Primary Cultured Glioma Cells Research Articles

EXTH-78. A NOVEL BISPECIFIC S100A4/TFR ANTIBODY TO REPROGRAM THE GLIOMA TUMOR MICROENVIRONMENT AND TARGET GLIOMA STEM CELLS

Abstract GBM (glioblastoma) is the most common and aggressive primary brain tumor in adults. While immunotherapy is a promising treatment modality for GBM, most clinical trials have thus far only provided a modest benefit to GBM patients. We propose that enhancing the efficacy of immunotherapy for GBM requires approaches that increase T cell infiltration and reprogram the myeloid cells to a more pro-inflammatory state. S100A4 is a major regulator of stemness, epithelial-mesenchymal transition, and a critical regulator of immune suppressive myeloid and T cell phenotypes in GBM. Targeting S100A4 in either glioma or stromal cells is sufficient to reprogram the tumor immune landscape and extend survival of glioma bearing mice, indicating that S100A4 is a promising immunotherapy target. We recently developed a S100A4 blocking monoclonal antibody; however, this antibody does not cross the blood brain barrier (BBB) efficiently to confer significant therapeutic benefit, limiting its efficacy in treating GBM. To overcome this challenge, we generated a bispecific S100A4-TfR antibody (BsA) that allows robust BBB penetration, and tested this in a pre-clinical syngeneic, orthotopic mouse model of GBM. Using ELISA, confocal immunofluorescent microscopy, flow cytometry, and bulk RNA sequencing, we report that systemic administration of the S100A4-TfR BsA results in increased immune cell infiltration with a preferential increase in CD4 T cell infiltration, and potent immune remodeling of the tumor microenvironment to a more pro-inflammatory state. Furthermore, the BsA is taken up by glioma cells intracellularly, which is correlated with depletion of intracellular S100A4 and decreased nuclear SOX2 localization, which would reduce stemness of glioma cells. Functionally, in vitro BsA treatment of primary glioma cell cultures shows decreased S100A4 expression, proliferation, and self-renewal. Our results suggest that S100A4-TfR BsA is a novel dual activity inhibitor to simultaneously reprogram the GBM immune landscape and reduce glioma stemness.

Multi-omics and pharmacological characterization of patient-derived glioma cell lines

Glioblastoma (GBM) is the most common brain tumor and remains incurable. Primary GBM cultures are widely used tools for drug screening, but there is a lack of genomic and pharmacological characterization for these primary GBM cultures. Here, we collect 50 patient-derived glioma cell (PDGC) lines and characterize them by whole genome sequencing, RNA sequencing, and drug response screening. We identify three molecular subtypes among PDGCs: mesenchymal (MES), proneural (PN), and oxidative phosphorylation (OXPHOS). Drug response profiling reveals that PN subtype PDGCs are sensitive to tyrosine kinase inhibitors, whereas OXPHOS subtype PDGCs are sensitive to histone deacetylase inhibitors, oxidative phosphorylation inhibitors, and HMG-CoA reductase inhibitors. PN and OXPHOS subtype PDGCs stably form tumors in vivo upon intracranial transplantation into immunodeficient mice, whereas most MES subtype PDGCs fail to form tumors in vivo. In addition, PDGCs cultured by serum-free medium, especially long-passage PDGCs, carry MYC/MYCN amplification, which is rare in GBM patients. Our study provides a valuable resource for understanding primary glioma cell cultures and clinical translation and highlights the problems of serum-free PDGC culture systems that cannot be ignored.

Effects of the IDH1 R132H Mutation on the Energy Metabolism: A Comparison between Tissue and Corresponding Primary Glioma Cell Cultures.

The R132H mutation in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) is the most important prognostic factor for the survival of glioma patients. Subsequent studies led to the discovery of a panel of enzymes mainly involved in glutamate anaplerosis and aerobic glycolysis that change in abundance as a result of the IDH1 mutation. To further study these changes, appropriate glioma models are required that accurately mimic in vivo metabolism. To investigate how metabolism is affected by in vitro cell culture, we here compared surgically obtained snap-frozen glioma tissues with their corresponding primary glioma cell culture models with a previously developed targeted mass spectrometry proteomic assay. We determined the relative abundance of a panel of metabolic enzymes. Results confirmed increased glutamate use and decreased aerobic glycolysis in resected IDH1 R132H glioma tissue samples. However, these metabolic profiles were not reflected in the paired glioma primary cell cultures. We suggest that culture conditions and tumor microenvironment play a crucial role in maintaining the in vivo metabolic situation in cell culture models. For this reason, new models that more closely resemble the in vivo microenvironment, such as three-dimensional cell co-cultures or organotypic multicellular spheroid models, need to be developed and investigated.

