Oncogene AEG 1 Promotes Glioma Induced Neurodegeneration by Increasing Glutamate Excitotoxicity

2011;71:6514-6523. Published OnlineFirst August 18, 2011.Cancer Res Seok-Geun Lee, Keetae Kim, Timothy P. Kegelman, et al. Neurodegeneration by Increasing Glutamate Excitotoxicity

Promotes Glioma-InducedAEG-1Oncogene

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Therapeutics, Targets, and Chemical Biology

Oncogene AEG-1 Promotes Glioma-InducedNeurodegeneration by Increasing Glutamate Excitotoxicity

Seok-Geun Lee1, Keetae Kim3, Timothy P. Kegelman3, Rupesh Dash3, Swadesh K. Das3,Jung Kyoung Choi1, Luni Emdad3,4, Eric L. Howlett3,5, Hyun Yong Jeon3, Zhao Zhong Su3,Byoung Kwon Yoo3, Devanand Sarkar3,4,5, Sung-Hoon Kim1, Dong-Chul Kang2, and Paul B. Fisher3,4,5

AbstractAggressive tumor growth, diffuse tissue invasion, and neurodegeneration are hallmarks of malignant glioma.

Although glutamate excitotoxicity is considered to play a key role in glioma-induced neurodegeneration, themechanism(s) controlling this process is poorly understood. Astrocyte elevated gene-1 (AEG-1) is an oncogenethat is overexpressed in several types of human cancers, including more than 90% of brain tumors. In addition,AEG-1 promotes gliomagenesis, particularly in the context of tumor growth and invasion, 2 primary char-acteristics of glioma. In the present study, we investigated the contribution of AEG-1 to glioma-inducedneurodegeneration. Pearson correlation coefficient analysis in normal brain tissues and samples from gliomapatients indicated a strong negative correlation between expression of AEG-1 and a primary glutamatetransporter of astrocytes EAAT2. Gain- and loss-of-function studies in normal primary human fetal astrocytesand T98G glioblastoma multiforme cells revealed that AEG-1 repressed EAAT2 expression at a transcriptionallevel by inducing YY1 activity to inhibit CBP function as a coactivator on the EAAT2 promoter. In addition, AEG-1–mediated EAAT2 repression caused a reduction of glutamate uptake by glial cells, resulting in induction ofneuronal cell death. These findings were also confirmed in samples from glioma patients showing that AEG-1expression negatively correlated with NeuN expression. Taken together, our findings suggest that AEG-1contributes to glioma-induced neurodegeneration, a hallmark of this fatal tumor, through regulation of EAAT2expression. Cancer Res; 71(20); 6514–23. �2011 AACR.

Introduction

Tumors of the central nervous system (CNS) are the mostprevalent solid neoplasms of childhood and the secondleading cancer-related cause of death in adults betweenthe ages of 20 and 39 years (1, 2). Gliomas, the most commonbrain tumors of the adult CNS, originate from neuroepithe-lial tissue and are classified morphologically as astrocytic,

oligodendroglial, ependymal, and choroid plexus tumors(2–4). Astrocytomas, composed predominantly of neoplasticastrocytes, account for 80% to 85% of all gliomas and arestaged as low grade (grade I) to high grade (grade IV) on thebasis of nuclear atypia, mitotic activity, endothelial hyper-plasia, and necrosis (4). Glioblastoma multiforme (grade IVastrocytoma; GBM) is an extremely aggressive, invasive, anddestructive malignancy with a proliferation rate that is 2 to5 times faster than grade III tumors (2, 5). Extensive surgicalresection is not curative due to the highly invasive capacityof GBM cells into normal brain parenchyma (3). Moreover,glioblastoma multiforme is largely resistant to currentlyavailable treatments that are based on cytotoxic approachestargeting replicating DNA, such as chemotherapy or radio-therapy (6–8). In addition to uncontrolled proliferation anddiffuse tissue invasion, neurodegeneration is another attri-bute of malignant gliomas (2, 9–11).

The mechanisms of glioma-induced neurodegeneration arepoorly understood although excitotoxic levels of glutamateplay a key role in this phenomenon (10, 11). Although gluta-mate is a major neurotransmitter implicated in most aspectsof normal brain functions, it is a potent neurotoxin at highconcentration, indicating that glutamate must be constantlyremoved for maintenance at a low level (12, 13). The excitatoryamino acid transporters 1 and 2 (EAAT1 and EAAT2), pre-dominantly expressed on astrocytes, are responsible for the

Authors' Affiliations: 1Cancer Preventive Material Development ResearchCenter, Institute of Oriental Medicine, College of Oriental Medicine, KyungHee University, Seoul; 2Ilsong Institute of Life Science, Hallym University,Anyang, Kyonggi-do, Republic of Korea; 3Department of Human andMolecular Genetics, 4VCU Institute of Molecular Medicine, 5VCU MasseyCancer Center, Virginia Commonwealth University, School of Medicine,Richmond, Virginia

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Paul B. Fisher, Department of Human andMolecular Genetics, and VCU Institute of Molecular Medicine, VirginiaCommonwealth University School of Medicine, Sanger Hall 11-015, 1101East Marshall Street, Richmond, VA 23298. Phone: 804-828-9632; Fax:804-827-1124; E-mail: [email protected] or Seok-Geun Lee, Cancer Pre-ventive Material Development Research Center, Institute of Oriental Med-icine, College of Oriental Medicine, Kyung Hee University, Seoul, Republicof Korea; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-0782

�2011 American Association for Cancer Research.

