Glioblastoma Tissue Specimens Research Articles

An AMP-activated protein kinase-PGC-1α axis mediates metabolic plasticity in glioblastoma.

Glioblastoma, the most frequent primary malignant brain tumour in adults, is characterised by profound yet dynamic hypoxia and nutrient depletion. To sustain survival and proliferation, tumour cells are compelled to acquire metabolic plasticity with the induction of adaptive metabolic programs. Here, we interrogated the pathways necessary to enable processing of nutrients other than glucose. We employed genetic approaches (stable/inducible overexpression, CRISPR/Cas9 knockout), pharmacological interventions with a novel inhibitor of AMP-activated protein kinase (AMPK) in glioblastoma cell culture systems and a proteomic approach to investigate mechanisms of metabolic plasticity. Moreover, a spatially resolved multiomic analysis was employed to correlate the gene expression pattern of PGC-1α with the local metabolic and genetic architecture in human glioblastoma tissue sections. A switch from glucose to alternative nutrients triggered an activation of AMPK, which in turn activated PGC-1α-dependent adaptive programs promoting mitochondrial metabolism. This sensor-effector mechanism was essential for metabolic plasticity with both functional AMPK and PGC-1α necessary for survival and growth of cells under nonglucose nutrient sources. In human glioblastoma tissue specimens, PGC-1α-expression correlated with nonhypoxic tumour niches defining a specific metabolic compartment. Our findings reveal a cell-intrinsic nutrient sensing and switching mechanism. The exposure to alternative fuels triggers a starvation signal that subsequently is passed on via AMPK and PGC-1α to induce adaptive programs necessary for broader spectrum nutrient metabolism. The integration of spatially resolved transcriptomic data confirms the relevance of PGC-1α especially in nonhypoxic tumour regions. Thus, the AMPK-PGC-1α axis is a candidate for therapeutic inhibition in glioblastoma. KEY POINTS/HIGHLIGHTS: AMPK activation induces PGC-1α expression in glioblastoma during nutrient scarcity. PGC-1α enables metabolic plasticity by facilitating metabolism of alternative nutrients in glioblastoma. PGC-1α expression is inversely correlated with hypoxic tumour regions in human glioblastomas.

Intratumoral thrombosis as a histological biomarker for predicting epidermal growth factor receptor alteration and poor prognosis in patients with glioblastomas.

Intratumoral thrombosis is a specific finding in glioblastomas and considered the origin of palisading necrosis. Its distribution and contribution to the glioblastoma pathophysiology and systemic thrombosis are obscure, although deep vein thrombosis is a common complication in glioblastoma cases. Clinicopathological and genetic analyses were performed on 97 glioblastoma tissue specimens to elucidate the role of thrombotic events and associated molecular abnormalities. Morphologically, intratumoral thrombosis was observed more frequently in vessels composed of single-layered CD34-positive endothelium and/or αSMA-positive pericytes in the tumor periphery, compared to microvascular proliferation with multi-channeled and pericyte-proliferating vessels in the tumor center. Intratumoral thrombosis was significantly correlated with the female sex, high preoperative D-dimer levels, and epidermal growth factor receptor (EGFR) amplification. The presence of one or more thrombi in 20 high-power fields was a predictive marker of EGFR amplification, with a sensitivity of 81.5% and specificity of 52.6%. RNA sequencing demonstrated that the group with many thrombi had higher EGFR gene expression levels than the group with few thrombi. The tumor cells invading along the vessels in the tumor periphery were positive for wild-type EGFR but negative for EGFRvIII, whereas the cells around the microvascular proliferation (MVP) in the tumor center were positive for both wild-type EGFR and EGFRvIII. Intratumoral thrombosis is an independent poor prognostic factor. Aberrant but exquisitely regulated EGFR can induce thrombosis in non-MVP vessels in the tumor invasion area and then promote palisading necrosis, followed by hypoxia, abnormal angiogenesis, and further tumor cell invasion.

