Panel Of Glioblastoma Multiforme Cell Lines Research Articles

Silencing GMPPB Inhibits the Proliferation and Invasion of GBM via Hippo/MMP3 Pathways.

Glioblastoma multiforme (GBM) is a highly aggressive malignancy and represents the most common brain tumor in adults. To better understand its biology for new and effective therapies, we examined the role of GDP-mannose pyrophosphorylase B (GMPPB), a key unit of the GDP-mannose pyrophosphorylase (GDP-MP) that catalyzes the formation of GDP-mannose. Impaired GMPPB function will reduce the amount of GDP-mannose available for O-mannosylation. Abnormal O-mannosylation of alpha dystroglycan (α-DG) has been reported to be involved in cancer metastasis and arenavirus entry. Here, we found that GMPPB is highly expressed in a panel of GBM cell lines and clinical samples and that expression of GMPPB is positively correlated with the WHO grade of gliomas. Additionally, expression of GMPPB was negatively correlated with the prognosis of GBM patients. We demonstrate that silencing GMPPB inhibits the proliferation, migration, and invasion of GBM cells both in vitro and in vivo and that overexpression of GMPPB exhibits the opposite effects. Consequently, targeting GMPPB in GBM cells results in impaired GBM tumor growth and invasion. Finally, we identify that the Hippo/MMP3 axis is essential for GMPPB-promoted GBM aggressiveness. These findings indicate that GMPPB represents a potential novel target for GBM treatment.

Anticancer effects of ABTL0812, a clinical stage drug inducer of autophagy-mediated cancer cell death, in glioblastoma models.

Glioblastoma multiforme (GBM) is the most malignant adult brain tumor. Current standard of care treatments have very limited efficacy, being the patients´ overall survival 14 months and the 2-year survival rate less than 10%. Therefore, the treatment of GBM is an urgent unmet clinical need. The aim of this study was to investigate in vitro and in vivo the potential of ABTL0812, an oral anticancer compound currently in phase II clinical stage, as a novel therapy for GBM. We showed that ABTL0812 inhibits cell proliferation in a wide panel of GBM cell lines and patient-derived glioblastoma stem cells (GSCs) with half maximal inhibitory concentrations (IC50s) ranging from 15.2 µM to 46.9 µM. Additionally, ABTL0812 decreased GSCs neurosphere formation. GBM cells aggressiveness is associated with a trans-differentiation process towards a less differentiated phenotype known as proneural to mesenchymal transition (PMT). ABTL0812 was shown to revert PMT and induce cell differentiation to a less malignant phenotype in GBM cell lines and GSCs, and consequently reduced cell invasion. As previously shown in other cancer types, we demonstrated that the molecular mechanism of action of ABTL0812 in glioblastoma involves the inhibition of Akt/mTORC1 axis by overexpression of TRIB3, and the activation of endoplasmic reticulum (ER) stress/unfolded protein response (UPR). Both actions converge to induce autophagy-mediated cell death. ABTL0812 anticancer efficacy was studied in vivo using subcutaneous and orthotopic intra-brain xenograft tumor models. We demonstrated that ABTL0812 impairs tumor growth and increases disease-free survival and overall survival of mice. Furthermore, the histological analysis of tumors indicated that ABTL0812 decreases angiogenesis. Finally, we investigated the combination of ABTL0812 with the standard of care treatments for GBM radiotherapy and temozolomide in an orthotopic model, detecting that ABTL0812 potentiates the efficacy of both treatments and that the strongest effect is obtained with the triple combination of ABTL0812+radiotherapy+temozolomide. Overall, the present study demonstrated the anticancer efficacy of ABTL0812 as single agent and in combination with the GBM standard of care treatments in models of glioblastoma and supports the clinical investigation of ABTL0812 as a potential novel therapy for this aggressive brain tumor type.

