Potential Therapeutic Target For Glioblastoma Research Articles

Mirna-383-5p Functions as an Anti-oncogene in Glioma through the Akt/mTOR Signaling Pathway by Targeting VEGFA.

Previously, we have screened 59 differentially expressed miRNAs and 419 mRNAs in the glioblastoma samples that have been compared to the peritumoral tissues using bioinformatics analyses, which included miRNA-383-5p and vascular endothelial growth factor A (VEGFA). miRNA-383-5p and VEGFA/Akt/mTOR pathway play important regulatory roles in the malignant biological behavior of glioma. Glioma cell lines, U87 and U251, were collected for in vitro experiments. miRNA-383-5p and VEGFA expression levels were detected with qRT-PCR and WB. The protein expressions of Akt, mTOR, and VEGFR in U87 and U251 were detected with WB. The effect of miRNA-383-5p on the VEGFA activity was verified by dual-luciferase reporter assay. CCK-8 was used to examine the U87 and U251 cells' inhibition. Flow cytometry and transwell assays were used to detect cell apoptosis and invasion, respectively. Our research data indicated overexpression of miRNA-383-5p to suppress malignant biological behavior, which was manifested as promoting the apoptosis of U87 and U251 cells and inhibiting invasion, proliferation, and metastasis. VEGFA is one of the downstream target genes of miRNA-383- 5p. miRNA-383-5p could inhibit the expression of VEGFA and Akt/mTOR signaling pathways. Overexpression of VEGFA can reverse the inhibitory effect of miRNA-383-5p and reactivate the Akt/mTOR signaling pathway. Our results indicate that miRNA-383-5p functions as an anti-oncogene by inhibiting the VEGFA/Akt/mTOR signaling pathway in glioma cells. These data provide potential therapeutic targets for glioblastoma.

Resveratrol ameliorates glioblastoma inflammatory response by reducing NLRP3 inflammasome activation through inhibition of the JAK2/STAT3 pathway

ObjectivesThe aim of this study was to investigate the anti-tumor effect of resveratrol (RSV) on glioblastoma (GBM) and its specific mechanism in improving the inflammatory response of the tumor microenvironment. The tumor microenvironment of GBM is highly neuroinflammatory, inducing tumor immunosuppression. Therefore, ameliorating the inflammatory response is an important focus for anti-tumor research.MethodsThe anti-tumor effect of RSV on GBM was demonstrated through in vitro cellular assays, including CCK-8, EdU, PI staining, Transwell, wound healing assay, and flow cytometry. Potential mechanisms of RSV's anti-GBM effects were identified through network pharmacological analysis. In addition, the relationship of RSV with the JAK2/STAT3 signaling pathway and the inflammasome NLRP3 was verified using Western blot.ResultsRSV significantly inhibited cell viability in GBM cell lines LN-229 and U87-MG. Furthermore, it inhibited the proliferation and invasive migration ability of GBM cells, while promoting apoptosis. Network pharmacological analysis revealed a close association between the anti-GBM effects of RSV and the JAK/STAT signaling pathway, as well as inflammatory responses. Western blot analysis confirmed that RSV inhibited the over-activation of the inflammasome NLRP3 through the JAK2/STAT3 signaling pathway. Partial reversal of RSV's inhibition of inflammasome NLRP3 was observed with the addition of the JAK/STAT agonist RO8191.ConclusionsIn vitro, RSV can exert anti-tumor effects on GBM and improve the inflammatory response in the GBM microenvironment by inhibiting the activation of the JAK2/STAT3 signaling pathway. These findings provide new insights into potential therapeutic targets for GBM.

LncRNA LUCAT1 Promotes Glioblastoma Progression by Enhancing HIF1α Activity.

