Glioblastoma Invasion Research Articles

COL1A2 inhibition suppresses glioblastoma cell proliferation and invasion.

An extracellular matrix such as collagen is an essential component of the tumor microenvironment. Collagen alpha-2(I) chain (COL1A2) is a chain of type I collagen whose triple helix comprises two alpha-1 chains and one alpha-2 chain. The authors' proteomics data showed that COL1A2 is significantly higher in the blood of patients with glioblastoma (GBM) compared with healthy controls. COL1A2 has many different functions in various types of cancers. However, the functions of COL1A2 in GBM are poorly understood. In this study, the authors analyzed the functions of COL1A2 and its signaling pathways in GBM. Surgical specimens and GBM cell lines (T98, U87, and U251) were used. The expression level of COL1A2 was examined using GBM tissues and normal brain tissues by quantitative real-time polymerase chain reaction. The clinical significance of these levels was evaluated using Kaplan-Meier analysis. Small interfering RNA (siRNA) and small hairpin RNA of COL1A2 were transfected into GBM cell lines to investigate the function of COL1A2 in vitro and in vivo. Flow cytometry was introduced to analyze the alteration of cell cycles. Western blot and immunohistochemistry were performed to analyze the underlying mechanisms. The expression level of COL1A2 was upregulated in GBM compared with normal brain tissues. A higher expression of COL1A2 was correlated with poor progression-free survival and overall survival. COL1A2 inhibition significantly suppressed cell proliferation in vitro and in vivo, likely due to G1 arrest. The invasion ability was notably deteriorated by inhibiting COL1A2. Cyclin D1, cyclin-dependent kinase 1, and cyclin-dependent kinase 4, which are involved in the cell cycle, were all downregulated after blockade of COL1A2 in vitro and in vivo. Phosphoinositide 3-kinase inhibitor reduced the expression of COL1A2. Although downregulation of COL1A2 decreased the protein kinase B (Akt) phosphorylation, Akt activator can phosphorylate Akt in siRNA-treated cells. This finding suggests that Akt phosphorylation is partially dependent on COL1A2. COL1A2 plays an important role in driving GBM progression. COL1A2 inhibition attenuated GBM proliferation by promoting cell cycle arrest, indicating that COL1A2 could be a promising therapeutic target for GBM treatment.

Bioconjugation of COL1 protein on liquid-like solid surfaces to study tumor invasion dynamics.

Tumor invasion is likely driven by the product of intrinsic and extrinsic stresses, reduced intercellular adhesion, and reciprocal interactions between the cancer cells and the extracellular matrix (ECM). The ECM is a dynamic material system that is continuously evolving with the tumor microenvironment. Although it is widely reported that cancer cells degrade the ECM to create paths for migration using membrane-bound and soluble enzymes, other nonenzymatic mechanisms of invasion are less studied and not clearly understood. To explore tumor invasion that is independent of enzymatic degradation, we have created an open three-dimensional (3D) microchannel network using a novel bioconjugated liquid-like solid (LLS) medium to mimic both the tortuosity and the permeability of a loose capillary-like network. The LLS is made from an ensemble of soft granular microgels, which provides an accessible platform to investigate the 3D invasion of glioblastoma (GBM) tumor spheroids using in situ scanning confocal microscopy. The surface conjugation of the LLS microgels with type 1 collagen (COL1-LLS) enables cell adhesion and migration. In this model, invasive fronts of the GBM microtumor protruded into the proximal interstitial space and may have locally reorganized the surrounding COL1-LLS. Characterization of the invasive paths revealed a super-diffusive behavior of these fronts. Numerical simulations suggest that the interstitial space guided tumor invasion by restricting available paths, and this physical restriction is responsible for the super-diffusive behavior. This study also presents evidence that cancer cells utilize anchorage-dependent migration to explore their surroundings, and geometrical cues guide 3D tumor invasion along the accessible paths independent of proteolytic ability.

Anti-seed PNAs targeting multiple oncomiRs for brain tumor therapy.

