Established Glioblastoma Cell Lines Research Articles

Lipid Peroxidation Plays an Important Role in Chemotherapeutic Effects of Temozolomide and the Development of Therapy Resistance in Human Glioblastoma

BACKGROUND: Glioblastoma (GBM) is the most malignant primary brain tumor. Relapse occurs regularly, and the clinical behavior seems to be due to a therapy-resistant subpopulation of glioma-initiating cells that belong to the group of cancer stem cells. Aldehyde dehydrogenase (ALDH) has been identified as a marker for this cell population, and we have shown previously that ALDH1A3-positive GBM cells are more resistant against temozolomide (TMZ) treatment. However, it is still unclear how ALDH expression mediates chemoresistance. MATERIALS AND METHODS: ALDH1A3 expression was analyzed in 112 specimens from primary and secondary surgical resections of 56 patients with GBM (WHO grade IV). All patients received combined adjuvant radiochemotherapy. For experimental analysis, CRISPR-Cas9–induced knockout cells from three established GBM cell lines (LN229, U87MG, T98G) and two glioma stem-like cell lines were investigated after TMZ treatment. RESULTS: ALDH1A3 knockout cells were more sensitive to TMZ, and oxidative stress seemed to be the molecular process where ALDH1A3 exerts its role in resistance against TMZ. Oxidative stress led to lipid peroxidation, yielding active aldehydes that were detoxified by ALDH enzymatic activity. During the metabolic process, autophagy was induced leading to downregulation of the enzyme, but ALDH1A3 is upregulated to even higher expression levels after finishing the TMZ therapy in vitro. Recurrent GBMs show significantly higher ALDH1A3 expression than the respective samples from the primary tumor, and patients suffering from GBM with high ALDH1A3 expression showed a shorter median survival time (12 months vs 21 months, P < .05). CONCLUSION: Oxidative stress is an important and clinically relevant component of TMZ-induced therapeutic effects. Cytotoxicity seems to be mediated by aldehydes resulting from lipid peroxidation, and ALDH1A3 is able to reduce the number of toxic aldehydes. Therefore, we present a molecular explanation of the role of ALDH1A3 in therapeutic resistance of human GBM cells.

Monotherapy efficacy of blood-brain barrier permeable small molecule reactivators of protein phosphatase 2A in glioblastoma.

Glioblastoma is a fatal disease in which most targeted therapies have clinically failed. However, pharmacological reactivation of tumour suppressors has not been thoroughly studied as yet as a glioblastoma therapeutic strategy. Tumour suppressor protein phosphatase 2A is inhibited by non-genetic mechanisms in glioblastoma, and thus, it would be potentially amendable for therapeutic reactivation. Here, we demonstrate that small molecule activators of protein phosphatase 2A, NZ-8-061 and DBK-1154, effectively cross the in vitro model of blood–brain barrier, and in vivo partition to mouse brain tissue after oral dosing. In vitro, small molecule activators of protein phosphatase 2A exhibit robust cell-killing activity against five established glioblastoma cell lines, and nine patient-derived primary glioma cell lines. Collectively, these cell lines have heterogeneous genetic background, kinase inhibitor resistance profile and stemness properties; and they represent different clinical glioblastoma subtypes. Moreover, small molecule activators of protein phosphatase 2A were found to be superior to a range of kinase inhibitors in their capacity to kill patient-derived primary glioma cells. Oral dosing of either of the small molecule activators of protein phosphatase 2A significantly reduced growth of infiltrative intracranial glioblastoma tumours. DBK-1154, with both higher degree of brain/blood distribution, and more potent in vitro activity against all tested glioblastoma cell lines, also significantly increased survival of mice bearing orthotopic glioblastoma xenografts. In summary, this report presents a proof-of-principle data for blood–brain barrier—permeable tumour suppressor reactivation therapy for glioblastoma cells of heterogenous molecular background. These results also provide the first indications that protein phosphatase 2A reactivation might be able to challenge the current paradigm in glioblastoma therapies which has been strongly focused on targeting specific genetically altered cancer drivers with highly specific inhibitors. Based on demonstrated role for protein phosphatase 2A inhibition in glioblastoma cell drug resistance, small molecule activators of protein phosphatase 2A may prove to be beneficial in future glioblastoma combination therapies.