Hypoxia is associated with poor prognosis in many cancers including glioblastoma (GBM). Glioma stem-like cells (GSCs) often reside in hypoxic regions and serve as reservoirs for disease progression. Long non-coding RNAs (lncRNAs) have been implicated in GBM. However, the lncRNAs that modulate GSC adaptations to hypoxia are poorly understood. Identification of these lncRNAs may provide new therapeutic strategies to target GSCs under hypoxia. lncRNAs induced by hypoxia in GSCs were identified by RNAseq. LUCAT1 expression was assessed by qPCR, RNAseq, Northern blot, single molecule FISH in GSCs, and interrogated in IvyGAP, TCGA, and CGGA databases. LUCAT1 was depleted by shRNA, CRISPR/Cas9, and CRISPR/Cas13d. RNAseq, Western blot, immunohistochemistry, co-IP, ChIP, ChIPseq, RNA immunoprecipitation, and proximity ligation assay were performed to investigate mechanisms of action of LUCAT1. GSC viability, limiting dilution assay, and tumorigenic potential in orthotopic GBM xenograft models were performed to assess the functional consequences of depleting LUCAT1. A new isoform of Lucat1 is induced by HIF1α and NRF2 in GSCs under hypoxia. LUCAT1 is highly expressed in hypoxic regions in GBM. Mechanistically, LUCAT1 formed a complex with HIF1α and its co-activator CBP to regulate HIF1α target gene expression and GSC adaptation to hypoxia. Depletion of LUCAT1 impaired GSC self-renewal. Silencing LUCAT1 decreased tumor growth and prolonged mouse survival in GBM xenograft models. A HIF1α-LUCAT1 axis forms a positive feedback loop to amplify HIF1α signaling in GSCs under hypoxia. LUCAT1 promotes GSC self-renewal and GBM tumor growth. LUCAT1 is a potential therapeutic target in GBM.

MODL-02. DEVELOPMENT OF RODENT GLIOBLASTOMA MODELS OF MGMT-MEDIATED TEMOZOLOMIDE RESISTANCE

Abstract O6-methylguanine-DNA methyltransferase (MGMT) is an enzyme that repairs O6-methylguanine DNA damage lesions generated by alkylators such as temozolomide (TMZ), the standard chemotherapeutic for glioblastoma (GBM) treatment. High MGMT expression leads to TMZ resistance and is correlated with lower survival, making it an important prognostic biomarker and potential therapeutic target. Xenograft models with human GBM cell lines require implantation into immunodeficient animals, precluding the study of MGMT-dependent alkylator sensitivity in the setting of an intact tumor immune microenvironment. In this study, we developed rat and mouse glioma models of MGMT overexpression and characterized response to alkylator therapy. RG2 rat glioma and SB28 mouse glioma cells, which have low endogenous MGMT levels, were transduced with cDNA expressing FLAG-tagged MGMT to generate an isogenic pair. RG2 MGMT+ and SB28 MGMT+ cells showed robust expression of MGMT, which was effectively abrogated by MGMT inhibitors. In vitro cell viability assays showed that MGMT overexpression resulted in increased resistance to TMZ compared to parental cells, and this resistance was reversed when cells were concurrently treated with the MGMT inhibitor lomeguatrib. To model MGMT overexpression in vivo, luciferase-expressing RG2 MGMT+ cells were implanted into the brains of syngeneic Fischer 344 rats. Rats were treated with TMZ at 50mg/kg daily x 5 days via intraperitoneal injection. Tumor growth was measured by bioluminescence imaging and rats were monitored for survival and toxicity. RG2 MGMT+ tumors were highly resistant to TMZ, as treatment did not improve survival or delay tumor growth. Overall, our newly developed isogenic rat and mouse glioma models recapitulate MGMT-dependent TMZ sensitivity and provide useful preclinical tools for studying MGMT as a biomarker and potential therapeutic target in GBM. In addition, these syngeneic models facilitate studies on the immune modulation of chemotherapeutic agents in the context of MGMT expression and allows for testing of chemoimmunotherapy combinations.

The Semaphorin 3A-Neuropilin-1 Pathway Promotes Clonogenic Growth of Glioblastoma via Activation of TGFβ Signaling.

Glioblastoma (GBM) is the most lethal brain cancer with a dismal prognosis. Stem-like GBM cells (GSCs) are a major driver of GBM propagation and recurrence, thus understanding the molecular mechanisms that promote GSCs may lead to effective therapeutic approaches. Through in vitro clonogenic growth-based assays, we determined mitogenic activities of the ligand molecules that are implicated in neural development. We have identified that Semaphorin 3A (Sema3A), originally known as an axon guidance molecule in the central nervous system, promotes clonogenic growth of GBM cells but not normal neural progenitor cells (NPCs). Mechanistically, Sema3A binds to its receptor Neuropilin-1 (NRP1) and facilitates an interaction between NRP1 and TGF receptor 1 (TGFR1), which in turn leads to activation of canonical TGF signaling in both GSCs and NPCs. TGF signaling enhances self-renewal and survival of GBM tumors through induction of key stem cell factors, but it evokes cytostatic responses in NPCs. Blockage of the Sema3A-NRP1 axis via shRNA-mediated knockdown of Sema3A or NRP1 impeded clonogenic growth and TGF pathway activity in GSCs and inhibited tumor growth in vivo. Taken together, these findings suggest that the Sema3A-NRP1-TGFR1 signaling axis is a critical regulator of GSC propagation and a potential therapeutic target for GBM.