Histone 2A Family Member J Drives Mesenchymal Transition and Temozolomide Resistance in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor and has a poor prognosis and is poorly sensitive to radiotherapy or temozolomide (TMZ) chemotherapy. Therefore, identifying new biomarkers to predict therapeutic responses of GBM is urgently needed. By using The Cancer Genome Atlas (TCGA) database, we found that the upregulation of histone 2A family member J (H2AFJ), but not other H2AFs, is extensively detected in the therapeutic-insensitive mesenchymal, IDH wildtype, MGMT unmethylated, or non-G-CIMP GBM and is associated with poor TMZ responsiveness independent of radiation. Similar views were also found in GBM cell lines. Whereas H2AFJ knockdown diminished TMZ resistance, H2AFJ overexpression promoted TMZ resistance in a panel of GBM cell lines. Gene set enrichment analysis (GSEA) revealed that H2AFJ upregulation accompanied by the activation of TNF-α/NF-κB and IL-6/STAT3-related pathways is highly predicted. Luciferase-based promoter activity assay further validated that the activities of NF-κB and STAT3 are causally affected by H2AFJ expression in GBM cells. Moreover, we found that therapeutic targeting HADC3 by tacedinaline or NF-κB by ML029 is likely able to overcome the TMZ resistance in GBM cells with H2AFJ upregulation. Significantly, the GBM cohorts harboring a high-level H2AFJ transcript combined with high-level expression of TNF-α/NF-κB geneset, IL-6/STAT3 geneset or HADC3 were associated with a shorter time to tumor repopulation after initial treatment with TMZ. These findings not only provide H2AFJ as a biomarker to predict TMZ therapeutic effectiveness but also suggest a new strategy to combat TMZ-insensitive GBM by targeting the interaction network constructed by TNF-α/NF-κB, IL-6/STAT3, HDAC3, and H2AFJ.

Identification of a panel of genes as a prognostic biomarker for glioblastoma.

BackgroundGlioblastoma multiforme (GBM) is a fatal disease without effective therapy. Identification of new biomarkers for prognosis would enable more rational selections of strategies to cure patients with GBM and prevent disease relapse.MethodsSeven datasets derived from GBM patients using microarray or next generation sequencing in R2 online database (http://r2.amc.nl) were extracted and then analyzed using JMP software. The survival distribution was calculated according to the Kaplan-Meier method and the significance was determined using log-rank statistics. The sensitivity of a panel of GBM cell lines in response to temozolomide (TMZ), salinomycin, celastrol, and triptolide treatments was evaluated using MTS and tumor-sphere formation assay.FindingsWe identified that CD44, ATP binding cassette subfamily C member 3 (ABCC3), and tumor necrosis factor receptor subfamily member 1A (TNFRSF1A) as highly expressed genes in GBMs are associated with patients' poor outcomes and therapy resistance. Furthermore, these three markers combined with MGMT, a conventional GBM marker, can classify GBM patients into five new subtypes with different overall survival time in response to treatment. The four-gene signature and the therapy response of GBMs to a panel of therapeutic compounds were confirmed in a panel of GBM cell lines.InterpretationThe data indicate that the four-gene panel can be used as a therapy response index for GBM patients and potential therapeutic targets. These results provide important new insights into the early diagnosis and the prognosis for GBM patients and introduce potential targets for GBM therapeutics.FundBaylor Scott & White Health Startup Fund (E.W.); Collaborative Faculty Research Investment Program (CFRIP) of Baylor University, Baylor Scott & White Health, and Baylor College of Medicine (E.W., T.S., J.H.H.); NIH R01 NS067435 (J.H.H.); Scott & White Plummer Foundation Grant (J.H.H.); National Natural Science Foundation of China 816280007 (J.H.H. and Fu.W.).