178P Glioblastoma multiforme cells down-regulate TNFα-mediated antitumor killer activity of dendritic cells

Monocyte-derived dendritic cells (DCs) of glioma patients are characterized by a defect in their functional activity, including their ability to kill tumor cells. The current study was aimed to analyze the production of soluble mediators by primary glioma cell cultures, as well as to elucidate the correlation between the cytokine-producing activity of glioma cells and TNFα-mediated cytotoxic activity of autologous DCs.

DDRE-17. TARGETING GLIOBLASTOMA’S GALACTOSE SCAVENGING PATHWAY

Abstract BACKGROUND We have recently shown that GBM use D-galactose (Gal) as a substrate, in vitro and in vivo. Gal is imported via Glut3 and/or Glut14 and metabolized through the Leloir pathway. We investigated 4-deoxy-4-fluorogalactose (4DFG) as the lead compound in a family of galactose-based antimetabolites. 4DFG is a potent chemotherapeutic in monotherapy and can bolster existing therapies. METHODS We examined the alteration of glioma metabolism in vitro and in vivo induced by 4DFG. 1H/13C-NMR and optical probes were used to interrogate the effects of 4DFG on glycolysis and mitochondrial respiration in primary glioma cell cultures. Labeled lectins were used to assay for the disruption of glycan synthesis induced by 4DFG. An intracranial model of primary GBM was used to assess efficacy and toxicity in vivo. RESULTS NMR reveals that at physiological concentrations of glucose, low concentrations of 4DFG (5 μM) is able to inhibit glycolytic and mitochondrial flux by approximately 12%, p< 0.05. Analysis using lectins shows a collapse in general glycan synthesis, but most especially in the incorporation of both Gal and GalNAc sugars. In nude mice with intracranial primary GBM, six treatments of 4DFG increased survival from 23 to 50 days, p< 0.002. DISCUSSION The ability of GBM to scavenge galactose allows us to target the Glut3/14 import and Leloir metabolic pathway using galactose-based anti-metabolites. Our first-generation compound is highly effective as a monotherapy, inhibiting glucose metabolism and glycan synthesis.

St8sia1-deficiency in mice alters tumor environments of gliomas, leading to reduced disease severity.

ABSTRACTGanglioside GD3/GD2 are over-expressed in various neuroectoderm-derived tumors. Previous studies indicated that GD3 is involved in the enhancement of cancer properties such as rapid growth and increased invasiveness. However, little is known about the functions of GD3/GD2 in glioma cells and glioma microenvironments. To clarify the functions of GD3/GD2 in gliomas, we used a mouse glioma model based on the RCAS/Gtv-a system. At first, we compared the gliomas size between wild-type (WT) and GD3 synthase (GD3S) knockout (KO) mice, showing a less malignant histology and slower tumor growth in GD3S-KO mice than in WT mice. Immunohistochemistry of glioma sections from WT and GD3S-KO mice revealed that reactive microglia/macrophages showed different localization patterns between the two genetic types of mice. CD68+ cells were more frequently stained inside glioma tissues of GD3S-KO mice, while they were stained mainly around glioma tissues in WT mice. The number of CD68+ cells markedly increased in tumor tissues of GD3S-KO mice at 2 weeks after injection of transfectant DF-1 cells. Furthermore, CD68+ cells in GD3S(-/-) glioma tissues expressed higher levels of inducible nitric oxide synthase. We observed higher expression levels of pro-inflammatory cytokine genes in primary-cultured glioma cells of WT mice than in GD3S-KO mice. DNA microarray data also revealed differential expression levels of various cytokines and chemokines in glioma tissues between WT and GD3S-KO mice. These results suggest that expression of GD3S allows glioma cells to promote polarization of microglia/macrophages towards M2-like phenotypes by modulating the expression levels of chemokines and cytokines.

