Properties Of Glioma Stem Cells Research Articles

The Bioinformatics Identification of Potential Protein Glycosylation Genes Associated with a Glioma Stem Cell Signature

Glioma stem cells (GSCs) contribute to the pathogenesis of glioblastoma (GBM), which is the most malignant form of glioma. The implications and underlying mechanisms of protein glycosylation in GSC phenotypes and GBM malignancy are not fully understood. The implication of protein glycosylation and the corresponding candidate genes on the stem cell properties of GSCs and poor clinical outcomes in GBM were investigated, using datasets from the Gene Expression Omnibus, The Cancer Genome Atlas, and the Chinese Glioma Genome Atlas, accompanied by biological validation in vitro. N-linked glycosylation was significantly associated with GSC properties and the prognosis of GBM in the integrated bioinformatics analyses of clinical specimens. N-linked glycosylation was associated with the glioma grade, molecular biomarkers, and molecular subtypes. The expression levels of the asparagine-linked glycosylation (ALG) enzyme family, which is essential for the early steps in the biosynthesis of N-glycans, were prominently associated with GSC properties and poor survival in patients with GBM with high stem-cell properties. Finally, the oxidative phosphorylation pathway was primarily enriched in GSCs with a high expression of the ALG enzyme family. These findings suggest the role of N-linked glycosylation in the regulation of GSC phenotypes and GBM malignancy.

Emerging Role of Glioma Stem Cells in Mechanisms of Therapy Resistance.

Since their discovery at the beginning of this millennium, glioma stem cells (GSCs) have sparked extensive research and an energetic scientific debate about their contribution to glioblastoma (GBM) initiation, progression, relapse, and resistance. Different molecular subtypes of GBM coexist within the same tumor, and they display differential sensitivity to chemotherapy. GSCs contribute to tumor heterogeneity and recapitulate pathway alterations described for the three GBM subtypes found in patients. GSCs show a high degree of plasticity, allowing for interconversion between different molecular GBM subtypes, with distinct proliferative potential, and different degrees of self-renewal and differentiation. This high degree of plasticity permits adaptation to the environmental changes introduced by chemo- and radiation therapy. Evidence from mouse models indicates that GSCs repopulate brain tumors after therapeutic intervention, and due to GSC plasticity, they reconstitute heterogeneity in recurrent tumors. GSCs are also inherently resilient to standard-of-care therapy, and mechanisms of resistance include enhanced DNA damage repair, MGMT promoter demethylation, autophagy, impaired induction of apoptosis, metabolic adaptation, chemoresistance, and immune evasion. The remarkable oncogenic properties of GSCs have inspired considerable interest in better understanding GSC biology and functions, as they might represent attractive targets to advance the currently limited therapeutic options for GBM patients. This has raised expectations for the development of novel targeted therapeutic approaches, including targeting GSC plasticity, chimeric antigen receptor T (CAR T) cells, and oncolytic viruses. In this review, we focus on the role of GSCs as drivers of GBM and therapy resistance, and we discuss how insights into GSC biology and plasticity might advance GSC-directed curative approaches.

Overexpression of PRKCH promotes tumorigenesis in patients with glioma and influences glioma stem cell properties.

PRKCH is a member of the PKC family with the potential to regulate cell proliferation and differentiation. Glioma is the most common primary tumor of the central nervous system, with a high recurrence rate and poor prognosis. Recent studies have demonstrated that PRKCH can promote the proliferation of glioma cells. The purpose of this study was to investigate the promoting effect and possible mechanism of PRKCH on glioma development. Tumor tissue and paracancerous tissue were collected from 160 glioma patients treated at the General Hospital of Northern Theater Command. The expression level of PRKCH was detected by immunohistochemistry and immunoblotting. Univariate/multivariate analysis and log-rank analysis, as well as prognosis and survival analysis, were performed using SPSS26 software. The PRKCH overexpression model was constructed in vitro to study the effect of PRKCH expression on the characteristics of glioma stem cells. The expression of PRKCH in glioma tissues was higher than that in adjacent tissues. PRKCH expression level is an independent prognostic factor in glioma patients, promoting poor prognosis and shorter survival in glioma patients. Furthermore, overexpression of PRKCH in glioma stem cells significantly increased stem cell properties and enhanced cell viability. Downregulation of PRKCH significantly inhibited the progression of glioma stem cells. PRKCH promotes the development of gliomas and may be a therapeutic target for gliomas.

