GBM Cell Growth Research Articles

Glioma-derived exosome Lncrna Agap2-As1 promotes glioma proliferation and metastasis by mediating Tgf-β1 secretion of myeloid-derived suppressor cells

BackgroundGlioma (GBM) is the most prevalent malignancy worldwide with high morbidity and mortality. Exosome-mediated transfer of long noncoding RNA (lncRNA) has been reported to be associated with human cancers, containing GBM. Meanwhile, myeloid-derived suppressor cells (MDSCs) play a vital role in mediating the immunosuppressive environments in GBM. ObjectivesThis study is designed to explore the role and mechanism of exosomal (Exo) lncRNA AGAP2-AS1 on the MDSC pathway in GBM. MethodsAGAP2-AS1, microRNA-486-3p (miR-486-3p), and Transforming growth factor beta-1 (TGF-β1) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation, apoptosis, migration, and invasion were detected by 5-ethynyl-2′-deoxyuridine (EdU), flow cytometry, and Transwell assays. E-cadherin, Vimentin, CD9, CD81, and TGF-β1 protein levels were examined using Western blot. Exosomes were detected by a transmission electron microscope (TEM). Binding between miR-486-3p and AGAP2-AS1 or TGF-β1 was predicted by LncBase or TargetScan and then verified using a dual-luciferase reporter assay. ResultsAGAP2-AS1 was highly expressed in GBM tissues and cells. Functionally, AGAP2-AS1 absence or TGF-β1 knockdown repressed tumor cell growth and metastasis. Furthermore, Exo-AGAP2-AS1 from GBM cells regulated TGF-β1 expression via sponging miR-486-3p in MDSCs. Exo-AGAP2-AS1 upregulation facilitated GBM cell growth and metastasis via the MDSC pathway. ConclusionExo-AGAP2-AS1 boosted GBM cell development partly by regulating the MDSC pathway, hinting at a promising therapeutic target for GBM treatment.

Long noncoding RNA UNC5B-AS1 suppresses cell proliferation by sponging miR-24-3p in glioblastoma multiforme

BackgroundGlioblastoma multiforme (GBM) is the most common primary CNS tumor, characterized by high mortality and heterogeneity. However, the related lncRNA signatures and their target microRNA (miRNA) for GBM are still mostly unknown. Therefore, it is critical that we discover lncRNA markers in GBM and their biological activities.Materials and methodsGBM-related RNA-seq data were obtained from the Cancer Genome Atlas (TCGA) database. The “edger” R package was used for differently expressed lncRNAs (DELs) identification. Then, we forecasted prospective miRNAs that might bind to lncRNAs by Cytoscape software. Survival analysis of those miRNAs was examined by the starBase database, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the miRNAs’ target genes was conducted by the Gene Set Enrichment Analysis (GSEA) database and R software. Moreover, the proliferative ability of unc-5 netrin receptor B antisense RNA 1 (UNC5B-AS1) cells was evaluated by Cell Counting Kit-8 (CCK-8) analysis. Mechanistically, the regulatory interaction between UNC5B-AS1 and miRNA in GBM biological processes was studied using CCK-8 analysis.ResultsOur results indicated that overexpression of UNC5B-AS1 has been shown to suppress GBM cell growth. Mechanistically, miR-24-3p in GBM was able to alleviate the anti-oncogenic effects of UNC5B-AS1 on cell proliferation.ConclusionThe discovery of the novel UNC5B-AS1-miR-24-3p network suggests possible lncRNA and miRNA roles in the development of GBM, which may have significant ramifications for the analysis of clinical prognosis and the development of GBM medications.

An End-to-end In-Silico and In-Vitro Drug Repurposing Pipeline for Glioblastoma.

Our study aims to address the challenges in drug development for glioblastoma, a highly aggressive brain cancer with poor prognosis. We propose a computational framework that utilizes machine learning-based propensity score matching to estimate counterfactual treatment effects and predict synergistic effects of drug combinations. Through our in-silico analysis, we identified promising drug candidates and drug combinations that warrant further investigation. To validate these computational findings, we conducted in-vitro experiments on two GBM cell lines, U87 and T98G. The experimental results demonstrated that some of the identified drugs and drug combinations indeed exhibit strong suppressive effects on GBM cell growth. Our end-to-end pipeline showcases the feasibility of integrating computational models with biological experiments to expedite drug repurposing and discovery efforts. By bridging the gap between in-silico analysis and in-vitro validation, we demonstrate the potential of this approach to accelerate the development of novel and effective treatments for glioblastoma.