PLK3 promotes the proneural-mesenchymal transition in glioblastoma via transcriptional regulation of C5AR1.

Accumulating evidence suggests that polo-like kinase 3 (PLK3) plays an essential role in tumor cells and induces cell proliferation and may have implications for the prognosis of various cancers. We sought to define the role of PLK3-dependent proneural-mesenchymal transition (PMT) in the glioblastoma (GBM) therapy. We analyzed the expression data for PLK3 by using the TCGA database. PLK3 expression in GBM cell lines was determined by qRT-PCR and Western blotting. PLK3 levels were modulated using Lentivirus infection, and the effects on symptoms, tumor volume, and survival in mice intracranial xenograft models were determined. Irradiation (IR) was performed to induce PMT. PLK3 expression was significantly elevated in mesenchymal subtype GBM and promoted tumor proliferation in GBM. Additionally enriched PLK3 expression could be associated with poor prognosis in GBM patients compared with those who have lower PLK3 expression. Mechanically, PLK3-dependent PMT induced radioresistance in GBM cells via transcriptional regulation of complement C5a receptor 1 (C5AR1). In therapeutic experiments conducted in vitro, targeting PLK3 by using small molecule inhibitor decreased tumor growth and radioresistance of GBM cells both in vitro and in vivo. PLK3-C5AR1 axis induced PMT thus enhanced radioresistance in GBM and could become a novel potential therapeutic target for GBM.

KIF18A interacts with PPP1CA to promote the malignant development of glioblastoma.

Glioblastoma (GBM), which has poor prognosis and low 5-year survival rate, is the most common primary central nervous system malignant tumour in adults. Kinesin family member 18A (KIF18A) plays an important role in multiple tumours and is potential therapeutic target for GBM. Therefore, the present study investigated the role of KIF18A in GBM. The expression level and survival prognosis of KIF18A and protein phosphatase 1 catalytic subunit α (PPP1CA) in GBM patients were analysed using the Chinese Glioma Genome Atlas (CGGA) database. Reverse transcription-quantitative PCR and western blot analysis were applied to measure the expression of KIF18A and PPP1CA in normal and GBM cell lines. KIF18A expression was inhibited through cell transfection with a KIF18A-targeting short hairpin RNA. Cell proliferation was detected with the Cell Counting Kit-8 assay. Flow cytometry was used to detect cell cycle changes. Transwell and wound healing assays were used to measure cell invasion and migration. Western blotting was utilized for the detection of invasion- and migration-related proteins MMP9 and MMP2. Biological General Repository for Interaction Datasets and GeneMANIA databases were used to analyse the interaction between KIF18A and PPP1CA. The correlation between PPP1CA and KIF18A was examined using data from the CGGA database. Immunoprecipitation was used to demonstrate the binding relationship between KIF18A and PPP1CA. PPP1CA was overexpressed using cell transfection technology and its mechanism was further examined. The results demonstrated that KIF18A was upregulated in GBM cells compared with normal microglia HMC3. Compared with that in sh-NC group, silencing of KIF18A reduced cell proliferation, induced G2/M cycle arrest and inhibited the migration and the invasion of A172 GBM cells by interacting with PPP1CA. In conclusion, KIF18A interacted with PPP1CA to promote the proliferation, cycle arrest, migration and invasion of GBM cells.

Cancer Stem Cell and Aggressiveness Traits Are Promoted by Stable Endothelin-Converting Enzyme-1c in Glioblastoma Cells.

