Pathogenesis Of GBM Research Articles

Bioinformatics analysis of gene expression profiles to diagnose crucial and novel genes in glioblastoma multiform

Therefore, the current study aimed to diagnose the genes associated in the pathogenesis of GBM. The differentially expressed genes (DEGs) were diagnosed using the limma software package. The ToppFun was used to perform pathway and Gene Ontology (GO) enrichment analysis of the DEGs. Protein-protein interaction (PPI) networks, extracted modules, miRNA-target genes regulatory network and miRNA-target genes regulatory network were used to obtain insight into the actions of DEGs. Survival analysis for DEGs carried out. A total of 701 DEGs, including 413 upregulated and 288 downregulated genes, were diagnosed between U1118MG cell line (PK 11195 treated with 1 h exposure) and U1118MG cell line (PK 11195 treated with 24 h exposure). The up-regulated genes were enriched in superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis, cell cycle, cell cycle process and chromosome. The down-regulated genes were enriched in folate transformations I, biosynthesis of amino acids, cellular amino acid metabolic process and vacuolar membrane. The current study screened the genes in PPI network, extracted modules, miRNA-target genes regulatory network and miRNA-target genes regulatory network with higher degrees as hub genes, which included MYC, TERF2IP, CDK1, EEF1G, TXNIP, SLC1A5, RGS4 and IER5L Survival suggested that low expressed NR4A2, SLC7 A5, CYR61 and ID1 in patients with GBM was linked with a positive prognosis for overall survival. In conclusion, the current study could improve our understanding of the molecular mechanisms in the progression of GBM, and these crucial as well as new molecular markers might be used as therapeutic targets for GBM.

Abstract 1424: Elucidating the microRNA-203 specific biological processes in glioblastoma cells from high-throughput RNA-sequencing

Abstract Glioblastoma (GBM) is the most common primary malignant intracranial adult brain tumor. Allelic deletion on chromosome 14q play an essential role in GBM pathogenesis, and this chromosome 14q site was thought to harbor multiple tumor suppressor gene associated with GBM, a region that also encodes microRNA-203 (miR-203). This study was conducted to identify gene expression profile changes associated with mir-203 expression by high-throughput RNA sequencing. Enrichment analyses for gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed that mir-203 expression had a strong, negative effect on a number of fundamental and interconnected biological processes involved in cell growth and proliferation. The biological processes mostly influenced were p53 signaling pathway, FoxO signaling pathway, DNA replication, cell cycle, MAPK signaling pathway and Apoptosis. In total, 847 upregulated and 345 downregulated differentially expressed genes (DEGs) were identified in control versus miR203 expressing glioma cells. After GO enrichment, the downregulated DEGs and miR-203 predicted target genes such as BCL-2, SPARC, PDGFA, CREB were found to be mainly enriched in cell cycle regulation and apoptosis processes, whereas the upregulated DEGs, such as CCND1, E2F1 were involved in the DNA replication and cell cycle regulation of gene expression. We also demonstrate that mir-203 expression suppressed BCL-2 protein and mRNA by western blotting and qRT-PCR analysis. Moreover, co-transfection experiments using a luciferase-based reporter assay demonstrated that mir-203 directly regulated BCL-2 expression and BCL-2 overexpression suppressed mir-203 mediated cell death. Knowledge gained through this profiling study can facilitate targeted therapeutic interventions. Citation Format: Bhavesh K. Ahir, Herbert H. Engelhard, Sajani S. Lakka. Elucidating the microRNA-203 specific biological processes in glioblastoma cells from high-throughput RNA-sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1424.

EZH2 suppression in glioblastoma shifts microglia toward M1 phenotype in tumor microenvironment