DDIS-18. NANOCELL-MEDIATED DELIVERY OF MIR-34A COUNTERACTS TEMOZOLOMIDE RESISTANCE IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastomas show marked intra- and inter-tumor heterogeneity and are strongly resistant to both radio- and chemo-therapy, which are standard therapeutic modalities for this tumor. MicroRNAs (miRNAs) have the potential to serve as effective therapeutics for glioblastoma as they modulate the activity of multiple signaling pathways. METHODS Glioblastoma cultures were transfected with miR-34a or control miRNA mimics to assess biological function and therapeutic potential in vitro. miR-34a was packaged into bacterially-derived nanocells and administered intravenously for delivery to orthotopic patient-derived glioblastoma xenografts in mice. RESULTS Overexpression of miR-34a strongly reduced the activation status of the three core signaling networks that have been found to be deregulated in the vast majority of glioblastoma tumors, the receptor tyrosine kinase, p53 and Rb networks. miR-34a transfection also inhibited the survival of multiple established glioblastoma cell lines as well as primary patient-derived xenograft cultures representing the proneural, mesenchymal and classical subtypes. Transfection of miR-34a synergized with temozolomide (TMZ) in in vitro cultures of glioblastoma cells with primary TMZ sensitivity, primary TMZ resistance and acquired TMZ resistance. Intravenous administration of bacterially-derived nanocells carrying miR-34a strongly enhanced TMZ sensitivity in an orthotopic patient-derived xenograft mouse model of glioblastoma. CONCLUSIONS miR-34a strongly sensitizes a wide range of glioblastoma cell cultures to TMZ, suggesting that combination therapy of TMZ with miR-34a may serve as a novel therapeutic approach for the treatment of glioblastoma tumors. Bacterially-derived nanocells are an effective vehicle for the delivery of miR-34a to glioblastoma tumors.

RDNA-14. RADIATION-INDUCED miR-4516 CONTRIBUTES TO RADIO-RESISTANCE AND PROMOTES AGGRESSIVE PHENOTYPE IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma is the most aggressive brain tumor with poor prognosis despite the best available treatment. MicroRNAs (miRNAs) are emerging as promising, novel prognostic biomarkers and therapeutic targets in glioblastoma. In a previous study, we demonstrated that miR-4516 predicts poor prognosis and functions as an oncogene in glioblastoma. Aim of the current study is to examine the role miR-4516 in radiation resistance and identify downstream targets contributing to this phenotype METHODS Radiosensitization was evaluated by cell viability and clonogenic assays. Cell apoptosis was evaluated using flow cytometry and immunoblotting. Potential targets of miR-4516 were identified using bioinformatic analysis (Targetscan and miRDB) and confirmed by luciferase reporter assays. Results were validated using immunoblotting. miR-4516 expression in glioblastoma cell lines after radiation treatment was quantified by qRT-PCR. RESULTS Expression of miR-4516 was increased up to 15 fold following radiation treatment, peaking at around 15min-60 min in primary and established glioblastoma cell lines including GBM 08-387, GBM 30 and U87-MG. Furthermore, inhibition of miR-4516 sensitized GBM 08-387, GBM30 and U87-MG cells to radiation in comparison to control groups as determined by cell viability and clonogenic assays. Further, miR-4516 inhibition induced apoptosis in these cell lines following radiation treatment. While conducting mechanistic studies, we found that the tumor-promoting function of miR-4516 was, in part, mediated by inhibition of p21 and PTPN14, two direct targets of miR-4516 CONCLUSION Our data suggest that radiation induces the expression of miR-4516 in glioblastoma cell lines. This miRNA plays a critical role in radio-resistance and promotes aggressive phenotypes in glioblastoma and therefore, functional analyses of its target pathways may uncover novel therapeutically vulnerable target(s) in glioblastoma. FUNDING: R01CA108633, R01CA169368, RC2CA148190, U10CA180850-01(NCI), Brain Tumor Funders Collaborative Grant, and OSU-CCC (all to AC). The Ton and Patricia Bohnenn Fund for Neuro_Oncology Research (to PR).

Encapsulation of Temozolomide in a Calixarene Nanocapsule Improves Its Stability and Enhances Its Therapeutic Efficacy against Glioblastoma.