Glioblastoma (GBM) is the most common and aggressive type of brain tumor due to its elevated recurrence following treatments. This is mainly mediated by a subpopulation of cells with stemness traits termed glioblastoma stem-like cells (GSCs), which are extremely resistant to anti-neoplastic drugs. Thus, an advancement in the understanding of the molecular processes underlying GSC occurrence should contribute significantly towards progress in reducing aggressiveness. High levels of endothelin-converting enzyme-1 (ECE1), key for endothelin-1 (ET-1) peptide activation, have been linked to the malignant progression of GBM. There are four known isoforms of ECE1 that activate ET-1, which only differ in their cytoplasmic N-terminal sequences. Isoform ECE1c is phosphorylated at Ser-18 and Ser-20 by protein kinase CK2, which increases its stability and hence promotes aggressiveness traits in colon cancer cells. In order to study whether ECE1c exerts a malignant effect in GBM, we designed an ECE1c mutant by switching a putative ubiquitination lysine proximal to the phospho-serines Lys-6-to-Arg (i.e., K6R). This ECE1cK6R mutant was stably expressed in U87MG, T98G, and U251 GBM cells, and their behavior was compared to either mock or wild-type ECE1c-expressing clone cells. ECE1cK6R behaved as a highly stable protein in all cell lines, and its expression promoted self-renewal and the enrichment of a stem-like population characterized by enhanced neurospheroid formation, as well as increased expression of stem-like surface markers. These ECE1cK6R-derived GSC-like cells also displayed enhanced resistance to the GBM-related chemotherapy drugs temozolomide and gemcitabine and increased expression of the ABCG2 efflux pump. In addition, ECE1cK6R cells displayed enhanced metastasis-associated traits, such as the modulation of adhesion and the enhancement of cell migration and invasion. In conclusion, the acquisition of a GSC-like phenotype, together with heightened chemoresistance and invasiveness traits, allows us to suggest phospho-ECE1c as a novel marker for poor prognosis as well as a potential therapeutic target for GBM.

Novel INHAT repressor drives glioblastoma growth by promoting ribosomal DNA transcription in glioma stem cells.

Cancer cells including cancer stem cells exhibit a higher rate of ribosome biogenesis than normal cells to support rapid cell proliferation in tumors. However, the molecular mechanisms governing the preferential ribosome biogenesis in glioma stem cells (GSCs) remain unclear. In this work, we show that the novel INHAT repressor (NIR) promotes ribosomal DNA (rDNA) transcription to support GSC proliferation and glioblastoma (GBM) growth, suggesting that NIR is a potential therapeutic target for GBM. Immunoblotting, immunohistochemical and immunofluorescent analysis were used to determine NIR expression in GSCs and human GBMs. Using shRNA-mediated knockdown, we assessed the role and functional significance of NIR in GSCs and GSC-derived orthotopic GBM xenografts. We further performed mass spectrometry analysis, chromatin immunoprecipitation, and other biochemical assays to define the molecular mechanisms by which NIR promotes GBM progression. Our results show that high expression of NIR predicts poor survival in GBM patients. NIR is enriched in the nucleoli of GSCs in human GBMs. Disrupting NIR markedly suppresses GSC proliferation and tumor growth by inhibiting rDNA transcription and pre-ribosomal RNA synthesis. In mechanistic studies, we find that NIR activates rDNA transcription to promote GSC proliferation by cooperating with Nucleolin (NCL) and Nucleophosmin 1 (NPM1), 2 important nucleolar transcription factors. Our study uncovers a critical role of NIR-mediated rDNA transcription in the malignant progression of GBM, indicating that targeting this axis may provide a novel therapeutic strategy for GBM.

CNSC-12. NEURONAL DIFFERENTIATION DRIVES THE ANTI-TUMOR EFFICACY OF MEK INHIBITION IN GLIOBLASTOMA

Abstract Multiple genomic aberrations and distinct molecular subtypes of glioblastoma (GBM) have been identified through multi-omics analyses including single-cell genomic and epigenomic analysis. Epidermal Growth Factor Receptor (EGFR) amplification is found in nearly 50% of GBM tumors. Several EGFR inhibitors have been tested in GBM but failed to demonstrate long-term therapeutic benefit, presumably due to target heterogeneity, escape signal pathways, and acquired resistance. Therefore, we investigated downstream signaling, mitogen-activated protein kinase (MEK1/2) as a potential therapeutic target for GBM. We utilized an unbiased high-throughput screening with a panel of glioma stem-like cells (GSCs) and identified that GBM cells harboring EGFR amplification showed anti-proliferative and apoptotic cell death (sensitive) to multiple MEK inhibitors. RNA-seq analysis of cells pre- and post-treatment with trametinib, a potent MEK inhibitor, revealed an upregulation of neurogenesis and neuronal differentiation genes such as β-III tubulin, ASCL1, DLL1, DLL3, NeuroD4, PAX6 and NCAM1. In addition, downregulation of the several MEK targets including erythroblast transformation specific (ETS) family genes, particularly ETV5 was observed. We demonstrated that MEK inhibition increased neuronal differentiation demonstrated by immunofluorescence, western blot, and RT-qPCR experiments using selected differentiation markers (β-III tubulin, ASCL1, DLL3, and NeuroD4). In addition, ETV-5 knockout in GSCs by CRISPR/Cas-9 promoted the expression of neuronal differentiation genes. Oral treatment of trametinib in an orthotopic GSC xenograft model significantly improved animal survival, in which 25-30% of mice are long-term survivors. This was accompanied by decreased MEK/ERK signal and cell proliferation in tumor tissues. Thus, we demonstrated here that MEK1/2 inhibition by trametinib induces neuronal differentiation mediated through downregulation of ETV-5 in GBM, a potential novel mechanism of action of MEK1/2 signal inhibition.