Lack of GD3 synthase (St8sia1) attenuates malignant properties of gliomas in genetically engineered mouse model.

High expression of gangliosides GD3 and GD2 is observed in human gliomas. The functions of GD3 and GD2 in malignant properties have been reported in glioma cells in vitro, but those functions have not yet been investigated in vivo. In this study, we showed that deficiency of GD3 synthase (GD3S, St8sia1) attenuated glioma progression and clinical and pathological features in a platelet‐derived growth factor B‐driven murine glioma model. Lack of GD3S resulted in the prolonged lifespan of glioma‐bearing mice and low‐grade pathology in generated gliomas. Correspondingly, they showed reduced phosphorylation levels of Akt, Erks, and Src family kinases in glioma tissues. A DNA microarray study revealed marked alteration in the expression of various genes, particularly in MMP family genes, in GD3S‐deficient gliomas. Re‐expression of GD3S restored expression of MMP9 in primary‐cultured glioma cells. We also identified a transcription factor, Ap2α, expressed in parallel with GD3S expression, and showed that Ap2α was critical for the induction of MMP9 by transfection of its cDNA and luciferase reporter genes, and a ChIP assay. These findings suggest that GD3S enhances the progression of gliomas by enhancement of the Ap2α‐MMP9 axis. This is the first report to describe the tumor‐enhancing functions of GD3S in vivo.

Borneol promotes apoptosis of Human Glioma Cells through regulating HIF-1a expression via mTORC1/eIF4E pathway.

The main reason for the failure of malignant glioma treatment is local tumor recurrence. Tumor cells in hypoxic microenvironment activate HIF-1 α transcription, and thus promoting tumor invasion and metastasis is one of the important reasons. In our previous study, we clearly established that borneol opens the blood-brain tumor barrier and its related mechanism. However, the effects of borneol itself on glioma proliferation have not yet been elucidated. Therefore, in this study, we evaluated the effect of borneol on glioma by constructing in vivo SD rat brain glioma model and in vitro human primary cultured glioma cell model. We found that borneol could suppress the proliferation of primary glioma cells and the tumor volume of SD rat brain glioma. Further, we measured the apoptosis effect induced by borneol in human primary cultured glioma cells. The results showed that the higher the concentration of borneol, the higher the apoptosis rate of human primary cultured glioma cells, but the effect was reversed after transfection of HIF-1 overexpression plasmid; In addition, borneol could downregulate the expression of Bcl-2 and upregulation the expression of Bax and caspase-3, similarly, the effect was also reversed after transfection of HIF-1 overexpression plasmid, suggesting that the apoptosis effect induced by borneol in human primary cultured glioma cells is mediated via HIF-1α. Moreover, the bioinformatics analysis of correlation between HIF-1α and apoptosis-related factors based on CGGA database showed that there was a positive correlation between the expression of eIF4E and HIF-1 α (P < 0.05), and in patients with high expression of eIF4E and HIF-1α had poor survival and prognosis (P<0.001). It was further discovered that in the human primary cultured glioma cells borneol regulated HIF-1a expression via mTORC1/eIF4E pathway. In conclusion, the findings of the present study suggest that HIF-1α may be a key factor in borneol induced apoptosis of glioma cells, and mTORC1 / eIF4E pathway is involved in the HIF-1α regulation by borneol in malignant glioma. Our results not only reveal the target and molecular mechanism and action of borneol leading to promote apoptosis in glioma cells, but also provide experimental basis and theoretical support for the clinical application of borneol.