The alkylating agent temozolomide (TMZ) is the first-line chemotherapeutic for glioblastoma (GBM), a common and aggressive primary brain tumor in adults. However, its poor stability and unfavorable pharmacokinetic profile limit its clinical efficacy. There is an unmet need to tailor the therapeutic window of TMZ, either through complex derivatization or by utilizing pharmaceutical excipients. To enhance stability and aqueous solubility, we encapsulated TMZ in a p-sulphonatocalix[4]arene (Calix) nanocapsule and used 1H-NMR, LC-MS, and UV-Vis spectroscopy to chart the stability of this novel TMZ@Calix complex according to FDA and European Medicines Agency guidelines. LC-MS/MS plasma stability assays were conducted in mice to further explore the stability profile of TMZ@Calix in vivo The therapeutic efficacy of TMZ@Calix was compared with that of unbound TMZ in GBM cell lines and patient-derived primary cells with known O6-methylguanine-DNA methyltransferase (MGMT) expression status and in vivo in an intracranial U87 xenograft mouse model. Encapsulation significantly enhanced the stability of TMZ in all conditions tested. TMZ@Calix was more potent than native TMZ at inhibiting the growth of established GBM cell lines and patient-derived primary lines expressing MGMT and highly resistant to TMZ. In vivo, native TMZ was rapidly degraded in mouse plasma, whereas the stability of TMZ@Calix was enhanced threefold with increased therapeutic efficacy in an orthotopic model. In the absence of new effective therapies, this novel formulation is of clinical importance, serving as an inexpensive and highly efficient treatment that could be made readily available to patients with GBM and warrants further preclinical and clinical evaluation.

Abstract 1787: Selective activation of a PI3K catalytic isoform by G protein-coupled receptors in glioblastoma

Abstract Objective: To determine how PI3K catalytic isoforms become dysregulated in glioblastoma. Background: Recurrence in glioblastoma (GBM) is common and even more resistant to treatment than initial GBM. The Sheng Lab has previously identified that PIK3CB expression levels, but not other PI3K subtypes, are higher in recurrent GBM and are associated with GBM recurrence in newly diagnosed individuals. PI3KCB encodes for PI3K catalytic subunit p110β, and PI3K/AKT pathway activation is a well-known promoter of cell survival. Understanding mechanisms of PIK3CB activation in GBM may identify new therapeutic targets and improve GBM survival. Current knowledge of PI3K isoform activity in GBM is insufficient and contradictory. Given previous studies, it was hypothesized that G-Coupled Protein Receptor (GPCR) promoters preferentially activate PI3K/AKT in PIK3CBhigh GBM cells. Further, that knockdown of PIK3CB, but not other PI3K isoforms, inhibits PI3K/AKT reactivation in PIK3CBhigh cell lines. Design/Methods: U87MG cells, an established p110βhigh GBM cell line, were starved overnight in serum free media, and then treated with growth factors, promoters or inhibitors of several previously hypothesized pathways. Cell lysates were collected and PI3K/AKT pathway activity was measured with immunoblotting. The ratio of pAKT/AKT/ACTB quantified PI3K/AKT pathway activity. Results: Overnight serum starvation depleted pAKT, and incubation with 1% FBS restored pAKT signaling in a time dependent manner. Neither EGF nor FGF-2 activated PI3K/AKT signaling. Gefitinib, an EGFR inhibitor, failed to inhibit reactivation when cells were co-treated with 1% FBS. However, GPCR stimulation by Lysophosphatidic acid (LPA) increased pAKT/AKT ratios in a dose dependent manner. More significantly, Gallein, a GPCR inhibitor, suppressed PI3K/AKT signaling when cells were co-treated with 1% FBS. Selective PI3K inhibitors showed little PI3K/AKT reactivation at multiple doses. Conclusions: These results suggest that GPCR signaling is responsible for PI3K/AKT pathway activation in p110βhigh cells. Citation Format: Abigail R. Winn, Zhi Sheng. Selective activation of a PI3K catalytic isoform by G protein-coupled receptors in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1787.

Viability of glioblastoma stem cells is effectively reduced by diisothiocyanate-derived mercapturic acids.

Glioblastoma is the most aggressive tumor of the central nervous system and is manifested by diffuse invasion of glioblastoma stem cells into the healthy tissue, chemoresistance and recurrence. Despite aggressive therapy, consisting of maximal surgical resection, radiotherapy and chemotherapy with temozolomide (Temodal®), life expectancy of patients with glioblastoma is typically less than 15 months. In general, natural isothiocyanates isolated from plants of the Cruciferae family are selectively cytotoxic to tumor cells. It has been demonstrated previously that diisothiocyanate-derived mercapturic acids are highly cytotoxic to colon cancer cells. In the present study, the application of diisothiocyanate-derived mercapturic acids led to a decrease in the viability of an established glioblastoma cell line, primary patient-derived sphere-cultured stem cell-enriched cell populations (SCs), and cells differentiated from SCs. Consequently, targeting glioblastoma cells by diisothiocyanate-derived mercapturic acids is a promising approach to restrict tumor cell growth and may be a novel therapeutic intervention for the treatment of glioblastoma.

