Expert opinion on translational research for advanced glioblastoma treatment

Malignant gliomas are known to be one of the most difficult diseases to diagnose and treat because of the infiltrative growth pattern,rapid progression,and poor prognosis.Many antitumor drugs are not ideal for the treatment of gliomas due to the blood-brain barrier.Temozolomide(TMZ)is a DNA alkylating agent that can cross the blood-brain barrier.As the only first-line chemotherapeutic drug for malignant gliomas at present,TMZ is widely utilized to provide a survival benefit;however,some patients are inherently insensitive to TMZ.In addition,patients could develop acquired resistance during TMZ treatment,which limits antitumor efficacy.To clarify the mechanism underlying TMZ resistance,numerous studies have provided multilevel solutions,such as improving the effective concentration of TMZ in tumors and developing novel small molecule drugs.This review discusses the in-depth mechanisms underlying TMZ drug resistance,thus aiming to provide possibilities for the establishment of personalized therapeutic strategies against malignant gliomas and the accelerated development and transformation of new targeted drugs.

Identification of the E2F1-RAD51AP1 axis as a key factor in MGMT-methylated GBM TMZ resistance

Objective:Epidermal growth factor receptor variant III(EGFRvIII)is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme(GBM).Temozolomide(TMZ)is a standard chemotherapeutic for GBM,but TMZ treatment benefits are compromised by chemoresistance.This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance.Methods:CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM.Western blot,realtime PCR,flow cytometry,and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1(RAD51AP1).Results:Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells.Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment.Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells.Knockdown of E2F1 increased sensitivity to TMZ.Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1,mediates TMZ resistance,and has a potential E2F1 binding site on the promoter.Knockdown of RAD51AP1 enhanced the sensitivity of TMZ;however,overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells.Furthermore,RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase(MGMT)expression.The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated,but not MGMT-unmethylated TMZ-treated GBM patients.Conclusions:Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment.RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair.Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.

Omics-based integrated analysis identified ATRX as a biomarker associated with glioma diagnosis and prognosis

Objective:ATRX is a multifunctional protein that is tightly regulated by and implicated in transcriptional regulation and chromatin remodeling.Numerous studies have shown that genetic alterations in ATRX play a significant role in gliomas.This study aims to further determine the relationship between ATRX and glioma prognosis and identify possible mechanisms for exploring the biological significance of ATRX using large data sets.Methods:We used The Cancer Genome Atlas(TCGA)database and 130 immunohistochemical results to confirm the difference in ATRX mutations in high-and low-grade gliomas.An online analysis of the TCGA glioma datasets using the cBioPortal platform was performed to study the relationship between ATRX mutations and IDH1,TP53,CDKN2 A and CDKN2 B mutations in the corresponding TCGA glioma dataset.In combination with clinical pathology data,the biological significance of the relationships were analyzed.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses and annotations of all adjacent genes in the network were performedin the Database for Annotation,Visualization and Integrated Discovery(DAVID)and R language.A protein-protein interaction(PPI)network was constructed,and the interactions of all adjacent nodes were analyzed by the String database and using Cytoscape software.Results:In the selected TCGA glioma datasets,a total of 2,228 patients were queried,21%of whom had ATRX alterations,which co-occurred frequently with TP53 and IDH1 mutations.ATRX alterations are associated with multiple critical molecular events,which results in a significantly improved overall survival(OS)rate.In low-grade gliomas,ATRX mutations are significantly associated with multiple important molecular events,such as ZNF274 and FDXR at mRNA and protein levels.A functional cluster analysis revealed that these genes played a role in chromatin binding and P53,and a link was observed between ATRX and IDH1 and TP53 in the interaction network.ATRX and TP53 are important nodes in the network and have potential links with the blood oxygen imbalance.Conclusions:ATRX mutations have clinical implications for the molecular diagnosis of gliomas and can provide diagnostic and prognostic information for gliomas.ATRX is expected to serve as a new therapeutic target.

