Published OnlineFirst August 18, 2011; DOI: 10.1158/0008-5472.

CAN-11-0782

Cancer
Therapeutics, Targets, and Chemical Biology Research

Oncogene AEG-1 Promotes Glioma-Induced

Neurodegeneration 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

Abstract
Aggressive 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, the
mechanism(s) controlling this process is poorly understood. Astrocyte elevated gene-1 (AEG-1) is an oncogene
that 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-induced
neurodegeneration. Pearson correlation coefficient analysis in normal brain tissues and samples from glioma
patients indicated a strong negative correlation between expression of AEG-1 and a primary glutamate
transporter of astrocytes EAAT2. Gain- and loss-of-function studies in normal primary human fetal astrocytes
and T98G glioblastoma multiforme cells revealed that AEG-1 repressed EAAT2 expression at a transcriptional
level 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 of
neuronal cell death. These findings were also confirmed in samples from glioma patients showing that AEG-1
expression negatively correlated with NeuN expression. Taken together, our findings suggest that AEG-1
contributes to glioma-induced neurodegeneration, a hallmark of this fatal tumor, through regulation of EAAT2
expression. Cancer Res; 71(20); 6514–23.
2011 AACR.

Introduction oligodendroglial, ependymal, and choroid plexus tumors

Tumors of the central nervous system (CNS) are the most
prevalent solid neoplasms of childhood and the second
leading cancer-related cause of death in adults between
the ages of 20 and 39 years (1, 2). Gliomas, the most common
brain tumors of the adult CNS, originate from neuroepithe-
lial tissue and are classified morphologically as astrocytic,
Authors' Affiliations: 1Cancer Preventive Material Development Research
Center, Institute of Oriental Medicine, College of Oriental Medicine, Kyung
Hee University, Seoul; 2Ilsong Institute of Life Science, Hallym University,
Anyang, Kyonggi-do, Republic of Korea; 3Department of Human and
Molecular Genetics, 4VCU Institute of Molecular Medicine, 5VCU Massey
Cancer Center, Virginia Commonwealth University, School of Medicine,
Richmond, Virginia
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Authors: Paul B. Fisher, Department of Human and
Molecular Genetics, and VCU Institute of Molecular Medicine, Virginia
Commonwealth University School of Medicine, Sanger Hall 11-015, 1101
East Marshall Street, Richmond, VA 23298. Phone: 804-828-9632; Fax:
804-827-1124; E-mail: pbfisher@vcu.edu or Seok-Geun Lee, Cancer Pre-
ventive Material Development Research Center, Institute of Oriental Med-
icine, College of Oriental Medicine, Kyung Hee University, Seoul, Republic
of Korea; E-mail: seokgeun@khu.ac.kr
doi: 10.1158/0008-5472.CAN-11-0782

2011 American Association for Cancer Research.
(2–4). Astrocytomas, composed predominantly of neoplastic
astrocytes, account for 80% to 85% of all gliomas and are
staged as low grade (grade I) to high grade (grade IV) on the
basis of nuclear atypia, mitotic activity, endothelial hyper-
plasia, and necrosis (4). Glioblastoma multiforme (grade IV
astrocytoma; GBM) is an extremely aggressive, invasive, and
destructive malignancy with a proliferation rate that is 2 to
5 times faster than grade III tumors (2, 5). Extensive surgical
resection is not curative due to the highly invasive capacity
of GBM cells into normal brain parenchyma (3). Moreover,
glioblastoma multiforme is largely resistant to currently
available treatments that are based on cytotoxic approaches
targeting replicating DNA, such as chemotherapy or radio-
therapy (6–8). In addition to uncontrolled proliferation and
diffuse tissue invasion, neurodegeneration is another attri-
bute of malignant gliomas (2, 9–11).
The mechanisms of glioma-induced neurodegeneration are
poorly understood although excitotoxic levels of glutamate
play a key role in this phenomenon (10, 11). Although gluta-
mate is a major neurotransmitter implicated in most aspects
of normal brain functions, it is a potent neurotoxin at high
concentration, indicating that glutamate must be constantly
removed for maintenance at a low level (12, 13). The excitatory
amino acid transporters 1 and 2 (EAAT1 and EAAT2), pre-
dominantly expressed on astrocytes, are responsible for the