High level EGFR amplification in a newly established glioblastoma cell line 170-MG-BA.

Glioblastoma multiforme is a highly invasive and incurable primary brain tumor. The most frequent genetic alteration therein is amplification of the epidermal growth factor receptor (EGFR) gene, the target of current clinical trials. However, EGFR amplification is poorly represented in glioblastoma cell lines. From the 30 cultures attempted herein, we were able to establish two glioblastoma permanent cell lines. The remaining cultures showed limited life span and underwent senescence between passage numbers (PN) 8 to 15. Our newly established glioblastoma cell lines, designated 170-MG-BA and 538-MG-BA, both originated between PN 3 and 5 when areas of smaller, more rapidly proliferating cells appeared. Both cell lines showed similar rates of growth, moderate morphological differences, cytoskeletal heterogeneity and multiple chromosome rearrangements. Analysis by molecular cytogenetics and comparative genomic hybridization (aCGH) revealed two copies of a stable marker chromosome in 170-MG-BA cells effecting focal amplification at 7q11 of the EGFR locus. Comparative RqPCR analysis confirmed that EGFR was uniquely highly expressed in 170-MG-BA cells. Combined targeted expression analysis and aCGH data excluded the recurrent EGFRvIII activating mutation. In contrast, EGFR expression in 538-MG-BA cells which lacked genomic EGFR amplification was not raised. Immunofluorescent staining showed high EGFR protein expression only in the 170-MG-BA cells. Cytogenetic, genomic and transcriptional analyses then confirmed high-level genomic amplification and transcriptional upregulation of wild type EGFR in 170-MG-BA; the first conventional cell line model for investigating the biology and targeted therapy of this key alteration in glioblastoma. Both cell lines are freely available from the DSMZ cell repository.

Abstract 881: Identifying the context of vulnerability to MLN4924 in glioblastoma (GBM)

Abstract Neddylation is a post-translational mechanism that marks proteins for degradation through activity of NEDD8 Activating Enzyme (NAE). NAE activates cullin-RING ligases (CRL), which ubiquitylate selected substrates and mark them for proteosomal degradation. MLN4924, or Pevonedistat, targets NAE and inhibits Neddylation and induces apoptosis in sensitive cells. To assess the preferential sensitivity of cell lines to MLN4924, we performed a 10-point drug dose response (DDR) assay on long-term established GBM cell lines. Efficacy of MLN4924 in glioma cell lines was evaluated by measuring cell viability (CellTiterGlo®) and cell cycle progression (flow cytometry with propidium iodide staining). To identify mechanism of differential response to MLN4924 treatment, cell cycle regulatory pathway and DNA damage were also examined by Western blotting. GB1 (IC50 = 0.28 μM) &amp; LN18 (IC50 = 0.19 μM) were established as sensitive and M059K (IC50 = 5.5 μM) &amp; SNU1105 (IC50 = 20.9 μM) as non-sensitive cell lines based on the IC50 values. Flow cytometry analysis of DNA content revealed significant arrest of cells in G2/M even at low doses of 100 nM of MLN4924 preferentially in GB1 and LN18. This was consistent with an increase in CRL substrates p21, p27 and WEE1 in GB1 and LN18 possibly contributing to the G2/M arrest. While CDT1 accumulation was observed starting at 2h post MLN4924 treatment in sensitive cell lines, it took upto 8h for CDT1 accumulation in the non-sensitive cell lines. Increases in CDT1 induced re-replication causing massive arrest in G2/M phase lead to increased DNA damage, validated by higher expression of γH2AX in the sensitive cell lines. Additionally, we also investigated the efficacy of MLN4924 against orthotopic glioma PDX models in vitro and in vivo to validate our findings. In a cohort of glioblastoma PDX models we discovered that GBM PDX models with lower Neddylation gene set enrichment score (GBM116, GBM59, SF7300) were markedly more vulnerable to MLN4924 than GBM PDX models with higher Neddylation gene set enrichment score (GBM91 and GBM102) in vitro and in vivo. Orthotopic PDX models of selected GBM revealed survival prolongation of GBM116, but minimal survival benefit to GBM102 tumors. Validation of the predictive markers of vulnerability to MLN4924 in additional PDX models will set the stage for prospective clinical trials of MLN4924 in glioblastoma patients. Citation Format: Leena Chaudhuri, Rita L. Bybee, Lauren K. Hartman, Sen Peng, Darren Finlay, Kristiina Vuori, Michael E. Berens, Harshil D. Dhruv. Identifying the context of vulnerability to MLN4924 in glioblastoma (GBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 881.