TMIC-44. TARGETING BRACHYURY REPROGRAMS ENDOTHELIAL TRANSDIFFERENTIATION OF GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor with a median survival of only 15 months and a 5-year survival of less than 5% despite the best available treatments. Tumor recurrence/progression and therapy resistance are major obstacles for GBM treatment. GBM is characterized by intensive vascular proliferation that is associated with tumor cell growth, invasion, resistance to chemo/radiotherapy, and decreased disease-free survival. Increasing data have shown the existence of cells with endothelial characteristics called tumor derived endothelial cells, which come from the transdifferentiation of GBM cells and, more specifically, from GBM stem cells (GSCs). However, the molecular mechanisms underlying this process remain largely unknown. GSCs are tightly regulated by distinct transcriptional programs that are driven by nuclear transcription factors. Brachyury is a transcription factor expressed in normal, undifferentiated embryonic notochord in the axial skeleton and plays an important role in stem cell development and differentiation during normal embryonic development. Here, we show that brachyury is highly expressed in patient-derived GBM cells. Functional studies demonstrate that brachyury is regulated by fibroblast growth factor receptor 1 (FGFR1)/mitogen-activated protein kinase (MAPK). Our studies further disclose that FGFR1/MAPK-directed brachyury activation promotes GSC survival and endothelial formation via vascular endothelial growth factor receptor 2 (VEGFR2), whereas pharmacological inhibition of FGFR1 and MAPK or shRNA-mediated downregulation of brachyury decreases GSC transdifferentiation into endothelial cells and suppresses GBM cell growth and stemness via VEGFR2. Our findings highlight the importance of FGFR1/MAPK-regulated brachyury activation in GSC transdifferentiation into endothelial cells via VEGFR2, and targeting the FGFR1-MAPK-brachyury-VEGFR2 signal pathway may represent a novel therapeutic strategy by reprogramming GSCs driving vascular transdifferentiation and tumor progression.

P10.08.A Lipogenesis inhibition by fatostatin shows effectiveness in glioblastoma models highly expressing fatty acid synthase (FASN)

Abstract Background Glioblastoma (GBM) is the most common and most aggressive primary brain tumour in adults. Despite intensive treatment, the medium overall survival of GBM patients remains below 20 months after diagnosis. Therefore, new tumour specific targets allowing successful therapeutic treatment are needed to distinctly enhance GBM patient overall survival. Reprogramming of lipid metabolism leading to highly upregulated anabolic pathways, like phosphatidylinositol-3-kinase (PI3K)/AKT pathway and subsequently lipogenesis, is a hallmark of cancer cells including glioblastomas to meet increased energy demands for proliferation and cell growth. Accordingly, lipogenesis inhibition might be a promising strategy to impede GBM cell growth. Therefore, it was aim of our study to analyse the effect of the lipogenesis inhibitor fatostatin on downstream targets e.g. fatty acid synthase (FASN) and its impact on cell growth in GBM cells in vitro. Material and Methods In a cohort of 52 GBM-derived cell lines, FASN mRNA was investigated by qRT-PCR. The impact of fatostatin on cell viability (IC50) was analysed in selected cell lines using cell viability assays (Cell Titer Glo®). To elucidate whether fatostatin exposure affects cell migration, wound healing assays were performed. The effect of fatostatin on cell cycle and apoptosis was analysed by flow cytometry. Additionally, expression of FASN and level of phosphorylated AKT, the active form of AKT serine/threonine kinase, were examined before and after fatostatin treatment with qRT-PCR (mRNA) and Western blot (protein), respectively. Results FASN mRNA was found to be expressed at varying levels in our GBM-derived cell line cohort (n=52). Compared to normal astrocytes, FASN mRNA expression was significantly upregulated in GBM cells (p=0.0127). Furthermore, high FASN mRNA expression was significantly associated with higher sensitivity against fatostatin (p=0.0352). Regarding corresponding recurrences, the response to the inhibitor was shown to be persistent. Accordingly, the effectiveness of fatostatin treatment resulted in substantial downregulation of AKT phosphorylation and consequently in the induction of apoptosis and G2/M arrest of the cell cycle. In addition, a significant inhibition of the migration potential by fatostatin treatment was observed. Conclusion In summary, FASN expression represents a promising biomarker and therapy target within the lipid metabolism indicated by a significant sensitivity to fatostatin in FASN overexpressing GBM cell lines. Consequently, lipogenesis inhibition by fatostatin might be a promising novel therapeutic option in glioblastoma.

Abstract 5647: PTEN loss is associated with elevation of cell cycle signature in human glioblastoma transcriptome