Glioblastoma (GBM) is one of the most lethal malignancies with poor survival and high recurrence rates. Here, we aimed to simultaneously target oncomiRs 10b and 21, reported to drive GBM progression and invasiveness. We designed short (8-mer) γ-modified peptide nucleic acids (sγPNAs), targeting the seed region of oncomiRs 10b and 21. We entrapped these anti-miR sγPNAs in nanoparticles (NPs) formed from a block copolymer of poly(lactic acid) and hyperbranched polyglycerol (PLA-HPG). The surface of the NPs was functionalized with aldehydes to produce bioadhesive NPs (BNPs) with superior transfection efficiency and tropism for tumor cells. When combined with temozolomide, sγPNA BNPs administered via convection-enhanced delivery (CED) markedly increased the survival (>120 days) of two orthotopic (intracranial) mouse models of GBM. Hence, we established that BNPs loaded with anti-seed sγPNAs targeting multiple oncomiRs are a promising approach to improve the treatment of GBM, with a potential to personalize treatment based on tumor-specific oncomiRs.

The Interleukin-11/IL-11 Receptor Promotes Glioblastoma Survival and Invasion under Glucose-Starved Conditions through Enhanced Glutaminolysis

Glioblastoma cells adapt to changes in glucose availability through metabolic plasticity allowing for cell survival and continued progression in low-glucose concentrations. However, the regulatory cytokine networks that govern the ability to survive in glucose-starved conditions are not fully defined. In the present study, we define a critical role for the IL-11/IL-11Rα signalling axis in glioblastoma survival, proliferation and invasion when cells are starved of glucose. We identified enhanced IL-11/IL-11Rα expression correlated with reduced overall survival in glioblastoma patients. Glioblastoma cell lines over-expressing IL-11Rα displayed greater survival, proliferation, migration and invasion in glucose-free conditions compared to their low-IL-11Rα-expressing counterparts, while knockdown of IL-11Rα reversed these pro-tumorigenic characteristics. In addition, these IL-11Rα-over-expressing cells displayed enhanced glutamine oxidation and glutamate production compared to their low-IL-11Rα-expressing counterparts, while knockdown of IL-11Rα or the pharmacological inhibition of several members of the glutaminolysis pathway resulted in reduced survival (enhanced apoptosis) and reduced migration and invasion. Furthermore, IL-11Rα expression in glioblastoma patient samples correlated with enhanced gene expression of the glutaminolysis pathway genes GLUD1, GSS and c-Myc. Overall, our study identified that the IL-11/IL-11Rα pathway promotes glioblastoma cell survival and enhances cell migration and invasion in environments of glucose starvation via glutaminolysis.

Glioblastoma Spheroid Invasion through Soft, Brain-Like Matrices Depends on Hyaluronic Acid-CD44 Interactions.

Increased secretion of hyaluronic acid (HA), a glycosaminoglycan abundant in the brain extracellular matrix (ECM), correlates with worse clinical outcomes for glioblastoma (GBM) patients. GBM cells aggressively invade the brain parenchyma while encountering spatiotemporal changes in their local ECM, including HA concentration. To investigate how varying HA concentrations affect GBM invasion, patient-derived GBM cells are cultured within a soft, 3D matrix in which HA concentration is precisely varied and cell migration observed. Data demonstrate that HA concentration can determine the invasive activity of patient-derived GBM cells in a biphasic and highly sensitive manner, where the absolute concentration of HA at which cell migration peaked is specific to each patient-derived line. Furthermore, evidence that this response relies on phosphorylated ezrin, which interacts with the intracellular domain of HA-engaged CD44 to effectively link the actin cytoskeleton to the local ECM is provided. Overall, this study highlights CD44-HA binding as a major mediator of GBM cell migration that acts independently of integrins and focal adhesion complexes and suggests that targeting HA-CD44-ezrin interactions represents a promising therapeutic strategy to prevent tumor cell invasion in the brain.