CancerResearch

Cancer Res; 71(20) October 15, 20116514





clearance of excitotoxic levels of glutamate from synapses, andan impaired glutamate uptake by glial cells causes widespreadneurodegeneration and lethal epilepsy (14–16). Furthermore,a number of studies have reported that glioma cells releasehigh levels of glutamate, which causes neuronal cell death andpromotes malignant glioma progression (17–20).Astrocyte elevated gene-1 (AEG-1) is a multifunctional

oncogene that is overexpressed in a variety of human cancers,although it was originally isolated as a novel HIV-1- and TNF-a–induced transcript from primary human fetal astrocytes(PHFA; refs. 21, 22). As a target of Ras, AEG-1 activates multipleoncogenic signaling pathways including phosphoinositide-3kinase (PI3K)–Akt, mitogen-activated protein kinase (MAPK),Wnt, and NF-kB involved in regulation of proliferation, inva-sion, chemoresistance, angiogenesis, and metastasis (21, 23–30). In particular, AEG-1 showed higher expression, comparedwith that in normal brain tissues, in tumors of the CNS, suchas neuroblastoma, glioblastoma multiforme, and oligodendro-glioma (28, 30–32). Gain- and loss-of-function studies inglioma cells revealed crucial roles in proliferation and invasiveability of glioma cells (28, 31). In addition, in vivo experimentsusing orthotopic glioma models confirmed the role of AEG-1in glioma progression (28, 31). Furthermore, we observed aninteresting inverse correlation between the expression levelsof AEG-1 and EAAT2. AEG-1 expression is elevated followingHIV-1 and TNF-a treatment of astrocytes, whereas EAAT2expression is downregulated (22, 33–36). In the setting ofglioma progression, AEG-1 gradually increases as astrocytesevolve into malignant glioma while, in parallel, EAAT2 ex-pression decreases (11, 17, 18, 28, 31, 32, 37). Both HIV-1infection and glioma progression are associated with neuro-degenerative changes, and glutamate excitotoxicity is one ofthe predominant mechanisms mediating neurodegeneration.On the basis of these considerations, we presently investigatedthe role of AEG-1 in glioma-induced neurodegeneration with afocus on regulation of the glutamate transporter and itsconcomitant control of glutamate levels.

Materials and Methods

Tissue array and immunostainingImmunofluorescence analyses in human glioma tissue

arrays (GL806) obtained from Tissue Array Networks wereconducted as previously described (28). Anti-AEG-1, anti-EAAT1, and anti-EAAT2 antibodies were described (34, 38)and the anti-NeuN antibody was purchased from Millipore.Images were captured with a confocal laser scanningmicroscope LSM multiphton 510 META (Zeiss), and ana-lyzed using ImageJ (NIH). For analyzing localization of AEG-1, PHFA cells seeded onto 4-well chamber slides weretransfected with pcDNA, AEG-1, or each AEG-1 deletionconstruct. Two days later, the cells were fixed and immu-nostaining was carried out with anti-HA antibody (Covance)as described (27).

Cell linesNormal PHFAs, human glioma cell lines H4 (neuroglioma),

T98G (glioblastoma multiforme), and U251-MG (neuronal

glioblastoma) cells were previously described (26, 28). PC-12(rat pheochromocytoma) cells were purchased from theAmerican Type Tissue Culture and Collection and culturedin Dulbecco's modified Eagle medium (DMEM) with 5% FBSand 10% heat-inactivated horse serum at 37�C. The normalcontrol sh (NCsh), AEG-1sh-2, and AEG-1sh-4 cell lines wereestablished by transfection with control short-hairpin RNA(shRNA), AEG-1 shRNA #2, and AEG-1 shRNA #4 expressionplasmids (SA Biosciences; catalogue no. KH18459H) in T98Gcells, respectively, and selected with hygromycin.

Recombinant adenovirus, siRNA, and plasmidsBoth Ad.vec and Ad.AEG-1 have been previously des-

cribed (26). Control and YY1 siRNAs were purchased fromSanta Cruz Biotechnology. The expression plasmids of AEG-1and AEG-1 deletion mutants tagged with hemagglutinin (HA)were described previously (25). The N0-deletion mutants N1–N5 include amino acids 71–582, 101–582, 205–582, 232–582,and 262–582, respectively. The C0-deletion mutants C1–C4include amino acids 1–513, 1–404, 1–356, and 10-289, respec-tively. The 50-deletion mutants of the human EAAT2 promot-er–reporter (EAAT2Pro) constructs and NF-kB–Luc havebeen described previously (25, 35, 38). The EAAT2Pro-954mYY1 and EAAT2Prom-954mNFkB1 constructs weremade using the QuickChange Site-Directed MutagenesisKit (Stratagene) in the context of the EAAT2Pro-954 con-struct. The sequences used for PCR primers include: EAAT2Pro-954mYY1, 50-TCGGAGCCCCCGGAGCTCCCCGCCAAG-CATTATCCCCGCG-30, and EAAT2Prom-954mNFkB1, 50-TCGGAGCCCCCGGAGCTCAAAGCCAAGCGCCATCCCCG-CG-30. The mutated sequences are underlined.