BackgroundGlioblastoma multiforme (GBM) induces tumor immunosuppression through interacting with tumor-infiltrating microglia or macrophages (TAMs) with an unclear pathogenesis. Enhancer of zeste homolog 2 (EZH2) is abundant in GBM samples and cell lines and is involved in GBM proliferation, cell cycle, and invasion, whereas its association with innate immune response is not yet reported. Herein, the aim of this study was to investigate the role of EZH2 in GBM immune.MethodsCo-culturing models of human/murine GBM cells with PBMC-derived macrophages/primary microglia were employed. EZH2 mRNAs and function were suppressed by siEZH2 and DZNep. Real-time PCR and flow cytometry were used to determine levels of microglia/macrophages markers. The fluorescence-labeled latex beads and flow cytometry were utilized to evaluate phagocytic abilities of microglia. CCK8 assay was performed to assess microglia proliferation.ResultsEZH2 inhibition led to significant reduction of TGFβ1-3 and IL10 and elevation of IL1β and IL6 in human and murine GBM cells. More importantly, EZH2 suppression in GBM cells resulted in significant increase of M1 markers (TNFα and iNOS) and decrease of a pool of M2 markers in murine microglia. The proportion of CD206+ cells was decreased in PBMC-derived macrophages as co-incubated with EZH2-inhibited GBM cells. Functional researches showed that phagocytic capacities of microglia were significantly ameliorated after EZH2 inhibition in co-culturing GBM cells and microglia proliferation was declined after addition of TGFβ2 antibodies to co-incubated GBM cells with EZH2 inhibition. Besides, we found that EZH2 suppression in GBM cells enhanced co-culturing microglia engulfment through activation of iNOS.ConclusionsOur data demonstrates that EZH2 participates in GBM-induced immune deficient and EZH2 suppression in GBM can remodel microglia immune functions, which is beneficial for understanding GBM pathogenesis and suggests potential targets for therapeutic approaches.

Abstract 3440: MicroRNA-mediated modulation of radioresistance of glioma stem cells

Abstract Glioblastoma (GBM) is the most common primary tumor of the central nervous system, with its deadly nature owing to a high rate of invasion and angiogenesis. A key player in the GBM pathogenesis is a distinct subpopulation of tumor cells which are capable of self-renewal, highly angiogenic and possess the potential for extensive proliferation and multi-lineage differentiation. This cell population termed Glioma Stem Cells (GSCs) is highly resistant to the chemo- and radiotherapies and plays an important role in tumor initiation, progression, and recurrence. In response to radiation therapy, GSCs are capable of triggering the activation of multiple signaling pathways including Wnt, Notch, and Hedgehog, and their DNA damage repair system is proactive. Taken together, the efforts to foster therapeutic interventions will prove futile, without first targeting the GSC population. The miRNA network adds a dimension of regulatory control, which serves to maintain pluripotency and reprograms multiple stemness and radioresistance promoting pathways. However, not much information is available that could provide mechanistic insights regarding how specific miRNAs modulate radioresistance pathways. Our initial efforts involving a global screening of microRNA profiles in different GSCs after radiation treatment resulted in identification of multiple microRNAs that were significantly altered in response to radiation. Based on our preliminary data, we selected 7 microRNAs as potential candidates, with each having several targets in Notch, Wnt, PI3K/Akt, and Hedgehog signaling pathways. In order to unfold the role of each of identified microRNAs, we used specific lentiviral constructs to create overexpression/knockdown of each microRNA in GSCs. Using these stable GSC clones, we carried out multiple in vitro studies to analyze the effects of the respective alteration in relation to radioresistance and stemness phenotypes. Other phenotypic studies included growth kinetics, cell proliferation, invasiveness, apoptosis and cell cycle. We also carried out an extensive in silico analysis to identify the potential targets of these microRNAs. This was followed by analyzing various downstream components of the target genes. Additionally, we carried out a context-based assessment of the overall effects to probe if there is an integration of one or more pathways that could lead to radiation sensitivity. Our in vitro findings also revealed their roles in regulating stemness and metastatic ability in vivo. Lastly, our study provides a basis for understanding the role of miRNAs in radioresistance phenotype and furthermore, it may lead towards another tool to inhibit GSC proliferation and/or survival. Funding: R01CA108633, R01CA169368, RC2CA148190, U10CA180850-01 (NCI), Brain Tumor Funders Collaborative Grant, and The Ohio State University CCC (all to AC). Citation Format: Rajbir Singh, Kamalakannan Palanichamy, Saikh Jaharul Haque, Arnab Chakravarti. MicroRNA-mediated modulation of radioresistance of glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3440. doi:10.1158/1538-7445.AM2017-3440

Abstract 2454: Genetic landscape of glioma reveals defective neuroactive ligand receptor interaction pathway as a poor prognosticator in glioblastoma patients