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 Published OnlineFirst August 18, 2011; DOI: 10.1158/0008-5472.CAN-11-0782
clearance of excitotoxic levels of glutamate from synapses, and
an impaired glutamate uptake by glial cells causes widespread
neurodegeneration and lethal epilepsy (14–16). Furthermore,
a number of studies have reported that glioma cells release
high levels of glutamate, which causes neuronal cell death and
promotes 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 multiple
oncogenic signaling pathways including phosphoinositide-3
kinase (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, compared
with that in normal brain tissues, in tumors of the CNS, such
as neuroblastoma, glioblastoma multiforme, and oligodendro-
glioma (28, 30–32). Gain- and loss-of-function studies in
glioma cells revealed crucial roles in proliferation and invasive
ability of glioma cells (28, 31). In addition, in vivo experiments
using orthotopic glioma models confirmed the role of AEG-1
in glioma progression (28, 31). Furthermore, we observed an
interesting inverse correlation between the expression levels
of AEG-1 and EAAT2. AEG-1 expression is elevated following
HIV-1 and TNF-a treatment of astrocytes, whereas EAAT2
expression is downregulated (22, 33–36). In the setting of
glioma progression, AEG-1 gradually increases as astrocytes
evolve into malignant glioma while, in parallel, EAAT2 ex-
pression decreases (11, 17, 18, 28, 31, 32, 37). Both HIV-1
infection and glioma progression are associated with neuro-
degenerative changes, and glutamate excitotoxicity is one of
the predominant mechanisms mediating neurodegeneration.
On the basis of these considerations, we presently investigated
the role of AEG-1 in glioma-induced neurodegeneration with a
focus on regulation of the glutamate transporter and its
concomitant control of glutamate levels.
Materials and Methods
Tissue array and immunostaining
Immunofluorescence analyses in human glioma tissue
arrays (GL806) obtained from Tissue Array Networks were
conducted 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 scanning
microscope 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 were
transfected with pcDNA, AEG-1, or each AEG-1 deletion
construct. Two days later, the cells were fixed and immu-
nostaining was carried out with anti-HA antibody (Covance)
as described (27).
Cell lines
Normal PHFAs, human glioma cell lines H4 (neuroglioma),
T98G (glioblastoma multiforme), and U251-MG (neuronal
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Role of AEG-1 in Glioma-Induced Neurodegeneration
glioblastoma) cells were previously described (26, 28). PC-12
(rat pheochromocytoma) cells were purchased from the
American Type Tissue Culture and Collection and cultured
in Dulbecco's modified Eagle medium (DMEM) with 5% FBS
and 10% heat-inactivated horse serum at 37
C. The normal
control sh (NCsh), AEG-1sh-2, and AEG-1sh-4 cell lines were
established by transfection with control short-hairpin RNA
(shRNA), AEG-1 shRNA #2, and AEG-1 shRNA #4 expression
plasmids (SA Biosciences; catalogue no. KH18459H) in T98G
cells, respectively, and selected with hygromycin.
Recombinant adenovirus, siRNA, and plasmids
Both Ad.vec and Ad.AEG-1 have been previously des-
cribed (26). Control and YY1 siRNAs were purchased from
Santa Cruz Biotechnology. The expression plasmids of AEG-1
and 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–C4
include 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 have
been described previously (25, 35, 38). The EAAT2Pro-
954mYY1 and EAAT2Prom-954mNFkB1 constructs were
made using the QuickChange Site-Directed Mutagenesis
Kit (Stratagene) in the context of the EAAT2Pro-954 con-
struct. The sequences used for PCR primers include: EAAT2
Pro-954mYY1, 50 -TCGGAGCCCCCGGAGCTCCCCGCCAAG-
CATTATCCCCGCG-30 , and EAAT2Prom-954mNFkB1, 50 -
TCGGAGCCCCCGGAGCTCAAAGCCAAGCGCCATCCCCG-
CG-30 . The mutated sequences are underlined.
Western blotting and immunoprecipitation assays
Whole-cell lysates were prepared, and coimmunoprecipita-
tion and Western blot analyses were carried out as described
previously (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 were
previously described (28).
Northern blotting, real-time PCR, and nuclear run-on
assays
Total RNA was extracted using the RNeasy Mini Kit (Qia-
gen). Real-time PCR (RT-PCR) was conducted using the ABI
7900 Fast Real-Time PCR System and TaqMan Gene Expres-
sion Assays for individual mRNAs (Applied Biosystems). The
nuclei were extracted using NP40 lysis buffer, and nuclear run-
on assays were conducted (38).
Transient transfection and luciferase assays
Cells were plated in 24-well plates, infected with Ad.vec
or Ad.AEG-1, and transfected with the indicated plasmids
and Renilla luciferase plasmid (Promega) together with
20 nmol/L of control or YY1 siRNA using LipofecAMINE
2000 (Invitrogen); luciferase activities were measured
using a Dual-Luciferase Reporter Assay Kit (Promega).
Firefly luciferase activity was normalized by Renilla luci-
ferase activity.
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Lee et al.