An agonistic anti-Tie2 antibody suppresses the normal-to-tumor vascular transition in the glioblastoma invasion zone

Tumor progression is intimately associated with the vasculature, as tumor proliferation induces angiogenesis and tumor cells metastasize to distant organs via blood vessels. However, whether tumor invasion is associated with blood vessels remains unknown. As glioblastoma (GBM) is featured by aggressive invasion and vascular abnormalities, we characterized the onset of vascular remodeling in the diffuse tumor infiltrating zone by establishing new spontaneous GBM models with robust invasion capacity. Normal brain vessels underwent a gradual transition to severely impaired tumor vessels at the GBM periphery over several days. Increasing vasodilation from the tumor periphery to the tumor core was also found in human GBM. The levels of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) showed a spatial correlation with the extent of vascular abnormalities spanning the tumor-invading zone. Blockade of VEGFR2 suppressed vascular remodeling at the tumor periphery, confirming the role of VEGF-VEGFR2 signaling in the invasion-associated vascular transition. As angiopoietin-2 (ANGPT2) was expressed in only a portion of the central tumor vessels, we developed a ligand-independent tunica interna endothelial cell kinase 2 (Tie2)-activating antibody that can result in Tie2 phosphorylation in vivo. This agonistic anti-Tie2 antibody effectively normalized the vasculature in both the tumor periphery and tumor center, similar to the effects of VEGFR2 blockade. Mechanistically, this antibody-based Tie2 activation induced VE-PTP-mediated VEGFR2 dephosphorylation in vivo. Thus, our study reveals that the normal-to-tumor vascular transition is spatiotemporally associated with GBM invasion and may be controlled by Tie2 activation via a novel mechanism of action.

Subtype-specific block of glioblastoma motility through dual inhibition of water and ion flux.

The aggressive invasion of glioma cells into healthy brain parenchyma makes glioblastoma multiforme (GBM) difficult to overcome with current treatment regimens, which are cytotoxic and not localised to the primary tumour site. Further limitations are imposed by extensive cellular heterogeneity within glioblastoma tumours, which can leave treatment-resistant cells intact and poised to initiate relapse. Targeting unique subsets of proteins implicated in cellular motility pathways, namely water and ion channels, could present a novel means of controlling glioblastoma invasion. Pharmacological inhibition of selected ion channels and aquaporin water channels that are upregulated in GBM decreased invasiveness of U87-MG and U251-MG glioblastoma cells in vitro at non-cytotoxic doses. Notably, combining ion channel blockers with aquaporin-1 inhibitor AqB013 evoked additive inhibition of invasion. These additive effects differed between cell lines, consistent with the differences in ion channel and water channel expression profiles in U87-MG and U251-MG.Impeding glioblastoma tumour invasion by concurrently targeting aquaporins and ion channels with functions in cellular motility pathways could constitute a powerful adjunct therapy when applied in parallel with existing therapies. Classes of channels that would be optimal targets for combined therapies could be tailored to GBM cancer cell subtypes. Administering specific combinations of aquaporin and ion channel antagonists at low doses could provide effective means of overcoming signalling pathway redundancy in GBM cells, exploiting the differences in channel expression patterns unique to each subtype while minimising toxicity in surrounding tissue. In response to ongoing calls for new therapeutic interventions for glioblastoma, this work proposes that simultaneous inhibition of water and ion movement in motile glioma cells can halt GBM cell spread and could augment the effectiveness of current standard practices used for eradicating primary tumours, constituting a promising addition to the targeted cancer therapy repertoire.

Aquaporins and Ion Channels as Dual Targets in the Design of Novel Glioblastoma Therapeutics to Limit Invasiveness.

Current therapies for Glioblastoma multiforme (GBM) focus on eradicating primary tumors using radiotherapy, chemotherapy and surgical resection, but have limited success in controlling the invasive spread of glioma cells into a healthy brain, the major factor driving short survival times for patients post-diagnosis. Transcriptomic analyses of GBM biopsies reveal clusters of membrane signaling proteins that in combination serve as robust prognostic indicators, including aquaporins and ion channels, which are upregulated in GBM and implicated in enhanced glioblastoma motility. Accumulating evidence supports our proposal that the concurrent pharmacological targeting of selected subclasses of aquaporins and ion channels could impede glioblastoma invasiveness by impairing key cellular motility pathways. Optimal sets of channels to be selected as targets for combined therapies could be tailored to the GBM cancer subtype, taking advantage of differences in patterns of expression between channels that are characteristic of GBM subtypes, as well as distinguishing them from non-cancerous brain cells such as neurons and glia. Focusing agents on a unique channel fingerprint in GBM would further allow combined agents to be administered at near threshold doses, potentially reducing off-target toxicity. Adjunct therapies which confine GBM tumors to their primary sites during clinical treatments would offer profound advantages for treatment efficacy.