TB-04 TERT PROMOTER MUTATION AS A SUSCEPTIBLE MOLECULAR MARKER OF BCNU LOCAL THERAPY

INTRODUCTIONAlkylating agents, including Temozolomide (TMZ) and CCNU (ACNU) have been widely accepted as a standard treatment in malignant gliomas. Several studies also demonstrated that BCNU wafer placement extended survival in glioblastoma patients. However, little study demonstrated gene-specific efficacy of BCNU local therapy in malignant gliomas. Herein, we investigated BCNU sensitivity for patient-derived primary cultured glioma cells.MATERIALS AND METHODSFrom January 2017 to July 2019, 58 gliomas (grade III, IV) were tested genomic analysis and ATP-based cell viability after BCNU treatment. IDH1/2 mutation and TERT promoter mutation status was determined by Sanger sequencing. MGMT methylation status were evaluated by methylation specific PCR.RESULTSOf 58 cases,10 cases (17.2%) and 32 (55.2%) cases harbored IDH1/2 mutation and TERT mutation (C228T, C250T), respectively. Among them, co-mutation was identified in 5/58 cases (8.6%). MGMT was methylated in 17/58 cases (29.3%). Interestingly, the presence of TERT promoter mutation was positively correlated with BCNU sensitivity, particularly in IDH1/2 wild-type tumors (p<0.05). In contrast, there was no significant relationship between TMZ sensitivity and IDH mutation/MGMT methylation status.CONCLUSIONAlthough sample size is small, our results imply TERT promoter mutations might be a predictive molecular marker for BCNU sensitivity in malignant gliomas. Since TERT mutations are located at two hot spot loci (C228T and C250T), vast majority of TERT promoter mutations can be evaluated during surgery, which may contribute tailored therapeutic strategy in malignant gliomas.

Anti-glioma Activity of Dapsone and Its Enhancement by Synthetic Chemical Modification.

The sulfone dapsone is an old antibiotic used for the treatment of mycobacterial and protozoal infections. We postulated before that dapsone might possess biological activity exceeding its anti-infectious properties and that it could potentially be repurposed for the treatment of glioma. To test this hypothesis, we treated established and primary cultured glioma cells with dapsone or several dapsone analogues which we previously synthesized (D2-D5) and determined effects on proliferation, anchorage-independent growth and migration. While dapsone and its synthetic analogues D2-D5 displayed only modest anti-proliferative activity, important neoplastic features such as anchorage-independent growth, clonogenic survival and directed migration were significantly inhibited by dapsone treatment. Moreover, dapsone analogues D3, D4 and D5 yielded even enhanced anti-glioma activity against different pro-neoplastic features. Overall these data suggest that dapsone provides activity against glioma which can be further enhanced by molecular modifications. These compounds could potentially serve as a therapeutic adjunct to the treatment of gliomas in a repurposing approach.

MiRNA expression profiling in human gliomas: upregulated miR-363 increases cell survival and proliferation.

The role of microRNAs (miRNAs) in glioma biology is increasingly recognized. To investigate the regulatory mechanisms governing the malignant signature of gliomas with different grades of malignancy, we analyzed miRNA expression profiles in human grade I-IV tumor samples and primary glioma cell cultures. Multiplex real-time PCR was used to profile miRNA expression in a set of World Health Organization (WHO) grade I (pilocytic astrocytoma), II (diffuse fibrillary astrocytoma), and IV (glioblastoma multiforme) astrocytic tumors and primary glioma cell cultures. Primary glioma cell cultures were used to evaluate the effect of transfection of specific miRNAs and miRNA inhibitors. miRNA microarray showed that a set of miRNAs was consistently upregulated in all glioma samples. miR-363 was upregulated in all tumor specimens and cell lines, and its expression correlated with tumor grading. The transfection of glioma cells with the specific inhibitor of miR-363 increased the expression level of tumor suppressor growth-associated protein 43 (GAP-43). Transfection of miR-363 induced cell survival, while inhibition of miR-363 significantly reduced glioma cell viability. Furthermore, miRNA-363 inhibition induced the downregulation of AKT, cyclin-D1, matrix metalloproteinase (MMP)-2, MMP-9, and Bcl-2 and upregulation of caspase 3. Together, these data suggest that the upregulation of miR-363 may play a role in malignant glioma signature.

P52: Glioma-derived soluble factors pose strong chemoattractants and partially change the cytotoxic activity of IFN-alpha-induced dendritic cells