Western blotting and immunoprecipitation assaysWhole-cell lysates were prepared, and coimmunoprecipita-

tion and Western blot analyses were carried out as describedpreviously (25, 38). The antibodies for YY1 (Santa Cruz Bio-technology), CBP (Abcam), and EF1a (Upstate) were pur-chased. Whole-cell lysates from human tissue samples werepreviously described (28).

Northern blotting, real-time PCR, and nuclear run-onassays

Total RNA was extracted using the RNeasy Mini Kit (Qia-gen). Real-time PCR (RT-PCR) was conducted using the ABI7900 Fast Real-Time PCR System and TaqMan Gene Expres-sion Assays for individual mRNAs (Applied Biosystems). Thenuclei were extracted using NP40 lysis buffer, and nuclear run-on assays were conducted (38).

Transient transfection and luciferase assaysCells were plated in 24-well plates, infected with Ad.vec

or Ad.AEG-1, and transfected with the indicated plasmidsand Renilla luciferase plasmid (Promega) together with20 nmol/L of control or YY1 siRNA using LipofecAMINE2000 (Invitrogen); luciferase activities were measuredusing a Dual-Luciferase Reporter Assay Kit (Promega).Firefly luciferase activity was normalized by Renilla luci-ferase activity.

Role of AEG-1 in Glioma-Induced Neurodegeneration

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Electrophoretic mobility shift assaysElectrophoretic mobility shift assays (EMSA) were con-

ducted as described (38). The sequences of oligonucleotideused as probe include 50-CGCCAAGCGCCATCCCCGCG-30

(the YY1 binding site is underlined) and the mutant oligonu-cleotide 50-CGCCAAGCATTATCCCCGCG-30 (the mutatedsequences are in bold type).

Chromatin immunoprecipitation assaysChromatin immunoprecipitation (ChIP) assays were con-

ducted using the ChIP-IT Kit (Active Motif). The primers forthe human EAAT2 promoter used are as following: sense, 50-ATCGCTCTCTCGGGGAAGCCA-30; antisense, 50-TAAGCCCT-TTAGCGCCTCAA-30.

Glutamate uptake assaysAssays to determine glutamate uptake were conducted as

described (38).

Cell viability assaysCells were treated with 1% heat-inactivated horse serum

containing 100 mmol/L of glutamate for 10 or less min-utes, and the conditioned media were collected. PC-12 cells(1 � 104 cells/well) were seeded in 24-well plates, andtreated with neuronal differentiation media [DMEM with1% heat-inactivated horse serum supplemented with1 mmol/L di-butyryl cAMP, Sigma; and 50 ng/mL of nervegrowth factor (NGF), Promega] for 3 to 6 days. Then, thePC-12–derived neuron cells were treated with conditioned

media for 1 day. Cell viability was measured by an MTTassay (24).

Statistical analysisData were presented as mean � SEM and analyzed for

statistical significance using the unpaired Student's t test.Pearson correlation coefficient (r) analysis was used to com-pare gene expressions between 2 genes.

Results

AEG-1 expression negatively correlates with EAAT2expression and the number of neuronal cells in gliomapatients

To examine a possible correlation between the expressionof AEG-1 and EAAT2 in glioma, we first carried out immu-nofluorescence staining of AEG-1 and EAAT2 in samplesfrom glioma patients using a tissue array containing 35 casesof glioma and 5 normal brain tissues in duplicate (Fig. 1A–C). The specificity of anti-AEG-1 and anti-EAAT2 antibodiesfor immunostaining was confirmed by competition witheach immunogen (Supplementary Fig. S1). Although theexpression of AEG-1 greatly increased in samples fromglioma patients compared with that in normal humanbrains, EAAT2 expression significantly decreased (Fig. 1Aand C). A scatter-plot and Pearson correlation coefficientanalysis revealed a strong negative correlation (r ¼ �0.725)between expression of AEG-1 and EAAT2 (Fig. 1B). However,analysis of expression patterns of EAAT1, another glial

Figure 1. Correlation among expression of AEG-1, EAAT2, and NeuN in samples from glioma patients. A, comparison of AEG-1 and EAAT2 expressionin samples from glioma patients compared with NC. B, correlation between AEG-1 and EAAT2 expression. C, expression of AEG-1, EAAT2, andNeuN. Scale bar, 100 mm. D, expression of AEG-1, EAAT1, and EAAT2 in 3 NB samples and 6 samples from glioblastoma multiforme patients.E, quantification and comparison of NeuN expression in samples from glioma patients compared with NC. F and G, correlation between NeuN and EAAT2(F) or AEG1 (G) expression analyzed using a scatter-plot. Data in graphs represent mean � SD. *, P < 0.01 versus NC. NC, normal cerebrum tissues;NB, normal brain tissue.

