Circular RNA circFGFR1 Functions as an Oncogene in Glioblastoma Cells through Sponging to hsa-miR-224-5p

Recently, increased studies have shown the important regulatory role of circular RNA (circRNA) in cancer progression and development, including glioblastoma (GBM). However, the function of circRNAs in glioblastoma is still largely unclear. Here, we state that circFGFR1 is elevated in glioma cells, resulting in aggravated glioma aggravated malignancy. The upregulation of circFGFR1 also promotes glioma growth in mouse xenograft models. Furthermore, CXCR4 level in glioma cells is positively correlated with circFGFR1 level, and higher CXCR4 expression is found in circFGFR1 overexpression groups. The effect of circFGFR1 on glioma malignancy is abolished in CXCR4 knockout cells. Then, RIP, RNA pull-down, and luciferase reporter assay results showed that hsa-miR-224-5p directly binds to circFGFR1 and CXCR4 mRNA. The CXCR4 3 ′ -untranslated region (UTR) activated luciferase activity was reduced with hsa-miR-224-5p transfection, while it is reversed when cotransfected with circFGFR1, indicating that circFGFR1 acts as a hsa-miR-244-5p sponge to increase CXCR4 expression. The hsa-miR-224-5p expression is negatively corrected with the glioma malignancy through inhibiting CXCR4 level. Besides, the circFGFR1-induced regulation in glioma malignancy is also abrogated in hsa-miR-224-5p knockout cells. Taken together, our findings suggest that circFGFR1 plays a critical role in the tumorigenic behaviors in glioma cells by upregulating CXCR4 expression via sponging to hsa-miR-224-5p. These findings provide a new perspective on circRNAs during GBM development.

Altered Elemental Distribution in Male Rat Brain Tissue as a Predictor of Glioblastoma Multiforme Growth—Studies Using SR-XRF Microscopy

Glioblastoma multiforme (GBM) is a particularly malignant primary brain tumor. Despite enormous advances in the surgical treatment of cancer, radio- and chemotherapy, the average survival of patients suffering from this cancer does not usually exceed several months. For obvious ethical reasons, the search and testing of the new drugs and therapies of GBM cannot be carried out on humans, and for this purpose, animal models of the disease are most often used. However, to assess the efficacy and safety of the therapy basing on these models, a deep knowledge of the pathological changes associated with tumor development in the animal brain is necessary. Therefore, as part of our study, the synchrotron radiation-based X-ray fluorescence microscopy was applied for multi-elemental micro-imaging of the rat brain in which glioblastoma develops. Elemental changes occurring in animals after the implantation of two human glioma cell lines as well as the cells taken directly from a patient suffering from GBM were compared. Both the extent and intensity of elemental changes strongly correlated with the regions of glioma growth. The obtained results showed that the observation of elemental anomalies accompanying tumor development within an animal’s brain might facilitate our understanding of the pathogenesis and progress of GBM and also determine potential biomarkers of its extension. The tumors appearing in a rat’s brain were characterized by an increased accumulation of Fe and Se, whilst the tissue directly surrounding the tumor presented a higher accumulation of Cu. Furthermore, the results of the study allow us to consider Se as a potential elemental marker of GBM progression.

TMEM158 promotes the proliferation and migration of glioma cells via STAT3 signaling in glioblastomas

Glioblastoma is the most common primary intracranial malignant tumor in adults and has high morbidity and high mortality. TMEM158 has been reported to promote the progression of solid tumors. However, its potential role in glioma is still unclear. Here, we found that TMEM158 expression in human glioma cells in the tumor core was significantly higher than that in noncancerous cells at the tumor edge using bioinformatics analysis. Cancer cells in patients with primary GBMs harbored significantly higher expression of TMEM158 than those in patients with WHO grade II or III gliomas. Interestingly, regardless of tumor grading, human glioma samples that were IDH1-wild-type (IDH1-WT) exhibited higher expression of TMEM158 than those with IDH1-mutant (IDH1-Mut). We also illustrated that TMEM158 mRNA expression was correlated with poor overall survival in glioma patients. Furthermore, we demonstrated that silencing TMEM158 inhibited the proliferation of glioma cells and that TMEM158 overexpression promoted the migration and invasion of glioma cells by stimulating the EMT process. We found that the underlying mechanism involves STAT3 activation mediating TMEM158-driven glioma progression. In vivo results further confirmed the inhibitory effect of the TMEM158 downregulation on glioma growth. Collectively, these findings further our understanding of the oncogenic function of TMEM158 in gliomas, which represents a potential therapeutic target, especially for GBMs.