Electrophoretic mobility shift assays
Electrophoretic mobility shift assays (EMSA) were con-
ducted as described (38). The sequences of oligonucleotide
used as probe include 50 -CGCCAAGCGCCATCCCCGCG-30
(the YY1 binding site is underlined) and the mutant oligonu-
cleotide 50 -CGCCAAGCATTATCCCCGCG-30 (the mutated
sequences are in bold type).
Chromatin immunoprecipitation assays
Chromatin immunoprecipitation (ChIP) assays were con-
ducted using the ChIP-IT Kit (Active Motif). The primers for
the human EAAT2 promoter used are as following: sense, 50 -
ATCGCTCTCTCGGGGAAGCCA-30 ; antisense, 50 -TAAGCCCT-
TTAGCGCCTCAA-30 .
Glutamate uptake assays
Assays to determine glutamate uptake were conducted as
described (38).
Cell viability assays
Cells 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, and
treated with neuronal differentiation media [DMEM with
1% heat-inactivated horse serum supplemented with
1 mmol/L di-butyryl cAMP, Sigma; and 50 ng/mL of nerve
growth factor (NGF), Promega] for 3 to 6 days. Then, the
PC-12–derived neuron cells were treated with conditioned
media for 1 day. Cell viability was measured by an MTT
assay (24).
Statistical analysis
Data 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 EAAT2
expression and the number of neuronal cells in glioma
patients
To examine a possible correlation between the expression
of AEG-1 and EAAT2 in glioma, we first carried out immu-
nofluorescence staining of AEG-1 and EAAT2 in samples
from glioma patients using a tissue array containing 35 cases
of glioma and 5 normal brain tissues in duplicate (Fig. 1A–
C). The specificity of anti-AEG-1 and anti-EAAT2 antibodies
for immunostaining was confirmed by competition with
each immunogen (Supplementary Fig. S1). Although the
expression of AEG-1 greatly increased in samples from
glioma patients compared with that in normal human
brains, EAAT2 expression significantly decreased (Fig. 1A
and C). A scatter-plot and Pearson correlation coefficient
analysis 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 expression
in samples from glioma patients compared with NC. B, correlation between AEG-1 and EAAT2 expression. C, expression of AEG-1, EAAT2, and
NeuN. 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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Role of AEG-1 in Glioma-Induced Neurodegeneration