Oncolytic HSV-1 suppresses cell invasion through downregulating Sp1 in experimental glioblastoma

Gliomas are highly aggressive intracranial tumors that are difficult to resect and have high lethality and recurrence rates. According to WHO grading criteria, glioblastoma with wild-type IDH1 has a poorer prognosis than WHO grade 4 IDH-mutant astrocytomas. To date, no effective therapeutic strategies have been developed to treat glioblastoma. Clinical trials have shown that herpes simplex virus (HSV)-1 is the safest and most efficacious oncolytic virus against glioblastoma, but the molecular antitumor mechanism of action of HSV-1 has not yet been determined. Deletion of the γ34.5 and ICP47 genes from a strain of HSV-1 yielded the oncolytic virus, oHSV-1, which reduced glioma cell viability, migration, and invasive capacity, as well as the growth of microvilli. Infected cell polypeptide 4 (ICP4) expressed by oHSV-1 was found to suppress the expression of the transcription factor Sp1, reducing the expression of host invasion-related genes. In vivo, oHSV-1 showed significant antitumor effects by suppressing the expression of Sp1 and invasion-associated genes, highly expressed in high-grade glioblastoma tissue specimens. These findings indicate that Sp1 may be a molecular marker predicting the antitumor effects of oHSV-1 in the treatment of glioma and that oHSV-1 suppresses host cell invasion through the ICP4-mediated downregulation of Sp1.

Knockdown of lncRNA HOXD-AS2 Improves the Prognosis of Glioma Patients by Inhibiting the Proliferation and Migration of Glioma Cells.

Increasing studies reported that long noncoding RNAs are involved in regulating glioma progression. However, the specific roles and mechanisms of lncRNAs in glioma remain unclear. Here, we sought to explore the functions of HOXD-AS2 in glioma progression. Gene expressions of lncRNAs in 5 normal brain tissue specimens and 5 glioblastoma tissue specimens were detected by gene expression profile chip technology. Bioinformatic analysis was performed to see whether differential expression of lncRNAs played any significant role in glioma occurrence and progression. The relationship between HOXD-AS2 level and clinical prognosis of the patients was analyzed. HOXD-AS2 was specifically interfered with by siRNA technology to observe its effects on U251 cell growth, proliferation, apoptosis, and invasion. The expression level of HOXD-AS2 gene in glioma was significantly higher than that in the normal brain tissue, which was related to the tumor grade. The level of HOXD-AS2 gene in patients with high-grade glioma was higher than that in patients with low-grade glioma. High expression of HOXD-AS2 gene was a risk factor for poor prognosis of glioma patients. Knocking down the expression of HOXD-AS2 in glioma cell line U251 arrested the cell cycle and reduced the cell proliferation. Furthermore, it could significantly reduce the migration ability of the cells but had no significant effect on the invasion. HOXD-AS2 is an oncogenic lncRNA associated with the poor prognosis of glioma. Knockdown of HOXD-AS2 may reduce the growth of glioma, which may provide a new avenue for treatment.

EXTH-40. MULTIDIMENSIONAL INTERROGATION OF GLIOBLASTOMA MICROENVIRONMENT TREATMENT RESPONSE IN EXPLANT CULTURES

Abstract The immune tumor microenvironment (iTME) of glioblastoma (GBM) contains microglial, macrophage, other myeloid cell populations and as adaptive immune cells. Recent therapeutic strategies for GBM aim at targeting iTME components to induce antitumoral immunity. A patient-tailored, ex vivo drug testing and response analysis platform would facilitate personalized therapy planning, provide insights into treatment-induced immune mechanisms in the iTME, and enable the discovery of biomarkers of response and resistance. Here, we generated patient-derived, live 3D GBM bioreactors from different tumor regions to assess iTME treatment responses to microglia modulators and immune checkpoint inhibitors. Intact GBM tissue specimens from the tumor center and periphery were cultured for 7 days in the presence or absence of anti-PD1, anti-CD47 antibodies or their combination. Tissues were analyzed by CODEX highly multiplexed microscopy using an immune-centered 54-marker panel, and changes in cytokine and chemokine levels in culture supernatants were investigated. A computational pipeline for integrative therapy response assessment was implemented. Explant cultures from n=8 IDH wt GBM were subjected to this integrative personalized analysis. Tissue integrity after 3D bioreactor cultures was comparable to tissue taken directly after surgery. FFPE CODEX workflow was feasible with adequate staining quality in bioreactor cultures. 850'000 single cells were segmented and clustered. Cellular composition between tumor center and the peripheral invasion zone differed significantly in immune phenotypes, cytokine profile and response to innate, adaptive or combinatorial local immunotherapies. Multiplexed cytokine analysis revealed IFNγ response signatures in a subset of center samples, whereas the peripheral invasion zone displayed a blunted cytokine response. This cytokine signature corresponded to cellular composition shifts within specific cellular neighborhoods. CD4 and CD8 T cells were invigorated and left their vascular niche. Our study demonstrates that local immunotherapies enable an active antitumoral immune response within the tumor center, and provides a multidimensional personalized framework for immunotherapy response assessment.