61 Functional characterization of ribosomal RNA methyltransferase NSUN5 in glioblastoma

Glioblastoma is the most common and malignant brain tumor with a median overall survival of 20.5 months. There is an urgent need to develop novel therapeutic strategies. Using a glioblastoma TCGA dataset, we have determined that high NSUN5 mRNA expression is strongly associated with poor survival in glioblastoma patients. NSUN5 is a ribosomal RNA (rRNA) cytosine methyltransferase. Human NSUN5 is located in chromosome 7 and is completely deleted in the Williams-Beurren syndrome, a complex neurodevelopmental disorder. However, RNA targets of NSUN5 in mammals and its role in cancer are unknown. The objective of this project is to determine whether elevated NSUN5 changes rRNA methylation pattern and thereby leads to pro-tumorigenic translational reprogramming and pro-tumorigenic phenotypes in glioblastoma. Western blotting showed that NSUN5 is expressed in 7 out of 9 established glioblastoma cell lines and in 8 out of 12 primary patient-derived glioblastoma cell lines. Bisulfite sequencing confirmed that NSUN5 methylates C3782 of human 28S rRNA in glioblastoma cells. Functionally, overexpression of NSUN5 increases, whereas NSUN5 knockout decreases global protein synthesis and sphere formation in glioblastoma cells. More importantly, mice bearing intracranial NSUN5-expressing U87 tumors survived for a shorter time than mice bearing tumors derived from U87 control cells. Our results suggest that NSUN5 methylates 28S rRNA and may enhance cancer stem cell phenotypes and tumor formation and/or progression in glioblastoma. Experiments are ongoing to determine whether NSUN5 promotes tumor formation and/or progression through translational reprogramming in glioblastoma. This study may help identify novel therapeutic targets for glioblastoma.

The critical role that STAT3 plays in glioma-initiating cells: STAT3 addiction in glioma.

Glioma-Initiating Cells (GICs) are thought to be responsible for tumor initiation, progression and recurrence in glioblastoma (GBM). In previous studies, we reported the constitutive phosphorylation of the STAT3 transcription factor in GICs derived from GBM patient-derived xenografts, and that STAT3 played a critical role in GBM tumorigenesis. In this study, we show that CRISPR/Cas9-mediated deletion of STAT3 in an established GBM cell line markedly inhibited tumorigenesis by intracranial injection but had little effect on cell proliferation in vitro. Tumorigenesis was rescued by the enforced expression of wild-type STAT3 in cells lacking STAT3. In contrast, GICs were highly addicted to STAT3 and upon STAT3 deletion GICs were non-viable. Moreover, we found that STAT3 was constitutively activated in GICs by phosphorylation on both tyrosine (Y705) and serine (S727) residues. Therefore, to study STAT3 function in GICs we established an inducible system to knockdown STAT3 expression (iSTAT3-KD). Using this approach, we demonstrated that Y705-STAT3 phosphorylation was critical and indispensable for GIC-induced tumor formation. Both phosphorylation sites in STAT3 promoted GIC proliferation in vitro. We further showed that S727-STAT3 phosphorylation was Y705-dependent. Targeted microarray and RNA sequencing revealed that STAT3 activated cell-cycle regulator genes, and downregulated genes involved in the interferon response, the hypoxia response, the TGFβ pathway, and remodeling of the extracellular matrix. Since STAT3 is an important oncogenic driver of GBM, the identification of these STAT3 regulated pathways in GICs will inform the development of better targeted therapies against STAT3 in GBM and other cancers.

FHL2 interacts with EGFR to promote glioblastoma growth.