glutamate transporter, indicated almost no difference
between 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 in
samples from glioblastoma multiforme patients compared
with normal brain tissues by Western blot analysis (Fig. 1D).
These results indicate an inverse correlation between
expressions of AEG-1 and EAAT2, but not EAAT1 in glioma.
Dysregulation of EAAT2 causes glutamate excitotoxicity,
which is implicated in various types of neurodegenerative
diseases and glioma-induced neurodegeneration (10–14, 17–
20, 37). Accordingly, we further quantified and compared the
expression of AEG-1 and EAAT2 with NeuN, a neuron-
specific marker in glioma patient samples. NeuN expression
was decreased in glioma samples compared with normal
cerebrum tissues, and a scatter-plot of the data and Pearson
correlation coefficient analysis showed a strong positive
correlation (r ¼ 0.798) between expression of EAAT2 and
NeuN (Fig. 1E and F). Furthermore, the comparison analysis
revealed a strong negative correlation (r ¼ 0.649) between
expression of AEG-1 and NeuN (Fig. 1G), indicating that
samples from glioma patients with their higher levels of
AEG-1 have fewer neurons. These observations and the
negative correlation between AEG-1 and EAAT2 expression
(Fig. 1B) together with previous studies showing that AEG-1
has 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 EAAT2
To determine whether AEG-1 could repress EAAT2 expres-
sion, we first confirmed the negative relationship between
expression of AEG-1 and EAAT2 in PHFA and human glioma
cell lines (Fig. 2A). Then, we analyzed PHFA cells infected with
Ad.vec or Ad.AEG-1. As shown in Figure 2B (left), overexpres-
sion of AEG-1 significantly reduced EAAT2 expression in
PHFA. In addition, EAAT2 mRNA was strongly reduced by
AEG-1 (Fig. 2B, middle). This reduction in EAAT2 mRNA
expression resulted from decreased transcription, as con-
firmed using nuclear run-on assays (Fig. 2B, right). These
observations document that AEG-1 negatively regulates
EAAT2 expression at a transcriptional level. To further confirm
the negative regulation of EAAT2 by AEG-1 in glioma, we
cloned AEG-1 knockdown glioma cell lines (NCsh, AEG-1sh-2,
and AEG-1sh-4) by stable transfection with a control, AEG-1
shRNA-2 or AEG-1 shRNA-4 plasmid in T98G cells, a highly
aggressive glioma cell line with high levels of AEG-1 and low
levels of EAAT2 (as shown in Fig. 2A). RT-PCR and Western
blot analyses verified AEG-1 knockdown in the clones. AEG-1
knockdown increased EAAT2 expression (Fig. 2C) and recov-
ery of AEG-1 expression in the knockdown cells reduced
EAAT2 expression (Supplementary Fig. S3), confirming that
AEG-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 of
infection (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 GAPDH mRNA served as a control (middle). Nuclear
run-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 lysates
from 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 were
transfected with EAAT2Pro–Luc. Transient transfection and luciferase assays were conducted at least 3 times in triplicate. Data: fold-normalized activity
relative to that of Ad-vec–infected PHFA was taken as 1. *, P < 0.01 versus Ad.vec.

