxicity against GBM. Here we hypothesize that misfolded protein response induced by pretreatment with proteasome inhibitors will sensitize GBM cells to NK cell therapy. Methods: IC50 doses of Bortezomib, Carfilzomib and MG132 proteasome inhibitors were determined by viability assays in P3, 2012-018 and P22 patient-derived GBM cells, as well as in normal human astrocytes (NHA). Phenotyping, time and dose dependent cytotoxicity/apoptosis and degranulation after combination therapy with Bortezomib and NK cells from healthy donors was conducted with flow cytometry and western blotting used to confirm protein signaling. Results: We demonstrated that Bortezomib is the most suitable proteasome inhibitor as it selectively killed GBM cells at low doses (IC50 39-42nM) while requiring higher doses (70nM) for NHA cells. In contrast, Carfilzomib and MG132 killed better the NHA than GBM cells. Bortezomib was more effective in combination treatment with NK cells, than monotherapy of Bortezomib or NK cells. The IC50 dose of Bortezomib was reduced by 40% from 42nM at 48hrs to 25nM at 24hrs when combined with effector:target (E:T) ratio 5:1 NK cells (p&lt0.05). E:T ratio of NK cells was also reduced by 75% in combination therapy, NK cell monotherapy killed only 45% of GBM cells at 20:1 ratio. The killing efficacy of combination treatment was not mediated by increased stress ligand expression, but Bortezomib attenuated autophagy and induced late apoptotic GBM cell death. Although Bortezomib did not increase degranulation or kill NK cells, importantly, it rather induced NK cell differentiation to mature CD56dimCD16-NKG2D+ subsets previously associated with increased natural cytotoxicity. Conclusion: Bortezomib was the best proteasome inhibitor against GBM that synergistically enhanced NK cell killing. Current work is investigating the mechanisms regulating the augmented differentiation and NK cell cytotoxicity during the combination therapy in vitro and in vivo in mice. Citation Format: Andrea Gras Navarro, Aminur Rahman, Marzieh Bahador, Martha Chekenya Enger. Bortezomib sensitizes glioblastoma for NK cell immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-195. doi:10.1158/1538-7445.AM2017-LB-195

MiR-140/BDNF axis regulates normal human astrocyte proliferation and LPS-induced IL-6 and TNF-α secretion.

Reactive astrocyte proliferation after spinal cord injury (SCI) contributes to glial scar formation that impedes axonal regeneration. The mechanisms underlying astrocyte proliferation upon injury remain partially understood. MicroRNAs (miRNAs) function as a major class of post-transcriptional gene expression regulators that participate in many biological processes. In this study, we focused on the functional role of miR-140 in normal human astrocyte (NHA) cell proliferation. Ectopic miR-140 expression significantly inhibited NHA cell viability and proliferation; miR-140 inhibition exerted the opposite function. Commonly, miRNAs exert functions through targeting downstream genes to inhibit their expression. In the present study, brain-derived neurotrophic factor (BDNF), a regulator of astrocyte proliferation and differentiation, confirmed as a direct target of miR-140 in NHA. Through binding to the 3'UTR of BDNF, miR-140 inhibited BDNF expression. BDNF overexpression significantly promoted NHA cell viability and proliferation; the regulatory effect of miR-140/BDNF on NHA proliferation was mediated by PI3K/AKT pathway. Moreover, we evaluated the functional role of miR-140 in Lipopolysaccharide (LPS)-induced in vitro injury model of astroglial cultures; a significantly up-regulated BDNF, interleukin (IL)-6 and tumor necrosis factor (TNF)-α expression in response to LPS stimulation was observed. After ectopic miR-140 expression, the promotive effect of LPS on BDNF, IL-6 and TGF-α expression was partially restored. Taken together, miR-140/BDNF axis regulates NHA proliferation through PI3K/AKT pathway; miR-140 could inhibit BDNF, IL-6 and TGF-α expression in LPS-induced in vitro injury model. MiR-140/BDNF might serve as a promising target in strategy against reactive astrocyte proliferation after SCI.

Mitochondrial DNA Mutations in Grade II and III Glioma Cell Lines Are Associated with Significant Mitochondrial Dysfunction and Higher Oxidative Stress.