Spastin Promotes the Migration and Invasion Capability of T98G Glioblastoma Cells by Interacting with Pin1 through Its Microtubule-Binding Domain.

Microtubule-severing protein Spastin has been shown to co-localize with actin in migratory glioblastoma cells and is linked to glioblastomas' migration and invasion capacity. However, the effectiveness of Spastin in glioblastoma migration and the molecular mechanism underpinning the orientation of Spastin towards actin filaments remain unknown. Here, we demonstrated that Spastin plays an active role in glioblastoma migration by showing a reduced migratory potential of T98G glioblastoma cells using real-time cell analysis (RTCA) in Spastin-depleted cells. Pull-down assays revealed that a cis-trans isomerase Pin1 interacts with Spastin through binding to the phosphorylated Pin1 recognition motifs in the microtubule-binding domain (MBD), and immunocytochemistry analysis showed that interaction with Pin1 directs Spastin to actin filaments in extended cell regions. Consequently, by utilizing RTCA, we proved that the migration and invasion capacity of T98G glioblastoma cells significantly increased with the overexpression of Spastin, of which the Pin1 recognition motifs in MBD are constitutively phosphorylated, while the overexpression of phospho-mutant form did not have a significant effect on migration and invasion of T98G glioblastoma cells. These findings demonstrate that Pin1 is a novel interaction partner of Spastin, and their interaction drives Spastin to actin filaments, allowing Spastin to contribute to the glioblastomas' migration and invasion abilities.

Flotillin-1 is a prognostic biomarker for glioblastoma and promotes cancer development through enhancing invasion and altering tumour microenvironment.

Flotillin-1(FLOT1) has long been recognized as a tumour-promoting gene in several types of cancer. However, the expression and function of FLOT1 in glioblastomas (GBM) has not been elucidated. Here, in this study, we find that the expression level of FLOT1 in GBM tissue was much higher than that in normal brain, and the expression was even higher in the more aggressive subtypes and IDH status of glioma. Kaplan-Meier survival revealed that high FLOT1 expression is closely associated with poor outcome in GBM patients. FLOT1 knockdown markedly reduced the proliferation, migration and invasiveness of GBM cells, while FLOT1 overexpression significantly increases GBM cell proliferation, migration and invasiveness. Mechanistically, FLOT1 expression may play a potential role in the microenvironment of GBM. Therefore, FLOT1 promotes GBM proliferation and invasion in vitro and in vivo and may serve as a biomarker of prognosis and therapeutic potential in the fight against GBM.

Abstract A045: Examining the role of RNA:DNA hybrids (r loops) in glioblastoma multiforme cell motility and invasion

Abstract Glioblastoma multiforme (GBM) is a highly aggressive brain cancer. The standard course of treatment is a combination of radiation and chemotherapy. Even with the dual treatment, the 5-year survival rate of patients with GBM is between 4-10%. A possible cause of the low survival rate for GBM patients is the presence of motile neoplastic cells with efficient DNA repair abilities. Thus, the two major obstacles to treating GBMs is 1) the tumors inherent invasive nature and 2) the recurrent tumors ability to acquire treatment resistance. As such, gaining a better understanding of the mechanisms mediating GBM recurrence would allow us to design better treatment strategies for patients. We have found that the 5’-3’ exoribonuclease XRN2 is upregulated in GBMs as compared to normal and other brain cancer types. While XRN2 is traditionally associated with the process of transcription termination, our published work has defined a role for XRN2 in the DNA damage response. Our group has shown that loss of XRN2 sensitizes to a variety of DNA damaging agents but in particular ionizing radiation. Previously, we have shown that XRN2 is required for DNA repair, specifically double stranded break (DSB) repair. Loss of XRN2 impairs both the non-homologous end-joining and homologous recombination DSB repair pathways. Loss of XRN2 also results in increased spontaneous DSB formation, chromosomal aberrations and RNA:DNA hybrid (R-loop) formation. In addition to a role in the DNA damage response, we found that loss of XRN2 is required for GBM cell motility, invasion through a matrix and decreased tumor volume in mouse models. Interestingly, R-loop formation can affect gene expression by modulating the access of genes to transcription factors, miRNA transcription, and methylation status of genes. To gain a better understanding of how XRN2 modulates gene expression, we conducted RNA-Seq analyses of GBM cell lines with and without XRN2 expression and found that XRN2 can regulate genes involved in cell movement, cell development and cellular organization programs. Consistent with our findings that XRN2 impairs DNA damage and tumor invasion; XRN2 loss results in R-loop formation in promoters of genes important for tumor invasiveness and DNA repair such as FOXM1 and Cyclin B1. These data suggest that the regulation of R-loop formation is an important determinant in tumor progression. Citation Format: Julio C. Morales, Tuyen T. Dang. Examining the role of RNA:DNA hybrids (r loops) in glioblastoma multiforme cell motility and invasion [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A045.