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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 as
previously shown (25; Supplementary Fig. S4), whereas it
repressed EAAT2 promoter activity (Fig. 2D). Taken together,
these results show that AEG-1 negatively regulates EAAT2
expression at a transcriptional level.
YY-1 is responsible for AEG-1–mediated EAAT2
repression
To determine the mechanism(s) by which AEG-1 regulates
transcription of EAAT2, a series of 50 -deletion mutants of the
EAAT2 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–mediated
repression in promoter activity (Fig. 3A). These results suggest
that transcription factors binding to the 37/+43 region are
capable of regulating a reduction of EAAT2 promoter activity
in response to AEG-1. Accordingly, we analyzed the 37/+44
region of the EAAT2 promoter using Transcription Element
Search System (www.cbil.upenn.edu/cgi-bin/tess) to identify
putative transcription factor binding sites potentially respon-
sible for AEG-1–mediated repression. This analysis located 2
putative 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 determine
which transcription factor was responsible for EAAT2 pro-
moter repression, we engineered 2 mutant EAAT2 promoter–
reporter plasmids containing a site-directed mutation of the
putative YY1 or NF-kB binding site in the 954 EAAT2
promoter construct (mYY1 and mNF-kB). These constructs
were 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 in
the YY1 binding site abolished the EAAT2 promoter response
to 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 the
role of YY1, we conducted EMSA using a 20-bp double-
stranded probe containing the YY1 binding site of the EAAT2
promoter. As shown in Figure 3C (lanes 2 and 3), the intensity
of 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 this
nucleoprotein complex, competition assays were conducted
using an unlabeled probe or a mutant probe containing
mutations 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 deletion
EAAT2 promoter–reporter construct. Data: fold-normalized activity relative to that of the 954 EAAT2Pro–Luc in Ad-vec–infected PHFA taken as 1. B, the
infected PHFA cells were transfected with EAAT2Pro-954, mNFkB1, or mYY1. Data: fold-normalized activity relative to that of the 954 EAAT2Pro–Luc in
Ad-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 EAAT2
promoter 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 probe
containing 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 were
cotransfected with EAAT2–Luc and control or YY1 siRNA. Data: fold-normalized activity relative to that of Ad-vec–infected and control siRNA-transected
PHFA was taken as 1. E, the infected and transfected PHFA cell lysates were immunoblotted with the indicated antibodies. All transient transfection
and luciferase assays were conducted at least 3 times in triplicate. Data in graphs present mean  SEM. *, P < 0.01 versus Ad.vec.

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Role of AEG-1 in Glioma-Induced Neurodegeneration

little effect on complex formation. In addition, supershift
experiments with an anti-YY1 antibody resulted in a retarded
mobility of the complex, whereas a control immunoglobulin G
(IgG) did not (Fig. 3C, lanes 6 and 7). In addition, YY1 siRNA
inhibited the AEG-1–mediated EAAT2 promoter repression
(Fig. 3D). These results were also confirmed by Western blot
analysis using PHFA cell lysates treated with Ad.AEG-1 and