Boosting of the enhanced permeability and retention effect with nanocapsules improves the therapeutic effects of cetuximab

Objective:The introduction of therapeutic antibodies(tAbs)into clinical practice has revolutionized tumor treatment strategies,but their tumor therapy efficiency is still far below expectations because of the rapid degradation and limited tumor accumulation of tAbs.Methods:We developed a nanocapsule-based delivery system to induce the self-augmentation of the enhanced permeability and retention(EPR)effect.This system constantly penetrated across the blood-tumor barrier into the tumor while avoiding the attack of tAbs by the immune system.The biodistribution and therapeutic effect were tested with single dose administration of nanocapsule-tAbs in vivo.Results:The accumulation of Nano(cetuximab)within subcutaneous PC9 tumors was gradually enhanced over 6 days after single dose administration,which was contrary to the biodistribution of native cetuximab.Nano(cetuximab)accumulated in tumor tissues via the EPR effect and released cetuximab.The released cetuximab acted on vascular endothelial cells to destroy the blood-tumor barrier and induce self-augmentation of the EPR effect,which in turn contributed to further tumor accumulation of long-circulating Nano(cetuximab).Compared with single dose administration of native cetuximab,Nano(cetuximab)showed an effective tumor suppressive effect for 3 weeks.Conclusions:The nanocapsule-based delivery system efficiently delivered tAbs to tum or tissues and released them to boost the EPR effect,which facilitated further tumor accumulation of the tAbs.This novel self-augmentation of the EPR effect facilitated by the biological characteristics of tAbs and nanotechnology contributed to the improvement of the therapeutic effect of tAbs,and stimulated new ideas for antibody-based tumor therapy.

TGFβ signaling-induced miRNA participates in autophagic regulation by targeting PRAS40 in mesenchymal subtype of glioblastoma

Objective:Mesenchymal subtype of glioblastoma(mesGBM)is a refractory disease condition characterized by therapeutic failure and tumor recurrence.Hyperactive transforming growth factor-β(TGF-β)signaling could be a signature event in mesGBM,which leads to dysregulation of downstream targets and contribute to malignant transformation.In this study we aimed to investigate the hyperactive TGFβsignaling-mediated pathogenesis and possible downstream targets for the development of novel therapeutic interventions for mesGBM.Methods:GBM-BioDP is an online resource for accessing and displaying interactive views of the TCGA GBM data set.Transcriptomic sequencing followed by bioinformatic analysis was performed to identify dysregulated microRNAs.Target prediction by MR-microT and dual luciferase reporter assay were utilized to confirm the predicted target of novel_miR56.CCK-8 assays was used to assesse cell viability.The miRNA manipulation was proceeded by cell transfection and lentivirus delivery.A plasmid expressing GFP-LC3 was introduced to visualize the formation of autophagosomes.Orthotopic GBM model was constructed forin vivo study.Results:TGFβ1 and TGFβreceptor type II(TβRII)were exclusively upregulated in mesGBM(P<0.01).Dysregulated miRNAs were identified after LY2109761(a TβRI/II inhibitor)treatment in a mesGBM-derived cell line,and novel_miR56 was selected as a promising candidate for further functional verification.Novel_miR56 was found to potentially bind to PRAS40 via seed region complementarity in the 3'untranslated region,and we also confirmed that PRAS40 is a direct target of novel_miR56 in glioma cells.In vitro,over expression of novel_miR56 in tumor cells significantly promoted proliferation and inhibited autophagy(P<0.05).The expression levels of P62/SQSTM was significantly increased accompanied by the decrease of BECN1 and LC3B-II/I,which indicated that autophagic activity was reduced after novel_miR56 treatment.In addition,over expression of novel_miR56 also promoted tumor growth and inhibited autophagyin vivo,which is associated with worse prognosis(P<0.05).Conclusions:In summary,we provide novel insight into TGFβsignaling-mediated pathogenesis in mesGBM and TGFβsignaling-induced novel_miR56 may be a novel target for mesGBM management.