Four-and-a-half LIM protein2 (FHL2) is a member of the LIM-only protein family, which plays a critical role in tumorigenesis. We previously reported that FHL2 is upregulated and plays an oncogenic role in glioblastoma (GBM), the most common and aggressive brain tumor. GBM is also marked by amplification of the epidermal growth factor receptor (EGFR) gene and its mutations, of which EGFRvIII is the most common and functionally significant. Here we report that FHL2 physically interacts with the wild-type EGFR and its mutated EGFRvIII form in GBM cells. Expression of FHL2 caused increased EGFR and EGFRvIII protein levels and this was due to an increase in protein stability rather than an increase in EGFR mRNA expression. In contrast, FHL2 knockdown using RNA interference reduced EGFR and EGFRvIII protein expression and the phosphorylation levels of EGFR and AKT. Consistent with these features, EGFR expression was significantly lower in mouse FHL2-null astrocytes, where reintroduction of FHL2 was able to restore EGFR levels. Using established GBM cell lines and patient-derived neurosphere lines, FHL2 silencing markedly induced cell apoptosis in EGFRvIII-positive cells. Targeting FHL2 significantly prevented EGFRvIII-positive GBM tumor growth in vivo. FHL2 expression also positively correlated with EGFR expression in GBM samples from patients. Taken together, our results demonstrate that FHL2 interacts with EGFR and EGFRvIII to increase their levels and this promotes glioma growth, representing a novel mechanism that may be therapeutically targetable.

ID2011 Potential strategies to improve Temozolomide/ radiation therapy of malignant gliomas by targeting glycolytic energy metabolism

The current standard treatment of Malignant Gliomas includes surgery, followed by Temozolomide (TMZ) - Radiotherapy. It leads to increased survival as compared to radiotherapy alone. However, haematological toxicities are also increased by the combination treatments. Therefore, it is important to carry out further preclinical studies, to develop more effective treatment for these tumors. 2-Deoxy-D-Glucose (2-DG), an inhibitor of glycolytic energy metabolism, has been shown earlier to differentially inhibit repair processes, growth and survival of cancer cells. It has been tolerated very well in combination with radiotherapy in clinical trials on Malignant Gliomas. In this study, we investigated the effects of combination of clinically relevant concentrations of 2-DG (0.5mM and 1mM) and TMZ (2µM and 5µM) ± Radiation (1Gy) on cell proliferation, total cellular damage (TCD) and colony formation in an established Glioblastoma cell line (U251MG), and primary cultures derived from malignant glioma tumor pieces. The monolayer cultures were grown on cover slips and stained with Acridine Orange (0.002%) after drug treatments and irradiation, for the study of cellular damage and death. Exponentially growing cells were exposed to drugs and Gamma irradiation. Drugs were removed 4 hrs after irradiation and cultures were processed for different assays of cell death and damage. Our results showed that combination of 2-DG with TMZ±Radiation significantly inhibited the cell proliferation up to 6 days. The TCD was significantly increased by the combination of drugs in primary as well as in established cell line. Cell proliferation as measured by MTT assay showed that drugs ± radiation significantly reduced proliferation response. Cell survival inhibition after combination of TMZ+2-DG+Gamma (2µM+0.5mM+1Gy) showed more than additive effects. These results suggest that targeting glycolytic energy metabolism by agents such as 2-DG can enhance the efficacy of TMZ+Radiation Therapy for Malignant Gliomas, without increasing toxic side effects

High Dose Ascorbate Causes Both Genotoxic and Metabolic Stress in Glioma Cells.

We have previously shown that exposure to high dose ascorbate causes double stranded breaks (DSBs) and a build-up in S-phase in glioblastoma (GBM) cell lines. Here we investigated whether or not this was due to genotoxic stress as well as metabolic stress generated by exposure to high dose ascorbate, radiation, ascorbate plus radiation and H2O2 in established and primary GBM cell lines. Genotoxic stress was measured as phosphorylation of the variant histone protein, H2AX, 8-oxo-7,8-dihydroguanine (8OH-dG) positive cells and cells with comet tails. Metabolic stress was measured as a decrease in NADH flux, mitochondrial membrane potential (by CMXRos), ATP levels (by ATP luminescence) and mitochondrial superoxide production (by mitoSOX). High dose ascorbate, ascorbate plus radiation, and H2O2 treatments induced both genotoxic and metabolic stress. Exposure to high dose ascorbate blocked DNA synthesis in both DNA damaged and undamaged cell of ascorbate sensitive GBM cell lines. H2O2 treatment blocked DNA synthesis in all cell lines with and without DNA damage. DNA synthesis arrest in cells with damaged DNA is likely due to both genotoxic and metabolic stress. However, arrest in DNA synthesis in cells with undamaged DNA is likely due to oxidative damage to components of the mitochondrial energy metabolism pathway.

Mesenchymal stem cells differentially affect the invasion of distinct glioblastoma cell lines.