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Cancer Res; 71(20) October 15, 2011 Cancer Research6516





glutamate transporter, indicated almost no differencebetween glioma and normal brain tissues and little corre-lation between expressions of AEG-1 and EAAT1 (Supple-mentary Fig. S2A–C). These results were also confirmed insamples from glioblastoma multiforme patients comparedwith normal brain tissues by Western blot analysis (Fig. 1D).These results indicate an inverse correlation betweenexpressions of AEG-1 and EAAT2, but not EAAT1 in glioma.Dysregulation of EAAT2 causes glutamate excitotoxicity,which is implicated in various types of neurodegenerativediseases and glioma-induced neurodegeneration (10–14, 17–20, 37). Accordingly, we further quantified and compared theexpression of AEG-1 and EAAT2 with NeuN, a neuron-specific marker in glioma patient samples. NeuN expressionwas decreased in glioma samples compared with normalcerebrum tissues, and a scatter-plot of the data and Pearsoncorrelation coefficient analysis showed a strong positivecorrelation (r ¼ 0.798) between expression of EAAT2 andNeuN (Fig. 1E and F). Furthermore, the comparison analysisrevealed a strong negative correlation (r ¼ �0.649) betweenexpression of AEG-1 and NeuN (Fig. 1G), indicating thatsamples from glioma patients with their higher levels ofAEG-1 have fewer neurons. These observations and thenegative correlation between AEG-1 and EAAT2 expression(Fig. 1B) together with previous studies showing that AEG-1has the ability to regulate promoter activity of its target gene(21, 25, 31, 34) suggested that AEG-1 in glioma might

negatively regulate EAAT2 expression and glutamate uptake,thereby causing neuronal cell death in patients with glioma.

AEG-1 represses expression of EAAT2To determine whether AEG-1 could repress EAAT2 expres-

sion, we first confirmed the negative relationship betweenexpression of AEG-1 and EAAT2 in PHFA and human gliomacell lines (Fig. 2A). Then, we analyzed PHFA cells infected withAd.vec or Ad.AEG-1. As shown in Figure 2B (left), overexpres-sion of AEG-1 significantly reduced EAAT2 expression inPHFA. In addition, EAAT2 mRNA was strongly reduced byAEG-1 (Fig. 2B, middle). This reduction in EAAT2 mRNAexpression resulted from decreased transcription, as con-firmed using nuclear run-on assays (Fig. 2B, right). Theseobservations document that AEG-1 negatively regulatesEAAT2 expression at a transcriptional level. To further confirmthe negative regulation of EAAT2 by AEG-1 in glioma, wecloned AEG-1 knockdown glioma cell lines (NCsh, AEG-1sh-2,and AEG-1sh-4) by stable transfection with a control, AEG-1shRNA-2 or AEG-1 shRNA-4 plasmid in T98G cells, a highlyaggressive glioma cell line with high levels of AEG-1 and lowlevels of EAAT2 (as shown in Fig. 2A). RT-PCR and Westernblot analyses verified AEG-1 knockdown in the clones. AEG-1knockdown increased EAAT2 expression (Fig. 2C) and recov-ery of AEG-1 expression in the knockdown cells reducedEAAT2 expression (Supplementary Fig. S3), confirming thatAEG-1 is a negative regulator of EAAT2. In addition, to

Figure 2. AEG-1 represses EAAT2 expression. A, AEG-1 and EAAT2 expression in various cell lines. B, PHFA cells were infected with multiplicity ofinfection (MOI) of 20 for Ad.vec or Ad.AEG-1 and Western blot analysis was carried out with the indicated antibodies, and EF1a served as an internal control(left). Using total RNAs from the infected cells, Northern blotting assays were conducted, and the levels of GAPDHmRNA served as a control (middle). Nuclearrun-on assays with nuclei from infected cells were conducted, and the transcription rate of GAPDH was used as a control (right). C, real-time PCR (RT-PCR)analysis of AEG-1 and EAAT2 mRNA expression in NCsh (normal control sh), AEG-1sh-2, or AEG-1sh-4 cells (left). *, P < 0.01 versus NCsh. Cell lysatesfrom NCsh, AEG-1sh-2, and AEG-1sh-4 were immunoblotted with the indicated antibodies (right). D, Ad.vec- or Ad.AEG-1–infected PHFA cells weretransfected with EAAT2Pro–Luc. Transient transfection and luciferase assays were conducted at least 3 times in triplicate. Data: fold-normalized activityrelative to that of Ad-vec–infected PHFA was taken as 1. *, P < 0.01 versus Ad.vec.







investigate the role of AEG-1 in regulating the EAAT2 pro-moter, an EAAT2 promoter–reporter plasmid (EAAT2Pro–Luc) was transiently transfected into Ad.vec- or Ad.AEG-1–infected PHFA cells. AEG-1 potently induced NF-kB activity aspreviously shown (25; Supplementary Fig. S4), whereas itrepressed EAAT2 promoter activity (Fig. 2D). Taken together,these results show that AEG-1 negatively regulates EAAT2expression at a transcriptional level.