Dendritic cells (DCs) are the most potent antigen-presenting cells, which play crucial role in initiation and maintenance of immune response. However, DCs of high-grade glioma patients are characterized by some functional impairment including reduced TNF α -mediated antitumor cytotoxic activity and low migratory activity. Tumor-produced soluble molecules can profoundly affect the differentiation and maturation of DCs and influence the functional activity of DCs. The aim of the present study was to determine the effect of supernatants of primary glioma cell lines on the migration and the cytotoxic activity of healthy donor DCs. The study included 11 healthy donors and 7 patients with histologically verified glioma Grade II (2 patients) and glioma Grade IV (5 patients). DCs were generated from peripheral blood monocytes of healthy donors in the presence of GM-CSF and IFN-alpha (IFN-DCs) followed by the addition of lipopolysaccharide (LPS Escherichia coli). Primary glioma cell cultures from tissue samples of studied patients were obtained by mechanical and enzymatic (0.3% collagenase IA) disaggregation followed by culturing in DMEM/F12 medium containing 10% FCS. 7-day supernatants were collected upon reaching the cellular subconfluence. Migratory capacity of donor IFN-DCs in response to supernatants (v/v 25%) of primary glioma cell cultures was measured in trans-well culture chamber. DCs that migrated to the bottom chamber with medium alone as a control or with supernatants were counted by FACS analysis using BD calibration beads. Cytotoxic activity of donor IFN-DCs against tumor cell line HEp-2 was studied using MTT-assay for 24 h at a ratio of DC: HEp-2 1:1. IFN-DCs were preincubated for 2 h with supernatants of primary glioma cell cultures. Migration index of donor IFN-DCs to lymphoid chemokine CCL19 (100 ng/ml) was an average of 6.5 ± 2.1. Migration capacity of IFN-DCs towards concentration gradient of primary glioma culture supernatants was significantly higher. Supernatants of low-grade glioma cultures as well as of high-grade glioma cultures effectively induced chemotaxis of IFN-DCs. Migration index in response to concentration gradient of primary glioma line-derived soluble factors (6 cell lines) ranged from 32.6 ± 9 to 48.4 ± 15.5. To assess the effect of supernatants of primary lines on the cytotoxic activity of IFN-DCs four primary high-grade glioma lines were used. The cytotoxicity of intact LPS-stimulated donor IFN-DCs against HEp-2 cells was an average of 34.8 ± 7.4%. Supernatant pretreating of IFN-DCs reduced the cytotoxic activity of DCs in 8 out of 16 experiments (with an average of 43%). In other 8 cases, supernatants of high-grade glioma lines, in contrast, possessed a mild stimulating activity and enhanced the cytotoxicity of DCs on average about of 34%. Thus, tumor cells in glioma patients produce chemoattractants capable of providing migration of DCs to tumor. At the same time glioma-derived soluble factors change the cytotoxic activity both ways. One can suppose thereupon that impairment of DC cytotoxic activity in glioma patients occurs not only due to tumor cell impact but because of unknown mechanisms.

NVP-BKM120 potentiates apoptosis in tumor necrosis factor-related apoptosis-inducing ligand-resistant glioma cell lines via upregulation of Noxa and death receptor 5.

We previously observed that glioma cells are differentially sensitive to TRAIL-induced toxicity. Based on our observation that TRAIL-resistant glioma cell lines typically exhibited high levels of Akt activation, we hypothesized that inhibition of Akt signaling using the PI3 kinase inhibitor NVP-BKM120 could promote TRAIL-induced apoptosis in gliomas. We assessed this combination in established and primary cultured glioma cells. Combination treatment led to significant cellular death when compared to either drug alone, but had no effect in normal human astrocytes, and demonstrated activation of the caspase cascade. This enhanced apoptosis appears dependent upon the loss of mitochondrial membrane potential and the release of Smac/DIABLO, AIF and cytochrome c into the cytosol. The upregulation of Noxa and sequestration of Mcl-1 by Noxa were important factors for cell death. Knockdown of Noxa abrogated apoptosis and suggested dependency on Noxa in combination-induced apoptosis. BKM120 upregulated cell surface expression of death receptor 5 (DR5), but did not increase levels of the other major TRAIL receptor, death receptor 4 (DR4). This study demonstrates that antagonizing apoptosis-resistance pathways, such as the PI3/Akt pathway, in combination with death receptor activation, may induce cell death in TRAIL-resistant glioma.

