Oncogene In Glioma Research Articles

KIF23 is an independent prognostic biomarker in glioma, transcriptionally regulated by TCF-4.

Kinesin family member 23 (KIF23), a nuclear protein and a key regulator of cellular cytokinesis, has been found to be overexpressed as an oncogene in glioma. However, the prognostic and clinicopathological features of glioma with KIF23 expression was not clear yet. Here, we analyzed KIF23 expression pattern by using whole genome mRNA expression microarray data from Chinese Glioma Genome Atlas (CGGA) database (http://www.cgga.org.cn), and found that KIF23 overexpression was significantly associated with high grade glioma as well as the higher mortality in survival analysis (log-rank test, p<0.01). The results of the three other validation datasets showed similar findings. Furthermore, KIF23 also served as an independent prognostic biomarker in glioma patients. Finally, functional assay showed that reduction of KIF23 suppressed glioma cell proliferation both in vivo and vitro. Additionally, we found that KIF23 was regulated by TCF-4 at transcriptionally level. Therefore, this evidence indicates KIF23 over-expression is associated with glioma malignancy and conferred a worse survival time in glioma, which suggests KIF23 is a new novel prognostic biomarker with potential therapeutic implications in glioma.

HULC long noncoding RNA silencing suppresses angiogenesis by regulating ESM-1 via the PI3K/Akt/mTOR signaling pathway in human gliomas.

Tumor angiogenesis plays a critical role in the tumor progression. Highly upregulated in liver cancer (HULC) is a long noncoding RNA (lncRNA) that acts as an oncogene in gliomas. We found that HULC, vascular endothelial growth factor (VEGF), and ESM-1 (endothelial cell specific molecule 1) expression and microvessel density were positively correlated with grade dependency in glioma patient tissues, and that HULC silencing suppressed angiogenesis by inhibiting glioma cells proliferation and invasion. This process induced anoikis and blocked the cell cycle at G1/S phase via the PI3K/Akt/mTOR signaling pathway, thus regulating the tumor-related genes involved in the above biological behavior in human glioma U87MG and U251 cells. However, these effects were reversed by ESM-1 overexpression, suggesting a mediating role of ESM-1 in the pro-angiogenesis effect of HULC. Our results define the mechanism of the pro-angiogenesis activity of HULC, which shows potential for application as a therapeutic target in glioma.

Insulator dysfunction and oncogene activation in IDH mutant gliomas.

Gain-of-function IDH mutations are initiating events that define major clinical and prognostic classes of gliomas1,2. Mutant IDH protein produces a novel onco-metabolite, 2-hydroxyglutarate (2-HG), that interferes with iron-dependent hydroxylases, including the TET family of 5′-methylcytosine hydroxylases3–7. TET enzymes catalyze a key step in the removal of DNA methylation8,9. IDH mutant gliomas thus manifest a CpG island methylator phenotype (G-CIMP)10,11, though the functional significance of this altered epigenetic state remains unclear. Here we show that IDH mutant gliomas exhibit hyper-methylation at CTCF binding sites, compromising binding of this methylation-sensitive insulator protein. Reduced CTCF binding is associated with loss of insulation between topological domains and aberrant gene activation. We specifically demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to aberrantly interact with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Treatment of IDH mutant gliomaspheres with demethylating agent partially restores insulator function and down-regulates PDGFRA. Conversely, CRISPR-mediated disruption of the CTCF motif in IDH wildtype gliomaspheres up-regulates PDGFRA and increases proliferation. Our study suggests that IDH mutations promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression.

MicroRNA-26a inhibits osteosarcoma cell proliferation by targeting IGF-1.

There are still controversies about the roles of microRNA-26a (miR-26a) in human malignancies, as it is a tumor suppressor in breast cancer, gastric cancer, and hepatocellular carcinoma, but is an oncogene in glioma and cholangiocarcinoma. Until now, the function of miR-26a in osteosarcoma remains largely elusive. Here, we found that miR-26a was downregualted in osteosarcoma tissues. Using in vitro and in vivo assays, we confirmed that miR-26a could inhibit the abilities of in vitro proliferation and suppress in vivo tumor growth in mouse model. Furthermore, we identified insulin-like growth factor 1 (IGF-1) as a novel and direct target of miR-26a and revealed that miR-26a exerted its tumor-suppressor function, at least in part, by inhibiting IGF-1 expression. These findings contribute to our understanding of the functions of miR-26a in osteosarcoma.