RGD Peptide-Conjugated Selenium Nanocomposite Inhibits Human Glioma Growth by Triggering Mitochondrial Dysfunction and ROS-Dependent MAPKs Activation

Chemotherapy is still one of the most common ways to treat human glioblastoma in clinic. However, severe side effects limited its clinic application. Design of cancer-targeted drugs with high efficiency and low side effect is urgently needed. Herein, silver nanoparticles (Ag NPs) and nano-selenium (Se NPs) conjugated with RGD peptides (Ag@Se@RGD NPs) to target integrin high-expressed glioma were designed. The results found that Ag@Se@RGD NPs displayed stable particle size and morphology in physiological condition, and induced significant integrin-targeted intracellular uptake. Ag@Se@RGD NPs in vitro dose-dependently inhibited U251 human glioma cells growth by induction of cells apoptosis through triggering the loss of mitochondrial membrane potential, overproduction of reactive oxygen species (ROS), and MAPKs activation. However, ROS inhibition dramatically attenuated Ag@Se@RGD NPs-induced MAPKs activation, indicating the significant role of ROS as an early apoptotic event. Importantly, Ag@Se@RGD NPs administration in vivov effectively inhibited U251 tumor xenografts growth by induction of apoptosis through regulation MAPKs activation. Taken together, our findings validated the rational design that Ag-Se NPs conjugated with RGD peptides was a promising strategy to combat human glioma by induction of apoptosis through triggering mitochondrial dysfunction and ROS-dependent MAPKs activation.

Long non-coding RNA FAM66C regulates glioma growth via the miRNA/LATS1 signaling pathway

Glioma is one of the most common primary intracranial carcinomas and typically associated with a dismal prognosis and poor quality of life. The identification of novel oncogenes is clinically valuable for early screening and prevention. Recently, the studies have revealed that long non-coding RNAs (lncRNAs) play important roles in the development and progression of cancers including glioma. The expression of lncRNA FAM66C is reduced in glioma cell lines and clinical samples compared to non-tumor samples. Knockdown of FAM66C in U87 and U251 cells significantly promoted cell proliferation and migration, respectively. Furthermore, the correlation between FAM66C and Hippo pathway regulators YAP1 and LATS1, along with the alteration of their protein expression level indicated that FAM66C regulated cell growth through this pathway. Moreover, luciferase assay demonstrated that another two noncoding RNAs, miR15a/miR15b, directly bonded to the 3′UTR of LATS1 to facilitated its transcriptional expression and inhibited cell growth. In addition, the luciferase activity of FAM66C was block by miR15a/miR15b, and the promotion of cell growth effects caused by FAM66C deficiency was attenuated by miR15a/miR15b mimics, further proved that FAM66C functioned as a competing endogenous RNA to regulate glioma growth via the miRNA/LATS1 signaling pathway.

D-Mannose Slows Glioma Growth by Modulating Myeloperoxidase Activity

Host immune response in the tumor microenvironment plays key roles in tumorigenesis. We hypothesized that D-mannose, a simple sugar with anti-inflammatory properties, could decrease oxidative stress and slow glioma progression. Using a glioma stem cell model in immunocompetent mice, we induced gliomas in the brain and tracked MPO activity in vivo with and without D-mannose treatment. As expected, we found that D-mannose treatment decreased the number of MPO+ cells and slowed glioma progression compared to PBS-treated control animals with gliomas. Unexpectedly, instead of decreasing MPO activity, D-mannose increased MPO activity in vivo, revealing that D-mannose boosted the MPO activity per MPO+ cell. On the other hand, D-glucose had no effect on MPO activity. To better understand this effect, we examined the effect of D-mannose on bone marrow-derived myeloid cells. We found that D-mannose modulated MPO activity via two mechanisms: directly via N-glycosylation of MPO, which boosted the MPO activity of each molecule, and indirectly by increasing H2O2 production, the main substrate for MPO. This increased host immune response acted to reduce tumor size, suggesting that increasing MPO activity such as through D-mannose administration may be a potential new therapeutic direction for glioma treatment.

NMNAT promotes glioma growth through regulating post-translational modifications of P53 to inhibit apoptosis

Gliomas are highly malignant brain tumors with poor prognosis and short survival. NAD+ has been shown to impact multiple processes that are dysregulated in cancer; however, anti-cancer therapies targeting NAD+ synthesis have had limited success due to insufficient mechanistic understanding. Here, we adapted a Drosophila glial neoplasia model and discovered the genetic requirement for NAD+ synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) in glioma progression in vivo and in human glioma cells. Overexpressing enzymatically active NMNAT significantly promotes glial neoplasia growth and reduces animal viability. Mechanistic analysis suggests that NMNAT interferes with DNA damage-p53-caspase-3 apoptosis signaling pathway by enhancing NAD+-dependent posttranslational modifications (PTMs) poly(ADP-ribosyl)ation (PARylation) and deacetylation of p53. Since PARylation and deacetylation reduce p53 pro-apoptotic activity, modulating p53 PTMs could be a key mechanism by which NMNAT promotes glioma growth. Our findings reveal a novel tumorigenic mechanism involving protein complex formation of p53 with NAD+ synthetic enzyme NMNAT and NAD+-dependent PTM enzymes that regulates glioma growth.

