Research Article
Integrative Genomic Analysis Identifies NDRG2 as a Candidate
Tumor Suppressor Gene Frequently Inactivated in Clinically
Aggressive Meningioma
1
2
3
4
5
Eriks A. Lusis, Mark A. Watson, Michael R. Chicoine, Meghan Lyman, Peter Roerig,
5
4
1
Guido Reifenberger, David H. Gutmann, and Arie Perry
1
Division of Neuropathology, Departments of 2Pathology and Immunology, 3Neurosurgery, and 4Neurology, Washington University School of
Medicine, St. Louis, Missouri; and 5Department of Neuropathology, Heinrich-Heine-University, Düsseldorf, Germany
Abstract
Although meningiomas are common central nervous system
tumors, little is known about the genetic events responsible
for malignant progression. In this study, we employed gene
expression profiling to identify transcripts whose expression
was lost in anaplastic (WHO grade III) versus benign (WHO
grade I) meningioma. Approximately 40% of genes downregulated in anaplastic meningioma were localized to chromosomes 1p and 14q. One specific gene located at 14q11.2,
NDRG2, was consistently down-regulated in grade III meningioma, a finding which we validated at both the transcript and
protein levels in independent sets of clinically and pathologically diverse meningiomas. Loss of NDRG2 expression was
also seen in a subset of lower-grade meningiomas, including
atypical meningiomas (WHO grade II) with clinically aggressive behavior. Furthermore, we found that the loss of NDRG2
expression was significantly associated with hypermethylation
of the NDRG2 promoter. Collectively, these data identify
NDRG2 as the first specific candidate tumor suppressor gene
on chromosome 14q that is inactivated during meningioma
progression. In addition, these findings highlight the utility of
combining genomic, epigenetic, and expression data to
identify clinically significant tumor biomarkers, and suggest
that NDRG2 expression will be a useful and functionally
relevant biomarker to predict aggressive behavior in patients
with meningioma. (Cancer Res 2005; 65(16): 7121-6)
Introduction
Meningiomas account for approximately one-fourth of all
primary central nervous system neoplasms. Although most are
benign, as many as 20% display clinically aggressive features,
leading to increased patient morbidity or mortality (1, 2). The
2000 WHO grading scheme has improved the ability to predict
clinical behavior (3), though significant variability remains,
particularly within the subset of atypical meningiomas (WHO
grade II). The most clearly established early genetic alteration in
sporadic meningioma is biallelic inactivation of the neurofibromatosis 2 (NF2) gene located on chromosome 22q, with
associated loss of merlin (schwannomin) expression. Recently,
we have shown that loss of expression of other protein 4.1 family
members and interacting proteins, such as 4.1B (DAL-1), 4.1R,
Requests for reprints: Arie Perry, Division of Neuropathology, Washington
University School of Medicine, Campus Box 8118, 660 South Euclid Avenue, St. Louis,
MO 63110. Phone: 314-362-9130; Fax: 314-362-4096; E-mail: [email protected].
I2005 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-0043
www.aacrjournals.org
and TSLC1 are also common (2, 4–6). However, there are few
known genetic changes associated with the malignant progression
of meningioma. Although losses of chromosomal arms 14q and 1p
are common in anaplastic tumors (WHO grade III) and are
generally associated with poor prognosis (7, 8), the relevant tumor
suppressor genes that map to these loci have not been identified.
Loss of heterozygosity studies have had limited success in
identifying minimal regions of deletion, in part, due to the fact
that the entire chromosome or chromosomal arm is lost in most
examples. In the current study, we used a strategy combining
expression profiling with known cytogenetic alterations, leading
to the identification of NDRG2 as a potential meningiomaassociated tumor suppressor gene on 14q11.2. We subsequently
validated its role in meningioma tumor progression using
multiple methods for measuring RNA and protein expression
within independent cohorts of clinically well-characterized
meningiomas. Lastly, hypermethylation of the CpG island within
the NDRG2 promoter region was shown to be the likely
mechanism of inactivation in the majority of meningiomas with
loss of NDRG2 expression.
