Glioblastoma Multiforme Invasion Research Articles

LGR6 promotes glioblastoma malignancy and chemoresistance by activating the Akt signaling pathway.

Chemoresistance is the primary cause of the poor outcome of glioblastoma multiforme (GBM) therapy. Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) is involved in the growth and proliferation of several types of cancer, including gastric cancer and ovarian cancer. Therefore, the aim of the present study was to investigate the role of LGR6 in GBM malignancy and chemoresistance. Cell counting kit-8 and Matrigel®-Transwell assays were conducted to assess GBM cell viability and invasion. The effect of LGR6 on cell cycle progression and activation of Akt signaling was analyzed by performing propidium iodide staining and western blotting, respectively. The results demonstrated that LGR6, a microRNA-1236-3p target candidate, promoted GBM cell viability and invasion, and mediated temozolomide sensitivity in SHG-44 and U251 GBM cells. In addition, LGR6 triggered the activation of the Akt signaling pathway during GBM progression. Collectively, the results of the present study suggested that LGR6 promoted GBM malignancy and chemoresistance, at least in part, by activating the Akt signaling pathway. The results may aid with the identification of a novel therapeutic target and strategy for GBM.

Cell Trapping via Migratory Inhibition within Density-Tuned Electrospun Nanofibers.

Cell migration is an essential bioprocess that occurs during wound healing and tissue regeneration. Abnormal cell migration is observed in various pathologies, including cancer metastasis. Glioblastoma multiforme (GBM) is an aggressive and highly infiltrative brain tumor. The white matter tracts are considered the preferred routes for GBM invasion and the subsequent spread throughout the brain tissue. In the present study, a platform based on electrospun nanofibers with a consistent alignment and controlled density was designed to inhibit cell migration. The observation of the cells cultured on the nanofibers with different fiber densities revealed an inverse correlation between the cell migration velocity and nanofiber density. This was attributed to the formation of focal adhesions (FAs). The FAs in the sparse fiber matrix were small, whereas those in the dense fiber matrix were large, aligned with the nanofibers, and distributed throughout the cells. A nanofiber-based platform with stepwise different fiber densities was designed based on the aforementioned observation. A time-lapse observation of the GBM cells cultured on the platform revealed a directional one-way migration that induced the entrapment of cells in the dense-fiber zone. The designed platform mimicked the structure of the white matter tracts and enabled the entrapment of migrating cells. The demonstrated approach is suitable for inhibiting metastasis and understanding the biology of invasion, thereby functioning as a promising therapeutic strategy for GBM.

The role of melatonin in angio-miR-associated inhibition of tumorigenesis and invasion in human glioblastoma tumour spheroids

Micro-RNA (miRNA)-based regulation of hypoxia, angiogenesis and tumour growth provides promising targets for effective therapy in malignant glioblastoma multiforme (GBM). Accumulating evidence suggests a potential role of melatonin in miRNA expression in cancer cells. Despite these findings, the melatonin–miRNA interaction in GBM and the effect of this interaction on GBM tumour development and invasion are not clearly understood. The aim of the present study was to evaluate the effects of melatonin on human GBM tumour spheroid tumorigenesis and invasion in vitro, and to analyse the interaction between 36 angio-miRNAs and the HIF1/VEGF/MMP9 axis, which is known to be associated with the antitumour effect of melatonin. We found that melatonin is able to selectively induce cell death in single-layer U87-MG cells (a GBM cell line) in a dose- and time-dependent manner, as characterized by MTT assay. The use of tumour spheroids and a Matrigel invasion assay revealed that melatonin impairs tumorigenesis, and it significantly reduced both the tumour spheroid area and invasion rate, especially at the 0.5 mM and 1 mM concentrations. This inhibition was accompanied by strong reductions in hypoxia-inducible factor 1-α (HIF1-α) and vascular endothelial growth factor (VEGF) gene expression and protein levels in GBM tumour spheroids. In addition, melatonin significantly reduced the relative gene expression and protein levels of matrix metalloproteinase-9 (MMP-9). This study revealed that six differentially expressed angio-miRs (miR-15b, miR-18a-5p, miR-23a-3p, miR-92a-3p, miR-130a-5p, miR-200b-3p) may play important roles in GBM tumorigenesis and invasion, and all respond to melatonin therapy. Our results suggest that melatonin inhibits tumorigenesis and invasion of human GBM tumour spheroids, possibly by suppressing HIF1-α/VEGF/MMP9 signalling via regulation of angio-miRNAs.