YY-1 is responsible for AEG-1–mediated EAAT2repression

To determine the mechanism(s) by which AEG-1 regulatestranscription of EAAT2, a series of 50-deletion mutants of theEAAT2 promoter–reporter construct were transfected into Ad.vec- or Ad.AEG-1–infected PHFA cells. Serial deletions from�964 to �37 showed a similar pattern of AEG-1–mediatedrepression in promoter activity (Fig. 3A). These results suggestthat transcription factors binding to the �37/+43 region arecapable of regulating a reduction of EAAT2 promoter activityin response to AEG-1. Accordingly, we analyzed the �37/+44region of the EAAT2 promoter using Transcription ElementSearch System (www.cbil.upenn.edu/cgi-bin/tess) to identifyputative transcription factor binding sites potentially respon-sible for AEG-1–mediated repression. This analysis located 2putative transcription factor binding sites, YY1 and NF-kB1,which are well-known transcription factors that function as

activators or repressors, depending on cellular binding con-text of a target promoter and the presence of other transcrip-tion factor binding sites in the promoter (39, 40). To determinewhich transcription factor was responsible for EAAT2 pro-moter repression, we engineered 2 mutant EAAT2 promoter–reporter plasmids containing a site-directed mutation of theputative YY1 or NF-kB binding site in the �954 EAAT2promoter construct (mYY1 and mNF-kB). These constructswere transfected into PHFA cells, and each promoter acti-vity was compared with that of the wild-type plasmid�954EAAT2Pro–Luc. As shown in Figure 3B, mutation inthe YY1 binding site abolished the EAAT2 promoter responseto AEG-1, whereas mutation in the NF-kB site had no effect.These experiments indicate that YY1 is responsible for AEG-1–mediated EAAT2 promoter repression. To further clarify therole of YY1, we conducted EMSA using a 20-bp double-stranded probe containing the YY1 binding site of the EAAT2promoter. As shown in Figure 3C (lanes 2 and 3), the intensityof a DNA–protein complex was significantly higher in Ad.AEG-1–infected PHFA nuclear extracts in comparison with Ad.vec–infected PHFA nuclear extracts. To further characterize thisnucleoprotein complex, competition assays were conductedusing an unlabeled probe or a mutant probe containingmutations in the YY1 binding site. As shown in Figure 3C,the cold probe (lane 4) completely competed with the DNA–protein complex, whereas the mutant probe (lane 5) had

Figure 3. YY1 is responsible for AEG-1–mediated EAAT2 repression. A, Ad.vec- or Ad.AEG-1–infected PHFA cells were transfected with each deletionEAAT2 promoter–reporter construct. Data: fold-normalized activity relative to that of the �954 EAAT2Pro–Luc in Ad-vec–infected PHFA taken as 1. B, theinfected PHFA cells were transfected with EAAT2Pro-954, mNFkB1, or mYY1. Data: fold-normalized activity relative to that of the �954 EAAT2Pro–Luc inAd-vec–infected PHFA was taken as 1. C, nuclear extracts from the infected PHFA cells were mixed with the probe containing the YY1 site of EAAT2promoter in the following order: 1, no extracts; 2, Ad.vec; 3, Ad.AEG-1; 4, Ad.AEG-1 þ cold wild probe (100�); 5, Ad.AEG-1 þ unlabeled mutant probecontaining mutated YY1 site (100�); 6, Ad.AEG-1 þ anti-YY-1 antibody; and 7, Ad.AEG-1 þ control immunoglobulin G (IgG). D, the infected cells werecotransfected with EAAT2–Luc and control or YY1 siRNA. Data: fold-normalized activity relative to that of Ad-vec–infected and control siRNA-transectedPHFA was taken as 1. E, the infected and transfected PHFA cell lysates were immunoblotted with the indicated antibodies. All transient transfectionand luciferase assays were conducted at least 3 times in triplicate. Data in graphs present mean � SEM. *, P < 0.01 versus Ad.vec.

Lee et al.






little effect on complex formation. In addition, supershiftexperiments with an anti-YY1 antibody resulted in a retardedmobility of the complex, whereas a control immunoglobulin G(IgG) did not (Fig. 3C, lanes 6 and 7). In addition, YY1 siRNAinhibited the AEG-1–mediated EAAT2 promoter repression(Fig. 3D). These results were also confirmed by Western blotanalysis using PHFA cell lysates treated with Ad.AEG-1 andYY1 siRNA (Fig. 3E). In total, these results indicate that AEG-1increases YY1 binding to the EAAT2 promoter, whichis responsible for AEG-1–mediated repression of EAAT2expression.