The role of mitochondria in tumorigenesis has regained much attention as it could dysregulate cellular energetics, oxidative stress and apoptosis. However, the role of mitochondria in different grade gliomasis still unknown. This study aimed to identify mitochondrial DNA (mtDNA) sequence variations that could possibly affect the mitochondrial functions and also the oxidative stress status. Three different grades of human glioma cell lines and a normal human astrocyte cell line were cultured in-vitro and tested for oxidative stress biomarkers. Relative oxidative stress level, mitochondria activity, and mitochondrial mass were determined by live cell imaging with confocal laser scanning microscope using CM-H2DCFDA, MitoTracker Green, and MitoTracker Orange stains. The entire mitochondrial genome was sequenced using the AffymetrixGeneChip Human Mitochondrial Resequencing Array 2.0. The mitochondrial sequence variations were subjected to phylogenetic haplogroup assessment and pathogenicity of the mutations were predicted using pMUT and PolyPhen2. The Grade II astrocytoma cells showed increased oxidative stress wherea high level of 8-OHdG and oxidative stress indicator were observed. Simultaneously, Grade II and III glioma cells showed relatively poor mitochondria functions and increased number of mutations in the coding region of the mtDNA which could be due to high levels of oxidative stress in these cells. These non-synonymous mtDNA sequence variations were predicted to be pathogenic and could possibly lead to protein dysfunction, leading to oxidative phosphorylation (OXPHOS) impairment, mitochondria dysfunction and could create a vicious cycle of oxidative stress. The Grade IV cells had no missense mutation but preserved intact mitochondria and excellent antioxidant defense mechanisms thus ensuring better survival. In conclusion, Grade II and III glioma cells demonstrated coding region mtDNA mutations, leading to mitochondrial dysfunction and higher oxidative stress.

Targeted inhibition of HDAC8 increases the doxorubicin sensitivity of neuroblastoma cells via up regulation of miR-137

Histone deacetylases (HDACs) have been suggested to be potential therapeutic targets for cancer treatment. Recent studies revealed that HDAC8 expression was associated with poor prognostic markers and poor overall survival rate of neuroblastoma (NB). Our present study revealed that among the four members of class I HDACs, HDAC8 is significantly over expressed in NB cells as compared with the normal fibroblast 3T3 cells or primary normal human astrocytes (NHA) cells. Targeted inhibition of HDAC8 by its specific siRNA (si-HDAC8) can inhibit the in vitro growth of NB cells. Furthermore, si-HDAC8 significantly increases the sensitivity of NB cells to doxorubicin (Dox). Silencing of HDAC8 can increase the expression of miR-137, which has been suggested to mediate the Dox sensitivity of NB cells. Knockdown of miR-137 can attenuate si-HDAC8 enhanced Dox sensitivity. Further, si-HDAC8 can also inhibit the expression of multi-drug resistance gene 1 (MDR1). While knockdown of miR-137 can attenuate si-HDAC8 induced down regulation of MDR1. Collectively, our data revealed that targeted inhibition of HDAC8 can suppress the growth of NB cells and increase Dox sensitivity via up regulation of miR-137 and suppression of MDR1. Therefor, combination of HDAC8 inhibitor will be helpful to elevate the treatment outcome of NB patients.

Hydrogel scaffolds promote neural gene expression and structural reorganization in human astrocyte cultures

Biomaterial scaffolds have the potential to enhance neuronal development and regeneration. Understanding the genetic responses of astrocytes and neurons to biomaterials could facilitate the development of synthetic environments that enable the specification of neural tissue organization with engineered scaffolds. In this study, we used high throughput transcriptomic and imaging methods to determine the impact of a hydrogel, PuraMatrix™, on human glial cells in vitro. Parallel studies were undertaken with cells grown in a monolayer environment on tissue culture polystyrene. When the Normal Human Astrocyte (NHA) cell line is grown in a hydrogel matrix environment, the glial cells adopt a structural organization that resembles that of neuronal-glial cocultures, where neurons form clusters that are distinct from the surrounding glia. Statistical analysis of next generation RNA sequencing data uncovered a set of genes that are differentially expressed in the monolayer and matrix hydrogel environments. Functional analysis demonstrated that hydrogel-upregulated genes can be grouped into three broad categories: neuronal differentiation and/or neural plasticity, response to neural insult, and sensory perception. Our results demonstrate that hydrogel biomaterials have the potential to transform human glial cell identity, and may have applications in the repair of damaged brain tissue.