Chemotherapy-Induced Degradation of Glycosylated Components of the Brain Extracellular Matrix Promotes Glioblastoma Relapse Development in an Animal Model.

Adjuvant chemotherapy with temozolomide (TMZ) is an intrinsic part of glioblastoma multiforme (GBM) therapy targeted to eliminate residual GBM cells. Despite the intensive treatment, a GBM relapse develops in the majority of cases resulting in poor outcome of the disease. Here, we investigated off-target negative effects of the systemic chemotherapy on glycosylated components of the brain extracellular matrix (ECM) and their functional significance. Using an elaborated GBM relapse animal model, we demonstrated that healthy brain tissue resists GBM cell proliferation and invasion, thereby restricting tumor development. TMZ-induced [especially in combination with dexamethasone (DXM)] changes in composition and content of brain ECM proteoglycans (PGs) resulted in the accelerated adhesion, proliferation, and invasion of GBM cells into brain organotypic slices ex vivo and more active growth and invasion of experimental xenograft GBM tumors in SCID mouse brain in vivo. These changes occurred both at core proteins and polysaccharide chain levels, and degradation of chondroitin sulfate (CS) was identified as a key event responsible for the observed functional effects. Collectively, our findings demonstrate that chemotherapy-induced changes in glycosylated components of brain ECM can impact the fate of residual GBM cells and GBM relapse development. ECM-targeted supportive therapy might be a useful strategy to mitigate the negative off-target effects of the adjuvant GBM treatment and increase the relapse-free survival of GBM patients.

Abstract 2885: Connexin 43-dependent miRNA transfer drives perivascular glioma invasion through dysregulation of astrocytes

Abstract Background: Glioblastoma multiforme (GBM) invasion occurs along vasculature, and intercellular communication between glioma cells and the astrocyte endfoot may manipulate astrocytes to repress cell-matrix adhesion through transfer of miRNAs. Intercellular communication, specifically through Connexin 43 (Cx43) gap junction (GJ) channels, has been confoundingly demonstrated to both limit and facilitate GBM invasion. In solid tumors, Cx43 between tumor cells is correlated with a low invasion and the GJs have long been postulated to be required for growth control. By contrast, functional Cx43 channels between GBM and non-cancerous cells is correlated with high invasiveness, and GJs have been proposed to provide the pathway through which GBM drives surrounding astrocytes into a tumor-permissive state. How GJs participate in the transfer of genetic regulatory molecules from GBM to astrocytes is incompletely understood and might involve direct diffusion through Cx43 channels, GJ-facilitated exosomal transfer, and biased GJ endocytosis. In this study we examine astrocyte miRNA expression after contact with GBM and used an ex vivo assay to assess Cx43-dependent invasion. Methods: Immortalized wild-type (WT) and connexin 43 (Cx43) knockout (KO) murine astrocytes were co-cultured for 24 hours with U87 GBM cells expressing cytosolic GFP. Cells were separated by FACS and collected for miRNAseq. For ex vivo invasion model, 350µm whole brain coronal slices (from WT and GFAP-Cre Cx43-/- C57BL/6J mice) were obtained via vibratome in high-sucrose ACSF and cultured for >12 hours in 20% serum recovery medium. At that time, 250-350µm U87 spheroids (4-8 per slice) expressing cytosolic mCherry (either WT or CRISPR-generated Cx43 KO U87 cell lines) were injected into the cerebral cortex. Slices were cultured for up to 72 hours, fixed, and cleared by SeeDB protocol. Invasion was imaged by confocal microscopy at 10x. Extent of perivascular invasion by the injected tumor spheroids was quantified using Sholl analysis to compare number and lengths of branches. Results: miRNASeq identified a cohort of >10 miRNAs that were upregulated in WT astrocytes, but not KO astrocytes, co-cultured with GBM. Some of these miRNAs are known to have elevated expression in GBM and have predicted cell adhesion protein targets. GBM pervascular invasion was substantially lower for WT-U87 spheroids in GFAP-Cre Cx43-/- slice when compared to WT-U87 in WT slice or Cx43 KO U87 in Cx43-/- slice. Conclusions: Specific miRNAs are transferred via Cx43-mediated mechanisms from GBM to astrocytes and may play a role in astrocyte-matrix adhesion at the endfoot, creating an opening for GBM invasion. Initial slice culture data indicate that functional GBM-GBM GJs inhibit peritumoral outgrowth, whereas astrocyte-GBM GJs are critical for invasion, presumably facilitating remodeling of host tissue by the miRNAs to accommodate invading GBM. Citation Format: Sean McCutcheon, David C. Spray. Connexin 43-dependent miRNA transfer drives perivascular glioma invasion through dysregulation of astrocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2885.