Mapping the Intratumoral Heterogeneity in Glioblastomas with Hyperspectral Stimulated Raman Scattering Microscopy.

Recent genomic studies on the glioblastoma (GBM) subtypes (e.g., mesenchymal, proneural, and classical) pave a way for effective clinical treatments of the recurrent brain tumor. However, identification of the GBM subtype is complicated by the intratumoral heterogeneity that results in coexistence of multiple subtypes within the tissue specimen. Here, we present the use of hyperspectral stimulated Raman scattering (SRS) microscopy for rapid, label-free molecular assessment of GBM intratumoral heterogeneity with submicron resolution. We develop a unique label-free Raman imaging diagnostic platform consisting of the spectral focusing hyperspectral SRS imaging of the large-area GBM tissue specimens, SRS images, and spectrum retrieval using the multivariate curve resolution algorithm and subtype classification based on the quadratic support vector machine model for rapid molecular subtyping of GBMs. Both the stain-free SRS histological images and 2D subtype maps can be obtained within 20-30 min which is superior to the days of the conventional single-cell RNA sequencing. While the SRS histology assesses the demyelination status as a new diagnostic feature, the SRS mapping provides a new insight into intratumoral heterogeneity across GBM tissue specimens. We find that the major proportions of the GBM tissues agree with the diagnostic results of the genomic analysis, but nontrivial portions of the remaining SRS image tiles in the specimens are found to belong to other molecular subtypes, implying the substantial degree of GBM heterogeneity. The rapid SRS imaging diagnostic platform developed has shown the ability of unveiling tumor heterogeneity in GBM tissues accurately, which would promote the improvement of the GBM-targeted therapy in near future.

Upregulation of miR-1825 inhibits the progression of glioblastoma by suppressing CDK14 though Wnt/\u03b2-catenin signaling pathway

BackgroundMounting evidences displayed that miRNAs play crucial roles in tumor initiation and development. However, the regulation and relevant mechanism of miR-1825 in glioblastoma (GBM) remain unclear.MethodsqRT-PCR was used to detect miR-1825 and CDK14 mRNA expression. Western blot was applied for testing protein levels (VEGF, E-cadherin, N-cadherin, vimentin, β-catenin, c-myc, p-c-Jun). MTT and transwell assays were used for detecting GBM cell progression, including cell viability, migration, and invasion.ResultsThe results showed that miR-1825 was decreased in GBM tissue specimens by qRT-PCR and it was confirmed as a prognostic marker of GBM by Kaplan-Meier survival analysis. Moreover, we also found that miR-1825 upregulation suppressed GBM cell viability, tumor growth, invasion, and migration. Furthermore, CDK14 was first identified as the direct target of miR-1825 by Luciferase reporter assay. CDK14 acted as an oncogene in GBM development by immunohistochemistry. In addition, Western blot analysis demonstrated that miR-1825 regulated Wnt/β-catenin signaling pathway in GBM development.ConclusionIn conclusion, miR-1825 upregulation suppressed GBM progression by targeting CDK14 through Wnt/β-catenin pathway.