AEG-1 directly interacts with YY-1 and CBPAEG-1 physically interacts with a coactivator, cyclic AMP-

responsive element binding protein (CREB)-binding protein(CBP), and YY1 interaction with CBP is 1 mechanism by whichYY1 negatively or positively regulates gene expression (25, 39).On the basis of this consideration, we hypothesized that AEG-1 might play a role as a bridge between YY1 and CBP on theEAAT2 promoter, causing YY1 to function as a negativeregulator of EAAT2 expression by inhibiting CBP. To examinethis possibility, we first established whether these proteinsdirectly physically interact with each other by immunopre-cipitation assays using Ad.vec- or Ad.AEG-1–infected PHFAcell lysates. As shown in Figure 4A, anti-HA, anti-YY1, and anti-CBP antibodies effectively immunoprecipitated YY1 and CBP,AEG-1 and CBP, and AEG-1 and YY1, respectively. To clarifywhether these associations occur on the EAAT2 promoter,PHFA cells were infected with Ad.vec or Ad.AEG-1, and ChIPassays were carried out using either control IgG or anti-HA,anti-YY1, or anti-CBP antibodies. Although only CBP wasassociated with the EAAT2 promoter in Ad.vec–infected PHFA

cells, all 3 of the proteins, AEG-1, YY1, and CBP as a complex,bound to the EAAT2 promoter in Ad.AEG-1–infected PHFAcells (Fig. 4B). These results were further confirmed in T98Gglioma cells that were highly expressing AEG-1. AEG-1, YY1,and CBP in NCsh cells were bound to the EAAT2 promoter as acomplex, but AEG-1 knockdown in these cells abolished AEG-1 as well as YY1 association with the EAAT2 promoter(Fig. 4B). These data indicate that AEG-1 increases YY1binding to the EAAT2 promoter and suggest that AEG-1 mightfunction as a bridge molecule between YY1 and CBP and thebasal transcription machinery, thus facilitating YY1 inhibitionof CBP function as a co-activator on the EAAT2 promoter.

To further clarify these interactions, we analyzed whichdomain of AEG-1 was responsible for the interactions by usingAEG-1 deletion constructs. As shown in Figure 4C, all C0-deletion mutants were associated with both YY1 and CBP aswere wild-type AEG-1. However, only the N1 construct amongthe N0-deletion mutants interacted with YY1, and none of theN0-deletion constructs physically interacted with CBP(Fig. 4C), suggesting that, in each region, amino acids 1–70and 71–100 of AEG-1 are responsible for interaction with CBPand YY1, respectively. We next examined whether theseinteractions would be crucial for AEG-1–mediated EAAT2repression. As shown in Figure 4D, as expected only wild-type AEG-1 repressed the EAAT2 promoter activity whereasnone of N'-deletion constructs induced repression. Intriguing-ly, all of the C'-deletion mutants failed to modify EAAT2promoter activity (Fig. 4D). A recent study suggested thatthe predominant nuclear localization signal of AEG-1 islocated at the end region of the C terminus (amino acids546–582; ref. 41), which is missing in all of the C0-deletionmutants we analyzed. In addition, we confirmed that AEG-1

Figure 4. AEG-1 interacts with CBP and YY-1 on the EAAT2 promoter. A, the Ad.vec (V)- or Ad.AEG-1 (A)-infected PHFA cell lysates were used forimmunoprecipitation (IP) analysis using anti-HA, anti-YY1, or anti-CBP antibody and the same antibodies for immunoblotting. B, the nuclear pellet containingchromatin isolated from the infected PHFA cells, NCsh, and AEG-1sh-2 cells were immunoprecipitated with control IgG, anti-AEG-1, anti-YY1, or anti-CBPantibody, and then the eluted DNAs were subjected to PCR. C, PHFA cells were transfected with control vector (pcDNA), full-length AEG-1, or each AEG-1deletion-expression construct as indicated. The cell lysates were immunoprecipitated and immunoblotted as indicated. D, PHFA cells were cotransfected withEAAT2–Luc and each AEG-1 deletion mutant. Data (mean � SEM): fold-normalized activity relative to that of PHFA cells transfected with pcDNA (denotedas "�") was taken as 1. *, P < 0.01 versus pcDNA. E, PHFA cells were transfected as indicated. The AEG-1 (HA) and 40,6-diamidino-2-phenylindolestaining were evaluated by confocal microscopy. Scale bar, 20 mm. V, Ad.vec; A, Ad.AEG-1.







and the N1 mutant were located in the nucleus, but the C1mutant that did not contain the nuclear localization signalwas present in the cytoplasm (Fig. 4E). Specificity of anti-HAantibody for immunostaining was confirmed by competitionwith HA peptide (Supplementary Fig. S5). Taken together,these results indicate that interactions among AEG-1, YY1,and CBP are crucial for AEG-1–mediated EAAT2 repression.

