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Abstract Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (EGFR-TKI) approved for patients with EGFR T790M resistance mutations as first- or second-line treatment of EGFR-positive patients. Resistance to Osimertinib will inevitably develop, and the underlying mechanisms are largely unknown. In this study, we discovered that acquired resistance to Osimertinib is associated with abnormal DNA damage response (DDR) in lung adenocarcinoma cells. We discovered that the polycomb protein Lethal(3) Malignant Brain Tumor-Like Protein 1 (L3MBTL1) regulates chromatin structure, thereby contributing to DDR and Osimertinib resistance. EGFR oncogene inhibition reduced L3MBTL1 ubiquitination while stabilizing its expression in Osimertinib-resistant cells. L3MBTL1 reduction and treatment with Osimertinib significantly inhibited DDR and proliferation of Osimertinib-resistant lung cancer cells in vitro and in vivo. L3MBTL1 binds throughout the genome and plays an important role in EGFR-TKI resistance. It also competes with 53BP1 for H4K20Me2 and inhibits the development of drug resistance in Osimertinib-resistant lung cancer cells in vitro and in vivo. Our findings suggest that L3MBTL1 inhibition is a novel approach to overcoming EGFR-TKI-acquired resistance.

Abstract Extracellular matrix (ECM) remodeling has been implicated in the tumor malignant progression and immune escape in glioblastoma (GBM). Runt-related transcription factor 1 (RUNX1) is a vital transcriptional factor for promoting tumorigenesis and invasion in mesenchymal subtype of GBM. But the correlation between RUNX1 and ECM genes expression and regulatory mechanism of RUNX1 on ECM genes expression remain poorly understood to date. In this study, by using integral analysis of chromatin immunoprecipitation-sequencing and RNA sequencing, we reported that RUNX1 positively regulated the expression of various ECM-related genes, including Fibronectin 1 (FN1), Collagen type IV alpha 1 chain (COL4A1), and Lumican (LUM), in GBM. Mechanistically, we demonstrated that RUNX1 interacted with Nucleophosmin 1 (NPM1) to maintain the chromatin accessibility and facilitate FOS Like 2, AP-1 Transcription Factor Subunit (FOSL2)-mediated transcriptional activation of ECM-related genes, which was independent of RUNX1’s transcriptional function. ECM remodeling driven by RUNX1 promoted immunosuppressive microenvironment in GBM. In conclusion, this study provides a novel mechanism of RUNX1 binding to NPM1 in driving the ECM remodeling and GBM progression.

Abstract 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.

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, real-time 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.

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.

Glioblastoma (GBM) is the most common and lethal type of primary malignant central nervous system (CNS) tumor with an extremely poor prognosis, and the mesenchymal subtype of GBM has the worst prognosis. Here, we found that lncRNA PRADX was overexpressed in the mesenchymal GBM and was transcriptionally regulated by RUNX1-CBFβ complex, overexpressed PRADX suppressed BLCAP expression via interacting with EZH2 and catalyzing trimethylation of lysine 27 on histone H3 (H3K27me3). Moreover, we showed that BLCAP interacted with STAT3 and reduced STAT3 phosphorylation, overexpressed PRADX activated STAT3 phosphorylation, and promoted ACSL1 expression via suppressing BLCAP expression, accelerating tumor metabolism. Finally, we determined that combined of ACSL1 and CPT1 inhibitors could reverse the accelerated cellular metabolism and tumor growth induced by PRADX overexpression in vivo and in vitro. Collectively, PRADX/PRC2 complex activated the STAT3 pathway and energy metabolism in relation to mesenchymal GBM progression. Furthermore, our findings provided a novel therapeutic strategy targeting the energy metabolism activity of GBM.

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 TGFβ signaling-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 for in vivo study. Results: TGFβ1 and TGFβ receptor 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 autophagy in vivo, which is associated with worse prognosis (P < 0.05). Conclusions: In summary, we provide novel insight into TGFβ signaling-mediated pathogenesis in mesGBM and TGFβ signaling-induced novel_miR56 may be a novel target for mesGBM management.

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 tumor 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.