Global research trends and hotspots on glioma stem cells.

BackgroundGlioma stem cells (GSCs) are a sub-population of cancer stem cells with capacity of self-renewal and differentiation. Accumulated evidence has revealed that GSCs were shown to contribute to gliomagenesis, distant metastasis as well as the resistance to radiotherapy and chemotherapy. As a result, GSCs were regarded as a promising therapeutic target in human glioma. The purpose of our study is to identify current state and hotspots of GSCs research by analyzing scientific publications through bibliometric methods.MethodsAll relevant publications on GSCs during 2003-2021 were extracted from the Science Citation Index Expanded of Web of Science Core Collection (WoSCC), and related information was collected and analyzed using Microsoft Excel 2016, GraphPad Prism 8 and VOSviewer software.ResultsA total of 4990 papers were included. The United States accounted for the largest number of publications (1852), the second average citations per item (ACI) value (67.54) as well as the highest H-index (157). Cancer Research was the most influential journal in this field. The most contributive institution was League of European Research Universities. RICH JN was the author with the most publications (109) and the highest H-index (59). All studies were clustered into 3 groups: “glioma stem cell properties”, “cell biological properties” and “oncology therapy”. The keywords “identification”, “CD133” and “side population” appeared earlier with the smaller average appearing years (AAY), and the keywords”radiotherapy” and “chemotherapy” had the latest AAY. The analysis of top cited articles showed that “temozolomide”, “epithelial-mesenchymal transition”, and “immunotherapy” emerged as new focused issues.ConclusionThere has been a growing number of researches on GSCs. The United States has always been a leading player in this domain. In general, the research focus has gradually shifted from basic cellular biology to the solutions of clinical concerns. “Temozolomide resistance”, “epithelial-mesenchymal transition”, and “immunotherapy” should be given more attention in the future.

Off the Clock: the Non-canonical Roles of Cyclin-Dependent Kinases in Neural and Glioma Stem Cell Self-Renewal.

Glioma stem cells (GSCs) are thought to drive growth and therapy resistance in glioblastoma (GBM) by "hijacking" at least a subset of signaling pathways active in normal neural stem cells (NSCs). Though the origins of GSCs still remain elusive, uncovering the mechanisms of self-renewing division and cell differentiation in normal NSCs has shed light on their dysfunction in GSCs. However, the distinction between self-renewing division pathways utilized by NSC and GSC becomes critical when considering options for therapeutically targeting signaling pathways that are specifically active or altered in GSCs. It is well-established that cyclin-dependent kinases (CDKs) regulate the cell cycle, yet more recent studies have shown that CDKs also play important roles in the regulation of neuronal survival, metabolism, differentiation, and self-renewal. The intimate relationship between cell cycle regulation and the cellular programs that determine self-renewing division versus cell differentiation is only beginning to be understood, yet seems to suggest potential differential vulnerabilities in GSCs. In this timely review, we focus on the role of CDKs in regulating the self-renewal properties of normal NSCs and GSCs, highlighting novel opportunities to therapeutically target self-renewing signaling pathways specifically in GBM.

Bioinformatics analysis of molecular pathways and key candidate biomarkers associated with human bone marrow hematopoietic stem cells (HSCs) micro-array gene expression data