Silencing of histone deacetylase 2 suppresses malignancy for proliferation, migration, and invasion of glioblastoma cells and enhances temozolomide sensitivity.

Histone deacetylases (HDACs) can regulate the progression of various cancers, while their roles in glioblastoma multiforme (GBM) are not well known. Our present study investigated the expression of class I HDACs (HDAC1, 2, 3, 8) in GBM U87, A172, U251, and LN229 cells and compared their levels with that in primary normal human astrocytes (NHA) cells. It showed that HDAC2 expression is significantly up-regulated in GBM cells. Silencing of HDAC2 via its specific siRNAs can suppress the in vitro proliferation, migration, and invasion of GBM U87 and A172 cells. Furthermore, silencing of HDAC2 can increase the sensitivity of GBM cells to temozolomide (TMZ), a standard-of-care during clinical GBM treatment. This might be due to that si-HDAC can significantly down-regulate the mRNA and protein expression of MRP1, while has no effect on ABCB1 and ABCG2. Schisandrin B (Sch B), a specific inhibitor of MRP1, can further increase the TMZ sensitivity in HDAC2-knocked down GBM cells. Collectively, our data revealed that targeted HDAC2 can suppress the malignancy of GBM cells and increase their sensitivity of TMZ via down-regulation of MRP1. It suggested that HDAC2 might be a potential target for GBM therapy and improvement in TMZ therapy efficiency.

IMST-14. IDH1 R132H MUTATION INHIBITS ANTI-GLIOMA IMMUNE RESPONSES THROUGH POST-TRANSCRIPTIONAL DOWN-REGULATION OF STAT1 AND TYPE-1 CHEMOKINES

We evaluated whether isocitrate dehydrogenase (IDH) mutations could directly alter the immune landscape of gliomas, possibly through production of the onco-metabolite, 2-hydroxygluterate (2HG). TCGA RNAseq data demonstrate reduced expression of CD8 T-cell, type-1 effector and chemokine genes in IDH-mutated (Mut) patients compared to IDH-wild-type (Wt) cases. Conversely, we observed no differences in B-cell, type-2 or regulatory T-cell-related genes between IDH-Wt and IDH-Mut cases. Normal human astrocyte (NHA) cells and syngeneic GL261 glioma models expressing the R132H mutated IDH1 (NHA-Mut and GL261-Mut, respectively) further demonstrated that the IDH1 mutation and 2HG directly reduce CXCL10 protein and migration of T-cells to the tumor site, which was reversed by an IDH-specific inhibitor, IDH-C35. Further, while mice receiving prophylactic peptide vaccination targeting glioma-associated antigens resulted in clearance of GL261-Wt tumors, vaccinated mice could not reject GL261-Mut tumors. Treatment of vaccinated mice bearing GL261-Mut tumors with IDH-C35 enhanced anti-tumor immunity and prolonged survival compared to mice receiving vaccine alone. Interestingly, both parental GL261 cells treated with 2HG and GL261-Mut cells demonstrated decreased amounts of STAT1 protein but not mRNA compared with untreated GL261-Wt cells, suggesting that STAT1, a known regulator of CXCL10, is regulated at the post-transcriptional level in IDH-Mut gliomas. Consistent with these findings, TCGA microRNA data demonstrated increased levels of miR-145, a known regulator of STAT1, in IDH-Mut compared with IDH-Wt cases. We next evaluated direct tumor extrinsic implications of the IDH1 mutation. 2HG, which is known to be present in the tumor microenvironment, directly induced a ~10 fold increase in miR-145-5p and reduced both STAT1 levels and CXCL10 secretion from primary and BV2 murine microglia. Our findings demonstrate both glioma-intrinsic and extrinsic mechanisms of immunosuppression in IDH-Mut gliomas and suggest that IDH inhibitors can be used to enhance T-cell migration to the tumor site in patients with IDH-Mut tumors.

Microfluidic profiling of apoptosis-related genes after treatment with BH3-mimetic agents in astrocyte and glioblastoma cell lines.