Overview of the molecular mechanisms of migration and invasion in glioblastoma multiforme.

Glioblastoma (GBM) is one of the most devastating cancers, with an approximate median survival of only 16 months. Although some new insights into the fantastic heterogeneity of this kind of brain tumor have been revealed in recent studies, all subclasses of GBM still demonstrate highly aggressive invasion properties to the surrounding parenchyma. This behavior has become the main obstruction to current curative therapies as invasive GBM cells migrate away from these foci after surgical therapies. Therefore, this review aimed to provide a relatively comprehensive study of GBM invasion mechanisms, which contains an intricate network of interactions and signaling pathways with the extracellular matrix (ECM). Among these related molecules, TGF-β, the ECM, Akt, and microRNAs are most significant in terms of cellular procedures related to GBM motility and invasion. Moreover, we also review data indicating that Musashi-1 (MSI1), a neural RNA-binding protein (RBP), regulates GBM motility and invasion, maintains stem cell populations in GBM, and promotes drug-resistant GBM phenotypes by stimulating necessary oncogenic signaling pathways through binding and regulating mRNA stability. Importantly, these necessary oncogenic signaling pathways have a close connection with TGF-β, ECM, and Akt. Thus, it appears promising to find MSI-specific inhibitors or RNA interference-based treatments to prevent the actions of these molecules despite using RBPs, which are known as hard therapeutic targets. In summary, this review aims to provide a better understanding of these signaling pathways to help in developing novel therapeutic approaches with better outcomes in preclinical studies.

Novel CTRP8‐RXFP1‐JAK3‐STAT3 axis promotes Cdc42‐dependent actin remodeling for enhanced filopodia formation and motility in human glioblastoma cells

C1q tumor necrosis factor‐related peptide 8 (CTRP8) is the least studied member of the C1Q‐TNF‐related peptide family. We identified CTRP8 as a ligand of the G protein‐coupled receptor relaxin family peptide receptor 1 (RXFP1) in glioblastoma multiforme (GBM). The CTRP8‐RXFP1 ligand–receptor system protects human GBM cells against the DNA‐alkylating damage‐inducing temozolomide (TMZ), the drug of choice for the treatment of patients with GBM. The DNA protective role of CTRP8 was dependent on a functional RXFP1‐STAT3 signaling cascade and targeted the monofunctional glycosylase N‐methylpurine DNA glycosylase (MPG) for more efficient base excision repair of TMZ‐induced DNA‐damaged sites. CTRP8 also improved the survival of GBM cells by upregulating anti‐apoptotic BCl‐2 and BCL‐XL. Here, we have identified Janus‐activated kinase 3 (JAK3) as a novel member of a novel CTRP8‐RXFP1‐JAK3‐STAT3 signaling cascade that caused an increase in cellular protein content and activity of the small Rho GTPase Cdc42. This is associated with significant F‐actin remodeling and increased GBM motility. Cdc42 was critically important for the upregulation of the actin nucleation complex N‐Wiskott–Aldrich syndrome protein/Arp3/4 and actin elongation factor profilin‐1. The activation of the RXFP1‐JAK3‐STAT3‐Cdc42 axis by both RXFP1 agonists, CTRP8 and relaxin‐2, caused extensive filopodia formation. This coincided with enhanced activity of ezrin, a key factor in tethering F‐actin to the plasma membrane, and inhibition of the actin filament severing activity of cofilin. The F‐actin remodeling and pro‐migratory activities promoted by the novel RXFP1‐JAK3‐STAT3‐Cdc42 axis were blocked by JAK3 inhibitor tofacitinib and STAT3 inhibitor STAT3 inhibitor VI. This provides a new rationale for the design of JAK3 and STAT3 inhibitors with better brain permeability for clinical treatment of the pervasive brain invasiveness of GBM.

