Glioblastoma Multiforme Specimens Research Articles

Glioblastoma cellular MAP4K1 facilitates tumor growth and disrupts T effector cell infiltration.

MAP4K1 has been identified as a cancer immunotherapy target. Whether and how cancer cell-intrinsic MAP4K1 contributes to glioblastoma multiforme (GBM) progression remains unclear. We found that MAP4K1 was highly expressed in the glioma cells of human GBM specimens. High levels of MAP4K1 mRNA were prevalent in IDH-WT and 1p/19q non-codeletion gliomas and correlated with poor prognosis of patients. MAP4K1 silencing inhibited GBM cell proliferation and glioma growth. Transcriptome analysis of GBM cells and patient samples showed that MAP4K1 modulated cytokine‒cytokine receptor interactions and chemokine signaling pathway, including IL-18R and IL-6R Importantly, MAP4K1 loss down-regulated membrane-bound IL-18R/IL-6R by inhibiting the PI3K-AKT pathway, whereas MAP4K1 restoration rescued this phenotype and therefore GBM cell proliferation. MAP4K1 deficiency abolished GBM cell pro-proliferation responses to IL-18, suggesting an oncogenic role of MAP4K1 via the intrinsic IL-18/IL-18R pathway. In addition, GBM cell-derived MAP4K1 impaired T-cell migration and reduced CD8+ T-cell infiltration in mouse glioma models. Together, our findings provide novel insight into the pathological significance of GBM cell-intrinsic MAP4K1 in driving tumor growth and immune evasion by remodeling cytokine-chemokine networks.

DNAR-04. BOTH P53 CODON 72 ARG/ARG AND PRO/ARG GENOTYPES IN GLIOBLASTOMA MULTIFORME PATIENTS HAVE A BETTER PROGNOSIS IN BEVACIZUMAB TREATMENT

Abstract BACKGROUND In glioblastoma multiforme (GBM), bevacizumab increased progression-free survival but it has failed to improve overall survival (OS) in controlled trials. This study, therefore, aimed to retrospectively analyze the polymorphisms of p53 codon 72 and the clinical characteristics of GBM specimens from Taiwanese patients. METHODS The polymorphisms of p53 codon 72 in 99 patients with GBM treated at Taichung Veterans General Hospital in Taiwan from 2007 to 2017 were analyzed using direct DNA sequencing and PCR-RFLP analysis. RESULTS We found that among these GBM patients, the polymorphisms of p53 codon 72 were not directly associated with the overall survival of GBM, both the Arg/Arg and Arg/Pro genotypes were associated with significant benefits in terms of overall survival in patients treated with CCRT plus bevacizumab compared to patients treated with CCRT alone. CONCLUSIONS This pilot study suggests that both the Arg/Arg and Arg/Pro genotypes of p53 codon 72 polymorphism may have value as independent prognostic or predictive parameters for bevacizumab treatment response and failure. Relatedly, the results of the study further demonstrate the utility of stratifying GBM patientsaccording to bevacizumab sensitivity.

ATF4-dependent fructolysis fuels growth of glioblastoma multiforme

Excessive consumption of fructose in the Western diet contributes to cancer development. However, it is still unclear how cancer cells coordinate glucose and fructose metabolism during tumor malignant progression. We demonstrate here that glioblastoma multiforme (GBM) cells switch their energy supply from glycolysis to fructolysis in response to glucose deprivation. Mechanistically, glucose deprivation induces expression of two essential fructolytic proteins GLUT5 and ALDOB through selectively activating translation of activating transcription factor 4 (ATF4). Functionally, genetic or pharmacological disruption of ATF4-dependent fructolysis significantly inhibits growth and colony formation of GBM cells in vitro and GBM growth in vivo. In addition, ATF4, GLUT5, and ALDOB levels positively correlate with each other in GBM specimens and are poor prognostic indicators in GBM patients. This work highlights ATF4-dependent fructolysis as a metabolic feature and a potential therapeutic target for GBM.