Abstract 5116: Inhibition of PI3K/AKT signaling by NVP-BKM120 promotes ABT-737-induced toxicity in established and primary cultured glioblastoma cells

Abstract Identification of therapeutic strategies that might enhance the efficacy of Bcl-2 inhibitor ABT-737 is of great interest in many cancers including glioma. Our recent study suggested that Akt is a crucial mediator of apoptosis sensitivity in response to ABT-737 in glioma cell lines. Inhibitors of PI3-K/Akt are currently being assessed clinically in patients with glioma, as PI3K/Akt inhibition would be expected to have many pro-apoptotic effects, we hypothesized that there may be unique synergy between PI3-K inhibitors and BH3-mimetics. We assessed the combination of the PI3K inhibitor BKM120 and the Bcl-2 family inhibitor ABT-737 in established and primary cultured glioma cells. We found that the combined treatment with these agents led to a significant activation of caspase-8, 3, PARP and cell death irrespective of PTEN status. The enhanced lethality observed with the combination also appears dependent upon the loss of mitochondrial membrane potential, release of cytochrome c, smac/DIABLO and apoptosis-inducing factor to the cytosol. Further study revealed that the upregulation of Noxa, truncation of Bid, activation of Bax and Bak caused by these inhibitors was the key factor for the synergy. In addition, we demonstrated the release of proapoptotic proteins Bim and Bak from Mcl-1. We found defects in chromosome segregation leading to multinuclear cells and loss of colony forming ability, suggesting the potential use of BKM120 as a promising agent to improve the anti-cancer activities of ABT-737. Citation Format: Esther P. Jane, Daniel R. Premkumar, Alejandro Morales, Kimberly A. Foster, Ian Pollack. Inhibition of PI3K/AKT signaling by NVP-BKM120 promotes ABT-737-induced toxicity in established and primary cultured glioblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5116. doi:10.1158/1538-7445.AM2014-5116

Abstract 5510: ABT-737 synergizes with Vorinostat to induce apoptosis, mediated by Noxa upregulation, Bax activation and mitochondrial dysfunction in PTEN-intact malignant human glioma cell lines

Abstract We have previously observed that glioma cells are differentially sensitive to ABT-737 and, when used as a single-agent, this drug failed to induce apopotsis. Identification of therapeutic strategies to enhance the efficacy of the Bcl-2 inhibitor ABT-737 in human glioma is of interest. Histone deacetylation inhibitors (HDACi) are currently being assessed clinically in patients with glioma, as regulation of epigenetic abnormalities is expected to produce pro-apoptotic effects. We hypothesized that cotreatment of glioma with a BH3-mimetic and HDACi may synergistically produce cell death. We assessed the combination of the Bcl-2 family inhibitor ABT-737 and the HDACi vorinostat in established and primary cultured glioma cells. We found that combined treatment with these agents led to significant cellular death when compared to either drug as a single agent and demonstrated activation of the caspase cascade. The enhanced apoptosis observed with the combination also appears dependent upon the loss of mitochondrial membrane potential, release of cytochrome c, smac/DIABLO and apoptosis-inducing factor to the cytosol. We observed that the upregulation of Noxa, truncation of Bid, and the activation of Bax caused by this combination were important factors for synergistic cell death. We demonstrated that Noxa functioned to occupy Mcl-1 in cells exposed to the combination, yet, contrary to studies in other malignancies, this combination in glioma occurred without the release of proapoptotic proteins Bim and Bak from Mcl-1. We showed this combination to be less effective in PTEN-deficient glioma cells. Both genetic manipulation of PTEN status and pharmacologic inhibition of PI3 kinase sensitized PTEN-deleted glioma cells to the combination. This study demonstrates that antagonizing apoptosis-resistance pathways, such as the Bcl-2 family, and regulating epigenetic aberrancies, such as histone regulation, may induce cell death. Moreover, we reiterate that therapies to target the PI3-kinase/Akt/mTor pathway may be additionally necessary in cancers lacking PTEN functionality. Citation Format: Kimberly A. Foster, Daniel R. Premkumar, Esther P. Jane, Alejandro Morales, Ian F. Pollack. ABT-737 synergizes with Vorinostat to induce apoptosis, mediated by Noxa upregulation, Bax activation and mitochondrial dysfunction in PTEN-intact malignant human glioma cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5510. doi:10.1158/1538-7445.AM2014-5510

MiR-181b sensitizes glioma cells to teniposide by targeting MDM2.