An identification of the molecular properties of glioma stem cells (GSCs) has led studies in clinical use, stem cell identification, and highly-effective usage. The GSE32719 contains a total expression of 54,676 genes of healthy human bone marrow HSCs in 14 young (20–31 years), 5 middle (42–61 years), and a lot of old (65–85 years) age groups. The researchers of this study described age-related changes in the human HSC population, using the gene expression profile of significance analysis to discover differentially expressed genes (DEGs) between each age group. The DEGs were subjected to significantly enriched biological processing that decoded the increase and functional decline in the HSC population. The GSE dataset analysis was conducted by the GEOquery package in Bioconductor. Using the Biobase and gplots packages, 453 DEGs were screened. DEGs analyses were conducted by gene ontology (GO) pathway enrichment and Kyoto Encylopedia of Genes and Genomes (KEGG) enrichment analysis. The Hippo signaling pathway was observed to be significant using the GO pathway enrichment analysis, which was previously reported as an effective pathway in cancers, including AML. A protein-protein interaction (PPI) network was constructed; then based on that, a subnetwork module analysis for the Hippo signaling pathway was made. Additionally, the GO pathway enrichment analysis revealed ‘cellular process’, ‘cellular metabolic process’, ‘metabolic process’, ‘biogenesis’, and ‘vasculogenesis biological processes’, which are involved in a wide of biological activities such as metabolic regulation, cell growth, and proliferation. Our findings offer silico evidence for candidate genes, such as the UBC, PTK2, and TCF7L2, that may be promising biomarkers for the translation approach associated with HSC population age-related diseases.

Regulation of Glioblastoma Stem Cell Properties and Tumor Invasion by G⍺12 Signaling

Glioblastoma multiforme (GBM), the most common malignant brain tumor, is associated with upregulation of a number of G‐protein coupled receptors. TCGA analysis of glioblastoma reveals that Gα12 mRNA levels are elevated in 28% of GBM patient tumors, the highest rate for all tumor types surveyed. There are concomitant alterations in expression of a subset of GPCRs that couple to Gα12. Gα12 signals through activation of RhoA and its downstream effectors. Our earlier studies revealed that gene programs induced through the RhoA‐regulated transcriptional co‐activators YAP and MRTF‐A contribute to glioma stem cell properties and their in vivo growth as orthotopic (brain) tumors (Yu et al., Oncogene 2018). To determine if Gα12 signaling pathways transduce the effects of known and orphan GPCRs activated in the tumor environment we knocked down Gα12 using shRNA in two human GBM cancer stem cell lines, GSC‐23 and HK‐281. Gα12 mRNA levels decreased by 60‐80% without compensatory upregulation of mRNA for the homologous Gα13. Loss of Gα12 significantly attenuated the stem cell properties of GSC‐23 cells as determined by limiting dilution assays and also reduced mRNA levels for multiple canonical stem cells genes (CCND1, NANONG, OCT4, SOX2 and NESTIN) by 35% to 60%. Tumor growth of GSC‐23 control and Gα12 knockdown (KD) cells was examined following orthotopic injection into mouse brain. Surprisingly, Gα12 deletion had no evident effect on tumor size nor did it prolong mouse survival. On the other hand, histopathological analyses revealed striking differences at the tumor border, indicative of diminished invasive properties and this was confirmed by immunohistochemical staining for the human nucleoli antigen. RNA sequencing and DESeq2 analysis in WT and KD tumors identified a number of genes encoding extra and intracellular proteins related to cell adhesion and migration dynamics which were differentially expressed in Gα12 KDs. Thrombospondin‐1 (Thbs‐1) was one of the most repressed genes in the Gα12 knockdown tumors and it was previously identified as a YAP‐dependent gene in these cells. Expression of Thbs‐1 and Gα12 is highly correlated in the more aggressive mesenchymal‐types of GBM by Gliovis and IvyGAP tools, and clinical reports show elevated Thbs‐1 as a marker of poor prognosis in GBM patients. To assess the effect of Gα12 and Thbs‐1 on glioblastoma stem cell migration we performed in vitro Transwell‐based assays. These studies demonstrated that both Gα12 and its transcriptional target Thbs‐1 are required for agonist‐stimulated GSC‐23 cell migration and invasion. We also demonstrated through chemogenetic activation of Gα12 by DREADD expression that Gα12 signaling in GSC‐23 cells is sufficient to drive Thbs‐1 expression and migration. Our findings indicate that Gα12 is a focal point for GPCR control of transcriptional programs for stemness and invasiveness in glioma stem cells derived tumors and suggest targeting this site as a therapeutic intervention to disrupt tumor cell invasion.

Identification and isolation of slow-cycling glioma stem cells.