Identification of a Panel of Genes as a Prognostic Biomarker for Glioblastoma

Glioblastoma multiforme (GBM) is a fatal disease without effective therapy. Identification of new biomarkers for prognosis would enable more rational selections of strategies to cure patients with GBM and prevent relapse. We analyzed a large amount of gene expression profiling data derived from GBM patients. We identified CD44, ATP binding cassette subfamily C member 3 (ABCC3), and TNRSF1A as highly expressed genes in GBMs. Furthermore, the expression of these three genes is closely associated with patient's short survival time and therapy resistance. Moreover, these three markers combining with MGMT, a conventional GBM marker, can classify GBM patients into 5 new subgroups with different overall survival time in response to treatment. The four-gene signature was then evaluated in a panel of GBM cell lines treated with traditional therapeutic compounds and the data indicate that the four-gene panel can be used as a therapy response index for GBM patients and potential therapeutic targets. These results provide important new insights into the early detections and prognosis for GBM patients and introduce potential targets for GBM therapeutics. Funding: This study is, in part, supported by NovoCure Inc, 160280 (E.F. and F.W.); Baylor Scott and White Startup funds (E.W.); NIH R01 NS067435 (J.H.H.), Scott & White Plummer Foundation Grant (J.H.H.); National Science Foundation in China 816280007 (J.H.H. and Fu. W.); Jiangsu Nature Foundation BK20151565, Jiangsu Traditional Chinese Medicine Foundation ZD201501, Jiangsu Six Talent Peak Project 2015YY006 (Fu. W.). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: Ethical approval of our experiments was given by the ethics committee of Baylor Scott and White Health.

ω-3 and ω-6 Fatty Acids Modulate Conventional and Atypical Protein Kinase C Activities in a Brain Fatty Acid Binding Protein Dependent Manner in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is a highly infiltrative brain cancer with a dismal prognosis. High levels of brain fatty acid binding protein (B-FABP) are associated with increased migration/infiltration in GBM cells, with a high ratio of arachidonic acid (AA) to docosahexaenoic acid (DHA) driving B-FABP-mediated migration. Since several protein kinase Cs (PKCs) are overexpressed in GBM and linked to migration, we explored a possible relationship between B-FABP and levels/activity of different PKCs, as a function of AA and DHA supplementation. We report that ectopic expression of B-FABP in U87 cells alters the levels of several PKCs, particularly PKCζ. Upon analysis of PKCζ RNA levels in a panel of GBM cell lines and patient-derived GBM neurospheres, we observed a trend towards moderate positive correlation (r = 0.624, p = 0.054) between B-FABP and PKCζ RNA levels. Analysis of PKC activity in U87 GBM cells revealed decreased typical PKC activity (23.4%) in B-FABP-expressing cells compared with nonexpressing cells, with no difference in novel and atypical PKC activities. AA and DHA modulated both conventional and atypical PKC activities in a B-FABP-dependent manner, but had no effect on novel PKC activity. These results suggest that conventional and atypical PKCs are potential downstream effectors of B-FABP/fatty acid-mediated alterations in GBM growth properties.

Abstract A104: AZD1390, a potent and selective orally bioavailable blood-brain barrier-penetrant ATM inhibitor, radiosensitizes and improves survival of orthotopic glioma and metastatic brain tumor models