Abstract 6058: The serine synthesis pathway contributes to the radiation-induced metabolic plasticity in glioblastoma multiforme

Abstract Purpose: Glioblastoma multiforme (GBM) remains a fatal disease despite aggressive treatment approaches. Post-surgical radiation therapy (RT) is the only treatment that significantly improves survival of GBM patients but recurrence is inevitable. Elucidating the mechanisms of resistance to RT could transform GBM patient outcomes. The purpose of this study was to elucidate the effect of radiation on the serine synthesis pathway (SSP) and the role of SSP in boosting antioxidant defenses in irradiated GBM cells and promoting radioresistance. Methods: We compared copy number alterations (CNA) and amplification of SSP enzymes (PHGDH, PSAT1, PSPH, SHMT1/2) in low-grade gliomas (LGG, n = 511) and GBM (n = 575) via TCGA pan-cancer analysis. In vitro HPLC/MS-based metabolomics assays were used to study the changes in overall metabolite levels induced by radiation in patient-derived GBM specimens. Targeted metabolomics with 13C-labeled glucose was used for tracking the fate of glucose towards SSP intermediates and antioxidant species. Gene expression levels after radiation were measured via molecular biology approaches. De novo SSP was targeted via pharmacological inhibition of PHGDH. Modified clonogenic, sphere forming assays (SFA) on gliomasphere cultures were used to determine radiosensitivity. Dependence on extracellular serine/glycine was determined via serine/glycine depletion experiments. Results: The TCGA analysis revealed increased CNAs in all the de novo SSP enzymes in GBM tumors relative to the LGGs. 82% of GBM tumors had CNAs or amplification of the PSPH gene, which codes for the ultimate enzyme in serine synthesis. In vitro metabolomics assays revealed that radiation increases the levels of serine, glycine and cystine in gliomaspheres, and this is accompanied by elevated levels of reduced glutathione (GSH). Targeted glucose metabolomics revealed radiation-induced upregulation of the de novoSSP, with a concomitant increase in gene expression of SSP enzymes. Downstream of the SSP, metabolomics data show that irradiated GBM cells elevate nucleotide and precursor levels, likely to support DNA damage repair mechanisms after radiation. The inhibition of PHGDH, the first rate-limiting enzyme in de novo SSP, significantly reduced GBM cell survival in vitro. Extracellular serine/glycine appeared to also be important contributors to radiation survival. Gene expression of SLC1A4, an amino acid transporter, was upregulated by radiation, while depletion of extracellular serine/glycine led to significant decreased survival in in vitro SFAs. Conclusion: Here we provide evidence that the de novo serine synthesis pathway is an important contributor to the metabolic reprogramming of GBM cells after radiation that promotes radiation resistance. These data call for further investigations to evaluate the radiosensitizing potential of targeting de novo SSP in GBM. Citation Format: Justine Bailleul, Yangjingyi Ruan, Erina Vlashi. The serine synthesis pathway contributes to the radiation-induced metabolic plasticity in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6058.

TAMI-58. TARGETING GLIOBLASTOMA BY ACTIVATING VIRAL SPECIFIC TISSUE-RESIDENT MEMORY T CELLS

Abstract Immunotherapy with the success of checkpoint blockade brings hope for cancer treatment with enduring or complete responses in various types of advanced malignancies, however, it has not benefited a number of so called “cold tumors”, such as glioblastoma multiforme (GBM), the most common and aggressive adult brain cancer. GBM is invariably lethal with a median survival of less than 15 months and characterized by a highly immunosuppressive tumor microenvironment, which desires new means of GBM immune-reactivation. Tissue-resident memory CD8+ T (TRM) cells are non-recirculating memory T cell subpopulation that resides permanently in peripheral tissues. TRM cells provide long-lived protective immunity against not only local pathogen infection but also tumor development through immediate effector function and recruitment of circulating immune cells upon antigen re-exposure. In this study, we found that memory T cells specific to some common human viruses that have infected almost everybody exist in clinical GBM specimens and can be activated by viral peptides, which are MHC I restricted CD8+ T cell epitopes derived from those viruses. In orthotopic GBM immunocompetent mouse models, we detected abundant virus specific TRM cells in the tumor microenvironment of GBMs intracranially established in mice that were previously exposed to the virus. Excitingly, intratumoral injection of viral peptides stimulated the reactivation of TRM cells, indicated by secretion of immuno-stimulatory cytokines, and demonstrated significant anti-tumor efficacy. This study provides proof of principle for TRM cell-based GBM therapy, which is a novel therapeutic paradigm with the translational potential for this deadly malignancy.