YY1 siRNA (Fig. 3E). In total, these results indicate that AEG-1
increases YY1 binding to the EAAT2 promoter, which
is responsible for AEG-1–mediated repression of EAAT2
expression.
AEG-1 directly interacts with YY-1 and CBP
AEG-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 which
YY1 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 the
EAAT2 promoter, causing YY1 to function as a negative
regulator of EAAT2 expression by inhibiting CBP. To examine
this possibility, we first established whether these proteins
directly physically interact with each other by immunopre-
cipitation assays using Ad.vec- or Ad.AEG-1–infected PHFA
cell 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 clarify
whether these associations occur on the EAAT2 promoter,
PHFA cells were infected with Ad.vec or Ad.AEG-1, and ChIP
assays were carried out using either control IgG or anti-HA,
anti-YY1, or anti-CBP antibodies. Although only CBP was
associated 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 PHFA
cells (Fig. 4B). These results were further confirmed in T98G
glioma cells that were highly expressing AEG-1. AEG-1, YY1,
and CBP in NCsh cells were bound to the EAAT2 promoter as a
complex, 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 YY1
binding to the EAAT2 promoter and suggest that AEG-1 might
function as a bridge molecule between YY1 and CBP and the
basal transcription machinery, thus facilitating YY1 inhibition
of CBP function as a co-activator on the EAAT2 promoter.
To further clarify these interactions, we analyzed which
domain of AEG-1 was responsible for the interactions by using
AEG-1 deletion constructs. As shown in Figure 4C, all C0 -
deletion mutants were associated with both YY1 and CBP as
were wild-type AEG-1. However, only the N1 construct among
the N0 -deletion mutants interacted with YY1, and none of the
N0 -deletion constructs physically interacted with CBP
(Fig. 4C), suggesting that, in each region, amino acids 1–70
and 71–100 of AEG-1 are responsible for interaction with CBP
and YY1, respectively. We next examined whether these
interactions would be crucial for AEG-1–mediated EAAT2
repression. As shown in Figure 4D, as expected only wild-
type AEG-1 repressed the EAAT2 promoter activity whereas
none of N'-deletion constructs induced repression. Intriguing-
ly, all of the C'-deletion mutants failed to modify EAAT2
promoter activity (Fig. 4D). A recent study suggested that
the predominant nuclear localization signal of AEG-1 is
located at the end region of the C terminus (amino acids
546–582; ref. 41), which is missing in all of the C0 -deletion
mutants 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 for
immunoprecipitation (IP) analysis using anti-HA, anti-YY1, or anti-CBP antibody and the same antibodies for immunoblotting. B, the nuclear pellet containing
chromatin isolated from the infected PHFA cells, NCsh, and AEG-1sh-2 cells were immunoprecipitated with control IgG, anti-AEG-1, anti-YY1, or anti-CBP
antibody, 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-1
deletion-expression construct as indicated. The cell lysates were immunoprecipitated and immunoblotted as indicated. D, PHFA cells were cotransfected with
EAAT2–Luc and each AEG-1 deletion mutant. Data (mean  SEM): fold-normalized activity relative to that of PHFA cells transfected with pcDNA (denoted
as "") 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-phenylindole
staining were evaluated by confocal microscopy. Scale bar, 20 mm. V, Ad.vec; A, Ad.AEG-1.