Up-regulation of SEPT7 Gene Inhibits Migration and Invasion of Human Glioma Cells in vitro and in vivo

The aim of this study was to explore the potential role of SEPT7 in glioma cell invasion. From in vitro experiments, we observed that the migratory and invasive abilities were inhibited in human glioblastoma U251MG and TJ899 cells after transfection with SEPT7 recombinant adenovirus constructs (Ad-SEPT7) as evaluated by Transwell assay, 3D Matrigel growth, 2D Matrigel growth and scratch assays. We further investigated the molecular events associated with the alteration of cell migration and invasion by immunohistochemistry and immuno uores-cence staining, Western blot and laser scanning confocal micros-copy analyses, and found the decreased expression of MMP2, MMP9, MT1-MMP and integrin αvβ3, increased expression of TIMP1 and TIMP2, and redistribution of intracellular cytoskel-eton tubulin-α. From in vivo study, it was demonstrated that the tumor growth rate in nude mice bearing xenograft subcutaneous U251 gliomas treated with Ad-SEPT7 was signi cantly slowed during the observation period of 4 weeks and the tumor volumes were much smaller than those in control and empty vector treated group. The expression of MMP2, MMP9, MT1-MMP and integrin αvβ3 was markedly inhibited while the expression of SEPT7, TIMP1, TIMP2 was upregulated in tumors treated with Ad-SEPT7. Taken together, these results suggest that SEPT7 plays an important role in glioma cell invasion and growth and it may be a candidate target for gene therapy of invasive gliomas.

Blockage of EGFR/AKT and mevalonate pathways synergize the antitumor effect of temozolomide by reprogramming energy metabolism in glioblastoma

Background Metabolism reprogramming plays a vital role in glioblastoma(GBM)progression and recurrence by producing enough energy for highly proliferating tumor cells.In addition,metabolic reprogramming is crucial for tumor growth and immune-escape mechanisms.Epidermal growth factor receptor(EGFR)amplification and EGFR-vIII mutation are often detected in GBM cells,contributing to the malignant behavior.This study aimed to investigate the functional role of the EGFR pathway on fatty acid metabolism remodeling and energy generation.Methods Clinical GBM specimens were selected for single-cell RNA sequencing and untargeted metabolomics analysis.A metabolism-associated RTK-fatty acid-gene signature was constructed and verified.MK-2206 and MK-803 were utilized to block the RTK pathway and mevalonate pathway induced abnormal metabolism.Energy metabolism in GBM with activated EGFR pathway was monitored.The antitumor effect of Osimertinib and Atorvastatin assisted by temozolomide(TMZ)was analyzed by an intracranial tumor model in vivo.Results GBM with high EGFR expression had characteristics of lipid remodeling and maintaining high cholesterol levels,supported by the single-cell RNA sequencing and metabolomics of clinical GBM samples.Inhibition of the EGFR/AKT and mevalonate pathways could remodel energy metabolism by repressing the tricarboxylic acid cycle and modulating ATP production.Mechanistically,the EGFR/AKT pathway upregulated the expressions of acyl-CoA synthetase short-chain family member 3(ACSS3),acyl-CoA synthetase long-chain family member 3(ACSL3),and long-chain fatty acid elongation-related gene ELOVL fatty acid elongase 2(ELOVL2)in an NF-κB-dependent manner.Moreover,inhibition of the mevalonate pathway reduced the EGFR level on the cell membranes,thereby affecting the signal transduction of the EGFR/AKT pathway.Therefore,targeting the EGFR/AKT and mevalonate pathways enhanced the antitumor effect of TMZ in GBM cells and animal models.Conclusions Our findings not only uncovered the mechanism of metabolic reprogramming in EGFR-activated GBM but also provided a combinatorial therapeutic strategy for clinical GBM management.