IKBIP, a novel glioblastoma biomarker, maintains abnormal proliferation of tumor cells by inhibiting the ubiquitination and degradation of CDK4

Sustained proliferative signaling is a crucial hallmark and therapeutic target in glioblastoma (GBM); however, new intrinsic regulators and their underlying mechanisms remain to be elucidated. In this study, I kappa B kinase interacting protein (IKBIP) was identified to be correlated with the progression of GBM by analysis of The Cancer Genome Atlas (TCGA) data. TCGA database analysis indicated that higher IKBIP expression was associated with high tumor grade and poor prognosis in GBM patients, and these correlations were subsequently validated in clinical samples. IKBIP knockdown induced G1/S arrest by blocking the Cyclin D1/CDK4/CDK6/CDK2 pathway. Our results showed that IKBIP may bind directly to CDK4, a key cell cycle checkpoint protein, and prevent its ubiquitination-mediated degradation in GBM cells. An in vivo study confirmed that IKBIP knockdown strongly suppressed cell proliferation and tumor growth and prolonged survival in a mouse xenograft model established with human GBM cells. In conclusion, IKBIP functions as a novel driver of GBM by binding and stabilizing the CDK4 protein. IKBIP could be a potential therapeutic target in GBM.

Circular RNA 0010117 promotes aggressive glioblastoma behavior by regulating the miRNA-6779-5p/SPEN axis

Noncoding RNAs (ncRNAs) play important roles in cancer biology, providing potential targets for cancer intervention. As a new class of endogenous noncoding RNAs, circular RNAs (circRNAs) have been recently identified in cell development and function, and certain types of pathological responses contribute to cancer progression, including glioblastoma. However, the potential mechanisms underlying the relationship between circRNAs and glioblastoma progression are still largely unknown. Methods: The expression and roles of circular RNA 0010117 (circ-0010117) were examined in vitro and in vivo. Quantitative RT‒PCR and western blotting were used to measure the expression of circRNA, miRNA, each gene, or related proteins. Cell biology experiments were performed to detect the biological function of circ-0010117 in glioblastoma cell lines. Moreover, bioinformatics analysis, luciferase reporter assays, and functional complementation analysis were carried out to investigate the target genes. Tumorigenesis was also evaluated by xenografting cells into nude mice. In this study, we found that circ-0010117 is downregulated in glioblastoma compared with corresponding paratumoural tissues. Subsequently, we observed that circ-0010117 can regulate aggressiveness in glioblastoma cells through miR-6779-5p. Furthermore, SPEN was verified as a direct target of miR-6779-5p and contributes to the circ-0010117 regulatory network. In addition, we identified that overexpression of circ-0010117 can suppress tumorigenesis in nude mice. Our findings indicate that circular RNA 0010117 promotes the aggressive behavior of glioblastoma by regulating the miRNA-6779-5p/SPEN axis. Our results provide a rationale for the use of circ-0010117 as a novel potential therapeutic target in glioblastoma.

Circular RNA VPS18 Promotes Glioblastoma Progression by Regulating miR-1229-3p/BCAT1 Axis.