Abstract Glioblastoma (GBM; grade IV), is highly proliferative, infiltrative and treatment refractory. Hence, understanding the complete genetic alteration profile of GBM would help us in identifying molecules or pathways that have strong implications in GBM pathogenesis, thus opening up avenues for targeted therapy. Recent large scale studies suggest that three pathways - receptor-tyrosine kinase, TP53 and RB, are significantly altered in GBM. However, even with the tremendous increase in our understanding of the tumor, advancement in therapeutics is minimal and the median survival still remains at 15 months. Hence, we need to elucidate novel altered molecules and pathways in GBM progression such that more effective therapeutic options can be explored. Here, we have carried out whole exome sequencing of grade II, grade III and GBM samples which revealed the mutation spectrum of glioma from our patient set. Further, we performed integrative analysis of mutated genes from our patient cohort as well as TCGA cohort (The Cancer Genome Atlas) to find out mutated pathways that predict survival in GBM patients. The most significant pathway - neuroactive ligand-receptor interaction pathway was explored further. Patients with mutations in one or more genes of this pathway had poor survival. The pathway comprises of G-protein coupled receptors, ion channels and ligands which functions in modulation of neural plasticity, memory processes, behavior etc. Of the enriched genes belonging to this pathway, Calcitonin Receptor (CALCR), which was highest mutated in GBM (2.75%), was taken up for further investigation. CALCR was found to be downregulated in GBM and mutation or downregulation of the gene was found to predict poor survival in patients. Functional studies through cell-line based experiments revealed CALCR is a tumor suppressor in GBM. The peptide hormone calcitonin (CT), a high affinity CALCR ligand, inhibited proliferation, migration and anchorage-independent growth of glioma cells expressing CALCR with a concomitant decrease in the phosphorylation levels of ERK, AKT and JNK signaling molecules. However, CT failed to do these functions in CALCR silenced glioma cells. Exogenous overexpression of CALCR in glioma cells expressing low levels of the receptor was found to inhibit proliferation, migration and anchorage independent growth and this effect was further augmented when CT was added. Further, introduction of tumor-derived mutations in CALCR led to the abrogation of its tumor suppressor function. Studies are ongoing to demonstrate the tumor suppressive nature of CALCR using in vitro astrocyte transformation and intracranial orthotopic mouse glioma model. Thus, our study finds CT-CALCR signaling axis is an important tumor suppressor pathway in glioma development and underscores the importance of using CT as a novel therapeutic molecule for GBM treatment. Citation Format: Jagriti Pal, Vikas Patil, Anupam Kumar, Kavneet Kaur, Chitra Sarkar, Kumaravel Somasundaram. Genetic landscape of glioma reveals defective neuroactive ligand receptor interaction pathway as a poor prognosticator in glioblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2454. doi:10.1158/1538-7445.AM2017-2454

Abstract 3535: Methylation regulates HEY1 expression in glioblastoma

Abstract Glioblastoma multiforme (GBM) remains one of the most lethal and difficult-to-treat cancers of the central nervous system despite the existence of multi-modal therapeutic approaches. The poor prognosis in GBM patients is due in part to its resistance to available treatments, which calls for identifying novel molecular therapeutic targets. In this study, we identified an interesting biomarker, Hairy/Enhancer-Of-Split related with YRPW Motif (HEY1), a mediator of Notch signaling, which contributes to the pathogenesis of GBM. Datamining studies and our immunohistochemistry and immunoblot analysis showed that HEY1 is highly expressed in GBM patient specimens. Recent studies indicate that methylation status may control the expression of cancer phenotype. Our studies using bisulphite sequencing on the patient samples showed that HEY1 promoter was hypermethylated in the normal brain when compared to GBM specimens. Treatment on the 4910 and 5310 xenograft cell lines with sodium butyrate (NaB) greatly decreased HEY1 expression owing to its promoter hypermethylation with a concomitant increase in DNMT1 expression confirming that promoter methylation regulate the HEY1 expression in GBM. NaB treatment also induced cell apoptosis as evaluated by flow cytometric analysis. Silencing HEY1 reduced invasion, migration and proliferation in 4910 and 5310 cells. Further, immunoblot and q-PCR analysis demonstrated the existence of a potential positive regulatory loop between HEY1 and p53. Additionally, DNA microarrays with HEY1 recombinant protein demonstrated good correlation with p53 and provided various bonafide targets of HEY1. Collectively, these studies suggest HEY1 as an important predictive marker for GBM, thus indicating a potential target for future GBM therapy. Citation Format: Maheedhara Reddy Guda, Andrew J. Tsung, Swapna Asuthkar, Collin M. Labak, Yining Lu, Sarah E. Martin, Prasad V. Durbaka, Kiran K. Velpula. Methylation regulates HEY1 expression in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3535. doi:10.1158/1538-7445.AM2017-3535