Gangliosides of Human Glioblastoma Multiforme: A Comprehensive Mapping and Structural Analysis by Ion Mobility Tandem Mass Spectrometry

Glioblastoma multiforme (GBM), a malignant, highly aggressive, grade IV brain tumor, which rapidly infiltrates into the nearby tissue, has drawn a significant amount of attention because of its poor prognosis and the limited treatment options available. In GBM, nearly all tumor cells exhibit aberrant cell-surface glycosylation patterns due to the alteration of their biosynthesis or postsynthesis modification process. Since gangliosides (GGs) are acknowledged as tumor-associated antigens, we have carried out here a comprehensive profiling of native ganglioside mixtures extracted and purified from GBM specimens. For this purpose, high performance ion mobility separation mass spectrometry (IMS MS) was thoroughly optimized to allow the discovery of GBM-specific structures and the assessment of their roles as tumor markers or possible associated antigens. GG separation by IMS according to the charge state, carbohydrate chain length, degree of sialylation, and ceramide composition led to the identification of no less than 160 distinct components, which represents 3-fold the number of structures identified before. The detected GGs and asialo-GGs were found characterized by a high heterogeneity in their ceramide and glycan compositions, encompassing up five Neu5Ac residues. The tumor was found dominated in equal and high proportions by GD3 and GT1 forms, with a particular incidence of C24:1 fatty acids in the ceramide. By the occurrence of only one mobility feature and the diagnostic fragment ions, the IMS tandem MS conducted using collision-induced dissociation (CID) disclosed for the first time the presence of GT1c(d18:1/24:1) newly proposed here as a potential GBM marker.

Microglia/macrophages express alternative proangiogenic factors depending on granulocyte content in human glioblastoma.

Myeloid cells are an inherent part of the microenvironment of glioblastoma multiforme (GBM). There is growing evidence for their participation in mechanisms of tumor escape, especially in the development of resistance following initially promising anti-VEGF/VEGFR treatment. Thus, we sought to define the capability of myeloid cells to contribute to the expression of proangiogenic molecules in human GBM. We investigated GBM specimens in comparison with anaplastic astrocytoma (WHO grade III) and epilepsy patient samples freshly obtained from surgery. Flow cytometric analyses revealed two distinct CD11b+ CD45+ cell populations in GBM tissues, which were identified as microglia/macrophages and granulocytes. Due to varied granulocyte influx, GBM samples were subdivided into groups with low (GBM-lPMNL) and high (GBM-hPMNL) numbers of granulocytes (polymorphonuclear leukocytes; PMNL), which were related to activation of the microglia/macrophage population. Microglia/macrophages of the GBM-lPMNL group were similar to those of astrocytoma specimens, but those of GBM-hPMNL tissues revealed an altered phenotype by expressing high levels of CD163, TIE2, HIF1α, VEGF, CXCL2 and CD13. Although microglia/macrophages represented the main source of alternative proangiogenic factors, additionally granulocytes participated by production of IL8 and CD13. Moreover, microglia/macrophages of the GBM-hPMNL specimens were highly associated with tumor blood vessels, accompanied by remodeling of the vascular structure. Our data emphasize that tumor-infiltrating myeloid cells might play a crucial role for limited efficacy of anti-angiogenic therapy bypassing VEGF-mediated pathways through expression of alternative proangiogenic factors. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

MicroRNA‑422a functions as a tumor suppressor in glioma by regulating the Wnt/β‑catenin signaling pathway via RPN2.