Cancer stem cells are defined as low-abundance, quiescent cells and are considered a major cellular source of tumor recurrence following therapy, which identifies these cells as important therapeutic targets for difficult-to-treat cancers, including high-grade gliomas. By contrast to the highly proliferative bulk tumor cells, glioma stem cells (GSC) are slow-cycling, and therefore less sensitive to DNA damaging cytotoxic drugs. GSC are also less reliant on aerobic glycolytic metabolism, leading to inadequate clearing of GSC by chemotherapy and radiotherapy. The definition of GSC is based on the expression of specific stem cell protein markers. This method of GSC isolation is successful in isolating cell populations that can reliably recapitulate the tumor. However, cell populations that lack stem marker expression may also be capable of tumor recapitulation. Therefore, robust, reproducible methods for isolating GSC are required to identify and isolate cells with stem cell characteristics. Here, we provide a comprehensive and reproducible protocol for the isolation of slow-cycling GSC. Using this method, GSC isolated retain key characteristics of the cells in situ, including expression of genes associated with cell quiescence and invasive potential, compared to non-quiescent cell populations. Thus, isolation of GSC gated on cell proliferation offers a reliable alternative method for in vitro GSC identification, that adequately mirrors the physiological properties of GSC seen in vivo.

P11.46 Discovery of a new HDAC6 inhibitor for the treatment of glioblastoma

Abstract BACKGROUND Glioblastoma’s origin and development is not only associated to genetic alterations, but also to epigenetic changes. Indeed, an altered expression or activity of epigenetic enzymes such as histone deacetylases (HDAC) has been associated to cancer stem cell activity, which has been widely described as a major feature for therapy resistance and tumor recurrence. In particular, inhibition of HDAC6 is an increasingly attractive pharmacological strategy, due to its association with low toxicity. Thus, the aim of the present study was to determine the impact of a new HDAC6-selective-inhibitor in glioblastoma and glioma stem cells. MATERIAL AND METHODS To test the effect of QTX compound in glioblastoma and glioma stem cell lines, cell viability after 72h of treatment was studied by MTT assay. After evaluation of IC50, QTX in vitro activity was analyzed, focusing on proliferation, apoptosis and stemness of U87-MG cell line and confirmed in a patient-derived glioma stem cell line. In vivo antitumor effect was evaluated using U87-MG cells xenografted in immunocompromised mice; after tumor formation, 5 mice were randomly selected as control group and another 5 for QTX treatment (intraperitoneal administration of 50 mg/kg; 5 days of dosing / 2 days off for 2 weeks). Mice weight was measured daily and tumor volume every two days. RESULTS We demonstrated that QTX reduces viability of all tested glioblastoma cells, even more greatly than normal astrocytes. Indeed, QTX diminishes proliferation and induces apoptosis in both conventional and patient-derived glioma cell lines. In particular, this effect was accompanied by a reduction of self-renewal properties of glioma stem cells. Interestingly, QTX in vitro activity was more effective comparing to the pan-inhibitor SAHA or the HDAC6-selective inhibitor Tubastatin A. Furthermore, QTX delayed tumor initiation and progression in vivo, without presenting significant side effects. CONCLUSION QTX compound presents a promising anti-tumor effect both in vitro and in vivo in glioblastoma, at least in part, inhibiting glioma stem cell activity.

VDAC2 interacts with PFKP to regulate glucose metabolism and phenotypic reprogramming of glioma stem cells