Prediction of Glioma Stemlike Cell Infiltration in the Non–Contrast-Enhancing Area by Quantitative Measurement of Lactate on Magnetic Resonance Spectroscopy in Glioblastoma

We previously reported that glioma stemlike cells (GSCs) exist in the area of the tumor periphery showing no gadolinium enhancement on magnetic resonance imaging. In the present work, we analyzed glucose metabolism to investigate whether lactate could be predictive of tumor invasiveness and of use in detection of the tumor invasion area in glioblastoma multiforme (GBM). The expression of lactate dehydrogenase A (LDH-A) and pyruvate dehydrogenase (PDH) was investigated in 20 patients. In GSC lines, LDH-A and PDH expression also was examined in parallel to assessments of mitochondrial respiration. We then investigated the relationship between lactate/creatine ratios in the tumor periphery measured by magnetic resonance spectroscopy, using learning-compression-model algorithms and phenotypes of GBMs. In 20 GBMs, high-invasive GBM expressed LDH-A at significantly higher expression than did low-invasive GBM, whereas low-invasive GBM showed significantly higher expression of PDH than did high-invasive GBM. The highly invasive GSC line showed higher expression of LDH-A and lower expression of PDH compared with low-invasive GSC lines. The highly invasive GSC line also showed the lowest consumption of oxygen and the lowest production of adenosine triphosphate. Lactate levels, as measured by magnetic resonance spectroscopy, showed a significant positive correlation with LDH-A transcript levels, permitting classification of the GBMs into high-invasive and low-invasive phenotypes based on a cutoff value of 0.66 in the lactate/creatine ratio. In the tumor periphery area of the highly invasive GBM, aerobic glycolysis was the predominant pathway for glucose metabolism, resulting in the accumulation of lactate. The level of lactate may facilitate prediction of the tumor-infiltrating area on GBM.

Quantitative proteomics analysis of glioblastoma cell lines after lncRNA HULC silencing

Glioblastoma multiforme (GBM) is a life-threatening brain tumor. This study aimed to identify potential targets of the long noncoding RNA (lncRNA) HULC that promoted the progression of GBM. Two U87 cell lines were constructed: HULC-siRNA and negative control (NC). Quantitative real-time PCR (qRT-PCR) was performed to validate the transfection efficiency of HULC silencing vector. Mass spectrometry (MS) was used to generate proteomic profiles for the two cell lines. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to distinguish HULC-related genes and pathway mapping. Colony formation, Transwell, and wound-healing assays were used to investigate the functional effects of HULC knockdown on GBM. We identified 112 up-regulated proteins and 24 down-regulated proteins from a total of 4360 quantified proteins. GO enrichment illustrated that these proteins were mainly involved in organelle structure, catalysis, cell movement, and material metabolism. KEGG pathway analysis indicated that some of these proteins were significantly enriched in tight junction, metabolic pathways, and arachidonic acid metabolism. In vitro experiments demonstrated that HULC knockdown inhibited GBM cell proliferation, invasion, and migration. Our KEGG analyses revealed that PLA2G4A was a shared protein in several enriched pathways. HULC silencing significantly down-regulated the expression of PLA2G4A. Knockdown of HULC changed the proteomic characteristics of GBM and altered the behaviors of GBM cells. Specifically, we identified PLA2G4A as an HULC target in GBM. This study provides a new perspective on the mechanisms and potential drug targets of GBM treatment.

AMOTL2‑knockdown promotes the proliferation, migration and invasion of glioma by regulating β‑catenin nuclear localization.

Glioblastoma multiforme (GBM) is the most prevalent type of malignant cancer in the adult central nervous system; however, its mechanism remains unclear. Angiomotin-like 2 (AMOTL2) is a member of the motin family of angiostatin-binding proteins. It has been reported as an oncogene in cervical and breast cancer, but its association with glioma remains unknown. The aim of the present study was to investigate AMOTL2-regulated processes in glioma cell lines using extensive in vitro assays and certain bioinformatics tools. These results revealed that AMOTL2 was downregulated in high-grade glioma cells and tissues, with patients with glioma exhibiting a high AMOTL2 expression having a higher survival rate. The results of the glioma cell phenotype experiment showed that AMOTL2 suppressed GBM proliferation, migration and invasion. In addition, immunoblotting, co-immunoprecipitation and immunofluorescence assays demonstrated that AMOTL2 could directly bind to β-catenin protein, the key molecule of the Wnt signaling pathway, and regulate its downstream genes by regulating β-catenin nuclear translocation. In conclusion, the present study demonstrated that AMOTL2 inhibited glioma proliferation, migration and invasion by regulating β-catenin nuclear localization. Thus, AMOTL2 may serve as a therapeutic target to further improve the prognosis and prolong survival time of patients with glioma.