Abstract PR10: A glycoprotein-mediated mechanical switch promotes glioma aggression

Abstract Glioblastoma multiforme (GBM) is a malignant brain tumor whose progression is associated with rampant extracellular matrix (ECM) remodeling. We recently found that ECM stiffness correlates with poor survival in human GBM specimens. Glycoproteins are the major constituent of normal brain ECM and many are overexpressed in brain tumors, yet the interplay between glycoproteins and mechanical signaling in GBM pathogenesis remains poorly understood. Here, we show that bulky glycoproteins and sugar-binding proteins are broadly upregulated in GBM relative to lower grade gliomas. Further, these genes are overexpressed in the mesenchymal (Mes) relative to the proneural (Pro) GBM subclass, the former of which is associated with treatment resistance and relapse. We took a specific interest in the hyaluronic acid (HA)-producing enzyme, HAS2, and the galactoside-binding lectin galectin-1 (Gal1) due to their ability to modulate tissue structure and rheology. Using mouse models of human GBM we showed that Mes tumors are enriched in HA and fibronectin, coincident with elevated ECM stiffness and mechanical signaling. These data suggest the possibility that aberrant glycoprotein expression drives GBM aggression through enhanced mechanical signaling resulting from tissue stiffening. Consistent with this hypothesis, by elevating mechanical signaling in Pro GBMs we induce a robust Mes-like transition and we see the opposite when reducing Gal1 expression or HA content in Mes tumors. Our data provides evidence of a feed-forward mechanism whereby mechanical signaling drives Gal1 and HA production which reinforce ECM stiffness, thus sustaining pro-tumorigenic mechanical signaling. This abstract is also presented as Poster A21. Citation Format: J Matthew Barnes, Yekaterina A. Miroshnikova, Jason C. Tung, Russel O. Bainer, Valerie M. Weaver. A glycoprotein-mediated mechanical switch promotes glioma aggression. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR10.

Abstract B36: NF-kappaB-dependent and -independent roles for NIK in regulating GBM cell invasion

Abstract The aggressive invasion of GBM cells into healthy brain tissue is a major factor contributing to the therapy resistance and poor prognosis of this malignancy. Our recent results demonstrated that the alternative, or noncanonical, NF-kappaB signaling pathway, predominates in the aggressive mesenchymal subtype of GBM. Additionally, NF-kappaB-inducing kinase (NIK), a key upstream activator of noncanonical NF-kappaB signaling, robustly promotes GBM cell invasion and pathogenesis. We demonstrate that expression of NIK is increased in response to exposure to collagen, a major extracellular matrix (ECM) protein present in GBM tumors, with NIK protein levels continuing to increase during invasion in 3D collagen matrices. Interestingly, NIK expression levels correlates with induction of membrane type-1 matrix metalloproteinase (MT1-MMP), a critical enzyme in many invasive tumors, functioning to degrade the basement membrane and ECM during cell migration and invasion. We provide evidence that NIK promotes MT1-MMP activation through increased phosphorylation at membrane protrusions and elevated enzymatic activity. Notably, the activation of MT1-MMP and the pro-invasive effects of NIK persist in the absence of the downstream NF-kappaB transcription factors RelA/p65 and RelB. Together, these data demonstrate a novel ability of NIK to promote GBM cell migration and invasion that is independent of NF-kappaB transcriptional activation functions. The results underscore the therapeutic potential of targeting noncanonical NF-kappaB signaling in these highly invasive tumors. Citation Format: Camille L. Duran, Linda Herrera de Lechuga, Dong W. Lee, Kayla J. Bayless, Raquel Sitcheran. NF-kappaB-dependent and -independent roles for NIK in regulating GBM cell invasion. [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 B36.