AEG-1 promotes glioma-induced neurodegeneration byblocking EAAT2 function

EAAT2 is the predominant glial glutamate transporter inthe brain and it functions to remove glutamate from thesynapse to prevent excitotoxicity. Dysregulation of EAAT2causes glutamate excitotoxicity, which is implicated in var-ious types of neurodegenerative diseases and glioma-in-duced neurodegeneration (10–14, 17–20, 37). In addition,we found a negative correlation between AEG-1 expressionand both EAAT2 and NeuN expression in glioma patients(Fig. 1). For these reasons, we hypothesized and investigatedwhether AEG-1–mediated EAAT2 repression is essential forglioma-induced neurodegeneration. We first measured glu-tamate uptake in PHFA cells infected with Ad.vec or Ad.AEG-1 to determine the functional significance of AEG-1–medi-ated EAAT2 repression in astrocytes. As shown in Figure 5A,AEG-1 decreased glutamate uptake in PHFA cells, suggestingthat the AEG-1–mediated EAAT2 repression impairs gluta-

mate uptake of astrocytes. In addition, AEG-1 knockdown inT98G cells increased the glutamate uptake in these cells(Fig. 5C). Expression of AEG-1 and EAAT2 in each gain-of-function and loss-of-function study was confirmed by West-ern blot analysis (Supplementary Fig. S6). These resultsindicate that AEG-1 causes glutamate excitotoxicity. Toexamine whether decreased uptake of glutamate causesneuronal cell death, we cultured PC-12–differentiated ratneuronal cells in conditioned media prepared from Ad.vec-or Ad.AEG-1–infected PHFA cells treated with glutamate.The conditioned media from Ad.vec–infected PHFA cellscontaining intact glial glutamate transporters did notcause neuronal cell death. In contrast, the conditionedmedia from Ad.AEG-1–infected cells induced severe neuro-nal cell death (Fig. 5B). Furthermore, AEG-1 knockdown inglioma cells inhibited glutamate-induced neuronal celldeath (Fig. 5D). However, these conditioned media fromglioma cells and AEG-1–overexpressing PHFA cells had nocytotoxic effect on PHFA cells (Supplementary Fig. S7),indicating that this glutamate excitotoxicity causes celldeath of neurons, but not astrocytes. Considered togetherwith results obtained from patient samples shown in Fig-ure 1, these observations indicate that AEG-1 repressesEAAT2 expression and glutamate uptake, thereby causingneuronal cell death in glioma patients. These provocativefindings highlight a novel mechanism by which gliomasinduce neurodegeneration as summarized in Figure 6.

Discussion

Brain tumors induce pathogenic changes by rapidly pro-liferating and invading surrounding normal tissues and bypromoting neuronal cell death through glutamate excitotoxi-city (10). Consequently, a primary therapeutic focus to limitbrain tumor-induced damage is by inhibiting cancer cellproliferation and invasion and potentially altering defectsin glutamate homeostasis. To achieve these objectivesrequires an enhanced understanding of the genetic and epi-genetic changes that promote development, progression, andpathogenesis of brain cancers. Previous studies have docu-mented that AEG-1 plays crucial roles in malignant gliomaprogression, and its expression level significantly correlateswith clinicopathologic stages of glioma (28, 31). In the presentstudy, we report that AEG-1 expression also significantlycorrelates with reduction of EAAT2 expression and neuronalcells in glioma patient samples. In addition, AEG-1 overex-pression in glioma impairs glutamate uptake by reducingEAAT2 expression, a primary glutamate transporter, culmi-nating in glioma-induced neurodegeneration. These newerdata when combined with previous studies suggest thatAEG-1 is involved in the majority of features of gliomaprogression, that is, rapid tumor growth, destructive invasionof surrounding normal brain tissue, and glioma-induced neu-rodegeneration. Accordingly, we hypothesize that AEG-1could be a primary regulator of glioma progression and thuscould be a potential therapeutic target for this fatal disease.Developing pharmacologic agents and/or small moleculedrugs that target AEG-1 for extinction would be predicted

Figure 5. AEG-1 induces neuronal cell death via inhibiting glutamateuptake in glial cells. A, glutamate uptake levels (pmol/mg protein/min) weremeasured in Ad.vec- or Ad.AEG-1–infected PHFA cells. *, P < 0.01 versusAd.vec. B, the differentiated PC-12 rat neuron cells were treated withconditioned media prepared from Ad.vec- or Ad.AEG-1–infected PHFAcells following treatment with 100 mmol/L glutamate. One day later, MTTassays were conducted. C, glutamate uptake levels (pmol/mg protein/min)were measured in NCsh and AEG-1sh cells. *, P < 0.01 versus NCsh. D, thedifferentiated PC-12 neuron cells were treated with conditioned mediaprepared from NCsh and AEG-1sh cells following treatment with 100mmol/L glutamate. One day later, MTT assays were conducted. *, P < 0.01in MTT assays (B and D) versus Mock-treated cells.

Lee et al.






to delimit the pathogenesis and toxicity of glioma. In addition,restoring glutamate transporter function in glioma might alsoprovide ameans of reducing neuronal damage in patients withmalignant glioma, particularly when combined with inhibitionof AEG-1.Gliomas release glutamate at levels that are neurotoxic