Plastic phenotype convention between glioma stem cells (GSCs) and non-stem tumor cells (NSTCs) significantly fuels glioblastoma heterogeneity that causes therapeutic failure. Recent progressions indicate that glucose metabolic reprogramming could drive cell fates. However, the metabolic pattern of GSCs and NSTCs and its association with tumor cell phenotypes remain largely unknown. Here we found that GSCs were more glycolytic than NSTCs, and voltage-dependent anion channel 2 (VDAC2), a mitochondrial membrane protein, was critical for metabolic switching between GSCs and NSTCs to affect their phenotypes. VDAC2 was highly expressed in NSTCs relative to GSCs and coupled a glycolytic rate-limiting enzyme platelet-type of phosphofructokinase (PFKP) on mitochondrion to inhibit PFKP-mediated glycolysis required for GSC maintenance. Disruption of VDAC2 induced dedifferentiation of NSTCs to acquire GSC features, including the enhanced self-renewal, preferential expression of GSC markers, and increased tumorigenicity. Inversely, enforced expression ofVDAC2 impaired the self-renewal and highly tumorigenic properties of GSCs. PFK inhibitor clotrimazole compromised the effect of VDAC2 disruption on glycolytic reprogramming and GSC phenotypic transition. Clinically, VDAC2 expression inversely correlated with glioma grades (Immunohistochemical staining scores of VDAC2 were 4.7 ± 2.8, 3.2 ± 1.9, and 1.9 ± 1.9 for grade II, grade III, and IV, respectively, p < 0.05 for all) and the patients with high expression of VDAC2 had longer overall survival than those with low expression of VDAC2 (p = 0.0008). In conclusion, we demonstrate that VDAC2 is a new glycolytic regulator controlling the phenotype transition between glioma stem cells and non-stem cells and may serves as a new prognostic indicator and a potential therapeutic target for glioma patients.

Abstract 4887: Mitochondrial SOD2 is the mainstay to protect the stemness-featured glioblastoma cells against drug-induced reactive oxygen stress

Abstract Glioblastoma (GBM) is a highly aggressive primary brain tumor with presence of stemness-featured cells that are prone to survive from cancer treatment. We reported Sp1-induced SOD2 was associated with the process to acquire temozolomide (TMZ) resistance that the significance remained to be elucidated. Resistant models were established through long-term coincubation with TMZ in GBM cell lines, which expressed significantly higher stemness properties in extreme limiting dilution analysis (ELDA) and serial transplantation. These cells also showed increased expression of SOD2. Enrichment of stemness-featured cells by spheroid culturing or by CD133-positive cells selection resulted in increased SOD2, suggesting their associations. The impact of the protein to glioma stem cells (GSCs) was then studied through SOD2 RNA interference (RNAi), displaying downregulated stemness features in the knockdown models. Functional studies of the resistant cells showed superior scavenging ability in TMZ- or H2O2-induced ROS that would be dysfunctioned in pretreatment with SOD2 RNAi. In addition, co-treatment with the reagents over the knockdown cells resulted in enhancement of apoptosis as well as inhibition in tumor spheroid culture and cell clonogenic formation, suggesting re-sensitization to the drug. By establishing in vivo model from patient derived xenograft implantation that was obtained from clinical resistant disease, the mouse receiving combination treatment with TMZ and SOD inhibitor had lower tumor proliferation comparing to those receiving TMZ alone. These data indicate that there was an association between SOD2 and the stemness features, suggesting the protein to play important roles in regulation of GSCs behavior. Down-regulation of SOD2 levels may thus be a potential therapeutic strategy for modulating stemness properties of GSCs, making these cells more susceptive to the chemo-therapeutic agent. Citation Format: Kwang-Yu Chang, Chia-Hung Chien, Jui-Mei Chu, Pei-Hsuan Chung, Ming-Sheng Liu, Jian-Ying Chuang. Mitochondrial SOD2 is the mainstay to protect the stemness-featured glioblastoma cells against drug-induced reactive oxygen stress [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 4887.

A unique epigenomic landscape defines the characteristics and differentiation potentials of glioma stem cells

A new study reveals comprehensive and unique epigenetic properties of glioma stem cells, leading to novel molecular insights and therapeutic potentials toward glioblastoma multiforme treatment.