Abstract ATM plays a central role in the detection, signalling, and repair of DNA double-strand breaks (DSB), the most cytotoxic lesion induced by ionizing radiation (IR) and certain chemotherapies. ATM is also activated by reactive oxygen species (ROS) induced by cellular exposures to IR. Genetic ablation and pharmacologic inhibition of ATM is associated with extreme hypersensitivity of glioblastoma multiforme (GBM) tumor cells to IR, especially those with checkpoint defects such as p53 abrogations. GBM is the most common and lethal form of brain tumor. Median survival of patients is 12-15 months, despite surgery, fractionated radiotherapy, and temozolomide standard of care. Poor survival is attributed to an inability to excise all tumor tissue (if operable), dissemination of disease into regions with an intact blood-brain barrier (BBB), and an intrinsic radio- and chemo-resistance. With ATM activity robustly upregulated in GBM stem cells, ATM represents an attractive radiosensitization target. Here, we describe the activity of AZD1390, a potent, selective, and orally bioavailable ATM inhibitor optimized for BBB penetration in preclinical model species. AZD1390 demonstrates exquisitely potent cellular inhibition of ATM activity (IC50 0.78 nM) with >1000-fold activity over closely related (PIKKs) and distant kinases. We confirm target and pathway engagement by Western blot and imaging pan-nuclear and discreet pATM foci staining (IC50 0.6-3nM). Radiosensitization of a panel of GBM cell lines and NCI-H2228 lung cells was confirmed in antiproliferation and clonogenic assays (IC50 3 nM). DEF37 of 2.7 was seen in p53 mutant GBM cells dosed at 3nM and p53 mutant GBM cell lines were more radiosensitized than wild type cells. Radiosensitization was confirmed in vivo in mouse orthotopic NCI-H2228 lung tumor models implanted directly into brain or via carotid artery injection showing dose-dependent tumor growth inhibition and remarkable increases in survival of mice when dosing AZD1390 PO an hour before four daily fractions of 2.5 Gy IR to the whole head. 20 mg/kg QD or BID gave the best survival benefit that correlated with bioluminescent tumor growth inhibition. Doses lower than 2 mg/kg were not efficacious, suggesting free brain PK cover over ATM IC50 of 3 hours or more are required for efficacy. Efficacy was also achieved in a dose-dependent manner in orthotopic GL261 murine GBM syngeneic models dosed in combination with either whole head radiotherapy or stereotactic beam radiotherapy. A PK PD efficacy relationship was establish by correlating AZD1390 free brain PK levels, phospho-ATM/Rad50 detection in tumor by IHC, and tumor growth inhibition and survival. Significant brain exposure was observed in a nonhuman primate PET study utilizing 11C-labelled AZD1390, further supporting the ability of the compound to efficiently cross the BBB. With confirmation that AZD1390 is not a substrate for human efflux transporters and having favorable pharmacokinetic and pharmacodynamic properties, AZD1390 is now in early clinical development for use as a radiosensitizer in central nervous system malignancies. Citation Format: Steve T. Durant, Kurt G. Pike, Nicola Colclough, Lucy Riches, Antonio Garcia-Trinidad, Thomas Hunt, Stephanie Ling, Jonathan Stott, Ian Barrett, Li Zheng, Yingchun Wang, Kan Chen, Tianwei Zhang, Venkatesh Pilla Reddy, Andrew Sykes, Peter Johnstrom, Gemma Jones, Andrew Pierce, Jeremy Karlin, Jenna Kahn, Jasmine Allen, Kristoffer Valerie, Ruth Illingworth, Martin Pass. AZD1390, a potent and selective orally bioavailable blood-brain barrier-penetrant ATM inhibitor, radiosensitizes and improves survival of orthotopic glioma and metastatic brain tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A104.

Abstract A26: Inhibition of MDM2 and AKT signaling networks synergize to activate Forkhead box O-class transcription factors and promote cell death in mutant p53 GBM cells