LEF1-AS1 is implicated in the malignant development of glioblastoma via sponging miR-543 to upregulate EN2

Glioblastoma (GBM) has been regarded as the most aggressive disease in the nervous system. Accumulating literatures have illustrated the crucial role of competing endogenous RNAs (ceRNAs) network in the pathogenesis and progression of various tumors. The promoting effect of LEF1-AS1 on GBM development has been previously identified. This study attempted to explore the underlying mechanism of LEF1-AS1 in GBM. Data of clinical GBM patients was downloaded from TCGA and GEO databases. The proliferative ability, clonogenic vitality, invasive, and migratory capabilities of GBM cells were measured using Cell counting kit-8 (CCK-8), colony formation and transwell assays. Luciferase reporter gene analysis was performed to verify the correlations between LEF1-AS1/EN2 and miR-543. qRT-PCR and western blotting were implemented to evaluate the mRNA and protein levels, respectively. Our results consolidated that LEF1-AS1 was highly expressed in GBM tissue specimens and its up-regulation induced unfavorable prognosis. The loss/gain-of-function analyses verified that LEF1-AS1 promoted the GBM cell malignant behaviors. Mechanically, LEF1-AS1 acted as a ceRNA for miR-543 and positively regulated engrailed homeobox 2 (EN2) expression. Down-regulation of miR-543 elevated GBM cell malignant behaviors, which was reversed by LEF1-AS1 knockdown. Meanwhile, the LEF1-AS1 inhibition could arrest the promoting effect of high-regulated EN2 on GBM cell aggressiveness and vice versa. In conclusion, our findings identified LEF1-AS1 as a ceRNA for miR-543 and showed that EN2 was positively regulated by LEF1-AS1. The LEF1-AS1/miR-543/EN2, as a novel ceRNA network, was implicated in the progression of GBM, which provided a novel insight for GBM treatment.

IMMU-17. TARGETING GLIOBLASTOMA WITH DNAM-1-BASED CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS

Abstract BACKGROUND Genetically engineered T cells that express a chimeric antigen receptor (CAR) have shown powerful anti-tumor activity in extracranial malignancies. This concept is now also being explored against glioblastoma. However, many single target antigens used for CAR cell therapy are non-homogeneously expressed. We assessed the therapeutic potential of CAR T cells targeting 2 antigens which are homogeneously expressed by glioma cells which reduces the probability of tumor immune escape due to antigen loss. METHODS We analyzed the expression of CD112 and CD155, which are ligands to the activating immune cell receptor DNAX Accessory Molecule-1 (DNAM-1), in a panel of mouse and human glioma cell lines as well as in human glioblastoma samples and generated glioma cells with a CD112 or CD155 knock-out. To exploit the specific binding properties of DNAM-1, we generated first or second-generation CAR T cells that use DNAM-1 as an antigen-binding domain and investigated their anti-tumor activity in vitro and in vivo using syngeneic orthotopic mouse glioma models. RESULTS CD112 and CD155 are homogeneously expressed in mouse and human glioma cell lines as well as human glioblastoma tissue specimens. CRISPR/Cas9-mediated knock-out of CD112 or CD155 affected the migration of glioma cells, but had no impact on the proliferation or susceptibility to irradiation or temozolomide. DNAM-1-based CAR T cells exerted high cytolytic activity and secretion of various effector cytokines in vitro. Upon intravenous administration, DNAM-1-based CAR T cells did not exert significant toxicity, homed to the tumor site in the brain and prolonged the survival of orthotopic glioma-bearing mice with durable anti-tumor responses in a fraction of mice. CONCLUSION CD112 and CD155 represent attractive targets for glioma immunotherapy using genetically engineered immune cells. Based on the data obtained from our preclincal assessment of DNAM-1-based CAR T cells, this immunotherapeutic strategy might also be explored in human glioma patients.