Microglia-Derived Olfactomedin-like 3 Promotes Pro-Tumorigenic Microglial Function and Malignant Features of Glioma Cells

Under the influence of transforming growth factor-beta (TGFβ), glioma-associated microglia produce molecules that promote glioma growth and invasion. Olfactomedin-like 3 (Olfml3), a novel, secreted glycoprotein, is known to promote several non-CNS cancers. While it is a direct TGFβ1 target gene in microglia, the role of microglia-derived OLFML3 in glioma progression is unknown. Here, we tested the hypotheses that microglial Olfml3 is integral to the pro-tumorigenic glioma-associated microglia phenotype and promotes glioma cell malignancy. Using an Olfml3 knockout microglial cell line (N9), we demonstrated that Olfml3 is a direct target gene of all TGFβ isoforms in murine microglia. Moreover, loss of Olfml3 attenuated TGFβ-induced restraint on microglial immune function and production of cytokines that are critical in promoting glioma cell malignancy. Importantly, microglia-derived OLFML3 directly contributes to glioma cell malignancy through increased migration and invasion. While exposure to conditioned medium (CM) from isogenic control microglia pre-treated with TGFβ increased mouse glioma cell (GL261) migration and invasion, this effect was abolished with exposure to CM from TGFβ-treated Olfml3-/- microglia. Taken together, our data suggest that Olfml3 may serve as a gatekeeper for TGFβ-induced microglial gene expression, thereby promoting the pro-tumorigenic microglia phenotype and glioma cell malignancy.

Endothelial cell-specific reduction of heparan sulfate suppresses glioma growth in mice

Purpose Heparan sulfate (HS) is one of the factors that has been suggested to be associated with angiogenesis and invasion of glioblastoma (GBM), an aggressive and fast-growing brain tumor. However, it remains unclear how HS of endothelial cells is involved in angiogenesis in glioblastoma and its prognosis. Thus, we investigated the effect of endothelial cell HS on GBM development. Methods We generated endothelial cell-specific knockout of Ext1, a gene encoding a glycosyltransferase and essential for HS synthesis, and murine GL261 glioblastoma cells were orthotopically transplanted. Two weeks after transplantation, we examined the tumor progression and underlying mechanisms. Results The endothelial cell-specific Ext1 knockout (Ext1CKO) mice exhibited reduced HS expression specifically in the vascular endothelium of the brain capillaries compared with the control wild-type (WT) mice. GBM growth was significantly suppressed in Ext1CKO mice compared with that in WT mice. After GBM transplantation, the survival rate was significantly higher in Ext1CKO mice than in WT mice. We investigated how the effect of fibroblast growth factor 2 (FGF2), which is known as an angiogenesis-promoting factor, differs between Ext1CKO and WT mice by using an in vivo Matrigel assay and demonstrated that endothelial cell-specific HS reduction attenuated the effect of FGF2 on angiogenesis. Conclusions HS reduction in the vascular endothelium of the brain suppressed GBM growth and neovascularization in mice.

CSIG-17. IMMUNE MODULATORY ROLE OF TYPE I INTERFERON IN SYNGENEIC MOUSE GLIOMA MODELS

Glioblastoma is characterized by a poor prognosis and a challenging phenotype for drug development. Although multimodal treatment, including surgery, radio- and chemotherapy is applied, the overall survival remains just above one year. Numerous clinical trials have studied targeted therapies against commonly deregulated pathways, but an efficient targeted drug is yet to be discovered. Likewise, immunotherapy has not been shown to be active. A subset of glioma tumor cells demonstrates stem-like properties; these cells are commonly referred to as glioma initiating cells (GIC). These types of cells are pluripotent and can by definition initiate and recapitulate glioma growth in experimental animals in vivo. Furthermore, these cells are often resistant to conventional therapies. Interferon β (IFN-β) is an immunomodulatory molecule with anti-cancer properties. We have previously shown that IFN-β greatly reduces sphere-formation capability of GIC. It was also confirmed that IFN-β sensitized resistant GIC to irradiation or the chemotherapeutic agent, temozolomide (TMZ). IFN-β treatment significantly prolonged survival in a xenograft model with GIC cells. In the current project, we want to use syngeneic mouse models to study the immunomodulatory effects of type I IFNs. Preliminary results indicate that abrogation of IFN signalling in tumor cells by CRISPR/Cas9 technology prolonged survival in mice only in cell lines which have substantial baseline autocrine IFN signalling. On the contrary, we did not observe a difference in survival when wild-type tumor cells were implanted in either IFNAR1 deficient or proficient hosts. Flow cytometry analysis will elucidate changes in immune cell recruitment and infiltration upon IFN signalling disruption. Moreover, we explore different treatments in combination with IFN-β as there are indications that TMZ or radiotherapy can have synergistic effects with stimulation of interferon type I signalling.