Abstract Glioblastoma is the most lethal type of brain cancer with universally poor prognosis. Despite advanced treatment, the cancer recurs frequently. Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene whose mutations have been found to be one of the most frequent events in GBM. PTEN acts as a negative regulator of the phosphoinositide 3-kinase (PI3K) pathway. GBM transcriptome data from The Cancer Genome Atlas (TCGA) revealed a positive enrichment of cell cycle and PI3K pathway. Moreover, weighted gene co-expression network analysis (WGCNA) on TCGA GBMLGG cohort revealed a cell cycle-related module (‘cell-cycle’ module), which was shown to be positively correlated with PTEN mutation status. This ‘cell-cycle’ module also showed a positive correlation with glioma aggressiveness.We then utilized a nestin-cre;Pten-fl/fl mouse model to investigate the consequences of PTEN loss that may be involved in GBM. Based on our preliminary data, transcriptomic profiling of primary astrocytes derived from Pten knockout mice revealed an overlapping enrichment of cell cycle related pathways with human GBM. Four cell cycle genes, RCC1, RCC2, NEK2, and CHK2, were validated to be significantly upregulated in Pten-null astrocytes by qRT-PCR. Regulator of chromatin condensation 1 (RCC1) and Regulator of chromatin condensation 2 (RCC2) both belong to the RCC1 family. Accumulating evidence demonstrated that both RCC1 and RCC2 are involved in tumorigenesis of multiple cancers and cell cycle progression. By siRNA inhibition, we demonstrated that silencing of either RCC1 or RCC2 in glioblastoma cell lines suppresses GBM cell growth. Moreover, we found that RCC2 is essential for both G1-S and G2-M transition. Additionally, RCC2 silencing and a PI3K inhibitor LY294002 inhibit glioblastoma cell viability in a synergistic manner. In summary, PTEN deficiency leads to the alteration of cell cycle progression, which may promote tumorigenesis of glioblastoma. RCC1 and RCC2 have been demonstrated to be potential targets regulated by PTEN and affects cell viability. RCC2 was shown to be a key regulator of cell cycle progression. To further explore the RCC1 and RCC2 roles, we will find the potential downstream target of RCC1 and RCC2 and its potential therapeutic effect in vivo. Funding: This work is supported by a General Research Fund grant (#14113519) from the University Grants Committee of Hong Kong. Citation Format: Tian Liu, Penelope Mei-Yu Or, Chi-Wai Wong, Stanley Kwok-Kuen Cheung, Yubing Wang, Yiwei Wang, Wayne Lut-Heng Ho, Andrew M. Chan. PTEN loss is associated with elevation of cell cycle signature in human glioblastoma transcriptome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5647.

HOXA-AS2 enhances GBM cell malignancy by suppressing miR-2116-3p thereby upregulating SERPINA3

BackgroundGlioblastoma (GBM) is malignant, demanding more attention to the improvement of the diagnosis and therapy. LncRNAs have been implicated in the malignancy of GBM cells.MethodsHOXA-AS2, miR-2116-3p and SERPINA3 expression levels in GBM tissues and cell lines were detected by qRT-PCR. Western blotting was performed to detect the protein levels of Bax and Bcl-2. Dual-luciferase reporter assay was for detection of relationship among these factors, together with RIP and RNA pull-down. CCK-8, EdU, wound healing and transwell assays were for detection of the role of HOXA-AS2, miR-2116-3p and SERPINA3 in cell viability, proliferation, migration and invasion in GBM, respectively.ResultsHOXA-AS2 and SERPINA3 showed higher level in GBM tissues and cell lines. Low level of HOXA-AS2 attenuated GBM cell growth in vitro. Moreover, the anti-tumor impact of silenced HOXA-AS2 was restored by miR-2116-3p inhibitor, but its tumor-promotional effect could be reversed by silenced SERPINA3.ConclusionHOXA-AS2 enhanced GBM cell malignancy through sponging miR-2116-3p and releasing SERPINA3, which might shed light on the diagnosis and therapy for GBM in the future.

Pharmacological inhibition of serine synthesis enhances temozolomide efficacy by decreasing O6-methylguanine DNA methyltransferase (MGMT) expression and reactive oxygen species (ROS)-mediated DNA damage in glioblastoma

AbstractGlioblastoma (GBM) is the most malignant primary tumor in the central nervous system of adults. Temozolomide (TMZ), an alkylating agent, is the first-line chemotherapeutic agent for GBM patients. However, its efficacy is often limited by innate or acquired chemoresistance. Cancer cells can rewire their metabolic programming to support rapid growth and sustain cell survival against chemotherapies. An example is the de novo serine synthesis pathway (SSP), one of the main branches from glycolysis that is highly activated in multiple cancers in promoting cancer progression and inducing chemotherapy resistance. However, the roles of SSP in TMZ therapy for GBM patients remain unexplored. In this study, we employed NCT503, a highly selective inhibitor of phosphoglycerate dehydrogenase (PHGDH, the first rate-limiting enzyme of SSP), to study whether inhibition of SSP may enhance TMZ efficacy in MGMT-positive GBMs. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flowcytometry and colony formation assays demonstrated that NCT503 worked synergistically with TMZ in suppressing GBM cell growth and inducing apoptosis in T98G and U118 cells in vitro. U118 and patient-derived GBM subcutaneous xenograft models showed that combined NCT503 and TMZ treatment inhibited GBM growth and promoted apoptosis more significantly than would each treatment alone in vivo. Mechanistically, we found that NCT503 treatment decreased MGMT expression possibly by modulating the Wnt/β-catenin pathway. Moreover, intracellular levels of reactive oxygen species were elevated especially when NCT503 and TMZ treatments were combined, and the synergistic effects could be partially negated by NAC, a classic scavenger of reactive oxygen species. Taken together, these results suggest that NCT503 may be a promising agent for augmenting TMZ efficacy in the treatment of GBM, especially in TMZ-resistant GBMs with high expression of MGMT.Pharmacological inhibition of the serine synthesis pathway with a highly selective inhibitor NCT503 synergistically works with temozolomide in inhibiting O6-methylguanine DNA methyltransferase (MGMT)-positive glioblastoma cell growth and inducing apoptosis both in vitro and in vivo. Mechanistically, NCT503 treatment reduces MGMT expression possibly via Wnt/βcatenin pathway. Reactive oxygen species-mediated DNA damage is at least partially involved. Combinational administration of NCT503 and TMZ may represent a novel and promising treatment strategy to enhance TMZ efficacy in patients with MGMT-high glioblastoma.