Abstract PR06: CSF1 signaling is a potential therapeutic target for glioma

Abstract Current therapies for high-grade gliomas only modestly extend survival. Given that intrinsic characteristics of glioma cancer stem cells (gCSCs) make them hard to effectively target with current therapies, cells in the tumor microenvironment such as microglia/macrophages are being investigated as therapy targets. We previously identified a microglial/macrophage growth factor, Macrophage colony stimulating factor (Csf1), as a candidate glioma oncogene in a mouse somatic mutagenesis screen. CSF1 overexpression, CSF1 receptor (CSF1R) overexpression, and phosphorylated CSF1R have also been detected in human gliomas. We are using genetic and pharmacological strategies to investigate the role of CSF1 signaling in gliomagenesis in immunocompetent autochthonous mouse models to examine the therapeutic utility of targeting the CSF1R signaling axis. To determine the relevant CSF1R ligand(s) in human gliomas, we performed semi-quantitative RT-PCR on patient-derived gCSCs to examine expression of splice variants encoding membrane bound or secreted CSF1 as well as the additional CSF1R ligand IL34. We found that gCSCs primarily express the mRNA splice variant encoding secreted CSF1, but a splice variant encoding a membrane bound form was also detected. Most gCSCs lines did not express detectable levels of IL34 mRNA. We therefore developed a tissue-specific secreted CSF1 overexpressing transgenic model to study the effect of modulating CSF1 levels in the central nervous system. Since CSF1 is a growth factor for microglia, we previously studied the effects of increased CSF1 levels on microglial phenotypes and found that CSF1 overexpression increased microglial numbers and promoted microglial proliferation in vivo, but did not inherently polarize microglia toward a M2-like phenotype. Moreover, CSF1 signaling is required for survival of adult microglia in vivo, since inhibition of CSF1 signaling by the small molecule inhibitor PLX3397 induced microglial apoptosis. We now show that CSF1 overexpression accelerates gliomagenesis in a genetic model of spontaneous gliomagenesis driven by activated Ras. Both high- and low-grade gliomas form in mice with activated Ras, but CSF1 overexpression causes a dramatic increase in the percentage of tumors that are high-grade. Additionally, gliomas do not form in mice expressing activated Ras that are also CSF1-deficient. Therefore our data in this in vivo model indicate that CSF1 signaling plays a fundamental role in glioma development. Additional studies are underway to determine the impact of CSF1 on microglial and glioma phenotypes, and to explore how to best target CSF1 signaling for glioma therapy. Citation Format: Ishani De, Megan D. Steffen, Emily Sokn, Clayton Patros, Suzanne Litscher, Paul A. Clark, John S. Kuo, Fausto J. Rodriguez, Lara S. Collier. CSF1 signaling is a potential therapeutic target for glioma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr PR06.

Overexpressed KDM5B is associated with the progression of glioma and promotes glioma cell growth via downregulating p21

Epigenetic alterations such as aberrant expression of histone-modifying enzymes have been implicated in tumorigenesis. Upregulation of lysine (K)-specific demethylase 5B (KDM5B) has been reported in a variety of malignant tumors. However, the impact of KDM5B in glioma remains unclear. The objective of this study was to investigate the expression and prognostic value of KDM5B in glioma. In clinical glioma samples, we found that KDM5B expression was significantly upregulated in cancer lesions compared with normal brain tissues. Kaplan–Meier analysis showed that patients with glioma and higher KDM5B expression tend to have shorter overall survival time. By silencing or overexpressing KDM5B in glioma cells, we found that KDM5B could promote cell growth both in vitro and in vivo. Moreover, we demonstrated that KDM5B promoted glioma proliferation partly via regulation of the expression of p21. Our study provided evidence that KDM5B functions as a novel tumor oncogene in glioma and may be a potential therapeutic target for glioma management.

Abstract 5336: Targeting glioma growth and invasiveness by microRNA-1.