MicroRNAs (miRs), which act as crucial regulators of oncogenes and tumor suppressors, have been confirmed to play a significant role in the initiation and progression of various malignancies, including glioma. The present study analyzed the expression and roles of miR-422a in glioma, and reverse transcription-quantitative PCR confirmed that miR-422a expression was significantly lower in glioblastoma multiforme (GBM) samples and cell lines compared with the low-grade glioma samples and the H4 cell line, respectively. miR-422a overexpression suppressed proliferation and invasion, and induced apoptosis in LN229 and U87 cell lines. Luciferase reporter assay, western blotting and RNA immunoprecipitation analysis revealed that ribophorin II (RPN2) is a direct functional target of miR-422a. Additionally, the overexpression of RPN2 partially reversed the miR-422a-mediated inhibitory effect on the malignant phenotype. Mechanistic investigation demonstrated that the upregulation of miR-422a inhibited β-catenin/transcription factor 4 transcriptional activity, at least partially through RPN2, as indicated by in vitro and in vivo experiments. Furthermore, RPN2 expression was inversely correlated with miR-422a expression in GBM specimens and predicted patient survival in the Chinese Glioma Genome Atlas, UALCAN, Gene Expression Profiling Interactive Analysis databases. In conclusion, the present data reveal a new miR-422a/RPN2/Wnt/β-catenin signaling axis that plays critical roles in glioma tumorigenesis, and it represents a potential therapeutic target for GBM.

Both p53 codon 72 Arg/Arg and pro/Arg genotypes in glioblastoma multiforme are associated with a better prognosis in bevacizumab treatment

BackgroundIt has previously been shown that bevacizumab, when added to chemotherapy, improved overall survival in several cancers. In glioblastoma multiforme (GBM), bevacizumab increased progression-free survival and it is widely used for tumor recurrence, though it has failed to improve overall survival (OS) in controlled trials. However, an effective biomarker for predicting the prognosis of bevacizumab treatment has yet to be identified. This study, therefore, aimed to retrospectively analyze the polymorphisms of p53 codon 72 and the clinical characteristics of GBM specimens from Taiwanese patients.MethodsThe polymorphisms of p53 codon 72 in 99 patients with GBM treated at Taichung Veterans General Hospital in Taiwan from 2007 to 2017 were analyzed using direct DNA sequencing and PCR-RFLP analysis.ResultsWe found that among these GBM patients, the distribution of codon 72 polymorphisms was 28.3% for proline homozygotes (Pro/Pro), 38.4% for arginine homozygotes (Arg/Arg), and 33.3% for proline/arginine heterozygotes (Pro/Arg). Although the polymorphisms of p53 codon 72 were not directly associated with the overall survival of GBM, both the Arg/Arg and Arg/Pro genotypes were associated with significant benefits in terms of overall survival in patients treated with CCRT plus bevacizumab compared to patients treated with CCRT alone.ConclusionsThis pilot study suggests that both the Arg/Arg and Arg/Pro genotypes of p53 codon 72 polymorphism may have value as independent prognostic or predictive parameters for bevacizumab treatment response and failure. Relatedly, the results of the study further demonstrate the utility of stratifying GBM patients according to bevacizumab sensitivity.

CCR2 of Tumor Microenvironmental Cells Is a Relevant Modulator of Glioma Biology.

Glioblastoma multiforme (GBM) shows a high influx of tumor-associated macrophages (TAMs). The CCR2/CCL2 pathway is considered a relevant signal for the recruitment of TAMs and has been suggested as a therapeutic target in malignant gliomas. We found that TAMs of human GBM specimens and of a syngeneic glioma model express CCR2 to varying extents. Using a Ccr2-deficient strain for glioma inoculation revealed a 30% reduction of TAMs intratumorally. This diminished immune cell infiltration occurred with augmented tumor volumes likely based on increased cell proliferation. Remaining TAMs in Ccr2-/- mice showed comparable surface marker expression patterns in comparison to wildtype mice, but expression levels of inflammatory transcription factors (Stat3, Irf7, Cox2) and cytokines (Ifnβ, Il1β, Il12α) were considerably affected. Furthermore, we demonstrated an impact on blood vessel integrity, while vascularization of tumors appeared similar between mouse strains. The higher stability and attenuated leakiness of the tumor vasculature imply improved sustenance of glioma tissue in Ccr2-/- mice. Additionally, despite TAMs residing in the perivascular niche in Ccr2-/- mice, their pro-angiogenic activity was reduced by the downregulation of Vegf. In conclusion, lacking CCR2 solely on tumor microenvironmental cells leads to enhanced tumor progression, whereby high numbers of TAMs infiltrate gliomas independently of the CCR2/CCL2 signal.