Growth suppression of glioblastoma multiforme cell line response to the combination of Temozolomide and Nimotuzumab

Background: Researches towards Glioblastoma multiforme therapy (GBM) has been performed, especially in finding the tumor’s genetic cellular expression. The overexpression of EGFR, frequently found in human malignancies, is known to significantly impact cancer cells hallmark traits, such as increased cell survival, proliferation and invasion. This research is performed to figure out the synergistic effect of temozolomide, the main chemotherapy regimen used, and the monoclonal antibody anti-EGFR nimotuzumab in inhibiting the growth of GBM cells in vitro.Methods: We performed cell cultures using GBM U87MG cells and administered nimotuzumab 1000µg/ml and temozolomide 20 µg/ml in different sequences and timings. Inhibition of GBM cell growth is evaluated by measuring Ki-67 and ?H2AX levels using flowcytometry and tested with one-way ANOVA. Data were analyzed using SPSS version 21 for Windows.Results: Levels of Ki-67 were in the control group (23.17±1.72), nimotuzumab monotherapy (15.43±1.70), temozolomide monotherapy (14.80±1.37), simultaneous therapy (10.73±1.19), nimotuzumab 24 hours before temozolomide (10.57±1.05), and nimotuzumab 48 hours before temozolomide (14.47±1.37). The level of ?H2AX is measured in the control group (11.90±1.25), nimotuzumab monotherapy (29.33±1.91), temozolomide monotherapy (28.13±1.58), simultaneous therapy (41.53±3.51), nimotuzumab 24 hours before temozolomide (39.56±2.06), and nimotuzumab 48 hours before temozolomide (35.93±3.56).Conclusion: This research shows that administration of nimotuzumab simultaneously with temozolomide and nimotuzumab 24 hours before temozolomide effectively inhibits the growth of U87MG GBM cells. Inhibition of EGFR expression before temozolomide administration is able to increase DNA damage and inhibit the proliferation of glioma cells.

Implication of Lactucopicrin in Autophagy, Cell Cycle Arrest and Oxidative Stress to Inhibit U87Mg Glioblastoma Cell Growth

In this study, we propose lactucopicrin (LCTP), a natural sesquiterpene lactone from Lactucavirosa, as a molecule able to control the growth of glioblastoma continuous cell line U87Mg. The IC50 of U87Mg against LCTP revealed a strong cytotoxic effect. Daily administration of LCTP showed a dose and time-dependent reduction of GBM cell growth and viability, also confirmed by inhibition of clonogenic potential and mobility of U87Mg cells. LCTP activated autophagy in U87Mg cells and decreased the phosphorylation of proliferative signals pAKT and pERK. LCTP also induced the cell cycle arrest in G2/M phase, confirmed by decrease of CDK2 protein and increase of p53 and p21. LCTP stimulated apoptosis as evidenced by reduction of procaspase 6 and the increase of the cleaved/full-length PARP ratio. The pre-treatment of U87Mg cells with ROS scavenger N-acetylcysteine (NAC), which reversed its cytotoxic effect, showed the involvement of LCTP in oxidative stress. Finally, LCTP strongly enhanced the sensitivity of U87Mg cells to canonical therapy Temozolomide (TMZ) and synergized with this drug. Altogether, the growth inhibition of U87Mg GBM cells induced by LCTP is the result of several synergic mechanisms, which makes LCTP a promising adjuvant therapy for this complex pathology.

Galangin (GLN) Suppresses Proliferation, Migration, and Invasion of Human Glioblastoma Cells by Targeting Skp2-Induced Epithelial-Mesenchymal Transition (EMT).