CRN2 binds to TIMP4 and MMP14 and promotes perivascular invasion of glioblastoma cells

CRN2 is an actin filament binding protein involved in the regulation of various cellular processes including cell migration and invasion. CRN2 has been implicated in the malignant progression of different types of human cancer. We used CRN2 knock-out mice for analyses as well as for crossbreeding with a Tp53/Pten knock-out glioblastoma mouse model. CRN2 knock-out mice were subjected to a phenotyping screen at the German Mouse Clinic. Murine glioblastoma tissue specimens as well as cultured murine brain slices and glioblastoma cell lines were investigated by immunohistochemistry, immunofluorescence, and cell biological experiments. Protein interactions were studied by immunoprecipitation, pull-down, and enzyme activity assays. CRN2 knock-out mice displayed neurological and behavioural alterations, e.g. reduced hearing sensitivity, reduced acoustic startle response, hypoactivity, and less frequent urination. While glioblastoma mice with or without the additional CRN2 knock-out allele exhibited no significant difference in their survival rates, the increased levels of CRN2 in transplanted glioblastoma cells caused a higher tumour cell encasement of murine brain slice capillaries. We identified two important factors of the tumour microenvironment, the tissue inhibitor of matrix metalloproteinase 4 (TIMP4) and the matrix metalloproteinase 14 (MMP14, synonym: MT1-MMP), as novel binding partners of CRN2. All three proteins mutually interacted and co-localised at the front of lamellipodia, and CRN2 was newly detected in exosomes. On the functional level, we demonstrate that CRN2 increased the secretion of TIMP4 as well as the catalytic activity of MMP14. Our results imply that CRN2 represents a pro-invasive effector within the tumour cell microenvironment of glioblastoma multiforme.

5‑Azacytidine treatment results in nuclear exclusion of DNA methyltransferase‑1, as well as reduced proliferation and invasion in human cytomegalovirus‑infected glioblastoma cells.

Glioblastoma (GBM) is the most aggressive form of brain tumor in adults, with a devastating outcome. Emerging evidence shows that human cytomegalovirus (HCMV) proteins and nucleic acids are present in GBM tissues. DNA methylation is important for the initiation and progression of cancer and is an established host response against invading nucleic acids. The expression and localization of DNA methyltransferase 1 (DNMT‑1) was assessed, and the effects of DNA methylation inhibitor 5‑azacytidine (5AZA) were analyzed in the context of the viral replication, proliferation and invasion capacities of HCMV‑infected GBM U343MG cells. In addition, the expression of various HCMV proteins and DNMT‑1 was examined in GBM tissue specimens obtained from five patients. DNMT‑1 was localized in the nucleus of cells expressing HCMV‑immediate early, whereas in cells expressing HCMV‑glycoprotein gB (gB), extranuclear/cytoplasmic localization was observed. This was also observed in vitro in U343MG cells. In addition, DNMT‑1 was localized to the extranuclear/cytoplasmic space of cells lining blood vessel walls within the GBM tumors. Treatment of infected U343MG cells with 5AZA did not affect viral replication, but reduced cell invasion and proliferation (P=0.05 and P<0.0001, respectively). However, 5AZA treatment of uninfected cells did not affect cell invasion (P=0.09), but proliferation was significantly reduced (P<0.0001). These findings may be of importance in further investigations aimed at using DNA methylation and viral inhibitors in GBM therapy.

Expression of long intergenic non-coding RNA, regulator of reprogramming, and its prognostic value in patients with glioblastoma.

Long intergenic non-coding RNA, regulator of reprogramming ( LINC-ROR) is a newly identified cytoplasmic long non-coding RNA (lncRNA), which has been found to be dysregulated in different cancers. The present work aimed to quantify LINC-ROR expression profile and assess the tumor proteins p53 and caspase 3 expressions in glioblastoma tissue specimens compared to non-cancer tissues, and to correlate these expression levels with the available clinicopathological and survival data. LINC-ROR relative expression in 57 glioblastoma cancer tissues and 10 non-cancer tissues was quantified by real-time polymerase chain reaction (qPCR). In addition, methylation-specific PCR of O-6-methylguanine-DNA methyltransferase ( MGMT) promoter and immunohistochemical expression of apoptosis related proteins: p53 and caspase 3 were performed. The up-regulation of LINC-ROR was encountered in 89.5% of patients. The higher expression of LINC-ROR was associated with poor disease progression-free and overall survival as well as a younger age of patients ( P=0.036). p53 protein was expressed only in glioblastoma but not in non-cancer tissues while caspase 3 was weakly expressed in most non-cancer tissues and in varying degrees in glioblastoma (24% weak, 30% moderate, and 16% strong expression). The Kaplan-Meier survival plot illustrated poor survival in glioblastoma patients with over-expressed LINC-ROR ( P=0.010) and down-regulated p53 ( P=0.002). Multivariate analysis showed that glioblastoma patients were clustered into two distinct groups based on LINC-ROR expression profile, p53 staining levels and patients' overall survival. LINC-ROR up-regulation may have a role in glioblastoma tumorigenesis and could be a potential prognostic marker for this fatal disease.