Antisense MMP-9 RNA inhibits malignant glioma cell growth in vitro and in vivo

The matrix-degrading metalloproteinases （MMPs）, particularly MMP-9, play important roles in the pathogenesis and development of malignant gliomas. In the present study, the oncogenic role of MMP-9 in malignant glioma cells was investigated via antisense RNA blockade in vitro and in vivo. TJ905 malignant glioma cells were transfected with pcDNA3.0 vector expressing antisense MMP-9 RNA （pcDNA-AS-MMP9）, which significantly decreased MMP-9 expression, and cell proliferation was assessed. For in vivo studies, U251 cells, a human malignant glioma cell line, were implanted subcutaneously into 4-to 6-week-old BALB/c nude mice. The mice bearing well-established U251 gliomas were treated with intratumoral pcDNA-AS-MMP9-Lipofectamine complex （AS-MMP-9-treated group）, subcutaneous injection of endostatin （endostatin-treated group）, or both （combined therapy group）. Mice treated with pcDNA （empty vector）-Lipofectamine served as the control group. Four or eight weeks later, the volume and weight of tumor, MMP-9 expression, microvessel density and proliferative activity were assayed. We demonstrate that pcDNA-AS-MMP9 significantly decreased MMP-9 expression and inhibited glioma cell proliferation. Volume and weight of tumor, MMP-9 expression, microvessel density and proliferative activity in the antisense-MMP-9-treated and therapeutic alliance groups were significantly lower than those in the control group. The results suggest that MMP-9 not only promotes malignant glioma cell invasiveness, but also affects tumor cell proliferation. Blocking the expression of MMP-9 with antisense RNA substantially suppresses the malignant phenotype of glioma cells, and thus can be used as an effective therapeutic strategy for malignant gliomas.

Systemic delivery of microRNA for treatment of brain ischemia

Brain ischemia is the second leading cause of death and the third leading cause of disability in the world.Systemic delivery of microRNA,a class of molecules that regulate the expression of cellular proteins associated with angiogenesis,cell growth,proliferation and differentiation,holds great promise for the treatment of brain ischemia.However,their therapeutic efficacy has been hampered by poor delivery efficiency of microRNA.We report herein a platform technology based on microRNA nanocapsules,which enables their effective delivery to the disease sites in the brain.Exemplified by microRNA-21,intravenous injection of the nanocapsules into a rat model of cerebral ischemia could effectively ameliorate the infarct volume,neurological deficit and histopathological severity.

MET fusions and splicing variants convergently defne a subgroup of glioma sensitive to MET inhibitors

Purpose:Our previous study has shown that PTPRZ1-MET(ZM)fusion is a viable target for MET inhibitors in gliomas.However,the diversity and prevalence of somatic MET alterations in difuse gliomas are still elusive and need to be extensively characterized for identifying novel therapeutic targets.Methods:Totally,1,350 glioma patients and 31 patient-derived cells were collected from the Chinese Glioma Genome Atlas(CGGA)and published data.All kinds of MET fusions and/or splicing variants(MET F/SVs)were identifed by bioinformatical methods.Single-cell RNA sequencing(scRNA-seq)were used for validation.In vitro experiments of drug resistance were conducted for the possibility of MET-targeted treatment.Results:MET F/SVs but not genomic amplifcation,were highly enriched in the secondary glioblastomas(sGBM)and marked worse prognosis.Further molecular and scRNA-seq analysis revealed that MET F/SVs were induced in the course of glioma evolution and highly associated with MET overexpression.Subsequent in vitro and the clinical study showed that cells and patients harboring MET F/SVs have better response to MET inhibitors.Conclusion:Our fndings expanded the percentage of gliomas with abnormal MET alterations and suggested that a subgroup of gliomas harboring MET F/SVs may beneft from MET-targeted therapy.

CACA guidelines for holistic integrative management of glioma

Glioma of the brain is a kind of tumor originating from neuroglial cells.It is the most common primary intracranial tumor,accounting for~30%of all central nervous system tumors and 80% of malignant brain tumors.Glioma is characterized by high disability and recurrence rates.The disease seriously threatens the life of patients,afects their quality of life,and brings a heavy economic and psychological burden to patients,families,and society.With the progression of molecular genetic testing technology and the completion of various clinical trials,the classifcation scheme for glioma is increasingly well established.Diagnosis and treatment regimens,including traditional and new regimens,are becoming increasingly specialized and standardized.The purpose is to develop a clinical diagnosis and treatment guideline for glioma in the Chinese population suitable for Chinese doctors and the general population based on domestic and international glioma research progress.Thus,domestic practitioners in the feld can obtain current information and provide better service to patients with glioma,promoting the development of domestic clinical medicine and basic research on glioma.