Fluoride in the Central Nervous System and Its Potential Influence on the Development and Invasiveness of Brain Tumours-A Research Hypothesis.

The purpose of this review is to attempt to outline the potential role of fluoride in the pathogenesis of brain tumours, including glioblastoma (GBM). In this paper, we show for the first time that fluoride can potentially affect the generally accepted signalling pathways implicated in the formation and clinical course of GBM. Fluorine compounds easily cross the blood-brain barrier. Enhanced oxidative stress, disruption of multiple cellular pathways, and microglial activation are just a few examples of recent reports on the role of fluoride in the central nervous system (CNS). We sought to present the key mechanisms underlying the development and invasiveness of GBM, as well as evidence on the current state of knowledge about the pleiotropic, direct, or indirect involvement of fluoride in the regulation of these mechanisms in various tissues, including neural and tumour tissue. The effects of fluoride on the human body are still a matter of controversy. However, given the growing incidence of brain tumours, especially in children, and numerous reports on the effects of fluoride on the CNS, it is worth taking a closer look at these mechanisms in the context of brain tumours, including gliomas.

CircNDC80 promotes glioblastoma multiforme tumorigenesis via the miR-139-5p/ECE1 pathway

BackgroundCircular RNAs (circRNAs) have been shown to be essential for the emergence and growth of different cancers. However, further research is required to validate the function of circRNA in glioblastoma (GBM).MethodsCircNDC80 expression in both normal brain tissues (NBTs) and glioma tissues was determined using real-time PCR. The impact of circNDC80 on GBM cell proliferation, migration, and invasion was then confirmed by CCK-8, colony formation, EdU incorporation, Transwell, and wound healing assays. To determine how circNDC80 affects the capacity of glioma stem cells (GSCs) to maintain their stemness and self-renewal, a CellTiter-Glo assay, clonogenic assay and extreme limiting dilution assay were utilized. To ascertain the impact of circNDC80 in vivo, intracranial xenograft models were established.ResultsWhen compared to NBT, glioblastoma tissue had a higher level of circNDC80 expression. In functional assays, circNDC80 promoted glioblastoma cell proliferation, migration, and invasion, while sustaining the stemness and fostering the self-renewal of glioma stem cells. In addition, a dual luciferase reporter assay and circRIP were used to verify that circNDC80 simultaneously affects the expression of ECE1 mRNA by sponging miR-139-5p, and a rescue experiment was used to verify the above results further.ConclusionsAccording to our research, circNDC80 is an oncogenic factor that promotes glioblastoma through the miR-139-5p/ECE1 pathway. This implies that circNDC80 may be employed as a novel therapeutic target and a possible predictive biomarker.

Sphingosine-1-Phosphate Recruits Macrophages and Microglia and Induces a Pro-Tumorigenic Phenotype That Favors Glioma Progression.

Glioblastoma is the most aggressive brain tumor in adults. Treatment failure is predominantly caused by its high invasiveness and its ability to induce a supportive microenvironment. As part of this, a major role for tumor-associated macrophages/microglia (TAMs) in glioblastoma development was recognized. Phospholipids are important players in various fundamental biological processes, including tumor-stroma crosstalk, and the bioactive lipid sphingosine-1-phosphate (S1P) has been linked to glioblastoma cell proliferation, invasion, and survival. Despite the urgent need for better therapeutic approaches, novel strategies targeting sphingolipids in glioblastoma are still poorly explored. Here, we showed that higher amounts of S1P secreted by glioma cells are responsible for an active recruitment of TAMs, mediated by S1P receptor (S1PR) signaling through the modulation of Rac1/RhoA. This resulted in increased infiltration of TAMs in the tumor, which, in turn, triggered their pro-tumorigenic phenotype through the inhibition of NFkB-mediated inflammation. Gene set enrichment analyses showed that such an anti-inflammatory microenvironment correlated with shorter survival of glioblastoma patients. Inhibition of S1P restored a pro-inflammatory phenotype in TAMs and resulted in increased survival of tumor-bearing mice. Taken together, our results establish a crucial role for S1P in fine-tuning the crosstalk between glioma and infiltrating TAMs, thus pointing to the S1P-S1PR axis as an attractive target for glioma treatment.