BackgroundAlthough the incidence of glioma is relatively low, it is the most malignant tumor of the central nervous system. The prognosis of high-grade glioma patient is very poor due to the difficulties in complete resection and resistance to radio-/chemotherapy. Therefore, it is worth investigating the molecular mechanisms involved in glioma drug resistance. MicroRNAs have been found to play important roles in tumor progression and drug resistance. Our previous work showed that miR-181b is involved in the regulation of temozolomide resistance. In the current study, we investigated whether miR-181b also plays a role in antagonizing the effect of teniposide.MethodsMiR-181b expression was measured in 90 glioma patient tissues and its relationship to prognosis of these patients was analyzed. Cell sensitivity to teniposide was tested in 48 primary cultured glioma samples. Then miR-181b stably overexpressed U87 cells were generated. The candidate genes of miR-181b from our previous study were reanalyzed, and the interaction between miR-181b and target gene MDM2 was confirmed by dual luciferase assay. Cell sensitivity to teniposide was detected on miR-181b over expressed and MDM2 down regulated cells.ResultsOur data confirmed the low expression levels of miR-181b in high-grade glioma tissues, which is related to teniposide resistance in primary cultured glioma cells. Overexpression of miR-181b increased glioma cell sensitivity to teniposide. Through target gene prediction, we found that MDM2 is a candidate target of miR-181b. MDM2 knockdown mimicked the sensitization effect of miR-181b. Further study revealed that miR-181b binds to the 3’-UTR region of MDM2 leading to the decrease in MDM2 levels and subsequent increase in teniposide sensitivity. Partial restoration of MDM2 attenuated the sensitivity enhancement by miR-181b.ConclusionsMiR-181b is an important positive regulator on glioma cell sensitivity to teniposide. It confers glioma cell sensitivity to teniposide through binding to the 3’-UTR region of MDM2 leading to its reduced expression. Our findings not only reveal the novel mechanism involved in teniposide resistance, but also shed light on the optimization of glioma treatment in the future.

Co-administration of ABT-737 and SAHA induces apoptosis, mediated by Noxa upregulation, Bax activation and mitochondrial dysfunction in PTEN-intact malignant human glioma cell lines.

We previously observed that glioma cells are differentially sensitive to ABT-737 and, when used as a single-agent, this drug failed to induce apoptosis. Identification of therapeutic strategies to enhance the efficacy of the Bcl-2 inhibitor ABT-737 in human glioma is of interest. Histone deacetylation inhibitors (HDACI) are currently being assessed clinically in patients with glioma, as regulation of epigenetic abnormalities is expected to produce pro-apoptotic effects. We hypothesized that co-treatment of glioma with a BH3-mimetic and HDACI may induce cellular death. We assessed the combination of ABT-737 and HDACI SAHA in established and primary cultured glioma cells. We found combination treatment led to significant cellular death when compared to either drug as single agent and demonstrated activation of the caspase cascade. This enhanced apoptosis also appears dependent upon the loss of mitochondrial membrane potential and the release of cytochrome c and AIF into the cytosol. The upregulation of Noxa, truncation of Bid, and activation of Bax caused by this combination were important factors for cell death and the increased levels of Noxa functioned to sequester Mcl-1. This combination was less effective in PTEN-deficient glioma cells. Both genetic and pharmacologic inactivation of the PI3K/Akt signaling pathway sensitized PTEN-deleted glioma cells to the combination. This study demonstrates that antagonizing apoptosis-resistance pathways, such as targeting the Bcl-2 family in combination with epigenetic modifiers, may induce cell death. These findings extend our previous observations that targeting the PI3K/Akt pathway may be additionally necessary to promote apoptosis in cancers lacking PTEN functionality.

Inhibition of phosphatidylinositol 3-kinase/AKT signaling by NVP-BKM120 promotes ABT-737-induced toxicity in a caspase-dependent manner through mitochondrial dysfunction and DNA damage response in established and primary cultured glioblastoma cells.