BackgroundGalangin (GLN), a pure natural flavonoid compound found in plants, has been shown to exert anti-cancer effects against multiple cancer types, including glioma. However, its underlying molecular mechanism remains unclear. Epithelial-to-mesenchymal transition (EMT) performs an important function in the genesis and development of cancer. Skp2, a pivotal component of SCFSkp2 E3 ubiquitin ligase, has been shown to function as an oncogene in GBM invasion that contributes to the EMT process. Thus, we explored whether GLN inhibited Skp2-mediated EMT and the mechanism underlying the Skp2 degradation pathway.MethodsCCK-8 assay, wound healing assay and transwell assay were used to examine cell proliferation, migration, and invasion after treatment with or without GLN. RT-PCR and Western blotting analysis were performed to evaluate mRNA and protein expression, respectively. Co-immunoprecipitation was conducted to detect ubiquitinated Skp2 levels in vitro and in vivo after GLN treatment. Bioluminescence imaging was performed to examine the intracranial tumor size of U87 xenograft mice. Microscale thermophoresis (MST) experiment was used to detect interactions between Skp2 and GLN.ResultsGLN suppressed GBM cell growth, migration, and invasion, and also downregulated the expression of Skp2 and mesenchymal markers (Zeb1, N-cadherin, snail, vimentin) in vitro. Moreover, the overexpression of Skp2 in GBM cells decreased the effect of GLN on EMT. Furthermore, we demonstrated that GLN degraded skp2 protein through the ubiquitination proteasome pathway and directly interacted with skp2 protein, as shown through the MST assay.ConclusionThis study is the first to identify Skp2 as a novel target of GLN for the treatment of GBM and report of Skp2 protein degradation in a ubiquitination proteasome pathway. Results from our study indicated the potential of GLN for the treatment of GBM through ubiquitin-mediated degradation of Skp2.

Therapeutic targeting of CD38 enhances temozolomide activity in glioblastoma cells and modulates their tumor microenvironment by eliciting an enhanced CD8+ T cell response

Abstract Glioblastoma (GBM) is an aggressive primary malignant brain tumor with poor prognosis. Surgical resection followed by chemoradiotherapy (temozolomide [TMZ] + radiation) results in a median survival of only 12-15 months. Recently, immunotherapy with targeted antibodies, therapeutic vaccination and adoptive cell therapy offers a promising option for treating GBM. Herein we observed that CD38, a catalytic receptor and regulator of NAD homeostasis, is expressed on GBM cells and their microenvironment in primary patient-derived GBM tissues. Blacher et al have shown that blocking CD38 enzymatic functions results in reduced GBM cell growth. This evidence prompted us to investigate the antitumor and immune engaging effects of a clinical grade anti CD38 therapeutic monoclonal antibody (Daratumumab/DARA) in preclinical models of GBM. We demonstrated that DARA significantly enhanced the direct apoptotic effect of TMZ and induced ADCC/CDC of GBM cells, irrespective of the presence of microglia. In an in vitro 3D simulated GBM + T cell coculture system, DARA induced activation (CD69/GITR upregulation/decreased PD1) and enhanced proliferation of CD8+ T cells/cTL activity against GBM cells. DARA also resulted in increased IFNγ and decreased IL-10; suggesting an improved antitumor cytokine milieu and ensued in downregulated inflammatory cytokine associated JAK2/STAT3 signaling pathway. Finally, we tested TMZ+DARA in a human GBM xenograft in vivo model and noted significantly prolonged survival of mice [TMZ+DARA vs. vehicle: 98.13±1.88 vs. 63.63±3.77 days, p<0.001]. Altogether, these observations led us to investigate the clinical activity of DARA+ TMZ in GBM patients in a phase I/II clinical trial.

RETRACTED ARTICLE: Dissecting the role of novel EZH2 inhibitors in primary glioblastoma cell cultures: effects on proliferation, epithelial-mesenchymal transition, migration, and on the pro-inflammatory phenotype

BackgroundGlioblastoma (GBM) is the most lethal and aggressive malignant primary brain tumor in adults. After surgical resection of the tumor, the patient typically should be subjected to chemotherapy (temozolomide, TMZ) and concomitant radiotherapy. Since the TMZ treatment does not lead to complete remission and often develops resistance, the identification of efficacious therapeutics is strongly to pursue. Among the epigenetic players, the H3K27 methyltransferase (MT) EZH2 (enhancer of zeste homologue 2) has been found overexpressed or mutated in several human cancers including gliomas, and its overexpression is associated with poor outcome in GBM. Two EZH2 inhibitors (EZH2i), UNC1999 and GSK343, suppressed GBM growth in vitro and in vivo indicating that EZH2i can be potential drugs against GBM.ResultsTwo new EZH2i, MC4040 and MC4041, were designed, prepared, and tested by us to determine their effects in primary GBM cell cultures. MC4040 and MC4041 displayed single-digit micromolar inhibition of EZH2, 10-fold less potency against EZH1, and no activity towards other MTs. In primary GBM cells as well as in U-87 GBM cells, the two compounds reduced H3K27me3 levels, and dose- and time-dependently impaired GBM cell viability without inducing apoptosis and arresting the cell cycle in the G0/G1 phase, with increased p21 and p27 levels. In combination with TMZ, MC4040 and MC4041 displayed stronger, but not additive, effects on cell viability. The potent clinical candidate as EZH2i tazemetostat, alone or in combination with TMZ, exhibited a similar potency of inhibition of GBM cell growth when compared to MC4040 and MC4041. At the molecular level, MC4040 and MC4041 reduced the VEGFR1/VEGF expression, reversed the epithelial-mesenchymal transition (EMT), and hampered cell migration and invasion attenuating the cancer malignant phenotype. Treatment of GBM cells with MC4040 and MC4041 also impaired the GBM pro-inflammatory phenotype, with a significant decrease of TGF-β, TNF-α, and IL-6, joined to an increase of the anti-inflammatory cytokine IL-10.ConclusionsThe two novel EZH2i MC4040 and MC4041 impaired primary GBM cell viability, showing even stronger effects in combination with TMZ. They also weakened the aggressive malignant phenotype by reducing angiogenesis, EMT, cell migration/invasion and inflammation, thus they may be considered potential candidates against GBM also for combination therapies.