Circular RNAs (circRNAs) have been verified to play important roles in malignant tumors, including glioblastoma. The aim of this study is to explore the biological roles and underlying mechanisms of circRNA vacuolar protein sorting 18 homolog (circVPS18) in glioblastoma. A quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the expression of circVPS18, microRNA (miR)-1299-3p, and branched-chain amino acid transaminase 1 (BCAT1). In vitro experiments were conducted using 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, and tube formation assays, respectively. Western blot was conducted to examine all protein levels. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to confirm the interaction between miR-1229-3p and circVPS18 or BCAT1. The murine xenograft model was established to conduct in vivo assay. CircVPS18 and BCAT1 were highly expressed while miR-1229-3p was lowly expressed in glioblastoma tissues and cells. CircVPS18 knockdown inhibited glioblastoma progression by inhibiting cell proliferation, migration, invasion, and angiogenesis, and promoting cell apoptosis. Moreover, miR-1229-3p could be targeted by circVPS18; inhibition of miR-1229-3p could invert the suppressive effect of circVPS18 knockdown on glioblastoma tumorigenesis. Furthermore, BCAT1 was a target of miR-1229-3p; functionally, BCAT1 overexpression could reverse the inhibitory effects of miR-1229-3p upregulation on glioblastoma cell malignant phenotypes. Moreover, we also verified that circVPS18A could regulate BCAT1 expression by sponging miR-1229-3p. Additionally, circVPS18 silencing also restrained tumor growth and metastasis in vivo. CircVPS18 accelerated glioblastoma progression by miR-1229-3p/BCAT1 axis, providing a potential therapeutic target for glioblastoma.

Hypoxia-induced polypoid giant cancer cells in glioma promote the transformation of tumor-associated macrophages to a tumor-supportive phenotype.

AimsPolypoid giant cancer cells (PGCCs) represent a unique subgroup of stem‐like cells, acting as a critical factor in promoting the recurrence of various solid tumors. The effect of PGCCs on the tumor malignancy of glioma and its immune microenvironment remains unclear.MethodsBioinformatic analysis was performed to investigate the relationship between M2 tumor‐associated macrophages (TAMs) infiltration and survival of glioblastoma (GBM) patients. The spatial location of M2 TAMs in GBM was also investigated using the Ivy Glioblastoma Atlas Project (Ivy GAP) database. PGCCs were quantified in glioma of different grades. CoCl2 was used to induce PGCCs in cultures of A172 cells. PGCCs, and their progeny cells in cultures were further evaluated for morphological features, tumorsphere formation, and TAMs activation.ResultsThe magnitude of M2 TAMs infiltration is significantly correlated with poor survival in GBM patients. M2 TAMs were enriched in the perinecrotic zone (PNZ) of GBM and positively correlated with hypoxic levels. Increased PGCCs were detected in glioma specimens of higher grades. CoCl2 induced hypoxia and the transformation of A172 cultures into PGCCs, producing the progeny cells, PGCCs‐Dau, through asymmetric division. PGCCs and PGCCs‐Dau possessed tumor stem cell‐like features, while PGCCs‐Dau enhanced the polarization of TAMs into an M2 phenotype with relevance to immunosuppression and malignancy in GBM.ConclusionsPGCCs promote malignancy and immune‐suppressive microenvironment in GBM. PGCCs or their progeny cells may be a potential therapeutic target for GBM.

Biophysical insights into OR2T7: Investigation of a potential prognostic marker for glioblastoma

Glioblastoma multiforme (GBM) is the most aggressive and prevalent form of brain cancer, with an expected survival of 12–15 months following diagnosis. GBM affects the glial cells of the central nervous system, which impairs regular brain function including memory, hearing, and vision. GBM has virtually no long-term survival even with treatment, requiring novel strategies to understand disease progression. Here, we identified a somatic mutation in OR2T7, a G-protein-coupled receptor (GPCR), that correlates with reduced progression-free survival for glioblastoma (log rank p-value = 0.05), suggesting a possible role in tumor progression. The mutation, D125V, occurred in 10% of 396 glioblastoma samples in The Cancer Genome Atlas, but not in any of the 2504 DNA sequences in the 1000 Genomes Project, suggesting that the mutation may have a deleterious functional effect. In addition, transcriptome analysis showed that the p38α mitogen-activated protein kinase (MAPK), c-Fos, c-Jun, and JunB proto-oncogenes, and putative tumor suppressors RhoB and caspase-14 were underexpressed in glioblastoma samples with the D125V mutation (false discovery rate < 0.05). Molecular modeling and molecular dynamics simulations have provided preliminary structural insight and indicate a dynamic helical movement network that is influenced by the membrane-embedded, cytofacial-facing residue 125, demonstrating a possible obstruction of G-protein binding on the cytofacial exposed region. We show that the mutation impacts the “open” GPCR conformation, potentially affecting Gα-subunit binding and associated downstream activity. Overall, our findings suggest that the Val125 mutation in OR2T7 could affect glioblastoma progression by downregulating GPCR-p38 MAPK tumor-suppression pathways and impacting the biophysical characteristics of the structure that facilitates Gα-subunit binding. This study provides the theoretical basis for further experimental investigation required to confirm that the D125V mutation in OR2T7 is not a passenger mutation. With validation, the aforementioned mutation could represent an important prognostic marker and a potential therapeutic target for glioblastoma.