Abstract 358: Integrative pathway analysis of molecular profiles of glioblastoma multiforme for predicting patient survival

Abstract Glioblastoma multiforme (GBM) is the most common form of malignant brain tumor in adults, characterized by median survival of one year and overall poor prognosis. Subtypes based on gene expression signatures have been reported; however, this classification varies based on molecular characteristics of patient samples, and mostly lacks prognostic utility. An important and challenging goal of stratifying patients by molecular markers is to identify functionally relevant sets of genes that are prognostically relevant. In this study we present a new computational approach that integrates multiple datasets to group patients based on their altered pathway characteristics and show prognostic differences within GBM patients (p<0.05). We first enumerate signaling paths from publicly available pathway databases, and calculate a score for signaling pathway segments utilizing gene expression profiles. Patient samples are clustered into groups based on these scores, and Kaplan-Meier curves based on their survival are plotted. Statistical assessment of the survival curves through a Log-Rank Test is calculated to observe prognostic utility of the groups based on pathway segments. We validated our approach on publicly available datasets (TCGA and GSE4271), and showed prognostic differences in patient groups (p<0.05). The two groups we have identified are different than commonly defined two GBM subtypes, namely proneural (PN) and mesenchymal (MES). While some PN tumors might carry features that might favor better prognosis, such as IDH1 mutations, these alterations are not generalizable. We performed gene set enrichment analysis (GSEA) to further investigate the pathway consequences of dysregulated genes in these patient groups, and identified differential enrichment (p<0.01) of various signaling pathways associated with GBM pathogenesis. Recently, Krishna et al. (Cancer Cell, 2013) identified NFKB pathway in aggressive GBM tumors that might have poor prognosis. Furthermore, our analysis identified genes enriched for the longer survivor patient group in PTEN dependent cell cycle arrest and apoptosis pathway, EGF Signaling Pathway, PDGF Signaling Pathway, IL2 Signaling Pathway, and mTOR signaling pathway. While some of key signaling genes (PTEN, PDGFRA, EGFR etc.) included in these pathways were previously associated with GBM subtypes and pathogenesis (Verhaak et al. Cancer Cell. 2010), we provide further utility for their prognostic function within these pathways. These results could serve as a means to improve prognosis and treatment options for future GBM patients as well as to provide opportunities for alternative interventions in multiple other cancers. Citation Format: Arda Durmaz, Douglas Brubaker, Gurkan Bebek. Integrative pathway analysis of molecular profiles of glioblastoma multiforme for predicting patient survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 358. doi:10.1158/1538-7445.AM2014-358

Abstract 3041: GPR56 promotes the adhesion of glioma stem-like cells to the perivascular niche and regulates cell fate

Abstract INTRODUCTION Gliobastomas (GBM) are the most aggressive and frequent human malignancies in the central nervous system. A small population of cells within the bulk of these tumors displays properties of normal neural stem cells (NSC). Accordingly, these cells are referred to as glioma stem-like initiating cells (GIC). Thus, deregulation of mechanisms that control NSC biology might contribute to the pathogenesis of GBM. NSC and GIC are localized to the perivascular space where they reside in close apposition to blood vessels (BV), which promote self-renewal and keep these cells in an undifferentiated state. GPR56 is an adhesion G-protein coupled receptor expressed in neural progenitors in the embryonic and adult brain and plays an indispensable role in cortical development. Knockout mice studies show that GPR56 mediates the attachment of radial glial endfeet to pial basement membrane (BM). Some of the components of the pial BM are also present in the extracellular matrix surrounding BV, such as laminin and collagen. Therefore, we hypothesized that GPR56 might mediate NSC/GIC localization to the perivascular niche thus contributing to their stem-related properties. EXPERIMENTAL PROCEDURES Here, we have compared the expression of GPR56 in neural progenitors versus more differentiated progeny. In addition, we have assessed the role of GPR56 in the neural stem-like properties of GIC, including their differentiation into more mesenchymal cell types such as endothelial cells or pericytes, by gain- and loss-of-function studies. RESULTS We have found that GPR56 is highly expressed in NSC and its expression is downregulated during differentiation. We have also observed that GPR56 is expressed in cells that reside in the perivascular niche. GPR56-knockdown GIC display impaired adhesion to endothelial cells (EC), whereas GPR56 overexpression increases their adhesion to EC. Furthermore, loss of GPR56 increases the ability of GIC to express endothelial/pericyte markers when co-cultured with EC and increases the CD44+ population, suggesting that GPR56 inhibits the acquisition of a mesenchymal-like phenotype. CONCLUSIONS Taken together, our findings indicate that GPR56 mediates the adhesion of GIC to EC. Moreover, GPR56 inhibits the acquisition of mesenchymal features. Therefore, we suggest that GPR56 might mediate the attachment of GIC to the perivascular niche while preserving their neural stem-like properties by preventing the acquisition of a mesenchymal-like phenotype. Citation Format: Marta Moreno, Stefanie Giera, Xianhua Piao, Nuria de la Iglesia. GPR56 promotes the adhesion of glioma stem-like cells to the perivascular niche and regulates cell fate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3041. doi:10.1158/1538-7445.AM2014-3041