The Intratumoral Heterogeneity Reflects the Intertumoral Subtypes of Glioblastoma Multiforme: A Regional Immunohistochemistry Analysis.

Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor in adults. Despite extensive therapy the prognosis for GBM patients remains poor and the extraordinary therapy resistance has been attributed to intertumoral heterogeneity of glioblastoma. Different prognostic relevant GBM tumor subtypes have been identified based on their molecular profile. This approach, however, neglects the heterogeneity within individual tumors, that is, the intratumoral heterogeneity. Here, we detected the regional immunoreactivity by immunohistochemistry and immunofluorescence using nine different markers on resected GBM specimens (IDH wildtype, WHO grade IV). We found repetitive expression profiles, that could be classified into clusters. These clusters could then be assigned to five pathophysiologically relevant groups that reflect the previously described subclasses of GBM, including mesenchymal, classical, and proneural subtype. Our data indicate the presence of tumor differentiations and tumor subclasses that occur within individual tumors, and might therefore contribute to develop adapted, individual-based therapies.

EGFRvIII-CAR-T Cells with PD-1 Knockout Have Improved Anti-Glioma Activity.

Glioblastoma multiforme (GBM) is the most malignant form of the brain tumors. EGFR variant III (EGFRvIII) is expressed in about 30% of GBM specimens, but not expressed in normal brain tissues. Therefore, EGFRvIII protein offers an ideal CAR-T therapeutic target for EGFRvIII-positive GBM patients. PD-L1 is expressed in a variety of cancer cells, including GBM. Tumor-associated PD-L1 can bind to PD-1 on T cells and promote apoptosis of T cells, thus suppressing the anti-cancer immune response. In our current studies, PD-1WT EGFRvIII-CAR-T cells and PD-1KD EGFRvIII-CAR-T cells were generated. Cytokine production and lytic activity of these two CAR-T cells against to PD-L1WT EGFRvIII+ U373 cells or PD-L1KO EGFRvIII+ U373 cells were evaluated. The results showed that PD-1KD EGFRvIII-CAR-T cells and PD-1WT EGFRvIII-CAR-T cells showed same levels of interferon-γ (IFN-γ) and interleukin-2 (IL-2) production as well as cytolytic activity against PD-L1KO EGFRvIII+ U373 cells; however, PD-1KD EGFRvIII-CAR-T cells exhibited higher levels of IFN-γ and IL-2 production as well as cytolytic activity against PD-L1+ EGFRvIII+ U373 cells than that of PD-1WT EGFRvIII-CAR-T cells. PD-1KD EGFRvIII-CAR-T cells also exhibited higher anti-glioma activity and longer survival in mice in vivo than that of PD-1WT EGFRvIII-CAR-T cells. Taken together, our findings indicate that PD-1 knockout enhances lytic activity of EGFRvIII-CAR-T cells against PD-L1+ EGFRvIII+ GBM cells. These might provide a new insight into strategy of GBM CAR-T cell therapy.

Specific glioblastoma multiforme prognostic-subtype distinctions based on DNA methylation patterns.

DNA methylation is an important regulator of gene expression, and plays a significant role in carcinogenesis in the brain. Here, we explored specific prognosis-subtypes based on DNA methylation status using 138 Glioblastoma Multiforme (GBM) samples from The Cancer Genome Atlas (TCGA) database. The methylation profiles of 11,637 CpG sites that significantly correlated with survival in the training set were employed for consensus clustering. We identified three GBM molecular subtypes, and their survival curves were distinct from each other. Furthermore, ten feature CpG sites were obtained on conducting a weighted gene co-expression network analysis (WGCNA) of the CpG sites. We were able to classify the samples into high- and low-methylation groups, and classified the prognosis information of the samples after cluster analysis of the training set samples using the hierarchical clustering algorithm. Similar results were obtained in the test set and clinical GBM specimens. Finally, we found that a positive relationship existed between methylation level and sensitivity to temozolomide (or radiotherapy) or anti-migration ability of GBM cells. Taken together, these results suggest that the model constructed in this study could help explain the heterogeneity of previous molecular subgroups in GBM and can provide guidance to clinicians regarding the prognosis of GBM.