Abstract Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Median survival is about 14 months, even with the most effective regimen of surgery followed by radiation and chemotherapy. GBM is characterized by the hallmarks of cellular heterogeneity, rapid proliferation, angiogenesis and extensive invasion. The dual feature of high proliferation and infiltration constitutes the major challenge for glioma therapy, as even extensive resection of the primary tumor mass leaves a significant number of tumor cells in the brain and leads to almost unavoidable recurrence. Despite abundance of data concerning glioma growth/angiogenesis, the mechanisms underlying the regulation of glioma cell motility and invasiveness are comparatively poorly understood. Established anti-glioma therapeutical modalities (radiation, chemotherapy) target preferentially rapidly proliferating rather than infiltrating glioma cells. Moreover invading cells are resistant to conventional therapies, and anti-angiogenic treatment was shown to induce invasiveness. Thus novel therapies aiming at blockade of glioma invasion, while curbing their proliferative potential are needed. MicroRNAs (miRs) are small, single stranded, non-coding RNAs that act as key regulators of gene expression, by reducing translation of target mRNAs with partial complementarity in their 3′UTRs. MiRs deregulation is a general feature of cancer, including GBM, and was associated with tumor suppressor and oncogenic activities. A number of studies have identified critical roles for miRs in the regulation of tumor cell invasion, migration and metastasis. In this study we report that miR-1 is down-regulated in glioma patients (relatively to adjacent tissue) and that it is down-regulated in migrating glioma cells in vitro (relatively to non-migrating glioma cells). Its forced expression reduces glioma cell growth and angiogenesis in vivo. MiR-1 inhibits migration in vitro and invasiveness in vivo. Additionally, we demonstrated impaired cell-cell adhesion and aggregation in glioma cells expressing miR-1. We identified Annexin A2, recognized oncogene in glioma, as a direct target of miR-1. Annexin A2 is up-regulated in various tumor types, including glioma. In vivo studies revealed a critical role for Annexin A2 in driving glioma growth, angiogenesis, progression and invasion. Our results show that the effects of miR-1 on glioma cells are consistent with Annexin A2 down-regulation, including a reduction in glioma cell proliferation and invasiveness, and re-expression of Annexin A2 rescues the effect of miR-1 expression. When glioma cells stably expressing miR-1 were injected intracranially, we observed significantly improved survival when compared to control cells. Our results suggest that miR-1 inhibits simultaneously glioma growth and invasiveness in vivo and as such may be a candidate for the therapeutic targeting of glioma cells. Citation Format: Jakub A. Godlewski, Agnieszka Bronisz, Yan Wang, E. Antonio Chiocca. Targeting glioma growth and invasiveness by microRNA-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5336. doi:10.1158/1538-7445.AM2013-5336

MiR-24 regulates the proliferation and invasion of glioma by ST7L via β-catenin/Tcf-4 signaling

MicroRNAs are strongly implicated as affecting glioma, but their specific roles and functions have yet to be fully elucidated. In this study, we defined the expression and function of miR-24, which we found to be upregulated in glioma samples and glioma cells by qRT-PCR. Downregulation of miR-24 in glioma cell lines inhibited proliferation and invasion and induced apoptosis. Using computational and expression analysis, ST7L was identified as a candidate target of miR-24. A reporter assay with the 3′UTR of ST7L cloned downstream of a luciferase gene showed increased luciferase activity in the absence of miR-24, providing strong evidence that miR-24 is a direct regulator of ST7L. Furthermore, we observed that restoration of ST7L activity resulted in effects that were similar to those from transfecting a miR-24 inhibitor into glioma cells. Mechanistic investigation revealed that the deletion of miR-24 suppressed β-catenin/Tcf-4 transcription activity by targeting ST7L. In conclusion, our study demonstrates that miR-24 upregulation is common in glioma and that suppression of miR-24 expression inhibits cell proliferation and invasion, suggesting that miR-24 may act as an oncogene in glioma.

The Putative Tumor Suppressor miR-524–5p Directly Targets Jagged-1 and Hes-1 in Glioma

Notch pathway plays critical role in stem cell maintenance and angiogenesis, as well as cell fate decisions of cancer. However, concrete mechanisms of notch pathway regulation in glioma were not well known, especially mediated by microRNAs. In this study, we identified a brain-specific miRNA, miR-524-5p, which was associated with the pathological grade and overall survival of gliomas. Restorated expression of miR-524-5p in glioma suppressed cell proliferation and invasion both in vitro and in vivo. Using bioinformatics and biological approaches, we found that Jagged-1 and Hes-1, two key components of notch pathway, were direct targets of miR-524-5p. Knocking down of Jagged-1 or Hes-1 partially phenocopied miR-524-5p re-expression, whereas forced expression of Jagged-1 or Hes-1 reversed the effects of miR-524-5p on proliferation and invasion of glioma. Moreover, miR-524-5p levels in glioma samples were inversely correlated with Jagged-1 and Hes-1 and their overexpressions were associated with poor survival. Thus, we have identified that miR-524-5p behaves as a tumor suppressor by negatively targeting Jagged-1 and Hes-1 and provides an additional option to inhibit this oncogene in gliomas.