Upregulated circular RNA circ_0074027 promotes glioblastoma cell growth and invasion by regulating miR-518a-5p/IL17RD signaling pathway

Glioblastoma (GBM) is one of the major cause of the cancer-related fatality worldwide. Several circular RNAs (circRNAs) have been observed to exert functions in GBM. The current study is aimed to explore the potential mechanism of circ_0074027 via miR-518a-5p and IL17RD in GBM progression. Circ_0074027 expression was determined in a cohort of 50 pairs of GBM specimens and five cell lines by qRT-PCR. In addition, the association between circ_0074027 expression and its clinical value was analyzed by Fisher’s exact test. Cell growth, clone formation, apoptosis, migration and invasion was evaluated after overexpress or knockdown the expression of circ_0074027 in GBM cells. Dual luciferase reporter assays were conducted to evaluate the relevant intermolecular target relationships. Circ_0074027 expression was evidently upregulated in GBM tissue specimens and cells compared to the adjacent non-tumorous tissues and NHA, respectively. The upregulation of circ_0074027 is related to clinical severity and exerts oncogenic functions in GBM. Moreover, circ_0074027 could sponge miR-518a-5p to release its suppression on IL17RD. Our findings provide evidence that circ_0074027 plays an oncogenic role in GBM by regulating miR-518a-5p/IL17RD signaling.

MicroRNA-485 inhibits malignant biological behaviour of glioblastoma cells by directly targeting PAK4.

Glioblastoma (GBM), which is characterised by rapid growth, cellular heterogeneity, angiogenesis, extensive invasion, hypoxia and necrosis, is the most common histological subtype of glioma in adults. MicroRNA(miRNA) dysregulation is a common feature of human cancers, including GBM. Previous studies have suggested that miRNAs are a novel class of regulatory molecules in various human cancers. Therefore, miRNAs may be investigated as a novel candidate and screening tool in the clinical diagnosis, therapy and prognosis of GBM. Recent accumulating evidence has demonstrated that miRNA‑485(miR‑485) is involved in the development and progression of several types of human cancers. However, the expression level, exact role and underlying mechanisms of miR‑485 in GBM remain unclear. In this study, miR‑485 was downregulated in both GBM tissue specimens and cell lines. miR‑485 overexpression inhibited GBM cell proliferation, colony formation, migration and invasion; increased apoptosis invitro; and reduced tumour growth invivo. In addition, p21‑activated kinase4 (PAK4) was demonstrated to be a direct and functional target of miR‑485 in GBM. Furthermore, PAK4 was upregulated in GBM tissues and negatively correlated with miR‑485 expression. Moreover, PAK4 knockdown exhibited a similar effect to miR‑485 overexpression in GBM cells. Enforced expression of PAK4 rescued miR‑485 tumour‑suppressor functions in GBM cells. miR‑485 inhibited the activation of the AKT and ERK signalling pathways in GBM. These results indicate that miR‑485 acts as a tumour suppressor in GBM by, at least partially, directly targeting PAK4 and regulating the AKT and ERK signalling pathways. Thus, miR‑485 may be a potential target for the treatment of patients with GBM.