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 Published OnlineFirst August 18, 2011; DOI: 10.1158/0008-5472.CAN-11-0782
Lee et al.
and the N1 mutant were located in the nucleus, but the C1
mutant that did not contain the nuclear localization signal
was present in the cytoplasm (Fig. 4E). Specificity of anti-HA
antibody for immunostaining was confirmed by competition
with 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 by
blocking EAAT2 function
EAAT2 is the predominant glial glutamate transporter in
the brain and it functions to remove glutamate from the
synapse to prevent excitotoxicity. Dysregulation of EAAT2
causes 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 expression
and both EAAT2 and NeuN expression in glioma patients
(Fig. 1). For these reasons, we hypothesized and investigated
whether AEG-1–mediated EAAT2 repression is essential for
glioma-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, suggesting
that the AEG-1–mediated EAAT2 repression impairs gluta-
Figure 5. AEG-1 induces neuronal cell death via inhibiting glutamate
uptake in glial cells. A, glutamate uptake levels (pmol/mg protein/min) were
measured in Ad.vec- or Ad.AEG-1–infected PHFA cells. *, P < 0.01 versus
Ad.vec. B, the differentiated PC-12 rat neuron cells were treated with
conditioned media prepared from Ad.vec- or Ad.AEG-1–infected PHFA
cells following treatment with 100 mmol/L glutamate. One day later, MTT
assays were conducted. C, glutamate uptake levels (pmol/mg protein/min)
were measured in NCsh and AEG-1sh cells. *, P < 0.01 versus NCsh. D, the
differentiated PC-12 neuron cells were treated with conditioned media
prepared from NCsh and AEG-1sh cells following treatment with 100
mmol/L glutamate. One day later, MTT assays were conducted. *, P < 0.01
in MTT assays (B and D) versus Mock-treated cells.
6520 Cancer Res; 71(20) October 15, 2011
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Research.
mate uptake of astrocytes. In addition, AEG-1 knockdown in
T98G 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 results
indicate that AEG-1 causes glutamate excitotoxicity. To
examine whether decreased uptake of glutamate causes
neuronal cell death, we cultured PC-12–differentiated rat
neuronal 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 cells
containing intact glial glutamate transporters did not
cause neuronal cell death. In contrast, the conditioned
media from Ad.AEG-1–infected cells induced severe neuro-
nal cell death (Fig. 5B). Furthermore, AEG-1 knockdown in
glioma cells inhibited glutamate-induced neuronal cell
death (Fig. 5D). However, these conditioned media from
glioma cells and AEG-1–overexpressing PHFA cells had no
cytotoxic effect on PHFA cells (Supplementary Fig. S7),
indicating that this glutamate excitotoxicity causes cell
death of neurons, but not astrocytes. Considered together
with results obtained from patient samples shown in Fig-
ure 1, these observations indicate that AEG-1 represses
EAAT2 expression and glutamate uptake, thereby causing
neuronal cell death in glioma patients. These provocative
findings highlight a novel mechanism by which gliomas
induce neurodegeneration as summarized in Figure 6.
Discussion
Brain tumors induce pathogenic changes by rapidly pro-
liferating and invading surrounding normal tissues and by
promoting neuronal cell death through glutamate excitotoxi-
city (10). Consequently, a primary therapeutic focus to limit
brain tumor-induced damage is by inhibiting cancer cell
proliferation and invasion and potentially altering defects
in glutamate homeostasis. To achieve these objectives
requires an enhanced understanding of the genetic and epi-
genetic changes that promote development, progression, and
pathogenesis of brain cancers. Previous studies have docu-
mented that AEG-1 plays crucial roles in malignant glioma
progression, and its expression level significantly correlates
with clinicopathologic stages of glioma (28, 31). In the present
study, we report that AEG-1 expression also significantly
correlates with reduction of EAAT2 expression and neuronal
cells in glioma patient samples. In addition, AEG-1 overex-
pression in glioma impairs glutamate uptake by reducing
EAAT2 expression, a primary glutamate transporter, culmi-
nating in glioma-induced neurodegeneration. These newer
data when combined with previous studies suggest that
AEG-1 is involved in the majority of features of glioma
progression, that is, rapid tumor growth, destructive invasion
of surrounding normal brain tissue, and glioma-induced neu-
rodegeneration. Accordingly, we hypothesize that AEG-1
could be a primary regulator of glioma progression and thus
could be a potential therapeutic target for this fatal disease.
Developing pharmacologic agents and/or small molecule
drugs that target AEG-1 for extinction would be predicted
Cancer Research
 Published OnlineFirst August 18, 2011; DOI: 10.1158/0008-5472.CAN-11-0782
A B
Neuron
Figure 6. Potential molecular
mechanism by which EAAT2
reduction mediated by AEG-1
promotes neurodegeneration in
human glioma. A, in normal
astrocytes, EAAT2 functions as a Glutamate
primary glutamate transporter. B,
in glioma cells, increased AEG-1
negatively regulates EAAT2
expression and induces glutamate
EAAT2
excitotoxicity and neuronal cell
death.
EAAT2
promoter