Abstract Background The EGFR-vIII mutation is the most common malignant event in GBM. Epigenetic reprogramming in EGFR-activated GBM has recently been suggested to downregulate the expression of tumour suppressor genes. Histone acetylation is important for chromatin structure and function. However, the role and biological function of H2AZK4/7AC in tumours have not yet been clarified. Results In our study, we found that EGFR-vIII negatively regulated H2AZK4/7AC expression though the PI3K/AKT-HDAC2 axis. Because HDAC1 and HDAC2 are highly homologous enzymes that usually form multi-protein complexes for transcriptional regulation and epigenetic landscaping, we simultaneously knocked out HDAC1 and HDAC2 and found that H2AZK4/7AC and H3K27AC were upregulated, which partially released EGFR-vIII-mediated inhibition of USP11, negative regulator of cell cycle. In addition, we demonstrated in vitro and in vivo that FK228 induced G1/S transition arrest in GBM with EGFR-vIII mutation. FK228 could enhance anti-tumour activity by upregulating expression of the tumour suppressor USP11 in GBM cells. Conclusions EGFR-vIII mutation downregulates H2AZK4/7AC and H3K27AC, inhibiting USP11 expression though the PI3K/AKT-HDAC1/2 axis. FK228 is an effective and promising treatment for GBM with EGFR-vIII mutation.

BackgroundImmunotherapy, especially checkpoint inhibitors targeting PD-1 or PD-L1, has revolutionized cancer therapy. However, PD-1/PD-L1 inhibitors have not been investigated thoroughly in glioblastoma (GBM). Studies have shown that polymerase 1 and transcript release factor (PTRF/Cavin-1) has an immune-suppressive function in GBM. Thus, the relationship between PTRF and PD-L1 and their role in immune suppression requires further investigation in GBM.MethodsWe used public databases and bioinformatics analysis to investigate the relationship between PTRF and PD-L1. We next confirmed the predicted relationship between PTRF and PD-L1 in primary GBM cell lines by using different experimental approaches. RIP-Seq, RIP, ChIP, and qRT-PCR were conducted to explore the molecular mechanism of PTRF in immunosuppression.ResultsWe found that PTRF stabilizes lncRNA NEAT1 to induce NF-κB and PD-L1 and promotes immune evasion in GBM. PTRF was found to correlate with immunosuppression in the public GBM databases. PTRF increased the level of PD-L1 in primary cell lines from GBM patients. We carried out RIP-Seq of GBM cells and found that PTRF interacts with lncRNA NEAT1 and stabilizes its mRNA. PTRF also promoted the activity of NF-κB by suppressing UBXN1 expression via NEAT1 and enhanced the transcription of PD-L1 through NF-κB activation. Finally, PTRF promoted immune evasion in GBM cells by regulating PD-1 binding and PD-L1 mediated T cell cytotoxicity.ConclusionsIn summary, our study identified the PTRF-NEAT1-PD-L1 axis as a novel immune therapeutic target in GBM.

ObjectiveATRX 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.MethodsWe 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, CDKN2A and CDKN2B 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.ResultsIn 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.ConclusionsATRX 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.

BackgroundCheckpoint blockade therapies targeting programmed death ligand 1 (PD-L1) and its receptor programmed cell death 1 promote T cell-mediated immune surveillance against tumors and have been associated with significant clinical benefit in cancer patients. The long-stranded non-coding RNA HOTAIR is highly expressed and associated with metastasis in a variety of cancer types and promotes tumor metastasis at least in part through association with the PRC2 complex that induces redirection to hundreds of genes involved in tumor metastasis. Here, we report that HOTAIR is an activator lncRNA of the NF-κB pathway and demonstrate that its apparent upregulation promotes inflammatory signaling and immune escape in glioma cells.MethodsBioinformatics analysis was used to elucidate the relationship between HOTAIR and NF-κB pathway in HOTAIR knockdown glioma cells. At the cytological level, protein hybridization and immunofluorescence were used to detect the response of proteins in the NF-κB signaling pathway to HOTAIR regulation. ChIP and ChIRP experiments identified HOTAIR target genes. Animal experiments verified alterations in inflammation and immune escape following HOTAIR knockdown and activity inhibition.ResultsHOTAIR activated the expression of proteins involved in NF-κB, TNFα, MAPK and other inflammatory signaling pathways. In addition, HOTAIR induced various proteins containing protein kinase structural domains and promoted the enrichment of proteins and complexes of important inflammatory signaling pathways, such as the TNFα/NF-κB signaling protein complex, the IκB kinase complex, and the IKKA-IKKB complex. In addition, HOTAIR aberrantly activated biological processes involved in glioma immune responses, T-cell co-stimulation and transcription initiation by RNA polymerase II. HOTAIR facilitated the induction of IκBα phosphorylation by suppressing the expression of the NF-κB upstream protein UBXN1, promoting NF-κB phosphorylation and nuclear translocation. In vivo, reduction of HOTAIR decreased PD-L1 protein expression, indicating that cells are more likely to be targeted by immune T cells.ConclusionIn conclusion, our results provide convincing evidence that lncRNA HOTAIR drives aberrant gene transcription and immune escape from tumor cells through the NF-κB pathway.