Glioblastoma(GB) is the most frequent and biologically the most aggressive primary brain tumor in adults. Standard treatment for newly diagnosed GB consists of surgical resection, radiotherapy and chemotherapy. Resistance to therapy is a major obstacle, even with optimal treatment with a survival median of only 12-15 months. The heterogeneity and treatment response of GB makes this tumor type a challenging area of research. The aim of our study was to study the response of normal human astrocyte(HA) and human GB (T98G) cell lines to apoptosis inhibitors invitro. ABT-737 is an inhibitor of anti-apoptotic proteins Bcl-2, Bcl-xL, Bcl-w, while MIM-1 is an Mcl-1 protein inhibitor. The viability of the cells was assayed biochemically using the cytotoxic methyl thiazolyl tetrazolium(MTT) assay. Changes in the expression of apoptosis-associated genes (n=93) in two human brain cell lines after treatment with the apoptosis inhibitors ABT-737 and MIM-1 (individually), between the apoptosis inhibitor treated group and the control group, were determined using a commercially pre-designed microfluidic array. Significant changes in apoptotic gene expression with more than a 2.0-fold difference in their expression levels were obtained in both cell lines; the most altered genes were in the HA cell line after MIM-1 treatment (n=42). These results contribute to the importance of apoptosis in normal and cancerous brain tissues and provide information on the effect of apoptosis inhibitors on cell viability and gene expression. Despite extensive investigations, a cure for GB is currently not available. The identification of an apoptotic gene panel and determining the sensitivity of normal and GB brain cells to individual apoptosis inhibitors could help to improve clinical practice and increase our understanding of brain tumor cell metabolism and apoptosis inhibitors in GB cells and astrocytes. Recognizing expression changes in pro-apoptotic and anti-apoptotic genes could contribute to the development of new treatments.

B-cell CLL/lymphoma 3 promotes glioma cell proliferation and inhibits apoptosis through the oncogenic STAT3 pathway.

Aberrant expression of oncogenes and/or tumor suppressors play fundamental roles in the pathogenesis of glioma. B-cell CLL/lymphoma 3 (BCL3) was previously found to be a putative proto-oncogene in human cancers and the decoy receptor DcR1 is induced in a p50/Bcl3-dependent manner and attenuates the efficacy of temozolomide in glioblastoma cells. However, its expression status, clinical significance and biological functions in glioma remain largely unknown. In the present study, the levels of BCL3 were overexpressed in glioma compared to normal brain tissues. Furthermore, high expression of BCL3 protein was confirmed by immunoblotting in glioma cells as compared with normal human astrocyte cell line. The positive expression of BCL3 was correlated with adverse prognostic features and reduced overall survival rate of glioma patients. BCL3 silencing resulted in prominent decreased proliferation, cell cycle arrest in G1 phase and increased apoptosis in U251 cells. In contrast, BCL3 overexpression in U87 cells remarkably facilitated proliferative ability and cell cycle progression and induced apoptosis. Invivo studies showed that BCL3 knockdown inhibited the tumor growth of U251 cells in a mouse xenograft model. Mechanistically, BCL3 positively regulated the abundance of STAT3, p-STAT3 and the downstream targets of STAT3 pathway including BCL2, MCL-1 and cyclinD1 in glioma cells. Furthermore, a positive correlation between BCL3 and STAT3 expression was observed in glioma specimens. Notably, we confirmed that STAT3 knockdown abolished the oncogenic roles of BCL3 in glioma. In conclusion, we suggest that BCL3 serves as an oncogene in glioma by modulating proliferation, cell cycle progression and apoptosis, and its oncogenic effects are mediated by the STAT3 signaling pathway.

Abstract 520: The role of the calpain pathway in early response and acquired resistance to radiation in glioblastoma