The Novel Protein, Olfactomedin‐like 3, Alters Microglia Biology and May Contribute to Glioma Progression

Despite decades of research, glioblastoma multiforme (GBM) remains a uniformly lethal brain tumor. Transforming growth factor-, in concert with glioma associated microglia (GAMS), promotes GBM growth and invasion. Although TGF- has a significant role in GBM progression, failed clinical trials suggest a complex role in GBM pathogenesis similar to non-CNS cancer. Thus, an improved understanding of the TGF- /GAM axis is critical to refine the therapeutic approach toward precise molecular targets. Olfactomedin-like 3 (OLFML3), a novel secreted glycoprotein, is elevated 9-fold in GBM. Although poorly studied within the context of GBM, OLFML3 contributes to non-CNS tumor progression via increased angiogenesis and cell growth pathways. Importantly, Olfml3 has been identified in mice as a direct target gene of TGF- in microglia. Therefore,we hypothesized that microglia-derived Olfml3 polarizes microglia cells toward a pro-tumorigenic phenotype and promotes tumor cell growth kinetics. We first demonstrated that Olfml3is a direct target gene of all three TGF- isoforms in microglia, but not a mouse glioma cell line (GL261). Using CRISPR/Cas9, we generated an Olfml3-knockout (Olfml3-/-) microglial cell line. Loss of Olfml3 altered microglial production of several pro-tumorigenic molecules. Most notably, secretion of key pro-tumorigenic molecules GM-CSF, IL-6, and CSF-1, was attenuated in Olfml3-/- microglia. Additionally, loss of Olfml3 attenuated microglia chemotaxis. Importantly, the effects of OLFML3 were non-cell autonomous. Following 48-hour treatment with increasing concentrations of rOLFML3, we observed a linear increase in migration of GL261. However, only a single concentration of rOLFML3 increased GL261 invasion, suggesting a concentration-dependent cellular response. In conclusion, Olfml3 is a direct target gene of TGF- in microglia, but not in glioma cells, suggesting that increased OLFML3 in human GBM may be microglia-derived. OLFML3 supports glioma progression via pro-tumorigenic microglia gene expression and promotion of glioma cell malignant phenotype. Further studies aim to elucidate microglia derived OLFML3 and its role in GBM progression in vivo.

Temozolomide Induces the Acquisition of Invasive Phenotype by O6-Methylguanine-DNA Methyltransferase (MGMT)+ Glioblastoma Cells in a Snail-1/Cx43-Dependent Manner

Glioblastoma multiforme (GBM) recurrences after temozolomide (TMZ) treatment result from the expansion of drug-resistant and potentially invasive GBM cells. This process is facilitated by O6-Methylguanine-DNA Methyltransferase (MGMT), which counteracts alkylating TMZ activity. We traced the expansion of invasive cell lineages under persistent chemotherapeutic stress in MGMTlow (U87) and MGMThigh (T98G) GBM populations to look into the mechanisms of TMZ-induced microevolution of GBM invasiveness. TMZ treatment induced short-term, pro-invasive phenotypic shifts of U87 cells, in the absence of Snail-1 activation. They were illustrated by a transient induction of their motility and followed by the hypertrophy and the signs of senescence in scarce U87 sub-populations that survived long-term TMZ stress. In turn, MGMThigh T98G cells reacted to the long-term TMZ treatment with the permanent induction of invasiveness. Ectopic Snail-1 down-regulation attenuated this effect, whereas its up-regulation augmented T98G invasiveness. MGMTlow and MGMThigh cells both reacted to the long-term TMZ stress with the induction of Cx43 expression. However, only in MGMThigh T98G populations, Cx43 was directly involved in the induction of invasiveness, as manifested by the induction of T98G invasiveness after ectopic Cx43 up-regulation and by the opposite effect after Cx43 down-regulation. Collectively, Snail-1/Cx43-dependent signaling participates in the long-term TMZ-induced microevolution of the invasive GBM front. High MGMT activity remains a prerequisite for this process, even though MGMT-related GBM chemoresistance is not necessary for its initiation.