Materials and Methods
Tissue specimens. Tissue specimens were collected by the Siteman
Cancer Center Tissue Procurement Facility and the Department of
Neuropathology, Heinrich-Heine-University, under approved protocols
from the Institutions’ Review Boards. Snap-frozen tissue specimens from
surgically resected meningiomas were histologically reviewed for specimen adequacy (at least 80% tumor) and subsequent 50 Am serial sections
from each banked frozen specimen were cut, placed immediately into
Trizol reagent (Invitrogen, Carlsbad, CA), and homogenized for RNA
preparation. Total RNA was isolated from Trizol homogenates using the
manufacturer’s protocol. RNA was then further purified using RNeasy spin
columns (Qiagen, Valencia, CA). For RNA isolation from paraffinembedded tissues, Ambion’s (Austin, TX) formalin-fixed paraffin-embedded
RNA isolation kit was used according to the manufacturer’s protocol. All
RNAs were quantified by UV absorbance at 260 and 280 nm and
qualitatively assessed using an RNA Nano Assay and Bioanalyzer 2100
(Agilent, Palo Alto, CA).
Microarray analysis. Five micrograms of tumor RNA was converted
to double-stranded cDNA using a dT-T7 promoter primer. Purified,
double-stranded cDNA was then used as a template to create
biotinylated aRNA. The labeled aRNA target was quantified, fragmented,
and 15 Ag was used for microarray hybridization. The complete target
preparation protocol was done according to the manufacturer’s
recommendations (Affymetrix, Santa Clara, CA) and as previously
published (9). Labeled targets were hybridized to Affymetrix U133A
and U133B GeneChip microarrays for 16 hours and washed following
standard protocols. Microarray images were processed using Affymetrix
Microarray Analysis Suite version 5.0. Each array was scaled so that the
average probe set hybridization signal intensity value (target intensity)
7121
Cancer Res 2005; 65: (16). August 15, 2005
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 2005 American Association for Cancer
Research.
Cancer Research
Figure 1. Histogram demonstrating the
location of 83 genes whose expression was
significantly lower (P < 0.01) in anaplastic
(WHO grade III) meningiomas relative to
benign (WHO grade I) meningiomas.
x -axis, the cytogenetic location; y -axis,
percentage of differentially expressed
probe sets located on each chromosome
arm, relative to the total number of probe
sets on that arm that are represented
on the GeneChip U133A and U133B
microarrays. Overrepresentation of probe
sets at chromosome 1p and 14q (black
column) was statistically significant
(1p, Z score = 1.7288, P = 0.0419; 14q,
Z score = 5.4000, P < 0.0001). NA, probe
sets for which chromosomal localizations
are not known.
was 1,500. Scaled data for each array was exported to the Siteman
Cancer Center Bioinformatics Server,6 merged with updated gene
annotation data for each probe set on the array, and downloaded for
further data visualization and analysis. The completely annotated,
MIAME-compliant data set can be found at the above noted URL. Basic
microarray data visualization, data filtering, and hierarchical clustering were
done using DecisionSite for Functional Genomics (Spotfire, Somerville, MA).
Average fold differences in gene expression between benign and anaplastic
meningiomas were calculated and Student’s t test (uncorrected for multiple
comparisons) was used to generate a list of differentially expressed genes
between the two tumor groups.