BackgroundThe main immune cells in GBM are tumor-associated macrophages (TAMs). Thus far, the studies investigating the activation status of TAM in GBM are mainly limited to bulk RNA analyses of individual tumor biopsies. The activation states and transcriptional signatures of TAMs in GBM remain poorly characterized.MethodsWe comprehensively analyzed single-cell RNA-sequencing data, covering a total of 16,201 cells, to clarify the relative proportions of the immune cells infiltrating GBMs. The origin and TAM states in GBM were characterized using the expression profiles of differential marker genes. The vital transcription factors were examined by SCENIC analysis. By comparing the variable gene expression patterns in different clusters and cell types, we identified components and characteristics of TAMs unique to each GBM subtype. Meanwhile, we interrogated the correlation between SPI1 expression and macrophage infiltration in the TCGA-GBM dataset.ResultsThe expression patterns of TMEM119 and MHC-II can be utilized to distinguish the origin and activation states of TAMs. In TCGA-Mixed tumors, almost all TAMs were bone marrow-derived macrophages. The TAMs in TCGA-proneural tumors were characterized by primed microglia. A different composition was observed in TCGA-classical tumors, which were infiltrated by repressed microglia. Our results further identified SPI1 as a crucial regulon and potential immunotherapeutic target important for TAM maturation and polarization in GBM.ConclusionsWe describe the immune landscape of human GBM at a single-cell level and define a novel categorization scheme for TAMs in GBM. The immunotherapy against SPI1 would reprogram the immune environment of GBM and enhance the treatment effect of conventional chemotherapy drugs.

Temozolomide resistance in glioblastoma is associated with MGMT overexpression. Here, the authors identify a lncRNA that is a competitive endogenous RNA for miR-20b-3p, which causes c-Met activation to modulate acetylation of histone H3 on MGMT promoter through Stat3/p300 complex to increase MGMT expression and temozolomide resistance.

Autophagy of mitochondria, termed mitophagy, plays an important role in cerebral ischemia-reperfusion (IR) injury, but the mechanism is not yet clear. Tissue-type plasminogen activator (tPA) is the most important thrombolytic drug in the clinical treatment of ischemic stroke and has neuroprotective effects. Here, we explored the effects of tPA on neuronal apoptosis and mitophagy following IR. We found that knocking out the tPA gene significantly aggravated brain injury and increased neuronal apoptosis and mitochondrial damage. Exposure of neurons to tPA reduced injury severity and protected mitochondria. Further studies demonstrated that this protective effect of tPA was achieved via regulation of FUNDC1-mediated mitophagy. Furthermore, we found that tPA enhanced the expression level of FUNDC1 by activating the phosphorylation of AMPK. In summary, our results confirm that tPA exerts neuroprotective effects by increasing the phosphorylation of AMPK and the expression of FUNDC1, thereby inhibiting apoptosis and improving mitochondrial function.

Glioblastoma (GBM) is a highly malignant and aggressive primary brain tumor mostly prevalent in adults and is associated with a very poor prognosis. Moreover, only a few effective treatment regimens are available due to their rapid invasion of the brain parenchyma and resistance to conventional therapy. However, the fast development of cancer immunotherapy and the remarkable survival benefit from immunotherapy in several extracranial tumor types have recently paved the way for numerous interventional studies involving GBM patients. The recent success of checkpoint blockade therapy, targeting immunoinhibitory proteins such as programmed cell death protein-1 and/or cytotoxic T lymphocyte-associated antigen-4, has initiated a paradigm shift in clinical and preclinical investigations, and the use of immunotherapy for solid tumors, which would be a potential breakthrough in the field of drug therapy for the GBM treatment. However clinical trial showed limited benefits for GBM patients. The main reason is drug resistance. This review summarizes the clinical research progress of immune checkpoint molecules and inhibitors, introduces the current research status of immune checkpoint inhibitors in the field of GBM, analyzes the molecular resistance mechanism of checkpoint blockade therapy, proposes corresponding re-sensitive strategies, and describes a reference for the design and development of subsequent clinical studies on immunotherapy for GBM.