Dual Specificity Kinase DYRK3 Promotes Aggressiveness of Glioblastoma by Altering Mitochondrial Morphology and Function.

Glioblastoma multiforme (GBM) is a malignant primary brain tumor with poor patient prognosis. Although the standard treatment of GBM is surgery followed by chemotherapy and radiotherapy, often a small portion of surviving tumor cells acquire therapeutic resistance and become more aggressive. Recently, altered kinase expression and activity have been shown to determine metabolic flux in tumor cells and metabolic reprogramming has emerged as a tumor progression regulatory mechanism. Here we investigated novel kinase-mediated metabolic alterations that lead to acquired GBM radioresistance and malignancy. We utilized transcriptomic analyses within a radioresistant GBM orthotopic xenograft mouse model that overexpresses the dual specificity tyrosine-phosphorylation-regulated kinase 3 (DYRK3). We find that within GBM cells, radiation exposure induces DYRK3 expression and DYRK3 regulates mammalian target of rapamycin complex 1 (mTORC1) activity through phosphorylation of proline-rich AKT1 substrate 1 (PRAS40). We also find that DYRK3 knockdown inhibits dynamin-related protein 1 (DRP1)-mediated mitochondrial fission, leading to increased oxidative phosphorylation (OXPHOS) and reduced glycolysis. Importantly, enforced DYRK3 downregulation following irradiation significantly impaired GBM cell migration and invasion. Collectively, we suggest DYRK3 suppression may be a novel strategy for preventing GBM malignancy through regulating mitochondrial metabolism.

Ciclopirox and bortezomib synergistically inhibits glioblastoma multiforme growth via simultaneously enhancing JNK/p38 MAPK and NF-\u03baB signaling

Ciclopirox (CPX) is an antifungal drug that has recently been reported to act as a potential anticancer drug. However, the effects and underlying molecular mechanisms of CPX on glioblastoma multiforme (GBM) remain unknown. Bortezomib (BTZ) is the first proteasome inhibitor-based anticancer drug approved to treat multiple myeloma and mantle cell lymphoma, as BTZ exhibits toxic effects on diverse tumor cells. Herein, we show that CPX displays strong anti-tumorigenic activity on GBM. Mechanistically, CPX inhibits GBM cellular migration and invasion by reducing N-Cadherin, MMP9 and Snail expression. Further analysis revealed that CPX suppresses the expression of several key subunits of mitochondrial enzyme complex, thus leading to the disruption of mitochondrial oxidative phosphorylation (OXPHOS) in GBM cells. In combination with BTZ, CPX promotes apoptosis in GBM cells through the induction of reactive oxygen species (ROS)-mediated c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) signaling. Moreover, CPX and BTZ synergistically activates nuclear factor kappa B (NF-κB) signaling and induces cellular senescence. Our findings suggest that a combination of CPX and BTZ may serve as a novel therapeutic strategy to enhance the anticancer activity of CPX against GBM.

CXCR4 antagonism sensitizes cancer cells to novel indole-based MDM2/4 inhibitors in glioblastoma multiforme

Glioblastoma Multiforme (GBM) is a highly invasive primary brain tumour characterized by chemo- and radio-resistance and poor overall survival. GBM can present an aberrant functionality of p53, caused by the overexpression of the murine double minute 2 protein (MDM2) and its analogue MDM4, which may influence the response to conventional therapies. Moreover, tumour resistance/invasiveness has been recently attributed to an overexpression of the chemokine receptor CXCR4, identified as a pivotal mediator of glioma neovascularization. Notably, CXCR4 and MDM2-4 cooperate in promoting tumour invasion and progression. Although CXCR4 actively promotes MDM2 activation leading to p53 inactivation, MDM2-4 knockdown induces the downregulation of CXCR4 gene transcription.Our study aimed to assess if the CXCR4 signal blockade could enhance glioma cells’ sensitivity to the inhibition of the p53-MDMs axis. Rationally designed inhibitors of MDM2/4 were combined with the CXCR4 antagonist, AMD3100, in human GBM cells and GBM stem-like cells (neurospheres), which are crucial for tumour recurrence and chemotherapy resistance. The dual MDM2/4 inhibitor RS3594 and the CXCR4 antagonist AMD3100 reduced GBM cell invasiveness and migration in single-agent treatment and mainly in combination. AMD3100 sensitized GBM cells to the antiproliferative activity of RS3594. It is noteworthy that these two compounds present synergic effects on cancer stem components: RS3594 inhibited the growth and formation of neurospheres, AMD3100 induced differentiation of neurospheres while enhancing RS3594 effectiveness preventing their proliferation/clonogenicity. These results confirm that blocking CXCR4/MDM2/4 represents a valuable strategy to reduce GBM proliferation and invasiveness, acting on the stem cell component too.