An attempt to understand glioma stem cell biology through centrality analysis of a protein interaction network

Biological networks can be analyzed using "Centrality Analysis" to identify the more influential nodes and interactions in the network. This study was undertaken to create and visualize a biological network comprising of protein-protein interactions (PPIs) amongst proteins which are preferentially over-expressed in glioma cancer stem cell component (GCSC) of glioblastomas as compared to the glioma non-stem cancer cell (GNSC) component and then to analyze this network through centrality analyses (CA) in order to identify the essential proteins in this network and their interactions. In addition, this study proposes a new centrality analysis method pertaining exclusively to transcription factors (TFs) and interactions amongst them. Moreover the relevant molecular functions, biological processes and biochemical pathways amongst these proteins were sought through enrichment analysis. A protein interaction network was created using a list of proteins which have been shown to be preferentially expressed or over-expressed in GCSCs isolated from glioblastomas as compared to the GNSCs. This list comprising of 38 proteins, created using manual literature mining, was submitted to the Reactome FIViz tool, a web based application integrated into Cytoscape, an open source software platform for visualizing and analyzing molecular interaction networks and biological pathways to produce the network. This network was subjected to centrality analyses utilizing ranked lists of six centrality measures using the FIViz application and (for the first time) a dedicated centrality analysis plug-in ; CytoNCA. The interactions exclusively amongst the transcription factors were nalyzed through a newly proposed centrality analysis method called "Gene Expression Associated Degree Centrality Analysis (GEADCA)". Enrichment analysis was performed using the "network function analysis" tool on Reactome. The CA was able to identify a small set of proteins with consistently high centrality ranks that is indicative of their strong influence in the protein protein interaction network. Similarly the newly proposed GEADCA helped identify the transcription factors with high centrality values indicative of their key roles in transcriptional regulation. The enrichment studies provided a list of molecular functions, biological processes and biochemical pathways associated with the constructed network. The study shows how pathway based databases may be used to create and analyze a relevant protein interaction network in glioma cancer stem cells and identify the essential elements within it to gather insights into the molecular interactions that regulate the properties of glioma stem cells. How these insights may be utilized to help the development of future research towards formulation of new management strategies have been discussed from a theoretical standpoint.

IDH1 mutation diminishes aggressive phenotype in glioma stem cells

The R132H mutation in isocitrate dehydrogenase 1 (IDH1-R132H) is associated with better prognosis in glioma patients. Glioma stem cells (GSCs) in glioma are believed to be responsible for glioma growth and maintenance. However, the relation between the R132H mutation and GSCs is not fully understood. In the present study, GSC markers were detected in patients with IDH1-R132H or wild-type IDH1 (IDH1-wt) by tissue microarray immunohistochemistry (TMA-IHC). The relationship between the expression patterns of GSC markers and the clinicopathological characteristics in glioma were analyzed. To confirm this mutation's role in GSCs, the IDH1-R132H in GSCs isolated from glioblastoma patients with IDH1 mutations was overexpressed by using lentiviral constructs invitro, and then the proliferation, differentiation, apoptosis, migration and invasion of the transfected GSCs were explored. At the molecular level, we detected Wnt/β-catenin signaling expression to verify its role in regulating the cellular properties of GSCs. The results showed that the positive rate of GSCs in patients with IDH1-R132H was significantly less than that in patients with IDH1-wt. The positive rate of GSCs was correlated with IDH1 mutation, TNM stage and poor overall survive. After transfection invitro, IDH1-R132H overexpression led to reduced GSCs proliferation, migration and invasion, inducing apoptosis and improving GSC differentiation, accompanied by a significant reduction in activity of β-catenin. Several mediators, effectors and targets of the Wnt/β-catenin signaling were downregulated. The data demonstrate that IDH1 mutation reduces the malignant progression of glioma by causing a less aggressive phenotype of GSCs which are involved in the Wnt/β‑catenin signaling.

Hinokitiol suppresses cancer stemness and oncogenicity in glioma stem cells by Nrf2 regulation.

Glioma is one of the lethal malignancies with poor prognosis. In addition, glioma stem cells (GSCs) have been considered as the crucial player that attributed to the tumorigenesis and drug resistance. In the current study, we investigated the therapeutic effect of hinokitiol, a natural bioactive compound of aromatic tropolone, on the characteristics of GSCs and the possible mechanism. U87MG and T98G glioma cells were used to isolate GSCs. CD133 positivity and ALDH1 activity of GSCs following hinokitiol treatment were assessed by flow cytometry analysis. Secondary sphere formation, migration, invasion, and colony-forming assays were performed to examine the self-renewal capacity and oncogenicity in GCS after hinokitiol administration. The expression of Nrf2 was evaluated by RT-PCR and western blot analyses. We demonstrated that hinokitiol effectively inhibited the CD133 positivity and ALDH1 activity along with the reduced self-renewal, migration, invasion, and colony formation properties of GSCs. In addition, hinokitiol repressed the gene and protein expression of Nrf2, which has been shown to be critical for those GSCs features. Furthermore, we showed that administration of exogenous Nrf2 counteracted the inhibitory effect of hinokitiol on self-renewal and invasiveness of GSCs. These evidences suggest that treatment of hinokitiol significantly attenuates the hallmarks of GSCs due to downregulation of Nrf2 expression. Hence, hinokitiol may serve as a promising agent for the therapy of glioma.