The role of protein arginine methyltransferases (PRMTs) in epigenetic regulation in cancer is still poorly understood. Here, the authors show that PRMT2 is highly expressed in Glioblastoma multiforme (GBM) and provide evidence that PRMT2 acts as a transcriptional co-activator for oncogenic gene expression programs, at least partly dependent on its H3R8me2a activity, in GBM pathogenesis.

Abstract Intratumor heterogeneity of tumor clones and an immunosuppressive microenvironment in cancer ecosystems contribute to inherent difficulties for tumor treatment. Recently, chimeric antigen receptor (CAR) T-cell therapy has been successfully applied in the treatment of B-cell malignancies, underscoring its great potential in antitumor therapy. However, functional challenges of CAR-T cell therapy, especially in solid tumors, remain. Here, we describe cancer-immunity phenotypes from a clonal-stromal-immune perspective and elucidate mechanisms of T-cell exhaustion that contribute to tumor immune evasion. Then we assess the functional challenges of CAR-T cell therapy, including cell trafficking and infiltration, targeted-recognition and killing of tumor cells, T-cell proliferation and persistence, immunosuppressive microenvironment and self-control regulation. Finally, we delineate tumor precision informatics and advancements in engineered CAR-T cells to counteract inherent challenges of the CAR-T cell therapy, either alone or in combination with traditional therapeutics, and highlight the therapeutic potential of this approach in future tumor precision treatment.

Abstract RNA is rarely used as a therapeutic target due to its flexible structure and instability. CRISPR‐Cas13a is a powerful tool for RNA knockdown, and the potential application of CRISPR‐Cas13a in cancer cells should be further studied. In this study, overexpression of LwCas13a by lentivirus in glioma cells reveals that crRNA‐EGFP induces a “collateral effect” after knocking down the target gene in EGFP‐expressing cells. EGFRvIII is a unique EGFR mutant subtype in glioma, and the CRISPR‐Cas13a system induces death in EGFRvIII‐overexpressing glioma cells. Bulk and single‐cell RNA sequencing analysis in U87‐Cas13a‐EGFRvIII cells confirm the collateral effect of the CRISPR‐Cas13a system. Furthermore, CRISPR‐Cas13a inhibits the formation of glioma intracranial tumors in mice. The results demonstrate the collateral effect of the CRISPR‐Cas13a system in cancer cells and the powerful tumor‐eliminating potential of this system.

Abstract Amplification of epidermal growth factor receptor (EGFR) and active mutant EGFRvIII occurs frequently in glioblastoma (GBM) and contributes to chemo/radio‐resistance in various cancers, especially in GBM. Elucidating the underlying molecular mechanism of temozolomide (TMZ) resistance in GBM could benefit cancer patients. A genome‐wide screening under a clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9 library is conducted to identify the genes that confer resistance to TMZ in EGFRvIII‐expressing GBM cells. Deep sgRNA sequencing reveals 191 candidate genes that are responsible for TMZ resistance in EGFRvIII‐expressing GBM cells. Notably, E2F6 is proven to drive a TMZ resistance, and E2F6 expression is controlled by the EGFRvIII/AKT/NF‐κB pathway. Furthermore, E2F6 is shown as a promising therapeutic target for TMZ resistance in orthotopic GBM cell line xenografts and GBM patient‐derived xenografts models. After integrating clinical data with paired primary–recurrent RNA sequencing data from 134 GBM patients who received TMZ treatment after surgery, it has been revealed that the E2F6 expression level is a predictive marker for TMZ response. Therefore, the inhibition of E2F6 is a promising strategy to conquer TMZ resistance in GBM.