P02.04 * MICRORNA-MEDIATED DOWN-REGULATION OF NKG2D LIGAND EXPRESSION REDUCES GLIOMA CELL IMMUNOGENICITY

Glioblastoma is a primary brain tumor with a dismal prognosis despite comprehensive therapeutic regimens. It is characterized by diffuse infiltration of the surrounding healthy brain tissue, well-adapted to hypoxic conditions and regarded as paradigmatic for tumor-associated immunosuppression. One of the major activating receptors of natural killer (NK) cells is NKG2D. It binds to at least 8 ligands (NKG2DL) which are induced after malignant transformation and cellular stress. Regulation of NKG2DL expression may be affected by endogenous RNA molecules known as microRNA (miRNA). Here, we aimed at characterizing the role of miRNA in the control of NKG2DL expression in glioma cells. We selected 6 miRNA that were described or predicted to target NKG2DL. Three of the miRNA candidates, miR-20a, miR-93 and miR-106b, were expressed in glioma cell lines and were also detected in glioblastoma tissue specimens. Silencing of these miRNA with locked nucleic acid (LNA) molecules resulted in an up-regulation of NKG2DL cell surface levels which translated into increased sensitivity to immune cell killing. This effect was reversed by neutralizing NKG2D antibodies, confirming that enhanced immune cell lysis upon miRNA silencing was mediated through the NKG2D system. We conclude that the expression of several miRNA may contribute to the immune escape of glioma cells at the level of the NKG2D system. Therapeutic targeting of miRNA that regulate NKG2DL levels may therefore represent a promising approach to allow for more potent immune responses against glioblastoma.

Suppression of G-protein–Coupled Receptor Kinase 3 Expression Is a Feature of Classical GBM That Is Required for Maximal Growth

G-protein-coupled receptor kinases (GRK) regulate the function of G-protein-coupled receptors (GPCR). Previously, we found that GPCR (CXCR4)-mediated astrocytoma growth was dependent upon abnormally sustained CXCR4 signaling and was correlated with decreased GRK-mediated receptor phosphorylation. As CXCR4 has also been implicated in the stimulation of high-grade glioma growth, we sought to determine whether dysregulation of GRK expression and/or function might also be present in high-grade gliomas. In an analysis of data from The Cancer Genome Atlas, we found that GRK3 expression is frequently decreased in glioblastoma (GBM) of the classical subtype, which possesses signature amplification or mutational activation of the epidermal growth factor (EGF) receptor. We tested the correlation between GRK3 expression and GBM subtypes, as well as the relationship between the activation of the EGF and other growth factor receptor pathways and GRK expression. In analyses of primary GBM tissue and RNA specimens, we found that GRK3 expression is correlated with established criteria for GBM subtyping including expression of EGF receptor, platelet-derived growth factor receptor (PDGFR)α, NF1, PTEN, CDKN2A, and neurofilament. We also found that established drivers of gliomagenesis, the EGF, PDGF, and TGF-β pathways, all regulate GRK expression. Coculture experiments, designed to mimic critical interactions between tumor and brain microvascular endothelial cells, showed that specifically increasing GRK3 expression reduced the trophic effect of endothelial cells on tumor cells. Together, these experiments show that GRK3 is a negative regulator of cell growth whose expression is preferentially reduced in GBM of the classical subtype as a consequence of activity in primary gliomagenic pathways.

The promoter hypermethylation status of GATA6, MGMT, and FHIT in glioblastoma.

Glioblastoma (GBM) is an aggressive and lethal cancer, accounting for the majority of primary brain tumors in adults. GBMs are characterized by large and small alterations in genes that control cell growth, apoptosis, angiogenesis, and invasion. Epigenetic alterations also affect the expression of cancer genes, either alone or in combination with genetic mechanisms. The current evidence suggests that hypermethylation of promoter CpG islands is a common epigenetic event in a variety of human cancers. A subset of GBMs is also characterized by a locus-specific and genome-wide decrease in DNA methylation. Epigenetic alterations are important in the molecular pathology of GBM. However, there are very limited data about these epigenetic alterations in GBM. Alterations in promoter methylations are important to understand because histone deacetylases are targets for drugs that are in clinical trial for GBMs. The aim of the current study was to investigate whether the promoter hypermethylation of putative tumor suppressor genes was involved in GBM. We examined the methylation status at the promoter regions of GATA6, MGMT, and FHIT using the methylation-specific polymerase chain reaction in 61 primary GBMs. Our results reveal that there is no promoter hypermethylation of FHIT in the examined GBM tissue specimens. In contrast, the promoter hypermethylation of GATA6 and MGMT was detected in 42.8 and 11.11% of GBMs, respectively. The frequency of MGMT promoter hypermethylation was low in the group of patients we evaluated. In conclusion, our study demonstrates that promoter hypermethylation of MGMT is a common event in GBMs, whereas GATA6 is epigenetically affected in GBMs. Furthermore, inactivation of FHIT by epigenetic mechanisms in GBM may not be associated with brain tumorigenesis.