Brahma-Related Gene-1 (BRG1) promotes the malignant phenotype of glioblastoma cells.

Glioblastoma multiforme (GBM) is an aggressive malignant brain tumour that is resistant to existing therapeutics. Identifying signalling pathways deregulated in GBM that can be targeted therapeutically is critical to improve the present dismal prognosis for GBM patients. In this report, we have identified that the BRG1 (Brahma‐Related Gene‐1) catalytic subunit of the SWI/SNF chromatin remodelling complex promotes the malignant phenotype of GBM cells. We found that BRG1 is ubiquitously expressed in tumour tissue from GBM patients, and high BRG1 expression levels are localized to specific brain tumour regions. Knockout (KO) of BRG1 by CRISPR‐Cas9 gene editing had minimal effects on GBM cell proliferation, but significantly inhibited GBM cell migration and invasion. BRG1‐KO also sensitized GBM cells to the anti‐proliferative effects of the anti‐cancer agent temozolomide (TMZ), which is used to treat GBM patients in the clinic, and selectively altered STAT3 tyrosine phosphorylation and gene expression. These results demonstrate that BRG‐1 promotes invasion and migration, and decreases chemotherapy sensitivity, indicating that it functions in an oncogenic manner in GBM cells. Taken together, our findings suggest that targeting BRG1 in GBM may have therapeutic benefit in the treatment of this deadly form of brain cancer.

MELK Inhibition Effectively Suppresses Growth of Glioblastoma and Cancer Stem-Like Cells by Blocking AKT and FOXM1 Pathways.

Glioblastoma multiforme (GBM) is a devastating disease yet no effective drug treatment has been established to date. Glioblastoma stem-like cells (GSCs) are insensitive to treatment and may be one of the reasons for the relapse of GBM. Maternal embryonic leucine zipper kinase gene (MELK) plays an important role in the malignant proliferation and the maintenance of GSC stemness properties of GBM. However, the therapeutic effect of targeted inhibition of MELK on GBM remains unclear. This study analyzed the effect of a MELK oral inhibitor, OTSSP167, on GBM proliferation and the maintenance of GSC stemness. OTSSP167 significantly inhibited cell proliferation, colony formation, invasion, and migration of GBM. OTSSP167 treatment reduced the expression of cell cycle G2/M phase-related proteins, Cyclin B1 and Cdc2, while up-regulation the expression of p21 and subsequently induced cell cycle arrest at the G2/M phase. OTSSP167 effectively prolonged the survival of tumor-bearing mice and inhibited tumor cell growth in in vivo mouse models. It also reduced protein kinase B (AKT) phosphorylation levels by OTSSP167 treatment, thereby disrupting the proliferation and invasion of GBM cells. Furthermore, OTSSP167 inhibited the proliferation, neurosphere formation and self-renewal capacity of GSCs by reducing forkhead box M1 (FOXM1) phosphorylation and transcriptional activity. Interestingly, the inhibitory effect of OTSSP167 on the proliferation of GSCs was 4-fold more effective than GBM cells. In conclusion, MELK inhibition suppresses the growth of GBM and GSCs by double-blocking AKT and FOXM1 signals. Targeted inhibition of MELK may thus be potentially used as a novel treatment for GBM.