Up-to-date knowledge regarding the biogenesis and functioning of microRNAs (miRNAs) has provided a much more comprehensive and concrete view of miRNA biology than anyone ever expected. Diverse genetic origins and biogenesis pathways leading to functional miRNAs converge on the synthesis of ≈21-nucleotide RNA duplex, almost all of which are processed from long premature sequences in a DICER- and/or DROSHA-dependent manner. Formerly, it was assumed that one mature strand of the duplex is preferentially selected for entry into the silencing complex, and the paired passenger strands (miRNA*) are subjected to degradation. However, given the consolidated evidence of substantial regulatory activity of miRNA* species, currently, this preconception has been overturned. Here, we see the caveat and opportunity toward exogenously manipulating the expression of premature miRNA, leading to simultaneous upregulation or downregulation of dual regulatory strands due to altered expressions. The caveat is the overlooked miRNA* interference while manipulating the expression of a target miRNA at the premature stage, wherein lies the opportunity. If the dual strands of a pre-miRNA function synergistically, the overlooked miRNA* interference may inversely optimize the therapeutic performance. Insightfully, targeting the premature miRNAs may serve as the “one-two punch” against diseases, especially cancers, and this has been discussed in detail in this review.

Greenhouse eggplant monocropping in China has contributed to the aggravation of soil-borne diseases, reductions in crop quality and yield, and the degradation of physical and chemical soil properties. Crop rotation is one effective way of alleviating the problems of continuous cropping worldwide; however, few studies have reported changes in soil bacterial community structures and physical and chemical soil properties after Brassica vegetables had been rotated with eggplant in greenhouses. In this experiment, mustard-eggplant (BFN) and oilseed rape-eggplant (BFC) rotations were studied to identify changes in the physicochemical properties and bacterial community structure in soil that was previously subject to monocropping. Samples were taken after two types of Brassica plants incorporated into soil for 15 days to compare with continually planted eggplant (control, CN) and chemical disinfection of soil (CF) in greenhouses. MiSeq pyrosequencing was used to analyze soil bacterial diversity and structure in the four different treatments. A total of 55,129 reads were identified, and rarefaction analysis showed that the soil treatments were equally sampled. The bacterial richness of the BFC treatment and the diversity of the BFN treatment were significantly higher than those of the other treatments. Further comparison showed that the bacterial community structures of BFC and BFN treatments were also different from CN and CF treatments. The relative abundance of several dominant bacterial genera in the BFC and BFN treatments (such as Flavobacteria, Stenotrophomonas, Massilia and Cellvibrio, which played different roles in improving soil fertility and advancing plant growth) was distinctly higher than the CN or CF treatments. Additionally, the total organic matter and Olsen-P content of the BFC and BFN treatments were significantly greater than the CN treatment. We conclude that Brassica vegetables-eggplant crop rotations could provide a more effective means of solving the problems of greenhouse eggplant monocultures.

The prognosis of glioma patients is usually poor, especially in patients with glioblastoma (World Health Organization (WHO) grade IV). The regulatory functions of microRNA (miRNA) on genes have important implications in glioma cell survival. However, there are not many studies that have investigated glioma survival by integrating miRNAs and genes while also considering pathway structure. In this study, we performed sample-matched miRNA and mRNA expression profilings to systematically analyze glioma patient survival. During this analytical process, we developed pathway-based random walk to identify a glioma core miRNA-gene module, simultaneously considering pathway structure information and multi-level involvement of miRNAs and genes. The core miRNA-gene module we identified was comprised of four apparent sub-modules; all four sub-modules displayed a significant correlation with patient survival in the testing set (P-values≤0.001). Notably, one sub-module that consisted of 6 miRNAs and 26 genes also correlated with survival time in the high-grade subgroup (WHO grade III and IV), P-value = 0.0062. Furthermore, the 26-gene expression signature from this sub-module had robust predictive power in four independent, publicly available glioma datasets. Our findings suggested that the expression signatures, which were identified by integration of miRNA and gene level, were closely associated with overall survival among the glioma patients with various grades.

The application of microRNAs (miRNAs) in the therapeutics of glioma and other human diseases is an area of intense interest. However, it's still a great challenge to interpret the functional consequences of using miRNAs in glioma therapy. Here, we examined paired deep sequencing expression profiles of miRNAs and mRNAs from human glioma cell lines after manipulating the levels of miRNAs miR-181d, -21, and -23b, as well as transcriptional regulators β-catenin, CBP, and STAT3. An integrated approach was used to identify functional miRNA-pathway regulatory networks (MPRNs) responding to each manipulation. MiRNAs were identified to regulate glioma related biological pathways collaboratively after manipulating the level of either post-transcriptional or transcriptional regulators, and functional synergy and crosstalk was observed between different MPRNs. MPRNs responsive to multiple interventions were found to occupy central positions in the comprehensive MPRN (cMPRN) generated by integrating all the six MPRNs. Finally, we identified a core module comprising 14 miRNAs and five pathways that could predict the survival of glioma patients and represent potential targets for glioma therapy. Our results provided novel insight into miRNA regulatory mechanisms implicated in therapeutic interventions and could offer more inspiration to miRNA-based glioma therapy.