Glioblastoma multiforme are an aggressive form of brain tumors that are characterized by distinct invasion of single glioblastoma cells, which infiltrate the brain parenchyma. This appears to be stimulated by the communication between cancer and stromal cells. Mesenchymal stem cells (MSCs) are part of the glioblastoma microenvironment, and their ‘cross-talk’ with glioblastoma cells is still poorly understood. Here, we examined the effects of bone marrow-derived MSCs on two different established glioblastoma cell lines U87 and U373. We focused on mutual effects of direct MSC/glioblastoma contact on cellular invasion in three-dimensional invasion assays in vitro and in a zebrafish embryo model in vivo. This is the first demonstration of glioblastoma cell-type-specific responses to MSCs in direct glioblastoma co-cultures, where MSCs inhibited the invasion of U87 cells and enhanced the invasion of U373. Inversely, direct cross-talk between MSCs and both of glioblastoma cell lines enhanced MSC motility. MSC-enhanced invasion of U373 cells was assisted by overexpression of proteases cathepsin B, calpain1, uPA/uPAR, MMP-2, -9 and -14, and increased activities of some of these proteases, as determined by the effects of their selective inhibitors on invasion. In contrast, these proteases had no effect on U87 cell invasion under MSC co-culturing. Finally, we identified differentially expressed genes, in U87 and U373 cells that could explain different response of these cell lines to MSCs. In conclusion, we demonstrated that MSC/glioblastoma cross-talk is different in the two glioblastoma cell phenotypes, which contributes to tumor heterogeneity.

Structural Optimization and Pharmacological Evaluation of Inhibitors Targeting Dual-Specificity Tyrosine Phosphorylation-Regulated Kinases (DYRK) and CDC-like kinases (CLK) in Glioblastoma.

The DYRK family contains kinases that are up-regulated in malignancy and control several cancer hallmarks. To assess the anticancer potential of inhibitors targeting DYRK kinases, we developed a series of novel DYRK inhibitors based on the 7-azaindole scaffold. All compounds were tested for their ability to inhibit DYRK1A, DYRK1B, DYRK2, and the structurally related CLK1. The library was screened for anticancer efficacy in established and stem cell-like glioblastoma cell lines. The most potent inhibitors (IC50 ≤ 50 nM) significantly decreased viability, clonogenic survival, migration, and invasion of glioblastoma cells. Target engagement was confirmed with genetic knockdown and the cellular thermal shift assay. We demonstrate that DYRK1A's thermal stability in cells is increased upon compound treatment, confirming binding in cells. In summary, we present synthesis, structure-activity relationship, and efficacy in glioblastoma-relevant models for a library of novel 7-azaindoles.

In Vitro/In Silico Study on the Role of Doubling Time Heterogeneity among Primary Glioblastoma Cell Lines.

The application of accurate cancer predictive algorithms validated with experimental data is a field concerning both basic researchers and clinicians, especially regarding a highly aggressive form of cancer, such as Glioblastoma. In an aim to enhance prediction accuracy in realistic patient-specific environments, accounting for both inter- and intratumoral heterogeneity, we use patient-derived Glioblastoma cells from different patients. We focus on cell proliferation using in vitro experiments to estimate cell doubling times and sizes for established primary Glioblastoma cell lines. A preclinically driven mathematical model parametrization is accomplished by taking into account the experimental measurements. As a control cell line we use the well-studied U87MG cells. Both in vitro and in silico results presented support that the variance between tumor staging can be attributed to the differential proliferative capacity of the different Glioblastoma cells. More specifically, the intratumoral heterogeneity together with the overall proliferation reflected in both the proliferation rate and the mechanical cell contact inhibition can predict the in vitro evolution of different Glioblastoma cell lines growing under the same conditions. Undoubtedly, additional imaging techniques capable of providing spatial information of tumor cell physiology and microenvironment will enhance our understanding regarding Glioblastoma nature and verify and further improve our predictability.

A novel NFIA-NFκB feed-forward loop contributes to glioblastoma cell survival.