EXTH-44. INHIBITION OF MerTK ACTIVATES GLIOBLASTOMA-ASSOCIATED MACROPHAGES AND INDUCES TUMOR CELL DEATH IN GLIOMA MICROENVIRONMENT

Abstract BACKGROUND Glioblastoma-associated macrophages and microglia (GAMs) are the predominant immune cells in the tumor microenvironment. Activation of MerTK, a receptor tyrosine kinase, triggers efferocytosis and polarizes GAMs to an immunosuppressive phenotype, promoting glioma growth. Our previous findings showed that UNC2371, a small-molecule inhibitor of MerTK, induced a less immunosuppressive phenotype of GAMs. Here, we investigate the role of MerTK inhibition on glioblastoma cells in the tumor microenvironment in vitro and in vivo. METHODS Cytotoxicity of UNC2371 in glioblastoma cells was determined by cell viability and colony formation assays. The protein expression of MerTK, AKT, and Erk were quantified by Western blotting in UNC2371-treated glioblastoma cells. A syngeneic GL261 mouse orthotopic glioblastoma model was used to evaluate the survival benefit of UNC2371 treatment. Fluorescent multiplex immunohistochemistry (IHC) was used to evaluate the expression of CD206, an anti-inflammatory marker on GAMs in murine brain tumor tissues. RESULTS UNC2371 inhibited GBM cell growth with an EC50 < 100 nM in both human U251 and mouse GL261 glioma cells, but not in GAMs. UNC2371-induced cell death and decreased cell proliferation were demonstrated by colony formation assays. UNC2371 decreased protein expression of phosphorylated MerTK, AKT, and Erk, which are essential for cell survival signaling, in U251 and GL261 cells. Furthermore, UNC2371 treatment prolonged survival in the mouse orthotopic GL261 glioblastoma model, suggesting that UNC2371 induces glioma cell death. A decreased of CD206+ GAMs was found in mice glioma tissues by fluorescent multiplex IHC, consistent with our previous findings in the in vitro cell-based assays. These data suggest that in addition to alleviate immunosuppression in the glioma microenvironment, UNC2371 directly inhibits GBM cell growth in vitro and in vivo. CONCLUSION Our findings suggest that UNC2371 has a therapeutic benefit via promoting GAM polarization towards proinflammatory status in the glioblastoma microenvironment and unexpectedly, inducing tumor cell death.

Induction Of XLF And 53BP1 Expression Is Associated With Temozolomide Resistance In Glioblastoma Cells.

IntroductionGlioblastoma (GBM) is the most commonly diagnosed primary brain tumor in adults. The 14.6 months median survival period of GBM patients is still palliative due to resistance to the first-line chemotherapeutic agent temozolomide (TMZ).MethodsThe cell growth inhibition effect was assessed using the SRB assay. The mRNA expression levels were examined using RT-qPCR. The protein expression levels were determined using Western blot analysis. The DNA repair by non-homologous end-joining (NHEJ) was quantified using NHEJ reporter assay. The TMZ-induced apoptosis was detected by the Caspase 3/7 activity kit. The DNA binding activity in cells was determined using chromatin fractionation assay. The 53BP1 inhibitor was identified using virtual screening followed by Western blot analysis. The synergy between TMZ and 53BP1 inhibitor in vivo was analyzed using a xenograft mouse model.ResultsWe found that non-homologous end joining (NHEJ), which is one of the major DNA double-strand break repair pathways, participates in acquired TMZ-resistance in GBM. Canonical NHEJ key factors, XLF and 53BP1, are upregulated in TMZ-resistant GBM cells. Depletion of XLF or 53BP1 in TMZ-resistant cells significantly improve the potency of TMZ against GBM cell growth. Importantly, we identified a small molecule HSU2018 to inhibit 53BP1 at nanomolar concentration. The combination of HSU2018 and TMZ generates excellent synergy for cell growth inhibition in TMZ-resistant GBM cells and xenograft.ConclusionOur data suggest that NHEJ is a novel mechanism contributing to TMZ-resistance, and its key factors may serve as potential targets for improving chemotherapy in TMZ-resistant GBM.