The lncRNA RP3-439F8.1 promotes GBM cell proliferation and progression by sponging miR-139-5p to upregulate NR5A2

BackgroundNuclear Receptor Subfamily 5 Group A Member 2 (NR5A2, LRH-1) is an oncogene in a wide range of cancer types. Bioinformatics analysis on glioblastoma multiforme (GBM) tumors has revealed that the miR-139-5p-NR5A2 axis may be putatively regulated by the long non-coding RNA (lncRNA) RP3-439F8.1. This led us to hypothesize the existence of a RP3-439F8.1-miR-139-5p-NR5A2 regulatory axis in GBM cells. MethodsGene expression analysis was performed in GBM tumor samples and normal controls from our hospital, the Cancer Genome Atlas Glioblastoma Multiforme (TCGA-GBM) cohort, and the Gene Expression Omnibus (GEO) database (GSE7696). Cell proliferation, apoptosis, Matrigel Transwell, colony formation, and cell cycle assays were performed in T98 G and U251 cells in vitro. An orthotopic U251 xenograft murine model was employed to test the effects of RP3-439F8.1 knockdown in vivo. ResultsNR5A2 was upregulated in the three independent GBM tumor cohorts. In vitro, NR5A2 overexpression enhanced GBM cell proliferation, colony formation, invasiveness, and G0-G1 cell cycle phase shift via co-activating β-catenin/TCF4 signaling, with no apparent effect upon apoptosis. In contrast, RP3-439F8.1 knockdown produced the opposite effects. RP3-439F8.1 knockdown reduced tumor progression in vivo, increasing overall survival in model mice. Further in vitro experiments revealed that RP3-439F8.1 acts as a competing endogenous RNA (ceRNA) to regulate NR5A2 by sponging the microRNA miR-139-5p. These findings were clinically validated by a positive correlation between RP3-439F8.1 and NR5A2 and a negative correlation between RP3-439F8.1 and miR-139-5p in GBM tumors. ConclusionsOur study supports a tumorigenic role for RP3-439F8.1 in GBM through the RP3-439F8.1/miR-139-5p/NR5A2 axis.

Fishing for Contact: Modeling Perivascular Glioma Invasion in the Zebrafish Brain.

Glioblastoma multiforme (GBM) is a highly invasive, central nervous system (CNS) cancer for which there is no cure. Invading tumor cells evade treatment, limiting the efficacy of the current standard of care regimen. Understanding the underlying invasive behaviors that support tumor growth may allow for generation of novel GBM therapies. Zebrafish (Danio rerio) are attractive for genetics and live imaging and have, in recent years, emerged as a model system suitable for cancer biology research. While other groups have studied CNS tumors using zebrafish, few have concentrated on the invasive behaviors supporting the development of these diseases. Previous studies demonstrated that one of the main mechanisms of GBM invasion is perivascular invasion, i.e., single tumor cell migration along blood vessels. Here, we characterize phenotypes, methodology, and potential therapeutic avenues for utilizing zebrafish to model perivascular GBM invasion. Using patient-derived xenolines or an adherent cell line, we demonstrate tumor expansion within the zebrafish brain. Within 24-h postintracranial injection, D54-MG-tdTomato glioma cells produce fingerlike projections along the zebrafish brain vasculature. As few as 25 GBM cells were sufficient to promote single cell vessel co-option. Of note, these tumor-vessel interactions are CNS specific and do not occur on pre-existing blood vessels when injected into the animal's peripheral tissue. Tumor-vessel interactions increase over time and can be pharmacologically disrupted through inhibition of Wnt signaling. Therefore, zebrafish serve as a favorable model system to study perivascular glioma invasion, one of the deadly characteristics that make GBM so difficult to treat.

Hsa_circ_0043278 functions as competitive endogenous RNA to enhance glioblastoma multiforme progression by sponging miR-638.

Circular RNAs have a critical function in the pathogenesis of many diseases and can function as competing endogenous RNA or miRNA sponges to inhibit miRNA and therefore upregulate the expression of target genes. However, little is known about the role of has_circRNA_0043278 (circ_0043278) in glioblastoma multiforme (GBM) and its potential downstream miRNA targets. This work validated that circ_0043278 is highly expressed in GMB cell lines and tissues, while knockdown circ_0043278 inhibited GBM cell migration, proliferation, and invasion in vitro and tumorigenesis in vivo. Dual-luciferase reporter assay determined that circ_0043278 directly sponged miR-638 to upregulate the expression of HOXA9, which can activate downstream Wnt/β-catenin signaling in GBM. Moreover, miR-638 inhibition reversed circ_0043278 silencing-induced impairment of malignant tumor behavior. These results showed that circ-0043278/miRNA-638/ Homeobox A9 (HOXA9) axis had a vital function in promoting GBM progression. Our findings may provide potential new targets for the diagnosis and therapy of GBM.