The nuclear factor I-A (NFIA) transcription factor promotes glioma growth and inhibits apoptosis in glioblastoma (GBM) cells. Here we report that the NFIA pro-survival effect in GBM is mediated in part via a novel NFIA-nuclear factor-kappaB (NFκB) p65 feed-forward loop. We examined effects of gain- and loss-of-function manipulations of NFIA and NFκB p65 on each other's transcription, cell growth, apoptosis and sensitivity to chemotherapy in patient-derived GBM cells and established GBM cell lines. NFIA enhanced apoptosis evasion by activating NFκB p65 and its downstream anti-apoptotic factors tumor necrosis factor receptor-associated factor 1 (TRAF1) and cellular inhibitor of apoptosis proteins (cIAPs). Induction of NFκB by NFIA was required to protect cells from apoptosis, and inhibition of NFκB effectively reversed the NFIA anti-apoptotic effect. Conversely, NFIA knockdown decreased expression of NFκB and anti-apoptotic genes TRAF1 and cIAPs, and increased baseline apoptosis. NFIA positively regulated NFκB transcription and NFκB protein level. Interestingly, NFκB also activated the NFIA promoter and increased NFIA level, and knockdown of NFIA was sufficient to attenuate the NFκB pro-survival effect, suggesting a reciprocal regulation between NFIA and NFκB in governing GBM cell survival. Supporting this, NFIA and NFκB expression levels were highly correlated in human GBM and patient-derived GBM cells. These data define a previously unknown NFIA-NFκB feed-forward regulation that may contribute to GBM cell survival.

Fetuin-A (alpha 2HS glycoprotein) modulates growth, motility, invasion, and senescence in high-grade astrocytomas.

Glioblastomas (high‐grade astrocytomas) are highly aggressive brain tumors with poor prognosis and limited treatment options. In the present studies, we have defined the role of fetuin‐A, a liver‐derived multifunctional serum protein, in the growth of an established glioblastoma cell line, LN229. We hereby demonstrate that these cells synthesize ectopic fetuin‐A which supports their growth in culture in the absence of serum. We have demonstrated that a panel of tissue microarray (TMA) of glioblastomas also express ectopic fetuin‐A. Knocking down fetuin‐A using shRNA approach in LN229, significantly reduced their in vitro growth as well as growth and invasion in vivo. The fetuin‐A knockdown subclones of LN229 (A and D) also had reduced motility and invasive capacity. Treatment of LN229 cells with asialofetuin (ASF), attenuated their uptake of labeled fetuin‐A, and induced senescence in them. Interestingly, the D subclone that had ~90% reduction in ectopic fetuin‐A, underwent senescence in serum‐free medium which was blunted in the presence of purified fetuin‐A. Uptake of labeled exosomes was attenuated in fetuin‐A knockdown subclones A and D. Taken together, the studies demonstrate the impact of fetuin‐A as significant node of growth, motility, and invasion signaling in glioblastomas that can be targeted for therapy.

Abstract B46: Inhibition of STAT3 enhances the radiosensitizing effect of Temozolomide in vitro and in vivo: Validation using patient-derived glioblastoma model

Abstract Background and purpose: Despite aggressive treatment with radiation therapy plus temozolomide (TMZ), the prognosis for glioblastoma remains poor. We investigated the potential of targeting signal transducer and activator of transcription-3 (STAT3) to improve the therapeutic outcome of glioblastoma. Material and methods: We evaluated the preclinical potential of a STAT3 inhibitor, Cpd188 combined with temozolomide and radiation in vitro assays using two established glioblastoma cell lines (U251, U87) and two patients-derived glioblastoma cell lines (GBL12, GBL28) and in vivo studies using nude mice bearing intracranial U251 xenografts. Results: Cpd188 potentiated the radiosensitizing effect of TMZ in U251 cell which has high levels of p-STAT3 expression. Increased radiosensitizing effects of TMZ were associated with impaired DNA damage repair, apoptosis and the reversion of epithelial-mesenchymal transition (EMT). Cpd188 delayed in vivo tumor growth both alone and in combination with radiation and TMZ. We also confirmed the radiosensitizing effect of Cpd188 of GBL28 cell which was originated from a patient with high level of STAT3 expression and unmethylated MGMT. Conclusion: Targeting STAT3 using Cpd188 could be a viable therapeutic approach to improve the outcome of current standard therapy for glioblastoma patients having high p-STAT3 expression regardless of MGMT methylation status. *Work supported by the grant (#2013R1A1A2074531) from the Ministry of Science, ICT &amp; Future Planning to In Ah Kim Citation Format: Tae Jin Han, Eun Jung Choi, Bong Jun Cho, Sang Hyuk Song, Sun Ha Paek, In Ah Kim{Authors}. Inhibition of STAT3 enhances the radiosensitizing effect of Temozolomide in vitro and in vivo: Validation using patient-derived glioblastoma model. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B46.