Silencing of the long non-coding RNA NEAT1 suppresses glioma stem-like properties through modulation of the miR-107/CDK6 pathway.

Developing novel strategies against glioma remains a significant challenge. Long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) significantly contributes to the progression of many human cancers, while glioma stem cells (GSCs) are believed to be responsible for tumor progression. However, whether NEAT1 alters the stem-like properties of GSC cells remains unknown. Using microbeads, CD133+ cells were isolated and were found to possess glioma stem cell properties. Using siRNA, NEAT1 was knocked down in the sorted CD133+ U87 glioma cells. We found higher NEAT1 RNA expression in CD133+ human glioma primary culture stem cells and CD133+ U87 cells via RT-PCR. Moreover, NEAT1 knockdown in the CD133+ U87 cells resulted in decreased colony formation, increased G1cell cycle arrest and apoptosis. In addition, these effects were accompanied by miR-107 activation and inactivation of CDK6 protein. These results strongly suggest that NEAT1 knockdown suppresses stem-like properties in glioma cells by modulating the miR‑107CDK6 pathway. Targeting NEAT1 may provide a novel therapeutic opportunity for developing a relapse-free treatment of glioma patients.

Epigenetic targeting of glioma stem cells: Short-term and long-term treatments with valproic acid modulate DNA methylation and differentiation behavior, but not temozolomide sensitivity.

Glioblastoma (GBM) is the most aggressive tumor of the central nervous system. GBM is a fatal tumor, incurable by conventional therapies. One of the factors underlying tumor recurrence and poor long-term survival is the presence of a cancer stem-like cell population, termed glioma stem cells (GSCs), which is particularly resistant to chemotherapy and radiotherapy and supports tumor self-renewal. The aim of the present study was to evaluate the impact and difference in effects of short-term and long‑term treatments with valproic acid (VPA), a histone deacetylase inhibitor, on seven GSC lines. We investigated for the first time the changes in the genome-wide DNA methylation profile and the differentiation behavior of GSCs induced by short-term and long-term VPA treatments. Moreover, we verified VPA sensitivity after long-term VPA pretreatment and, notably, the results provide evidence of a subpopulation more resistant to further VPA treatments. Finally, since short-term VPA treatment induced a reversal of the MGMT methylation status, we aimed to sensitize GSCs to temozolomide, the drug commonly used for this tumor, using this regimen. The overall data highlighted the heterogeneous behavior of GSC lines that is representative of tumor heterogeneity in GBM. The VPA effects were variable among these cell lines in terms of pro‑differentiating ability and DNA methylation switch. Here, we attempted to identify a suitable therapy for the eradication of the stem cell subpopulation, which is mandatory to achieve an effective treatment for this tumor. Differentiation-inducing and epigenetic therapies are the most promising approaches to affect the multiple properties of GSCs and, finally, defeat GBM.

LIM domain only 2 induces glioma invasion via cytosolic p27(KIP1).