Abstract A multi-targeted approach will be necessary to eradicate glioblastoma multiforme (GBM) cells due to the immense genetic heterogeneity associated with GBM. Mouse double minute-2 (MDM2) regulates multiple signaling pathways and is a promising therapeutic target in GBM. In wild type (wt) p53 cells, MDM2 binds to wtp53, ubiquitinates it, and negatively regulates p53-mediated downstream events. In wtp53 and mutant (mt) p53 cells, MDM2 binds to and sequesters p73α thereby blocking p73α-mediated signaling. Our objective in the present studies was to determine if the p73α-MDM2 axis could be exploited to increase death of mtp53 GBM cells. We utilized MDM2 antagonists nutlin3a or RG7112 to block protein-protein interactions between MDM2-p53 and MDM2-p73α. In a panel of GBM cell lines, TMZ resistance was reduced in both wt53 and mt53 cells in the presence of MDM2 antagonists. In mtp53 cells, siRNA knockdown of p73α indicated that sensitivity to treatment was dependent on p73α levels. Isobologram analysis indicated that while dose-ratios of TMZ to MDM2 antagonists were additive to synergistic in inhibiting growth of wtp53 GBM cells, this was not the case in mtp53 GBM cells (SF118, GBM43, gain-of-function-mtp53 R273H U373 and MHBT32). Analysis of intracellular targets in mtp53 GBM cells exposed to TMZ and MDM2 antagonists indicated that p73α and MDM2 expression increased by 24 hours post-treatment. In addition, AKT activity was increased or sustained in mtp53 GBM cells following treatment with TMZ in the absence or presence of MDM2 antagonists. Since increased AKT activity may render cells resistant to therapy, the AKT inhibitor GDC0068 was evaluated in combination with TMZ and RG7112. As a measure of AKT-downstream target modulation, phosphorylation status of the Forkhead box O-class (FoxO) transcription factors (TFs) was determined. In the non-phosphorylated state, FoxO TFs upregulate expression of proteins involved in cell-death pathways. While phospho-FoxO1/FoxO3a TFs were increased in TMZ/RG7112-treated mtp53 GBM cells compared to controls, it was decreased in GDC0068-, TMZ/GDC0068- and TMZ/RG7112/GDC0068-treated mtp53 GBM cells which is consistent with inactivation of AKT and activation of FoxO TFs. Isobologram analysis of mtp53 GBM cell growth indicated that combination RG7112 and GDC0068 inhibited growth in a synergistic manner even in the absence of TMZ. For in vivo studies, an intermittent dosing regimen of TMZ/RG7112/GDC0068 was developed to avoid normal tissue toxicity. GBM43 flank tumor growth was significantly inhibited in mice with tumors treated with RG7112/GDC0068 and inhibited to a larger extent by the triple combination TMZ/RG7112/GDC0068 compared to vehicle and single-agent exposure (n=9-10 mice per group; single agent vs GDC0068/RG7112 or TMZ/RG7112/GDC0068, p<0.05). The present data indicate that targeting the p73α-MDM2 and AKT-FoxO signaling networks inhibit mtp53 GBM cell growth and with an appropriate dosing schedule can be utilized in vivo with an acceptable toxicity profile. Citation Format: Mohammad Reza Saadatzadeh, Haiyan Wang, Jixin Ding, Barbara J. Bailey, Eva Tonsing-Carter, Shanbao Cai, Nimita Dave, Harlan E. Shannon, Aaron Cohen- Gadol, Karen E. Pollok. Inhibition of MDM2 and AKT signaling networks synergize to activate Forkhead box O-class transcription factors and promote cell death in mutant p53 GBM cells. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A26.

Abstract 40: Developmental profile and regulation of the glycolytic enzyme hexokinase 2 and its association with aerobic glycolysis

Abstract Proliferating embryonic and tumor tissues rely on aerobic glycolysis, or the metabolism of glucose to lactate under oxygenated conditions, to assist in the synthesis of biosynthetic precursors necessary for proliferation. The reliance on aerobic glycolysis may be mediated by the expression of specific metabolic enzymes. Mammalian Hexokinases HK1 and HK2 are 100kDa proteins that phosphorylate glucose to glucose-6-phosphate as the first step of the glycolytic pathway. In normal adult tissues, HK1 is ubiquitously expressed but is particularly prominent in the brain and kidney. HK2 is generally expressed at low levels within adipose tissue and skeletal tissue and negligently in normal brain. In this study, we wished to determine the ontogeny of these isoforms within the developing brain and determine how their expression relates to the extent of aerobic glycolysis in Glioblastoma Multiforme (GBM), a highly aggressive malignant brain tumor. Mining of existing published microarray data found strong expression of HK1 in 1, 2, 4 cell stage but a switch to stronger expression of HK2 in the blastocyst stage, previously reported to rely heavily on aerobic glycolysis. We performed HK2 immunohistochemistry in mice from gestational age E12, E16, post natal 1 month and 2 months. Our results demonstrated an age and cell specific HK1 and HK2 immunoreactivity in embryonic brain tissue with decreased expression of HK2 post-natally. Subsequently, we investigated the expression of HK1 and HK2 isoforms in a panel of GBM cell lines with varying levels of dependence on aerobic glycolysis, as measured by lactate levels and O2 consumption. HK2 but not HK1 expression was higher in GBM cells that had low O2 consumption and high extracellular lactate levels, supporting an association of HK2 with aerobic glycolysis. As HK2 expression is nearly silent in adult brain but expressed in fetal tissue and GBM cells, we hypothesized that DNA methylation/demethylation events may be playing important in its regulation. Adult normal human brain and GBM cell lines that had no HK2 expression were found to be methylated at CpG islands within intron 1 by bisulfite sequencing in contrast to fetal tissue and HK2-expressing GBM cells. The degree of methylation correlated with transcript expression in GBM cell lines. Treatment of U343 and A172 cell lines with 5-azacytidine restored HK2 transcript expression, supporting that HK2 maybe epigenetically regulated. Overall, our results demonstrate that the expression of the HK2 isoform, in contrast to HK1, may be particularly important in tissues relying on aerobic glycolysis for proliferation including embryonic tissue and GBMs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 40.