Identification of therapeutic strategies that might enhance the efficacy of B-cell lymphoma-2 (Bcl-2) inhibitor ABT-737 [N-{4-[4-(4-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-3-nitro-benzenesulfonamide] is of great interest in many cancers, including glioma. Our recent study suggested that Akt is a crucial mediator of apoptosis sensitivity in response to ABT-737 in glioma cell lines. Inhibitors of phosphatidylinositol 3-kinase (PI3K)/Akt are currently being assessed clinically in patients with glioma. Because PI3K/Akt inhibition would be expected to have many proapoptotic effects, we hypothesized that there may be unique synergy between PI3K inhibitors and Bcl-2 homology 3 mimetics. Toward this end, we assessed the combination of the PI3K/Akt inhibitor NVP-BKM120 [5-(2,6-dimorpholinopyrimidin-4-yl)-4-(trifluoromethyl)pyridin-2-amine] and the Bcl-2 family inhibitor ABT-737 in established and primary cultured glioma cells. We found that the combined treatment with these agents led to a significant activation of caspase-8 and -3, PARP, and cell death, irrespective of PTEN status. The enhanced lethality observed with this combination also appears dependent on the loss of mitochondrial membrane potential and release of cytochrome c, smac/DIABLO, and apoptosis-inducing factor to the cytosol. Further study revealed that the upregulation of Noxa, truncation of Bid, and activation of Bax and Bak caused by these inhibitors were the key factors for the synergy. In addition, we demonstrated the release of proapoptotic proteins Bim and Bak from Mcl-1. We found defects in chromosome segregation leading to multinuclear cells and loss of colony-forming ability, suggesting the potential use of NVP-BKM120 as a promising agent to improve the anticancer activities of ABT-737.

Phosphorylated 4E‐binding protein 1 (p‐4E‐BP1): a novel prognostic marker in human astrocytomas

To investigate the significance of the mammalian target of rapamycin (mTOR) pathway in astrocytic tumours, published information in this context being limited, especially regarding phosphorylated 4E-binding protein (p-4E-BP) 1. Paraffin-embedded tissue from 111 patients with astroglial tumours (grades II-IV) was investigated for the association of phosphorylated mTOR (p-mTOR) signalling components with phosphorylated extracellular signal-related kinase 1/2 (p-ERK1/2) and phosphorylated AKT (p-AKT) expression, clinicopathological features, angiogenesis, isocitrate dehydrogenase 1 (IDH1)-R132H, and survival. Expression was also quantified by western blot analysis in 12 cases and in three primary glioma cell cultures following rapamycin treatment. p-mTOR expression correlated with p-4E-BP1 expression and marginally with p-p70S6K expression. p-4E-BP1 expression increased with tumour grade. Rapamycin induced a decline in phosphorylation levels of all three proteins. Nuclear p-AKT and cytoplasmic p-ERK1/2 immunoexpression correlated with p-4E-BP1 expression, whereas cytoplasmic p-AKT expression correlated with p-p70S6K expression. All three proteins were associated with increased angiogenesis but not with IDH1-R132H expression status. p-mTOR adversely affected overall and disease-free survival in univariate analysis. In multivariate survival analysis, the presence of p-4E-BP1 predicted shortened overall survival in the entire cohort and glioblastomas. mTOR signalling components are differentially involved in the acquisition of a more aggressive and angiogenic phenotype in astrocytic tumours. Moreover, p-4E-BP1 emerges as a novel prognostic marker, which might aid in the selection of patients who are more likely to benefit from therapy with mTOR inhibitors.

Expression levels of Fas/Fas-L mRNA in human brain glioma stem cells

Despite advancements in research made over the past decades, the prognosis of gliomas remains dismal. Fas/Fas ligand (Fas-L)-related immunotherapy may be regarded as a treatment of choice as it can induce the apoptosis of glioma cells, and has shown promising results in experimental studies. Over the years, brain glioma stem cells (BGSCs) have been accepted as the origin of gliomas and determine their biological features. The theory of BGSCs has facilitated the study of gliomas. In this study, we conducted a series of assays to culture BGSCs from clinical samples and determined the mRNA expression levels of Fas/Fas-L in BGSCs. We also investigated the effects of Fas/Fas-L-related immunotherapy on the apoptosis of glioma cells. BGSCs were grown from samples of 8 patients suffering from gliomas and identified by the assessment of biological characteristics and immunocytochemistry. Total RNA was extracted and reverse-transcribed into cDNA, and the expression levels of Fas/Fas-L mRNA were determined by real-time RT-PCR, followed by statistical analysis. The results showed that the Fas and Fas-L mRNA expression levels in BGSCs were lower compared to those in primary cultured glioma cells, which were statistically significant (P<0.001). These results indicate that immunotherapy involving Fas/Fas-L may not eradicate the BGSCs, which would result in the relapse of glioma. However, further research is required to investigate the mechanisms involved and define the prospect of Fas-involved immunotherapy against gliomas.