Abstract 2608: EGF signaling activates a TAZ-driven oncogenic program in glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most aggressive and lethal primary brain tumor in humans. Hyperactivation of the signaling pathway driven by the epidermal growth factor (EGF) and its receptor (EGFR) are commonly occurring in GBM. The oncogenic transcriptional program driven by EGF signaling is still incompletely understood in GBM. Here, we found that EGF induces the upregulation and nuclear translocation of TAZ, an oncogenic transcriptional co-activator. TAZ and its paralog gene YAP are commonly known as downstream mediators of the Hippo signaling pathway. TAZ has been found as a driver of mesenchymal transition in GBM cells and promotes GBM radioresistance. Our results support that EGF induces TAZ transcription and the activation of TAZ gene targets, through the signaling axis including EGFR, STAT3 and ERK. EGFRvIII is the most frequently occurring EGFR mutation in GBM causing constitutively active EGFR. We found that EGFRvIII causes EGF-independent activation of TAZ and its downstream oncogenic targets. Next, we used next-generation sequencing to establish a genome-wide map of TAZ gene targets in GBM cells after EGF stimulation. The results suggest that EGF signaling activates a TAZ-driven oncogenic program including autocrine and paracrine factors that promote GBM growth, angiogenesis, stemness and immunoescape. Moreover, we found that TAZ targeting by shRNAs or the FDA-approved drug Verteporfin effectively inhibits the TAZ-driven oncogenic program activated by EGF stimulation or the EGFRvIII mutant. TAZ targeting also inhibits GBM cell growth and xenograft formation. In contrast, enforced TAZ induces the TAZ-driven oncogenic program and promotes GBM cell growth and xenograft formation. In conclusion, our results reveal a novel TAZ-driven oncogenic program that is activated by EGF signaling in GBM. Our discoveries strongly support TAZ as a therapeutic target for GBM and likely other human cancer with hyperactivation of EGF signaling. Our preclinical studies also provide a clinically translatable strategy for GBM therapy based on using the FDA-approved drug Verteporfin. Citation Format: Minling Gao, Yi Fu, Laterra John, Mingyao Ying. EGF signaling activates a TAZ-driven oncogenic program 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 2608.

PTPN12/PTP-PEST Regulates Phosphorylation-Dependent Ubiquitination and Stability of Focal Adhesion Substrates in Invasive Glioblastoma Cells.

Glioblastoma (GBM) is an invasive brain cancer with tumor cells that disperse from the primary mass, escaping surgical resection and invariably giving rise to lethal recurrent lesions. Here we report that PTP-PEST, a cytoplasmic protein tyrosine phosphatase, controls GBM cell invasion by physically bridging the focal adhesion protein Crk-associated substrate (Cas) to valosin-containing protein (Vcp), an ATP-dependent protein segregase that selectively extracts ubiquitinated proteins from multiprotein complexes and targets them for degradation via the ubiquitin proteasome system. Both Cas and Vcp are substrates for PTP-PEST, with the phosphorylation status of tyrosine 805 (Y805) in Vcp impacting affinity for Cas in focal adhesions and controlling ubiquitination levels and protein stability. Perturbing PTP-PEST-mediated phosphorylation of Cas and Vcp led to alterations in GBM cell-invasive growth in vitro and in preclinical mouse models. Collectively, these data reveal a novel regulatory mechanism involving PTP-PEST, Vcp, and Cas that dynamically balances phosphorylation-dependent ubiquitination of key focal proteins involved in GBM cell invasion.Significance: PTP-PEST balances GBM cell growth and invasion by interacting with the ATP-dependent ubiquitin segregase Vcp/p97 and regulating phosphorylation and stability of the focal adhesion protein p130Cas.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3809/F1.large.jpg Cancer Res; 78(14); 3809-22. ©2018 AACR.

Abstract 4831: DSF-Cu complex sensitizes patient-derived unmethylated MGMT expressing brain tumor cells to temozolomide