Identification of a peptide that interacts with Nestin protein expressed in brain cancer stem cells

Glioma stem cells (GSCs) are presumably major culprits for brain tumor initiation, progression, and recurrence after conventional therapies. Thus, selective targeting and eradication of GSCs may provide a promising and effective therapeutic approach. Here, we isolated a GSC-targeting (GSCT) peptide that demonstrated selective binding affinity for many undifferentiated GSCs using in vitro phage display technology. This GSCT peptide binds to isotypes of Nestin proteins specifically expressed in GSCs, enabling it to target Nestin-positive cells in human glioblastoma tissues. In human glioblastoma tissue specimens, the fluorescence-conjugated GSCT peptide could visualize putative GSC populations, showing its possible use as a diagnostic agent. GSCT peptide is also internalized into undifferentiated GSCs specifically in vitro, and moreover, intravenously injected GSCT peptide effectively penetrated into tissues, specifically accumulated in gliomas that arise from subcutaneous and orthotopic implantation, and predominantly targeted Nestin-positive cells in these tumors. Thus, our GSCT peptide may be useful for the development of more promising therapeutic and diagnostic modalities that target GSCs in brain tumors.

Overexpression of Heparin-Binding Growth-Associated Molecule in Malignant Glioma Cells

The highly invasive and angiogenic characteristics of malignant gliomas depend on the production of growth factors and angiogenic factors. Heparin-binding growth-associated molecule (HB-GAM) is a secreted growth factor that is mitogenic for endothelial cells. To examine the expression profile of HB-GAM in malignant glioma cells, messenger ribonucleic acid (mRNA) expression was analyzed in 10 malignant glioma cell lines, two glioblastoma tissue specimens, and two normal brain tissue specimens by the reverse transcription-polymerase chain reaction. HB-GAM mRNA was expressed in all specimens including normal brain tissue specimens. Western blot analysis revealed that HB-GAM protein contents in glioma cell lines and glioblastoma tissues were 1.8 to 6.3 times higher than those in normal brain tissues. The effect of neutralizing anti-platelet-derived growth factor (PDGF) antibody was also examined on the production of HB-GAM in malignant glioma cells, since malignant glioma cells secrete PDGF that upregulates HB-GAM expression. Treatment of U251 and T98G glioblastoma cells with the anti-PDGF antibody did not affect the HB-GAM production. These results suggest that HB-GAM is overexpressed in malignant glioma cells and is involved in tumor growth.

The Human Fn14 Receptor Gene Is Up-Regulated in Migrating Glioma Cells in Vitro and Overexpressed in Advanced Glial Tumors

Glioblastoma multiforme comprises the majority of human brain tumors. Patients with glioblastoma multiforme have poor survival rates, with an average life expectancy of <1 year. To assess possible mechanisms and to potentially target invasive glioma cells, we previously measured the gene expression profiles of glioma cells under migration-activated or passive states. One of the genes identified was Fn14, which encodes a cell surface receptor for the tumor necrosis factor superfamily member named TWEAK. In this study, we show that Fn14 gene expression is induced in migration-activated glioma cells in vitro and significantly increases according to tumor grade in vivo (P < 0.01), with highest levels in glioblastoma tissue specimens. The in situ expression pattern of Fn14 mRNA and protein was confined to primary glioma cells and the vascular endothelium, with no detection in adjacent normal brain. Conversely, TWEAK mRNA levels are low in glioblastoma samples relative to normal brain tissue. In addition, activation of the Fn14 receptor by addition of recombinant TWEAK resulted in increased glioma cell migration in vitro. These results suggest a positive role for TWEAK and Fn14 in glioma progression and indicate that Fn14 gene expression may serve as a marker for invasive glioma cells.