Quantitative reverse transcription-PCR analysis. Oligonucleotide
sequences corresponding to the NDRG2 gene transcript were designed
using Primer Express software (Applied Biosystems, Foster City, CA) and
are available on request from the authors. Five hundred nanograms of
total cellular RNA from tissue specimens was subjected to reverse
transcription using Omniscript reverse transcriptase (Qiagen) and oligodT, following the manufacturer’s protocol. After first strand synthesis, an
equivalent of 50 ng of starting total cellular RNA (1/10 of the cDNA
reaction) was added to two duplicate PCR reactions containing 1
SybrGreen master mix (Applied Biosystems), 100 nmol/L forward primer,
and 100 nmol/L reverse primer in a final volume of 20 AL. Each sample
was used in a single reaction that cycled at 95jC for 10 minutes (to
activate enzyme), followed by 40 cycles of 95jC for 30 seconds, and 60jC
for 1 minute on an ABI SDS 7000 sequence detection system (Applied
Biosystems). Fluorescent data were converted into cycle threshold (CT)
measurements using the SDS system software and exported to Microsoft
Excel. Thermal dissociation plots were examined for biphasic melting
curves, indicative of whether primer-dimer formation or other nonspecific
product could be contributing to amplification signal. The DDCT method
(10) was used to calculate fold expression, relative to the lowest tumor
grade in the specific analysis and using glyceraldehyde-3-phosphate
dehydrogenase and h-glucuronidase as reference transcripts. The MannWhitney rank sum and Kruskall-Wallis ANOVA by ranks were used to
compare relative expression levels between the different groups and
results <0.05 were considered significant.
6
http://bioinformatics.wustl.edu.
Cancer Res 2005; 65: (16). August 15, 2005
Immunohistochemistry. Immunohistochemistry using NDRG-specific
affinity-purified rabbit polyclonal antisera (1:10,000 dilution) was done on
formalin-fixed paraffin-embedded sections, using a recently developed
antibody (11). Paraffin blocks from surgically resected meningiomas were
retrieved from the surgical pathology files at Washington University
School of Medicine in St. Louis, MO. Positive controls included normal
human and rodent brain, as well as human leptomeningeal rolls prepared
from autopsy specimens as described previously (4). Cases with strong
diffuse staining were scored as +, those with either patchy immunoreactivity or decreased staining intensity compared with adjacent vessels were
scored as F (i.e., partial loss), and those with lack of tumoral staining in
>90% of the specimen were scored as (i.e., complete loss). Regions lacking
vascular staining were not scored due to the possibility of technical failure.
NDRG2 promoter methylation analysis. The NDRG2 promoter was
analyzed for CpG site methylation using sequencing of sodium bisulfitemodified DNA. Sodium bisulfite treatment of genomic DNA was carried out
as described by Herman et al. (12). Two overlapping fragments from the
NDRG2 promoter region (covering 32 CpG sites between nucleotides
20,564,110 and 20,562,576, Genbank accession no. NC_000014) were
PCR-amplified from sodium bisulfite–modified DNA using the following
primers: P1-sense, 5V-TTTTCGAGGGGTATAAGGAGAGTTTATTTT-3V and
P1-antisense, 5V-CCAAAAACTCTAACTCCTAAATAAACA-3V(320 bp product);
P2-sense, 5V-TTTAGGATATTGCGTTTTTTTTAAGTTTTTATTTT-3V and P2-antisense, 5V-AAAATTCCGACTCCCTCGTACCCAAAA-3V (335 bp product). PCR
amplification was carried out for 43 cycles and the PCR products were then
purified using the High Pure PCR Product Purification Kit (Roche, Penzberg,
Germany). Sequencing was done with the BigDye Cycle Sequencing Kit and an
ABI PRISM 377 semiautomated DNA sequencer (Applied Biosystems).
DNA extracted from four different normal leptomeningeal tissue specimens obtained at autopsy served as nonneoplastic references. As a positive
control, we methylated reference DNA in vitro using SssI (CpG) methylase
(New England Biolabs, Beverly, MA). To calculate the degree of CpG site
methylation in the NDRG2 promoter region, the methylation status at each
of the 32 CpG sites analyzed was semiquantitatively rated using the
following scale: 0, completely unmethylated; 1, a weak methylated signal
detectable in the sequence; 2, methylated signal approximately equal to
unmethylated signal; 3, methylated signal markedly stronger than unmethylated signal. For each tumor, a methylation score was then calculated by
adding the figures determined at the individual CpG sites. Based on this
7122
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 2005 American Association for Cancer
Research.