to delimit the pathogenesis and toxicity of glioma. In addition,
restoring glutamate transporter function in glioma might also
provide a means of reducing neuronal damage in patients with
malignant glioma, particularly when combined with inhibition
of 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 normal
astrocytes mainly due to a loss of the predominant astroglial
glutamate transporter EAAT2 (10, 16). This glutamate release
also promotes growth of malignant gliomas (20). We previ-
ously reported that TNF-a induces AEG-1 expression, and
AEG-1 functions as a coactivator for NF-kB by its direct
interaction with p65 (22, 25, 33). However, TNF-a reduces
EAAT2 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 have
now showed that AEG-1 employs YY1 to repress EAAT2
expression. TNF-a–mediated NF-kB repression is not a
general phenomenon in all contexts, because it can also
function as a positive regulator of NF-kB expression, and this
process is poorly understood. We previously observed that
TNF-a preferentially recruits N-myc to the EAAT2 promoter
resulting in a repression of NF-kB–mediated EAAT2 promoter
activation, 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 EAAT2
promoter resulting in reduction of EAAT2 expression. A
previous report showed that AEG-1 acts as a bridge molecule
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Research.
Role of AEG-1 in Glioma-Induced Neurodegeneration
Neuron
Neurotoxicity
Glutamate
AEG-1 EAAT2
EAAT2
promoter
facilitating interaction among NF-kB, CBP, and the basal
transcriptional 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 a
critical role as a link between YY1 and CBP on the EAAT2
promoter, causing YY1 to function as a negative regulator of
EAAT2 expression by inhibiting CBP. These results indicate
that interactions among AEG-1, YY1, and CBP are crucial for
AEG-1–mediated EAAT2 repression, and also suggest that
AEG-1 functions in the nucleus as a bona fide transcriptional
cofactor.
Excitotoxicity caused by impaired glutamate uptake by
glial cells has been implicated in various neurodegenerative
conditions such as ischemia, stroke, epilepsy, amyotrophic
lateral sclerosis, and HIV-associated dementia (41), in psy-
chiatric disorders like depression and schizophrenia, as well
as in certain forms of pain (12, 14–16). Although most studies
of 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-associated
dementia. A recent genome-wide association study implicat-
ed AEG-1 in migraine (43). In addition, in this article we
document that AEG-1 reduces expression of EAAT2 in astro-
cytes, causing neuronal cell death, suggesting that AEG-1
might also play crucial roles in neurodegenerative diseases,
not only in glioma-induced neurodegeneration. We have also
observed that AEG-1 expression is negatively correlated with
EAAT2 expression and neuronal cell survival in a transient
focal ischemia animal model (unpublished data), indicating
its role in ischemia associated with EAAT2 and glutamate
Cancer Res; 71(20) October 15, 2011 6521
 Published OnlineFirst August 18, 2011; DOI: 10.1158/0008-5472.CAN-11-0782
Lee et al.
excitotoxicity. In this context, our present studies are focused
on developing a conditional transgenic animal model to
express AEG-1 specifically in astrocytes using the GFAP
and nestin promoters, which would be beneficial in more
precisely defining AEG-1 functions in astrocytes for examin-
ing in vivo brain tumor development/progression as well as
for neurodegeneration/glioma-induced neurodegeneration.
In view of the assortment of effects of AEG-1 in the context
of brain tumors, such as glioblastoma multiforme, this gene
provides a viable target not only for delimiting the direct
pathogenesis of brain tumors but also for reducing the
indirect toxicity to neurons promoted by defects in glutamate
transport observed in glioblastoma multiforme. Further stud-
ies are warranted and some are currently in progress to test
these possibilities and to develop improved therapies for
glioblastoma multiforme and methods for ameliorating its
pathogenesis.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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We thank Professor David J. Volsky (Molecular Virology Division, St. Luke's-
Roosevelt Hospital Center, Columbia University, New York, NY) for providing
primary human fetal astrocytes. D. Sarkar is the Harrison Endowed Scholar in
the VCU Massey Cancer Center. P.B. Fisher holds the Thelma Newmeyer
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Grant Support
This study was financially supported by the NIH grants R01 CA134721, P01
CA104177, and P01 NS31492, the Thelma Newmeyer Corman Endowment, the
Samuel Waxman Cancer Research Foundation, and the National Foundation for
Cancer Research (PB. Fisher); the Goldhirsh Foundation for Brain Cancer
Research, 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 and
Technology.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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 Published OnlineFirst August 18, 2011; DOI: 10.1158/0008-5472.CAN-11-0782

Oncogene AEG-1 Promotes Glioma-Induced

Neurodegeneration by Increasing Glutamate Excitotoxicity
Seok-Geun Lee, Keetae Kim, Timothy P. Kegelman, et al.

Cancer Res 2011;71:6514-6523. Published OnlineFirst August 18, 2011.

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