Abstract LB-466: Genomic Loss of microRNA 491 contributes to IGFBP2 overexpression and stemness in GBM

Abstract Insulin-like growth factor-binding protein 2 (IGFBP2) is overexpressed in 80% of glioblastoma multiforme (GBM, WHO grade IV). Our mouse model experiments demonstrated that IGFBP2 is a glioma oncogene. However, the underlying mechanism for overexpression of IGFBP2 remains unknown. We examined a total of 388 GBM samples from the Cancer Genome Atlas (TCGA) and found that miR-491, which is located on chr9p21.3, is deleted in 57% of cases. TargetScan analysis revealed that there is a binding site of miR-491 on the 3′-UTR of the IGFBP2 gene. The miR-491 expression levels in GBM patients are inversely correlated with that of IGFBP2. We validated this regulatory relationship by overexpression of miR-491 and observed down-regulation of IGFBP-2 in both protein level and mRNA level in U251 and T98G glioma cell lines. Conversely, miR-491 inhibitors increased IGFBP2 in LN229 cells which express low level of endogenous IGFBP2. Luciferase reporter assay further proved that IGFBP2 is a direct target of miR-491. Moreover, ectopic expression of miR-491 inhibited cell migration and invasion of U251 and T98G as efficiently as knockdown of IGFBP2 and such inhibitory effects can be partially reversed by overexpression of IGFBP2-EGFP in U251, supporting the notion that IGFBP2 plays an important role in miR-491's functions. To investigate the role of miR-491 in stemness of GBM, we introduced miR-491 into 3 patient-derived glioma stem cell lines and found that miR-49 downregulated CDK6 and Bcl-xL and inhibited neurosphere formation and cell proliferation. These findings suggest that miR-491 acts as a tumor suppressor miR by coordinately attenuating multiple oncogenes in GBM. Our results provide new insights into the mechanism for oncogenic IGFBP2 overexpression and may provide new opportunities for therapeutic strategies for GBM treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-466. doi:1538-7445.AM2012-LB-466

Insulin-like growth factor-binding protein 2-driven glioma progression is prevented by blocking a clinically significant integrin, integrin-linked kinase, and NF-κB network

Insulin-like growth factor-binding protein 2 (IGFBP2) is increasingly recognized as a glioma oncogene, emerging as a target for therapeutic intervention. In this study, we used an integrative approach to characterizing the IGFBP2 network, combining transcriptional profiling of human glioma with validation in glial cells and the replication-competent ASLV long terminal repeat with a splice acceptor/tv-a glioma mouse system. We demonstrated that IGFBP2 expression is closely linked to genes in the integrin and integrin-linked kinase (ILK) pathways and that these genes are associated with prognosis. We further showed that IGFBP2 activates integrin β1 and downstream invasion pathways, requires ILK to induce cell motility, and activates NF-κB. Most significantly, the IGFBP2/integrin/ILK/NF-κB network functions as a physiologically active signaling pathway in vivo by driving glioma progression; interfering with any point in the pathway markedly inhibits progression. The results of this study reveal a signaling pathway that is both targetable and highly relevant to improving the survival of glioma patients.

Abstract 3963: MicroRNA-124 and microRNA-1, commonly lost in glioma, coordinately target sphingosine kinase-1 in glioma cells