ER stress and UPR activation in glioblastoma: identification of a noncanonical PERK mechanism regulating GBM stem cells through SOX2 modulation

Patients with aggressive brain tumors, named glioblastoma multiforme (GBM), have a poor prognoses. Here we explored if the ER stress/unfolded protein response (UPR) is involved in the pathophysiology of GBM and may provide novel therapeutic targets. Immunohistochemical analyses of a tissue microarray containing primary GBM specimens showed strong variability in expression of the UPR markers GRP78/BiP, XBP1, and ATF4. Interestingly, high ATF4 expression was associated with poor overall survival suggesting involvement of PERK signaling in GBM progression. In vitro experiments using patient-derived neurospheres, enriched for GBM stem cells (GSCs), showed high sensitivity for the ER stressor thapsigargin (Tg) mainly via PERK signaling. In contrast, neurospheres-derived differentiated GBM cells were less sensitive likely due to lower UPR activity as indicated by comparative transcriptional profiling. Tg and Tunicamycin strongly reduced neurosphere forming ability of GSCs that was linked with potent PERK-dependent downregulation of SOX2 protein. Interestingly, SOX2 downregulation occurred directly via PERK, not requiring downstream activation of the PERK-UPR pathway. Moreover, PERK inactivation resulted in aberrant serum-induced differentiation of GBM neurospheres accompanied by persistent SOX2 expression, delayed upregulation of GFAP and reduced cell adherence. In conclusion, we provide evidence that PERK signaling contributes to the prognoses of primary GBM patients and identified PERK as a novel regulator of SOX2 expression and GSC differentiation. The role of PERK appeared to be pleiotropic involving UPR-dependent, as well as novel identified noncanonical mechanisms regulating SOX2. ER stress and PERK modulation appear to provide promising therapeutic targets for therapy in GBM.

Prognostic significance of contactin 3 expression and associated genes in glioblastoma multiforme.

Contactin 3 (CNTN3) is a member of the contactin family that is primarily expressed in the nervous system. However, to the best of our knowledge, expression of contactin and its role in the development and progression of brain tumours has not been studied. Although glioblastoma multiforme (GBM) is the most common malignant brain tumour, advances in therapeutic options for patients with GBM have been modest due to an incomplete understanding of the molecular mechanisms underlying development and progression. The aim of the present study was to examine the correlation between CNTN3 and its associated genes and the clinical outcome in patients with GBM. CNTN3 and the expression levels of associated genes were analysed in GBM datasets obtained from the SAGE Anatomical viewer website, Gene Expression Omnibus, Oncomine and The Cancer Genome Atlas. CNTN3 was significantly downregulated in patients with GBM. Subsequently, the expression of CNTN3 was further validated using immunohistochemistry in a cohort of GBM specimens. The immunohistochemistry results were consistent with the in silico analyses. Kaplan-Meier analysis indicated that patients with lower expression levels of CNTN3 had a significantly shorter overall survival (OS) time compared with patients with higher levels of CNTN3 expression. Univariate and multivariate Cox regression analyses demonstrated that CNTN3 expression was an independent prognostic indicator in patients with GBM. Furthermore, gene set enrichment analysis revealed that CNTN3 was associated with the receptor tyrosine-protein kinase (ErbB) signalling pathway. In the ErbB signalling pathway, epidermal growth factor receptor (EGFR) was negatively correlated with CNTN3. Taken together, these data suggest that lower expression levels of CNTN3 may be an independent biomarker that predicts poor OS time in patients with GBM, and that EGFR expression in the ErbB pathway may be associated with CNTN3 expression.