Abstract Glioblastoma (GBM) is the most aggressive, malignant brain tumor that is highly heterogeneous and often develops resistance to conventional chemotherapy and radiation treatments. The median survival time of GBM patients is 12-15 months despite aggressive treatment modalities, and there is an urgent need for the development of novel treatment strategies. The goal of this study is to identify novel mechanisms of radioresistance in GBM through mass spectrometry-based phosphoproteomic profiling, with the objective that more effective therapies can be rationally developed in the future. We and others have previously reported that GBMs are characterized by deregulated signaling pathways that control critical cellular processes including survival, migration, and proliferation. Very little is known about acquired resistance mechanisms to radiation, specifically how radiation alters protein phosphorylation patterns in tumors on a global level, or how radiation-induced changes in protein phosphorylation contribute to treatment resistance. In order to identify radiation-induced changes in protein phosphorylation, we performed phosphoproteomic profiling of 6 GBM cell lines and 1 normal human astrocyte cell line both before and after radiation treatment in 3 independent experiments. Specifically, cells were collected before treatment and at 2 time-points following a 10 Gy radiation treatment. Following protein isolation and phosphopeptide enrichment, global phosphoproteomic analysis was performed by mass spectrometry. Relative quantitation and statistical analysis were performed to determine fold changes in phosphorylation at both post-radiation time-points. An average of 3,971 phosphopeptides mapping to 1,649 phosphoproteins were quantified for each independent experiment, and a total of 621 phosphoproteins were quantified across all 3 datasets. We identified a major component of the calpain signaling pathway that consistently shows an increase in phosphorylation following radiation treatment in GBM cell lines. The post-radiation increase in phosphorylation was confirmed in 2 independent experiments and observed across all 6 GBM cell lines. This is the first study linking the calpain signaling pathway to radiation response in GBM. Mechanistic studies using phosphomimetic and nonphosphorylatable mutants of the protein of interest will determine the effect of phosphorylation on calpain activity and radiation resistance. Results of this work will contribute to our understanding of how GBM tumors become resistant to radiation therapy, and may identify a novel target to overcome radiation resistance in GBM. Citation Format: Emily A. Bassett, Arnab Chakravarti. The role of the calpain pathway in early response and acquired resistance to radiation in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 520.

MiR-508-5p Inhibits the Progression of Glioma by Targeting Glycoprotein Non-metastatic Melanoma B.

Glioma is a severe and highly lethal brain cancer, a malignancy largely stemming from growing in a relatively restrained area of the brain. Hence, the understanding of the molecular regulation of the growth of glioma is critical for improving its treatment. MicroRNA has become a hotspot in research on diseases, especially in the initiation and progression of different types of cancer. However, the molecular function and mechanisms of miR-508-5p in gliomagenesis are still unclear. The aim of this study was to investigate miR-508-5p expression in glioma and determine its effects on proliferation. miR-508-5p expression levels, both in glioma cell lines and in tissue, were significantly lower than in a normal human astrocyte cell line or adjacent tissues. Cell growth was analyzed using a MTT assay and over-expression of miR-508-5p was found to decrease glioma cell growth. Moreover, a bioinformatic analysis was performed, showing that glycoprotein non-metastatic melanoma B (GPNMB) was a direct target for miR-508-5p in glioma cells. Furthermore, in vivo treatment with miR-508-5p reduced GPNMB protein levels in the tumor. Additionally, overexpression of GPNMB without 3'-UTR partially reversed the cell growth arrest induced by miR-508-5p over-expression in glioma cells. In conclusion, these results indicate that increased expression of miR-508-5p might be related to glioma progression, indicating a potential role of miR-508-5p for clinical therapy.

MiR-22 inhibits the proliferation, motility, and invasion of human glioblastoma cells by directly targeting SIRT1.

Recently, microRNAs (miRNAs), a kind of small and non-coding RNA, can target the downstream molecules. Increasing evidence demonstrates that miRNAs meditate the onset and progression of a variety of tumors. In the present study, we carried out gene transfection, western blot, and reverse transcription PCR (RT-PCR) to explore the role of miR-22 in glioblastoma tissues and cell lines. Here, we verified that the expression of miR-22 was downregulated in glioblastoma tissues and cells rather than matched non-tumor tissues and normal human astrocyte (NHA) cells (p < 0.001). By contrast, SIRT1 messenger RNA (mRNA) and protein were upregulated in glioblastoma tissues and cells (p < 0.001). In vitro miR-22 mimics interfered with cell proliferation, migration, and invasion of U87 and U251 cells. Mechanically, the 3'-untranslated regions (3'-UTRs) of SIRT1 were a direct target of miR-22, leading to the decreased expression of SIRT1 protein in U87 and U251 cells. Meanwhile, miR-22 mimics also inhibited the expression of epidermal growth factor receptor (EGFR) and matrix metallopeptidase 9 (MMP9). In conclusion, miR-22 inhibited cell proliferation, migration, and invasion via targeting the 3'-UTR of SIRT1 in the progression of glioblastoma and miR-22-SIRT1 pathway can be recommended as a potential target for treatment of glioblastoma.