Mesenchymal Stem-Like Cells Derived from the Ventricle More Effectively Enhance Invasiveness of Glioblastoma Than Those Derived from the Tumor.

Glioblastoma (GBM) is one of the most lethal human tumors with a highly infiltrative phenotype. Our previous studies showed that GBM originates in the subventricular zone, and that tumor-derived mesenchymal stem-like cells (tMSLCs) promote the invasiveness of GBM tumorspheres (TSs). Here, we extend these studies in terms of ventricles using several types of GBM patient-derived cells. The invasiveness of GBM TSs and ventricle spheres (VSs) were quantified via collagen-based 3D invasion assays. Gene expression profiles were obtained from microarray data. A mouse orthotopic xenograft model was used for in vivo experiments. After molecular and functional characterization of ventricle-derived mesenchymal stem-like cells (vMSLCs), we investigated the effects of these cells on the invasiveness of GBM TSs. We found that vMSLC-conditioned media (CM) significantly accelerated the invasiveness of GBM TSs and VSs, compared to the control and even tMSLC-CM. Transcriptome analyses revealed that vMSLC secreted significantly higher levels of several invasiveness-associated cytokines. Moreover, differentially expressed genes between vMSLCs and tMSLCs were enriched for migration, adhesion, and chemotaxis-related gene sets, providing a mechanistic basis for vMSLC-induced invasion of GBM TSs. In vivo experiments using a mouse orthotopic xenograft model confirmed vMSLC-induced increases in the invasiveness of GBM TSs. Although vMSLCs are non-tumorigenic, this study adds to our understanding of how GBM cells acquire infiltrative features by vMSLCs, which are present in the region where GBM genesis originates.

Long non-coding RNA DPP10-AS1 represses the proliferation and invasiveness of glioblastoma by regulating miR-24-3p/CHD5 signaling pathway

Long non-coding RNA DPP10-AS1 represses the proliferation and invasiveness of glioblastoma by regulating miR-24-3p/CHD5 signaling pathway

Inhibition of STAT3 signaling as critical molecular event in HUC-MSCs suppressed Glioblastoma Cells.

Objective: We investigated the effect of human umbilical cord mesenchymal stem cells (HUC-MSCs) supernatants on proliferation, migration, invasion, and apoptosis in glioblastoma (GBM) cell lines RG-2, U251, U87-MG, and LN-428, as well as their apoptosis and autophagy-mediated through IL-6/JAK2/STAT3 signaling pathway to explore the molecular mechanisms. Methods: In this study, RG-2, U251, U87-MG, and LN-428 cells were treated with 9 mg/ml HUC-MSCs supernatants. Their responses to HUC-MSCs supernatants treatment and the status of STAT3 signaling were analyzed by multiple experimental approaches to elucidate the importance of HUC-MSCs supernatants for GBM. Results: The results demonstrated that after treatment with HUC-MSCs supernatants, in vitro proliferation of RG-2, U251, U87-MG, and LN-428 cells were inhibited, and their sustained growth was also blocked. RG-2, U251, and U87-MG cells showed significant S phase accumulation, while LN-428 cells were blocked in G0/G1 phase. Their migratory invasive capacities were inhibited, and their apoptosis and autophagy ratios were increased. These effects were mediated through the IL-6/JAK2/STAT3 and its downstream signaling pathway. Conclusion: Our data showed that HUC-MSCs supernatants had anti-tumor effects on GBM cells. It inhibited the proliferation, migration, and invasion of GBM cells and promoted their apoptosis. Negative regulation of the IL-6/JAK2/STAT3 signaling pathway enhanced apoptosis and autophagy in tumor cells, thereby improving the therapeutic effect on GBM.