High-grade gliomas are considered the most malignant of brain tumors and have a poor prognosis. In a previous study, we showed that LIM domain only 2 (LMO2) regulates glioma stem cell properties and tumor angiogenesis and gave rise to highly invasive glioma xenografts. Glioma invasion in the surrounding parenchymal tissues is a major hurdle with respect to eliminating glioma by surgery. Invasive glioma cells are considered one of the main culprits for the recurrence of tumors after therapies. In the current study, we focused on determining the molecular mechanism(s) by which LMO2 regulates glioma cell migration and invasion. Forced expression of LMO2 in human U87MG glioma cells led to glioma invasion, as determined by in vivo xenograft assays and enhanced in vitro migration and invasion. LMO2 was associated with increased levels of cytosolic p27(Kip1) protein. LMO2 possibly induced the stabilization and augmented interactions between p27(Kip1) and RhoA. We knocked down the expression of p27(Kip1), which led to a decrease in LMO2-driven glioma cell migration and invasion. Taken together, our findings indicate that LMO2 promotes glioma cell migration and invasion by increasing the levels of cytosolic p27(Kip1).

Pterostilbene suppressed irradiation-resistant glioma stem cells by modulating GRP78/miR-205 axis

Glioblastoma multiforme (GBM) is the most aggressive type characterized by relapse and resistance even with the combination of radio- and chemotherapy. The presence of glioma stem cells (GSCs) has been shown to contribute to tumorigenesis, recurrence and treatment resistance. Particularly, CD133-positive glioma cells have been shown to represent the subpopulation that confers glioma radioresistance and suggested to be the source of tumor recurrence after radiation. Thus, a better understanding and the development of agents which target GSCs could potentially lead to a significant improvement in treating GBM patients. Here, we demonstrated that GRP78 (an antistress protein) was highly expressed in GBM cells along with β-catenin and Notch and correlated to the development of GSCs. CD133+ GSCs exhibited enhanced migration/invasion and self-renewal abilities. When GRP78 was silenced, GSC properties were suppressed and the sensitivity towards irradiation increased. In addition, the level of microRNA 205 appeared to be negatively associated with GRP78 expression. Our previous study indicated that pterostilbene (PT) possessed anticancer stem cell properties in hepatocellular carcinoma. Thus, we examined whether PT is also effective against GSCs. We found that PT-treated GSCs exhibited suppressed self-renewal and irradiation-resistant abilities. PT-mediated effects were associated with an increase of miR-205. Finally, we showed that PT treatment suppressed tumorigenesis in GSC xenograft mice. In conclusion, we provided evidence that GRP78/miR-205 axis played an important role in GSC maintenance and irradiation resistance. PT treatment suppressed GSC development via negatively modulating GRP78 signaling. PT may be considered for combined therapeutic agent to enhance irradiation efficacy in GBM patients.

CB-09 * THE CELL OF ORIGIN FOR GLIOBLASTOMA CONTRIBUTES TO THE PHENOTYPIC HETEROGENEITY OF GLIOMA STEM CELLS

Glioblastoma Multiforme (GBM) is the most frequent adult primary malignant brain tumor that remains incurable despite aggressive treatment. The cell of origin (COO) for GBM is unknown but assumed to be a glial stem or progenitor cell. GBM harbours hierarchical tumor cells called glioma stem cells (GSCs) that maintain tumor growth, drive tumor progression and cause tumor relapse due to their increased resistance to therapy. We have analyzed the significance of cellular origin for GBM development and GSC properties by comparing mouse GBMs and GSCs derived thereof induced in neural stem cells (NSCs), glial-restricted precursor cells (GPCs) or oligodendrocyte precursor cells (OPCs) by identical mutations. There were striking differences in GBM development and the phenotypes of GSCs and their response to drugs owing to the COO. Global gene expression analysis of mouse GSC lines displayed a clear separation due to COO and differential gene expression analysis identified a COO gene signature of 175 genes. Cross-species bioinformatics analyses were performed. First we analyzed the human cancer genome atlas (TCGA) GBM tissue samples and the mouse GSC expression data for a collection of TCGA GBM subtype signature genes. This showed that we could model both Proneural and Mesenchymal GBMs in mice by merely switching the COO. Next, we used the mouse COO gene signature to stratify a large number of newly established human glioma stem cell lines. This produced two groups of human GSCs; the NSC origin group and the progenitor cell (PC) origin group in which the mouse GPC- and OPC-derived genes were combined. Importantly, patient survival was significantly different between the NSC and PC COO groups with a better prognosis for the PC group patients. Thus, the cell of origin is essential for GBM biology and needs to be considered for more accurate patient stratification, target identification and drug discovery.