Abstract Introduction: O6 methylguanine DNA methyltransferase (MGMT) repairs the DNA damage caused by alkylating agents [temozolomide (TMZ)] leading to chemoresistence. Members of the aldehyde dehydrogenase (ALDH) family of isoenzymes serve as markers of cancer stem cells and contribute to chemotherapy resistance. Disulfiram (DSF) inhibits MGMT through ubiquitin mediated degradation; it is also a specific inhibitor of ALDH. We therefore, hypothesized that DSF through MGMT and ALDH inhibition decreases stemness and sensitizes GBM cells to TMZ. Methods: Normal astrocytes as well as established MGMT expressing GBM cell lines (LN18, T98G, U138, U118) and MGMT expressing (unmethylated) patient derived glioblastoma cells (ANII 7730 and ANII 7754) were treated with DSF/Cu +/- TMZ in various doses and combinations. We have also evaluated MGMT, ALDH levels and Sox2 expression. Cell viability, apoptotic assay and caspase 3/7 assay were used to evaluate inhibitory effect of various treatment combinations. Results: DSF and TMZ have a minimal effect on normal astrocyte growth. DSF alone inhibited MGMT in established MGMT expressing glioblastoma cells (5 to 10 µM). DSF alone was unable to inhibit MGMT in patient derived brain tumor cells (up to 20µM). Addition of copper (</=1µM) significantly inhibited MGMT (~ 90%) in patient derived GBM cells which correlated with significant growth inhibition even when DSF was used at low concentrations (</=1µM). This suggests that addition of copper to DSF may reduce any potential, dose dependent DSF related neurotoxicity. Temozolomide alone did not inhibit MGMT in established cell lines nor patient derived brain tumor cell lines (up to 1000µM). DSF+Cu further sensitized patient derived GBM cells to TMZ and significantly inhibited GBM cell growth (~ 95%) and did not inhibit normal astrocyte growth. Further DSF+Cu and DSF+Cu+TMZ combinations caused significant apoptotic cell death and significantly increased caspase 3/7 in patient derived unmethylated GBM cells. Similarly, combination of DSF+Cu and TMZ+DSF+Cu caused significant drop in ALDH activity in patient derived brain tumor cells compared to untreated controls and single agents. DSF induced ALDH and MGMT inhibition correlated with decrease in SOX2. MGMT expression canceled by CRISPR/Cas9 led to significant decrease in SOX2 expression in primary cell cultures. Conclusions: Our findings suggest that DSF/Cu treatment, a dual MGMT and ALDH inhibitor, suppresses stemness. Furthermore, our results confirm that combination of TMZ and DSF/Cu significantly inhibited glioblastoma cell growth compared to TMZ alone and untreated controls. Citation Format: George C. Bobustuc, Amin B. Kassam, Richard A. Rovin, Deborah Donohoe, Dmitry Bosenko, Santhi D. Konduri. DSF-Cu complex sensitizes patient-derived unmethylated MGMT expressing brain tumor cells to temozolomide [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 4831.

Reactive species balance via GTP cyclohydrolase I regulates glioblastoma growth and tumor initiating cell maintenance.

Depending on the level, differentiation state, and tumor stage, reactive nitrogen and oxygen species inhibit or increase cancer growth and tumor initiating cell maintenance. The rate-limiting enzyme in a pathway that can regulate reactive species production but has not been thoroughly investigated in glioblastoma (GBM; grade IV astrocytoma) is guanosine triphosphate (GTP) cyclohydrolase 1 (GCH1). We sought to define the role of GCH1 in the regulation of GBM growth and brain tumor initiating cell (BTIC) maintenance. We examined GCH1 mRNA and protein expression in patient-derived xenografts, clinical samples, and glioma gene expression datasets. GCH1 levels were modulated using lentiviral expression systems, and effects on cell growth, self-renewal, reactive species production, and survival in orthotopic patient-derived xenograft models were determined. GCH1 was expressed in GBMs with elevated but not exclusive RNA and protein levels in BTICs in comparison to non-BTICs. Overexpression of GCH1 in GBM cells increased cell growth in vitro and decreased survival in an intracranial GBM mouse model. In converse experiments, GCH1 knockdown with short hairpin RNA led to GBM cell growth inhibition and reduced self-renewal in association with decreased CD44 expression. GCH1 was critical for controlling reactive species balance, including suppressing reactive oxygen species production, which mediated GCH1 cell growth effects. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence, and worse survival. GCH1 expression in established GBMs is pro-tumorigenic, causing increased growth due, in part, to promotion of BTIC maintenance and suppression of reactive oxygen species.

Long Noncoding RNA NEAT1, Regulated by the EGFR Pathway, Contributes to Glioblastoma Progression Through the WNT/β-Catenin Pathway by Scaffolding EZH2

Purpose: Long noncoding RNAs have been implicated in gliomagenesis, but their mechanisms of action are mainly undocumented. Through public glioma mRNA expression data sets, we found that NEAT1 was a potential oncogene. We systematically analyzed the clinical significance and mechanism of NEAT1 in glioblastoma.Experimental Design: Initially, we evaluated whether NEAT1 expression levels could be regulated by EGFR pathway activity. We subsequently evaluated the effect of NEAT1 on the WNT/β-catenin pathway and its target binding gene. The animal model supported the experimental findings.Results: We found that NEAT1 levels were regulated by EGFR pathway activity, which was mediated by STAT3 and NFκB (p65) downstream of the EGFR pathway. Moreover, we found that NEAT1 was critical for glioma cell growth and invasion by increasing β-catenin nuclear transport and downregulating ICAT, GSK3B, and Axin2. Taken together, we found that NEAT1 could bind to EZH2 and mediate the trimethylation of H3K27 in their promoters. NEAT1 depletion also inhibited GBM cell growth and invasion in the intracranial animal model.Conclusions: The EGFR/NEAT1/EZH2/β-catenin axis serves as a critical effector of tumorigenesis and progression, suggesting new therapeutic directions in glioblastoma. Clin Cancer Res; 24(3); 684-95. ©2017 AACR.