Abstract 4948: Uncovering microRNA and transcription factor-mediated regulatory network in glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most common and lethal brain tumor in humans. Recent studies revealed that patterns of microRNA (miRNA) expression in GBM tissue samples are different from that in normal brain tissues, suggesting that a number of miRNAs play critical roles in the pathogenesis of GBM. However, little is yet known about which miRNAs play central roles in the pathology of GBM and their regulatory mechanisms of action. In this study, we systematically explored the combinatory regulation of miRNAs and transcription factors (TFs) impacting GBM genes. We compiled GBM-related miRNAs, genes, and TFs and then identified 1,128 3-node feed-forward loops (FFLs) and 805 4-node FFLs with statistical significance. These FFLs were converged to construct a comprehensive GBM-specific miRNA-TF mediated regulatory network and, from which, an essential GBM-specific regulatory network. To further identify critical miRNA components in the GBM regulatory network, we specifically extracted a subnetwork for Notch signaling pathway. Our follow up network and functional association analyses revealed that six miRNAs (miR-124, miR-137, miR-219-5p, miR-34a, miR-9, and miR-92b) might play important roles in GBM, including some supported by previous studies. This study has generated the first comprehensive miRNA-TF regulatory network for GBM, providing a valuable resource for further understanding the complex regulatory mechanisms in GBM. Our findings of important miRNAs in the Notch signaling pathway, with partial verification by previous studies, demonstrates that our network-based approach is promising for identifying new and important miRNAs in GBM and, potentially, other cancers. 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 4948. doi:1538-7445.AM2012-4948

Abstract 5621: MicroRNA 203 modulates glioma cell migration and invasion by targeting ROBO1

Abstract MicroRNAs (miRNAs) regulate mRNA stability and protein expression, and certain miRNAs have been demonstrated to act either as oncogenes or tumor suppressors. A potential role for miRNAs in malignancy has been suggested by the location of the genes for several miRNAs at sites of translocation breakpoints or deletions linked to a specific neoplastic state. Allelic deletion on chromosome 14q plays an important role in the pathogenesis of GBM and was thought to harbor multiple tumor suppressor genes associated with GBM, a region which also encodes mir-203. Real-time PCR and insitu hybridization showed decreased levels of mir203 expression in anaplastic astrocytoma and glioblastoma tissues and cell lines. Exogenous expression of mir-203 using a plasmid expressing mir-203 precursor (pmir-203) suppressed cell invasion and migration in SNB19 glioblastoma cell line and 4910 xenograft cells. We determined that one relevant target of miR-203 was ROBO1, since miR-203 expression decreased mRNA and protein levels as determined by RT-PCR and western blot analysis. Moreover, cotransfection experiments using luciferase-based transcription reporter assay have shown direct regulation of ROBO1 3α-UTR regions by miR-203 which was significantly abrogated when mutating the predicted miR-203 seed regions in the pMIR-REPORT UTR constructs, thereby validating the direct interaction between miR-203 and the ROBO1 3α-UTRs. Concordantly, over expressing ROBO1 expression construct with no mir-203 binding site blocked pmir-203 mediated suppression of migration and invasion. Taken together these studies demonstrate that up regulation of Robo1 in response to the decrease in mir-203 in glioma cells is responsible for glioma tumor cell migration and invasion. 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 5621. doi:1538-7445.AM2012-5621

An LXR Agonist Promotes Glioblastoma Cell Death through Inhibition of an EGFR/AKT/SREBP-1/LDLR–Dependent Pathway