Pharmacologic inhibition of cyclin-dependent kinases 4 and 6 arrests the growth of glioblastoma multiforme intracranial xenografts.

Activation of cyclin-dependent kinases 4 and 6 (cdk4/6) occurs in the majority of glioblastoma multiforme (GBM) tumors, and represents a promising molecular target for the development of small molecule inhibitors. In the current study, we investigated the molecular determinants and in vivo response of diverse GBM cell lines and xenografts to PD-0332991, a cdk4/6-specific inhibitor. In vitro testing of PD-0332991 against a panel of GBM cell lines revealed a potent G(1) cell cycle arrest and induction of senescence in each of 16 retinoblastoma protein (Rb)-proficient cell lines regardless of other genetic lesions, whereas 5 cell lines with homozygous inactivation of Rb were completely resistant to treatment. Short hairpin RNA depletion of Rb expression conferred resistance of GBM cells to PD-0332991, further demonstrating a requirement of Rb for sensitivity to cdk4/6 inhibition. PD-0332991 was found to efficiently cross the blood-brain barrier and proved highly effective in suppressing the growth of intracranial GBM xenograft tumors, including those that had recurred after initial therapy with temozolomide. Remarkably, no mice receiving PD-0332991 died as a result of disease progression while on therapy. Additionally, the combination of PD-0332991 and radiation therapy resulted in significantly increased survival benefit compared with either therapy alone. In total, our results support clinical trial evaluation of PD-0332991 against newly diagnosed as well as recurrent GBM, and indicate that Rb status is the primary determinant of potential benefit from this therapy.

Survivin enhances radiation resistance in primary human glioblastoma cells via caspase-independent mechanisms

The observed radioresistance of human glioblastoma multiforme (GBM) poses a major challenge, which, if overcome, may lead to significant advances in the management of this patient population. There is accumulating evidence from correlative studies that Survivin expression is associated with increased malignant potential of human gliomas. The purpose of this study was to investigate whether Survivin plays a direct role in mediating radiation resistance in primary human glioma cell lines, and, if so, investigating the underlying mechanisms. Our panel of GBM cell lines included two that were relatively radiation resistant (GM20 and GM21) and two that were more radiation sensitive (GM22 and GM23), which demonstrated differential levels of Survivin expression between the two groups. Through the use of adenoviral vectors containing either dominant-negative (pAd-S(T34A)) or wild-type Suvrivin (pAd-S(WT)), we were able to inactivate or overexpress Survivin, respectively. Our findings suggest that Survivin plays a critical role in mediating radiation resistance in primary GBM cells, in part through suppression of apoptotic cell death via a caspase-independent manner. We have identified novel mechanisms by which Survivin may enhance tumor cell survival upon radiation exposure such as regulation of double-strand DNA break repair and tumor cell metabolism, which were most evident in the radiation-resistant cell lines. These differences in Survivin function both in radiation-resistant vs radiation-sensitive cell lines and in the presence vs absence of radiation exposure warrant further investigation and highlight potentially important mechanisms of radiation resistance in these tumors.