Abstract Glioma (Glioblastoma multiforme, GBM) is the most common and the most aggressive form of brain tumor. As its Latin name implies, glioma is characterized by enormous heterogeneity and many different genes and cellular pathways responsible for glioma genesis and progression have been described. One of them is Sphingosine Kinase-1 (SPHK-1), a bona fide oncogene in glioma, whose marked up-regulation of expression and activity is responsible for the poor patient outcome due to escalating proliferation and invasiveness of glioma cells. MicroRNAs are short, non-coding regulators of the gene expression and deregulation of their expression is a frequent event in the formation and progression of many malignancies including glioma. Among these deregulated microRNAs, microRNA-124 is the most downregulated microRNA. It has been shown to promote neuronal differentiation and inhibit glioma cell proliferation and to target CDK6. Here we show that microRNA-1 is also severely downregulated in glioma patients and that it acts coordinately with microRNA-124 to target SPHK-1. We show that both microRNAs directly target the SPHK-1 3’UTR and that such targeting results in the decrease of both SPHK-1 expression and activity. We also demonstrate that overexpression of both microRNA-124 and microRNA-1 causes a significant increase in the frequency of apoptotic cells and marked decrease in glioma cell proliferation and invasiveness. Additionally, we show that microRNA-1 directly targets another glioma oncogene – Annexin2A (ANXA2). Targeting of ANXA2 by microRNA-1 impairs glioma cell-cell adhesion and glioma spheroid formation, which effect can be partially rescued by the overexpression of ANXA2 with a mutated microRNA-1 target site but not by wild type ANXA2. Our results provide another target of therapeutic importance for microRNA-124 in glioma cells. Here we show that microRNA-124, along with microRNA-1 can exert profound effect on glioma cells by inhibiting expression and activity of SPHK1 and that additional effects caused by microRNA-1 are likely mediated through targeting ANXA2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3963. doi:10.1158/1538-7445.AM2011-3963

HMGN5: a potential oncogene in gliomas

Gliomas are the most common primary brain tumors in the central nervous system and a leading cause of tumor-related death. High-mobility group nucleosome binding domain 5 (HMGN5/NSBP1), which is highly expressed in breast cancer and in hormone-induced mouse uterine adenocarcinoma, acts as a potential oncogene in gliomas. In this study, the role of HMGN5 in the proliferation of human glioma cells was investigated by lentivirus-mediated RNA interference (RNAi). The decrease in HMGN5 expression in human glioma U251 and U87 cells caused cell cycle arrest in the G1 phase and a delay in cell proliferation, as well as resulting in more apoptosis and an inhibition of clonogenic growth in soft agar in U251 cells; these results suggest that HMGN5 is required for tumorigenesis in vitro. Furthermore, HMGN5 was highly expressed in both high-grade and low-grade glioma tissue samples compared with normal brain tissues. Collectively, our data suggest that HMGN5 may play a critical role in the development of gliomas.

PLAGL2 Regulates Wnt Signaling to Impede Differentiation in Neural Stem Cells and Gliomas

A hallmark feature of glioblastoma is its strong self-renewal potential and immature differentiation state, which contributes to its plasticity and therapeutic resistance. Here, integrated genomic and biological analyses identified PLAGL2 as a potent protooncogene targeted for amplification/gain in malignant gliomas. Enhanced PLAGL2 expression strongly suppresses neural stem cell (NSC) and glioma-initiating cell differentiation while promoting their self-renewal capacity upon differentiation induction. Transcriptome analysis revealed that these differentiation-suppressive activities are attributable in part to PLAGL2 modulation of Wnt/beta-catenin signaling. Inhibition of Wnt signaling partially restores PLAGL2-expressing NSC differentiation capacity. The identification of PLAGL2 as a glioma oncogene highlights the importance of a growing class of cancer genes functioning to impart stem cell-like characteristics in malignant cells.

Knocking down Interferon Induced Transmembrane protein 1 (IFITM1) Inhibited Cell Proliferation and Invasion in Glioma Cells

Here we investigated the role of Interferon Induced Transmembrane Protein 1 (IFITM1) in glioma carcinogenesis. We determined the expression levels of IFITM1 in different glioma cell lines, examined its role in cell proliferation/cell migration &amp; invasion, and the corresponding molecular mechanism in glioma cells. Our results showed that IFITM1 mRNA level was elevated in four of five widely used glioma cell lines and positively correlated with their tumorigenic abilities. Suppression of IFITM1 by siIFITM1 inhibited the proliferation of U‐373 MG and U‐87 MG glioma cells lines in a time dependent manner. The growth inhibition effect of siIFITM1 was mediated not by apoptosis but G1 to S phase cell cycle arrest, which are mediated via decreased G1/S‐specific cylin‐D1, cyclin‐dependent kinase 2 (CDK2) and increased cyclin‐dependent kinase inhibitor 1B (p27kip1). Furthermore, IFITM1 knockdown significantly inhibit the migration and invasion of U‐373 MG and U‐87 MG cells, which was mediated by decreased MMP9 expression and activity. Taken together, these results suggested IFITM1 might be a potential candidate oncogene in glioma.