Suppression of RPL34 Inhibits Tumor Cell Proliferation and Promotes Apoptosis in Glioblastoma.

Accumulating evidence indicates Ribosomal protein 34 (RPL34) promotes tumor malignance and its expression is associated with poor prognosis in multiple cancer cells. However, the physiological role and biological mechanism of RPL34 in glioblastoma (GBM) remain unclear. Hence, this study aimed to investigate the expression and the role of RPL34 in GBM. A total of 59 glioma samples and 12 normal brains for epilepsy surgery were used to determine the underlying mechanisms and the biological behaviors of RPL34 in GBM. In this study, we identified that RPL34 expression was significantly (p < 0.05) enriched in GBM tumors compared with low-grade glioma and normal brain, and its expression was associated with poor survival. Additionally, RPL34 was functionally required for tumor proliferation in vitro. Mechanically, inhibition of RPL34 induced glioma cell apoptosis by activation of Bad/Caspase7/PARP signaling pathway. The RPL34 promotes cell survival in GBM and could be a potential therapeutic target for GBM.

CRB2 enhances malignancy of glioblastoma via activation of the NF-κB pathway

Glioblastoma (GBM) is one of the most lethal types of primary brain tumors in adults with a median survival of less than 15 months. Although comprehensive clinical treatment strategies including surgical resection followed by radiotherapy and chemotherapy are widely applied, the prognosis for GBM patients remains dismal. The Nuclear Factor-κB (NF-κB) signaling pathway is a complex network linking extracellular stimuli to cell survival and proliferation, and aberrant activation of NF-κB signaling has been implicated in the propagation of a wide range of cancers. However, the underlying mechanism of NF-κB activation still requires further investigation. Here, we report that crumbs homolog 2 (CRB2) is markedly up-regulated in human GBM relative to non-tumor tissues or normal astrocytes. Clinically, enriched CRB2 could be observed in high grade glioma with IDH IDH wild-type and 1p19q co-deletion and implied poor outcome in GBM. Consistent with this, malignant characteristics of GBM cells including proliferation, migration, invasion and tumorigenesis were significantly suppressed by lentivirus knock-down of CRB2. Furthermore, exogenous overexpression of CRB2 enhanced the malignant biological signatures of GBM cells as well as therapy resistance to temozolomide (TMZ). To further investigate the molecular mechanisms responsible, bioinformatics analysis was performed using 3 public databases, with the result that CRB2 was found to correlate closely with tumor necrosis factor α (TNFα)–NF–κB signaling. Mechanistically, elevated CRB2 increased the phosphorylation of IκB-kinase α (IKKα), thus activating NF-κB via reduction of Ikβ protein. Taken together, these data suggest that CRB2 might be a reliable prognostic biomarker and potential therapeutic target for GBM.

6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 acts as a protein kinase to regulate glioblastoma progression by activating the AKT/forkhead box O1 pathway.

Abnormal expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) is closely related to the occurrence and development of tumors, and PFKFB4 has been shown to function as a protein kinase. However, the molecular mechanisms through which PFKFB4 functions in glioblastoma (GBM) remain poorly understood. Accordingly, in this study, we assessed the roles of PFKFB4 in GBM. Compared to in adjacent tissues, PFKFB4 was highly expressed in GBM, and its expression level was negatively correlated with the overall survival time. In addition, knockdown of PFKFB4 inhibited the proliferation and invasion of GBM cells and promoted apoptosis. In a xenograft tumor model, tumor growth was inhibited by knockdown of PFKFB4 using short hairpin RNA. Further studies demonstrated that PFKFB4 is involved in regulating the AKT signaling pathway. Thus, PFKFB4 acts as a protein kinase to regulate GBM progression by activating the AKT/forkhead box O1 pathway, which may be a potential therapeutic target in GBM.

Exosomes Derived from Glioma Cells under Hypoxia Promote Angiogenesis through Up-regulated Exosomal Connexin 43.