It is well known that Notch signaling plays either oncogenic or tumor suppressive role in a variety of tumors, depending on the cellular context. However, in our previous study, we found that Notch1 was overexpressed while Notch2 downregulated in the majority of astrocytic gliomas with different grades as well as in glioblastoma cell lines U251 and A172. We had knocked down Notch1 by siRNA in glioblastoma cells, and identified that the cell growth and invasion were inhibited, whereas cell apoptosis was induced either in vitro or in vivo. For further clarification of the role of Notch2 in pathogenesis of gliomas, enforced overexpression of Notch2 was carried out with transfection of Notch2 expression plasmid in glioma cells and the cell growth, invasion and apoptosis were examined in vitro and in vivo in the present study, and siRNA targeting Notch1 was used as a positive control in vivo. The results showed that upregulating Notch2 had the effect of suppressing cell growth and invasion as well as inducing apoptosis, just the same as the results of knocking down Notch1. Meanwhile, the activity of core signaling pathway-EGFR/PI3K/AKT in astrocytic glioma cells was repressed. Thus, the present study reveals, for the first time, that Notch1 and Notch2 play different roles in the biological processes of astrocytic gliomas. Knocking down the Notch1 or enforced overexpression of Notch2 both modulate the astrocytic glioma phenotype, and the mechanism by which Notch1 and 2 play different roles in the glioma growth should be further investigated.

OBJECTIVES: Seizure is a common presenting symptom of primary brain tumors. There are no published studies regarding the roles of MicroRNA (miRNA) in tumor-related epilepsy. The authors set out to correlate miR-196b expression in low-grade glioma patients with pre-operative seizures and post-operative seizure control. METHODS: Twenty-three patients with WHO grade II astrocytomas and 83 similar patients for independent validation were included. Follow-up visits regarding seizure prognosis were scheduled at 6 months. MiRNA profiling was used to identify differentially expressed miRNAs. The most important miRNA was determined by quantitative reverse-transcriptase polymerase chain reaction (q-PCR) in the validation cohort. Gene ontology (GO) analysis and whole genome mRNA profiling was performed to investigate the underlying biological processes. RESULTS: Array results showed that 30 miRNAs were overexpressed and 10 miRNAs were underexpressed (with more than 2 fold change) in patients with pre-operative seizures. MiR-196b was validated in the independent validation cohort. Patients with good seizure prognosis exhibited low levels of miR-196b expression compared with those who had poor seizure prognosis in the group without pre-operative seizures. Biological processes that relate to transcription and cell cycles were over-represented in the miR-196b-associated gene expression signature. MiR-196b-associated gene expression profiling was characterized by enrichment of genes usually involved in cell proliferation. CONCLUSIONS: We have provided the first evidence that expression of miR-196b was associated with the occurrence of pre-operative seizures in low-grade gliomas, and may predict seizure prognosis in patients without pre-operative seizures. Targeted treatments that decrease endogenous levels of miR-196b might represent novel therapeutic strategies.

It has been reported that IDH1 (IDH1R132) mutation was a frequent genomic alteration in grade II and grade III glial tumors but rare in primary glioblastoma (pGBM). To elucidate the frequency of IDH1 mutation and its clinical significance in Chinese patients with pGBM, one hundred eighteen pGBMs were assessed by pyro-sequencing for IDH1 mutation status, and the results were correlated with clinical characteristics and molecular pathological factors. IDH1 mutations were detected in 19/118 pGBM cases (16.1%). Younger age, methylated MGMT promoter, high expression of mutant P53 protein, low expression of Ki-67 or EGFR protein were significantly correlated with IDH1 mutation status. Most notably, we identified pGBM cases with IDH1 mutation were mainly involved in the frontal lobe when compared with those with wild-type IDH1. In addition, Kaplan-Meier survival analysis revealed a highly significant association between IDH1 mutation and a better clinical outcome (p = 0.026 for progression-free survival; p = 0.029 for overall survival). However, in our further multivariable regression analysis, the independent prognostic effect of IDH1 mutation is limited when considering age, preoperative KPS score, extent of resection, TMZ chemotherapy, and Ki-67 protein expression levels, which might narrow its prognostic power in Chinese population in the future.