High expression of RAD18 in glioma induces radiotherapy resistance via down-regulating P53 expression

As a key regulator of DNA translesion synthesis (TLS) pathway, RAD18 is reported to be abnormally expressed in many kinds of cancers. In glioma, RAD18 was overexpressed in the primary and recurrent glioblastoma multiforme specimens, and its overexpression weakened ionizing radiation-induced apoptosis in glioma A172 cells. Moreover, A172 cells with mutational P53 also showed enhanced radiation resistance. And RAD18 activation induced by cyclin-dependent kinase 2 (CDK2) was repressed by P53. However, whether P53 involves in RAD18-induced radiation resistance remains unknown. Therefore, this study was conducted to explore the effects and mechanism of RAD18 in the radiation resistance of glioma and study P53 role in this process. Results showed that, RAD18 expression was obviously elevated in glioma tissues and cell lines such as U251, SHG-44, A172, U-87 MG and U-118 MG as compared with the normal brain tissues and neuroglia cells. Up-regulation of RAD18 in U-118 MG and A172 cells with lentivirus infection significantly increased cell growth and inhibited cell apoptosis, determined by CCK-8 and flow cytometry technologies. Besides, RAD18 overexpression enhanced cell growth and inhibited cell apoptosis after U-118 MG or A172 cells were irradiated at a dose of 4 Gy. On the contrary, silencing of endogenous RAD18 sensitized U-118 MG and A172 cells to radiation. Moreover, RAD18 and P53 proteins were co-located in the nucleus, and up-regulation of RAD18 decreased the expression of P53 protein and facilitated its nuclear export. Furthermore, cell growth promotion and cell apoptosis inhibition induced by RAD18 up-regulation were impaired when P53 expression was up-regulated under radiation condition. In a word, this study clarifies that RAD18 functions as a promoter in glioma progression and reduces glioma cells’ sensibility to radiation through down-regulating P53, which provides new strategies to overcome the radiation resistance in glioma.

Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs

Current progress in the field of next-generation transcriptome sequencing have contributed significantly to the study of various malignancies including glioblastoma multiforme (GBM). Differential sequencing of transcriptomes of patients and non-tumor controls has a potential to reveal novel transcripts with significant role in GBM. One such candidate group of molecules are long non-coding RNAs (lncRNAs) which have been proved to be involved in processes such as carcinogenesis, epigenetic modifications and resistance to various therapeutic approaches. To maximize the value of transcriptome sequencing, a proper protocol for library preparation from tissue-derived RNA needs to be found which would produce high quality transcriptome sequencing data and increase the number of detected lncRNAs. It is important to mention that success of library preparation is determined by the quality of input RNA, which is in case of real-life tissue specimens very often altered in comparison to high quality RNA commonly used by manufacturers for development of library preparation chemistry. In the present study, we used GBM and non-tumor brain tissue specimens and compared three different commercial library preparation kits, namely NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), SENSE Total RNA-Seq Library Prep Kit (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). Libraries generated using SENSE kit were characterized by the most normal distribution of normalized average GC content, the least amount of over-represented sequences and the percentage of ribosomal RNA reads (0.3–1.5%) and highest numbers of uniquely mapped reads and reads aligning to coding regions. However, NEBNext kit performed better having relatively low duplication rates, even transcript coverage and the highest number of hits in Ensembl database for every biotype of our interest including lncRNAs. Our results indicate that out of three approaches the NEBNext library preparation kit was most suitable for the study of lncRNAs via transcriptome sequencing. This was further confirmed by highly consistent data reached in an independent validation on an expanded cohort.

Crosslink between Temozolomide and PD-L1 immune-checkpoint inhibition in glioblastoma multiforme

BackgroundIn recent years, PD-1/PD-L1 immune checkpoint inhibitors have improved cancer therapy in many tumor types, but no benefit of immune checkpoint therapy has been found in glioblastoma multiforme (GBM). Based on the results of our earlier work, which showed a reduction of PD-L1 expression in patients treated with temozolomide (TMZ), we aimed to investigate the link between TMZ therapy and the immune control point target PD-L1.MethodsRNA-sequencing data from de-novo and recurrent glioblastoma were analyzed by AutoPipe algorithm. Results were confirmed either in a cell model by two primary and one established GBM cell line and specimens of de-novo and recurrent GBM. PD-L1 and pathway activation of the JAK/STAT pathway was analyzed by quantitative real-time PCR and western blot.ResultsWe found a significant downregulation of the JAK/STAT pathway and immune response in recurrent tumors. The cell model showed an upregulation of PD-L1 after IFNγ treatment, while additional TMZ treatment lead to a reduction of PD-L1 expression and JAK/STAT pathway activation. These findings were confirmed in specimens of de-novo and recurrent glioblastoma.ConclusionsOur results suggest that TMZ therapy leads to a down-regulation of PD-L1 in primary GBM cells. These results support the clinical findings where PD-L1 is significantly reduced in recurrent GBMs. If the target is diminished, it may also lead to impaired efficacy of PD-1/PD-L1 inhibitors such as nivolumab.