CHRM3 is a novel prognostic factor of poor prognosis and promotes glioblastoma progression via activation of oncogenic invasive growth factors.

Glioblastoma (GBM) is the most aggressive cancer of the brain and has a high mortality rate due to the lack of effective treatment strategy. Clarification of molecular mechanisms of GBM's characteristic invasive growth is urgently needed to improve the poor prognosis. Single-nuclear sequencing of primary and recurrent GBM samples revealed that levels of M3 muscarinic acetylcholine receptor (CHRM3) were significantly higher in the recurrent samples than in the primary samples. Moreover, immunohistochemical staining of an array of GBM samples showed that high levels of CHRM3 correlated with poor prognosis, consistent with The Cancer Genome Atlas database. Knockdown of CHRM3 inhibited GBM cell growth and invasion. An assay of orthotopic GBM animal model in vivo indicated that inhibition of CHRM3 significantly suppressed GBM progression with prolonged survival time. Transcriptome analysis revealed that CHRM3 knockdown significantly reduced an array of classic factors involved in cancer invasive growth, including MMP1/MMP3/MMP10/MMP12 and CXCL1/CXCL5/CXCL8. Taken together, CHRM3 is a novel and vital factor of GBM progression via regulation of multiple oncogenic genes and may serve as a new biomarker for prognosis and therapy of GBM patients.

Hypoxia-induced ROS aggravate tumor progression through HIF-1α-SERPINE1 signaling in glioblastoma.

Hypoxia, as an important hallmark of the tumor microenvironment, is a major cause of oxidative stress and plays a central role in various malignant tumors, including glioblastoma. Elevated reactive oxygen species (ROS) in a hypoxic microenvironment promote glioblastoma progression; however, the underlying mechanism has not been clarified. Herein, we found that hypoxia promoted ROS production, and the proliferation, migration, and invasion of glioblastoma cells, while this promotion was restrained by ROS scavengers N-acetyl-L-cysteine (NAC) and diphenyleneiodonium chloride (DPI). Hypoxia-induced ROS activated hypoxia-inducible factor-1α (HIF-1α) signaling, which enhanced cell migration and invasion by epithelial-mesenchymal transition (EMT). Furthermore, the induction of serine protease inhibitor family E member 1 (SERPINE1) was ROS-dependent under hypoxia, and HIF-1α mediated SERPINE1 increase induced by ROS via binding to the SERPINE1 promoter region, thereby facilitating glioblastoma migration and invasion. Taken together, our data revealed that hypoxia-induced ROS reinforce the hypoxic adaptation of glioblastoma by driving the HIF-1α-SERPINE1 signaling pathway, and that targeting ROS may be a promising therapeutic strategy for glioblastoma.

The role of branched-chain aminotransferase 1 in driving glioblastoma cell proliferation and invasion varies with tumor subtype.

Branched-chain aminotransferase 1 (BCAT1) has been proposed to drive proliferation and invasion of isocitrate dehydrogenase (IDH) wild-type glioblastoma cells. However, the Cancer Genome Atlas (TCGA) dataset shows considerable variation in the expression of this enzyme in glioblastoma. The aim of this study was to determine the role of BCAT1 in driving the proliferation and invasion of glioblastoma cells and xenografts that have widely differing levels of BCAT1 expression and the mechanism responsible. The activity of BCAT1 was modulated in IDH wild-type patient-derived glioblastoma cell lines, and in orthotopically implanted tumors derived from these cells, to examine the effects of BCAT1 expression on tumor phenotype. In cells with constitutively high BCAT1 expression and a glycolytic metabolic phenotype, inducible shRNA knockdown of the enzyme resulted in reduced proliferation and invasion by increasing the concentration of α-ketoglutarate, leading to reduced DNA methylation, HIF-1α destabilization, and reduced expression of the transcription factor Forkhead box protein M1 (FOXM1). Conversely, overexpression of the enzyme increased HIF-1α expression and promoted proliferation and invasion. However, in cells with an oxidative phenotype and very low constitutive expression of BCAT1 increased expression of the enzyme had no effect on invasion and reduced cell proliferation. This occurred despite an increase in HIF-1α levels and could be explained by decreased TCA cycle flux. There is a wide variation in BCAT1 expression in glioblastoma and its role in proliferation and invasion is dependent on tumor subtype.