(10, 11, 17, 20). Clearance of extracellular glutamate is marked-ly impaired in glioma cells compared with that in normalastrocytes mainly due to a loss of the predominant astroglialglutamate transporter EAAT2 (10, 16). This glutamate releasealso promotes growth of malignant gliomas (20). We previ-ously reported that TNF-a induces AEG-1 expression, andAEG-1 functions as a coactivator for NF-kB by its directinteraction with p65 (22, 25, 33). However, TNF-a reducesEAAT2 expression in an NF-kB–dependent manner (35, 42).These results suggest that NF-kB might be a signaling path-way in AEG-1–mediated EAAT2 repression. However, we havenow showed that AEG-1 employs YY1 to repress EAAT2expression. TNF-a–mediated NF-kB repression is not ageneral phenomenon in all contexts, because it can alsofunction as a positive regulator of NF-kB expression, and thisprocess is poorly understood. We previously observed thatTNF-a preferentially recruits N-myc to the EAAT2 promoterresulting in a repression of NF-kB–mediated EAAT2 promoteractivation, indicating a mechanism by which TNF-a over-comes intrinsic NF-kB–mediated activation through a path-way not involving the direct inhibition of NF-kB (42). Instead,we now showed that AEG-1 recruits YY1 to the EAAT2promoter resulting in reduction of EAAT2 expression. Aprevious report showed that AEG-1 acts as a bridge molecule

facilitating interaction among NF-kB, CBP, and the basaltranscriptional machinery, and therefore, functions as a coac-tivator facilitating the induction of NF-kB–dependent genes(25). In the present study, we document that AEG-1 plays acritical role as a link between YY1 and CBP on the EAAT2promoter, causing YY1 to function as a negative regulator ofEAAT2 expression by inhibiting CBP. These results indicatethat interactions among AEG-1, YY1, and CBP are crucial forAEG-1–mediated EAAT2 repression, and also suggest thatAEG-1 functions in the nucleus as a bona fide transcriptionalcofactor.

Excitotoxicity caused by impaired glutamate uptake byglial cells has been implicated in various neurodegenerativeconditions such as ischemia, stroke, epilepsy, amyotrophiclateral sclerosis, and HIV-associated dementia (41), in psy-chiatric disorders like depression and schizophrenia, as wellas in certain forms of pain (12, 14–16). Although most studiesof AEG-1 have focused on its functions in tumor progression,AEG-1 was originally isolated as a HIV-1–inducible gene(22, 33), implying a possible role for it in HIV-associateddementia. A recent genome-wide association study implicat-ed AEG-1 in migraine (43). In addition, in this article wedocument that AEG-1 reduces expression of EAAT2 in astro-cytes, causing neuronal cell death, suggesting that AEG-1might also play crucial roles in neurodegenerative diseases,not only in glioma-induced neurodegeneration. We have alsoobserved that AEG-1 expression is negatively correlated withEAAT2 expression and neuronal cell survival in a transientfocal ischemia animal model (unpublished data), indicatingits role in ischemia associated with EAAT2 and glutamate

Figure 6. Potential molecularmechanism by which EAAT2reduction mediated by AEG-1promotes neurodegeneration inhuman glioma. A, in normalastrocytes, EAAT2 functions as aprimary glutamate transporter. B,in glioma cells, increased AEG-1negatively regulates EAAT2expression and induces glutamateexcitotoxicity and neuronal celldeath.

Neuron

Neurotoxicity

EAAT2 promoter

EAAT2AEG-1EAAT2

promoter

EAAT2

Glutamate

NeuronA B

Glutamate







excitotoxicity. In this context, our present studies are focusedon developing a conditional transgenic animal model toexpress AEG-1 specifically in astrocytes using the GFAPand nestin promoters, which would be beneficial in moreprecisely defining AEG-1 functions in astrocytes for examin-ing in vivo brain tumor development/progression as well asfor neurodegeneration/glioma-induced neurodegeneration.In view of the assortment of effects of AEG-1 in the contextof brain tumors, such as glioblastoma multiforme, this geneprovides a viable target not only for delimiting the directpathogenesis of brain tumors but also for reducing theindirect toxicity to neurons promoted by defects in glutamatetransport observed in glioblastoma multiforme. Further stud-ies are warranted and some are currently in progress to testthese possibilities and to develop improved therapies forglioblastoma multiforme and methods for ameliorating itspathogenesis.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Professor David J. Volsky (Molecular Virology Division, St. Luke's-Roosevelt Hospital Center, Columbia University, New York, NY) for providingprimary human fetal astrocytes. D. Sarkar is the Harrison Endowed Scholar inthe VCU Massey Cancer Center. P.B. Fisher holds the Thelma NewmeyerCorman Endowed Chair in Cancer Research at the VCU Massey Cancer Centerand is an SWCRF investigator.

Grant Support

This study was financially supported by the NIH grants R01 CA134721, P01CA104177, and P01 NS31492, the Thelma Newmeyer Corman Endowment, theSamuel Waxman Cancer Research Foundation, and the National Foundation forCancer Research (PB. Fisher); the Goldhirsh Foundation for Brain CancerResearch, the Dana Foundation, and the McDonnell Foundation (D. Sarkar);the National Research Foundation Basic Science Research Program (2010-0008219; S.G. Lee); and the Medical Research Center Program (2011-0006220;S.G. Lee and S.H. Kim) of the Korean Ministry of Education, Science andTechnology.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 8, 2011; revised July 11, 2011; accepted July 18, 2011;published OnlineFirst August 18, 2011.

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Oncogene AEG 1 Promotes Glioma Induced Neurodegeneration by Increasing Glutamate Excitotoxicity