Identification of a novel role of IL-13R\u03b12 in human Glioblastoma multiforme: interleukin-13 mediates signal transduction through AP-1 pathway

BackgroundPreviously, we have demonstrated that Interleukin 13 receptor alpha 2 (IL-13Rα2) is overexpressed in approximate 78% Glioblastoma multiforme (GBM) samples. We have also demonstrated that IL-13Rα2 can serve as a target for cancer immunotherapy in several pre-clinical and clinical studies. However, the significance of overexpression of IL-13Rα2 in GBM and astrocytoma and signaling through these receptors is not known. IL-13 can signal through IL-13R via JAK/STAT and AP-1 pathways in certain cell lines including some tumor cell lines. Herein, we have investigated a role of IL-13/IL-13Rα2 axis in signaling through AP-1 transcription factors in human glioma samples in situ.MethodsWe examined the activation of AP-1 family of transcription factors (c-Jun, Fra-1, Jun-D, c-Fos, and Jun-B) after treating U251, A172 (IL-13Rα2 +ve) and T98G (IL-13Rα2 −ve) glioma cell lines with IL-13 by RT-qPCR, and immunocytochemistry (ICC). We also performed colorimetric ELISA based assay to determine AP-1 transcription factor activation in glioma cell lines. Furthermore, we examined the expression of AP-1 transcription factors in situ in GBM and astrocytoma specimens by multiplex-immunohistochemistry (IHC). Student t test and ANOVA were used for statistical analysis of the results.ResultsWe have demonstrated up-regulation of two AP-1 transcription factors (c-Jun and Fra-1) at mRNA and protein levels upon treatment with IL-13 in IL-13Rα2 positive but not in IL-13Rα2 negative glioma cell lines. Both transcription factors were also overexpressed in patient derived GBM specimens, however, in contrast to GBM cell lines, c-Fos is also overexpressed in patient derived specimens. Astrocytoma specimens showed lesser extent of immunostaining for IL-13Rα2 and three AP-1 factors compared to GBM specimens. By transcription factor activation assay, we demonstrated that AP-1 transcription factors (C-Jun and Fra-1) were activated upon treatment of IL-13Rα2 + GBM cell lines but not IL-13Rα2 − GBM cell line with IL-13. Our results demonstrate functional activity of AP-1 transcription factor in GBM cell lines in response to IL-13.ConclusionsThese results indicate that IL-13/IL-13Rα2 axis can mediate signal transduction in situ via AP-1 pathway in GBM and astrocytoma and may serve as a new target for GBM immunotherapy.

High expression of a novel splicing variant of VEGF, L-VEGF144 in glioblastoma multiforme is associated with a poorer prognosis in bevacizumab treatment

A previous study confirmed that a novel splicing variant of large vascular endothelial growth factor (L-VEGF) termed L-VEGF144, a nucleolus protein, is found in glioblastoma cells and specimens, but the actual biological function and clinical significance of L-VEGF144 remain unclear. In this study, we analyzed the expression of L-VEGF144 in 68 glioblastoma multiforme specimens using reverse transcriptase-polymerase chain reaction analysis. The results showed that the high expression of L-VEGF144 was associated with a poor prognosis in the bevacizumab plus concurrent chemoradiotherapy with temozolomide treatment. In addition, we constructed a series truncated and mutant form of L-VEGF144 to confirm that exon 6a of L-VEGF144 is able to engage in the nuclear importation and found that 8 lysines within exon 6a play a critical role in the nucleolus aggregation of L-VEGF144. Also, the transfection of the L-VEGF144 increased the number of nucleoli. Furthermore, the recombinant protein Flag-L-VEGF144 and commercial VEGF protein have similar growth stimulatory activities in terms of inducing glioblastoma cell proliferation in vitro. Taken together, these results indicated that the expression of L-VEGF144 could potentially serve as an independent indicator of poor prognosis in bevacizumab treatment.