Glioblastoma (GBM) is the most common malignant primary brain tumor of adults and one of the most lethal of all cancers. Epidermal growth factor receptor (EGFR) mutations (EGFRvIII) and phosphoinositide 3-kinase (PI3K) hyperactivation are common in GBM, promoting tumor growth and survival, including through sterol regulatory element-binding protein 1 (SREBP-1)-dependent lipogenesis. The role of cholesterol metabolism in GBM pathogenesis, its association with EGFR/PI3K signaling, and its potential therapeutic targetability are unknown. In our investigation, studies of GBM cell lines, xenograft models, and GBM clinical samples, including those from patients treated with the EGFR tyrosine kinase inhibitor lapatinib, uncovered an EGFRvIII-activated, PI3K/SREBP-1-dependent tumor survival pathway through the low-density lipoprotein receptor (LDLR). Targeting LDLR with the liver X receptor (LXR) agonist GW3965 caused inducible degrader of LDLR (IDOL)-mediated LDLR degradation and increased expression of the ABCA1 cholesterol efflux transporter, potently promoting tumor cell death in an in vivo GBM model. These results show that EGFRvIII can promote tumor survival through PI3K/SREBP-1-dependent upregulation of LDLR and suggest a role for LXR agonists in the treatment of GBM patients.

The Novel Hsp90 Inhibitor NXD30001 Induces Tumor Regression in a Genetically Engineered Mouse Model of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) has an abysmal prognosis. We now know that the epidermal growth factor receptor (EGFR) signaling pathway and the loss of function of the tumor suppressor genes p16Ink4a/p19ARF and PTEN play a crucial role in GBM pathogenesis: initiating the early stages of tumor development, sustaining tumor growth, promoting infiltration, and mediating resistance to therapy. We have recently shown that this genetic combination is sufficient to promote the development of GBM in adult mice. Therapeutic agents raised against single targets of the EGFR signaling pathway have proven rather inefficient in GBM therapy, showing the need for combinatorial therapeutic approaches. An effective strategy for concurrent disruption of multiple signaling pathways is via the inhibition of the molecular chaperone heat shock protein 90 (Hsp90). Hsp90 inhibition leads to the degradation of so-called client proteins, many of which are key effectors of GBM pathogenesis. NXD30001 is a novel second generation Hsp90 inhibitor that shows improved pharmacokinetic parameters. Here we show that NXD30001 is a potent inhibitor of GBM cell growth in vitro consistent with its capacity to inhibit several key targets and regulators of GBM biology. We also show the efficacy of NXD30001 in vivo in an EGFR-driven genetically engineered mouse model of GBM. Our findings establish that the Hsp90 inhibitor NXD30001 is a therapeutically multivalent molecule, whose actions strike GBM at the core of its drivers of tumorigenesis and represent a compelling rationale for its use in GBM treatment.

The role of a single nucleotide polymorphism of MDM2 in glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a 5-year survival rate of < 5%. Aberrant function of TP53 is common in GBM. Although mutational inactivation of p53 is found in many cases, there remain tumors in which genetic alterations of p53 are absent. Negative regulators of the TP53 pathway such as MDM2, which directly inhibits TP53 expression and activity, may influence the pathogenesis of GBM. To understand its potential function in gliomagenesis, the authors analyzed a novel single nucleotide polymorphism (SNP) in the MDM2 promoter that enhances MDM2 expression. The investigators isolated DNA from 98 patients with GBM and 102 healthy, cancer-free controls. A polymerase chain reaction analysis was performed to determine the MDM2 SNP309 genotype by using distinct primer pairs for the wild-type (T) and mutant (G) alleles. The frequency of the mutant MDM2 polymorphism was found to be higher (p = 0.0092) in patients with GBM (54.6%) compared with healthy controls (41.2%); the TT and GG genotypes were more common in healthy controls and patients with GBM (p = 0.0004 and p = 0.02, respectively). Although there was no association between the MDM2 SNP309 and overall survival, the GG genotype was associated with development of GBM at a younger age in patients with tumors harboring wild-type p53, which may mitigate the effect of the MDM2 SNP. Although the MDM2 SNP309 does not portend decreased survival, the increased incidence of the mutant G allele in patients with GBM and its influence on age of onset suggest a potential role in the molecular pathogenesis of GBM, and may be a therapeutic target.

Conspirators in a capital crime: co-deletion of p18INK4c and p16INK4a/p14ARF/p15INK4b in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is one of the most dreaded cancer diagnoses due to its poor prognosis and the limited treatment options. Homozygous deletion of the p16(INK4a)/p14(ARF)/p15(INK4b) locus is among the most common genetic alterations in GBM. Two recent studies have shown that deletion and mutation of another INK4 family member, p18(INK4c), also drives the pathogenesis of GBM. This minireview will discuss the known roles for p18(INK4c) in the initiation and progression of cancer and suggest opportunities for future studies.