Glioblastoma multiform (GBM) is a highly aggressive primary brain tumor. Exosomes derived from glioma cells under a hypoxic microenvironment play an important role in tumor biology including metastasis, angiogenesis and chemoresistance. However, the underlying mechanisms remain to be elucidated. In this study, we aimed to explore the role of connexin 43 on exosomal uptake and angiogenesis in glioma under hypoxia. U251 cells were exposed to 3% oxygen to achieve hypoxia, and the expression levels of HIF-1α and Cx43, involved in the colony formation and proliferation of cells were assessed. Exosomes were isolated by differential velocity centrifugation from U251 cells under normoxia and hypoxia (Nor-Exos and Hypo-Exos), respectively. Immunofluorescence staining, along with assays for CCK-8, tube formation and wound healing along with a transwell assay were conducted to profile exosomal uptake, proliferation, tube formation, migration and invasion of HUVECs, respectively. Our results revealed that Hypoxia significantly up-regulated the expression of HIF-1α in U251 cells as well as promoting proliferation and colony number. Hypoxia also increased the level of Cx43 in U251 cells and in the exosomes secreted. The uptake of Dio-stained Hypo-Exos by HUVECs was greater than that of Nor-Exos, and inhibition of Cx43 by 37,43gap27 or lenti-Cx43-shRNA efficiently prevented the uptake of Hypo-Exos by recipient endothelial cells. In addition, the proliferation and total loops of HUVECs were remarkably increased at 24 h, 48 h, and 10 h after Hypo-Exos, respectively. Notably, 37,43gap27, a specific Cx-mimetic peptide blocker of Cx37 and Cx43, efficiently alleviated Hypo-Exos-induced proliferation and tube formation by HUVECs. Finally, 37,43gap27 also significantly attenuated Hypo-Exos-induced migration and invasion of HUVECs. These findings demonstrate that exosomal Cx43 contributes to glioma angiogenesis mediated by Hypo-Exos, and suggests that exosomal Cx43 might serve as a potential therapeutic target for glioblastoma.

Overexpression of Cystine/Glutamate Antiporter xCT Correlates with Nutrient Flexibility and ZEB1 Expression in Highly Clonogenic Glioblastoma Stem-like Cells (GSCs)

Simple SummaryGlioblastoma (GBM) is the most aggressive form of glioma (WHO grade IV), and mounting evidence suggests that glioblastoma stem-like cells (GSCs) play an important role in tumor growth and response to therapy. In the current study, we sought to understand the metabolic dependencies of GSCs using high-resolution proton magnetic resonance spectroscopy (1H-NMR). In a defined experimental setting, we stratified in vitro GSC models into two subtypes (Gln/GluHigh, Gln/GluLow) and used diverse molecular approaches to perform comprehensive analyses in GSC neurosphere cultures and primary GBM samples.Cancer stem-like cells mediate tumor initiation, progression, and therapy resistance; however, their identification and selective eradication remain challenging. Herein, we analyze the metabolic dependencies of glioblastoma stem-like cells (GSCs) with high-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy. We stratify our in vitro GSC models into two subtypes primarily based on their relative amount of glutamine in relationship to glutamate (Gln/Glu). Gln/GluHigh GSCs were found to be resistant to glutamine deprivation, whereas Gln/GluLow GSCs respond with significantly decreased in vitro clonogenicity and impaired cell growth. The starvation resistance appeared to be mediated by an increased expression of the glutamate/cystine antiporter SLC7A11/xCT and efficient cellular clearance of reactive oxygen species (ROS). Moreover, we were able to directly correlate xCT-dependent starvation resistance and high Gln/Glu ratios with in vitro clonogenicity, since targeted differentiation of GSCs with bone morphogenic protein 4 (BMP4) impaired xCT expression, decreased the Gln/Glu ratio, and restored the sensitivity to glutamine starvation. Moreover, significantly reduced levels of the oncometabolites lactate (Lac), phosphocholine (PC), total choline (tCho), myo-inositol (Myo-I), and glycine (Gly) were observed in differentiated GSCs. Furthermore, we found a strong association between high Gln/Glu ratios and increased expression of Zinc finger E-box-binding homeobox 1 (ZEB1) and xCT in primary GBM tumor tissues. Our analyses suggest that the inhibition of xCT represents a potential therapeutic target in glioblastoma; thus, we could further extend its importance in GSC biology and stress responses. We also propose that monitoring of the intracellular Gln/Glu ratio can be used to predict nutrient stress resistance.