NDRG2 in Aggressive Meningiomas
score, the tumors were then subdivided into three groups: low/absent
promoter methylation (methylation score <20), moderate promoter
methylation (methylation score 21-30), and strong methylation (methylation
score >30).
Results and Discussion
To date, only a limited number of studies have examined the
transcriptome of meningiomas (13, 14). Therefore, we compared
gene expression in a set of 10 anaplastic and benign meningiomas
using high-density oligonucleotide microarrays. Initially, we
selected transcripts whose average expression between the two
tumor groups varied by at least 2-fold and by a standard t test,
showed a statistical difference in expression at the level of P <
0.01 (uncorrected for multiple comparisons). Using these criteria,
we identified 153 transcripts and subsequently focused on a
subset of 83 transcripts with reduced expression in anaplastic
meningiomas.
Transcripts were next mapped to their corresponding chromosomal locations, and the number of differentially expressed
transcripts localized to a specific chromosome arm was normalized
to the total number of probe sets on the array representing that
chromosomal arm (Fig. 1). After controlling for the unequal
representation of probes along the genome, the percentage of
down-regulated transcripts localized to chromosomes 1p and 14q
was statistically more frequent than that at any other cytogenetic
location. Thirty-four of the 83 genes (41%) with attenuated
expression in anaplastic meningiomas mapped to the 1p (P =
0.0419) or 14q (P < 0.0001) arms, both previously shown to be
frequently lost during meningioma progression.
This finding led us to further define the exact cytogenetic
location of these transcripts, hypothesizing that a cluster of downregulated transcripts might represent a common region of
chromosomal loss. The 21 transcripts on 14q and the 13
transcripts on 1p were mapped according to their absolute
nucleotide location, along with all other transcripts represented
on the microarray. Figure 2 illustrates the cytogenetic locations of
these 34 genes. Down-regulated transcripts were distributed
randomly along the 1p and 14q chromosomal arms, and were
not clustered at any single cytogenetic locus, arguing against a
single region of chromosome loss that would account for all of the
coordinate decreases in gene expression. We further reasoned that
potential tumor suppressor genes critical to progression would
exhibit biallelic inactivation, with a consequent complete loss of
gene expression. Accordingly, we identified one gene, NDRG2,
Figure 2. Cytogenetic locations of 34 genes with a statistically significant decrease in expression in anaplastic versus benign meningiomas. Each column represents
one tumor sample (five benign versus five anaplastic meningiomas), whereas each row represents a unique probe set on the microarray. Filled boxes indicate
probe sets scored detected (‘‘P’’) in the particular tumor by the Affymetrix Microarray Analysis Suite 5.0 software, whereas open boxes indicate probe sets whose signal
was below the threshold of detection. A, cytogenetic location of 21 genes on chromosomal arm 14q that are differently expressed. Arrow, loci where these transcripts
are found. B, cytogenetic location of 13 genes on chromosomal arm 1p that are differently expressed. Arrow, loci where these transcripts are found. MI, benign
meningioma (WHO grade I); MIII, anaplastic meningioma (WHO grade III).
www.aacrjournals.org
7123
Cancer Res 2005; 65: (16). August 15, 2005
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 2005 American Association for Cancer
Research.
Cancer Research
Figure 3. Differential NDRG2 expression in human meningiomas as measured by real-time reverse transcription-PCR. A, relative gene expression among 15 benign
(WHO grade I), 11 atypical (WHO grade II), and 8 anaplastic meningiomas (WHO grade III) shows significantly reduced NDRG2 mRNA expression in the anaplastic
meningiomas (Kruskall-Wallis ANOVA by ranks, P = 0.0015). B, relative NDRG2 mRNA expression in 16 atypical meningiomas (WHO grade II), stratified by clinical
behavior. Levels of NDRG2 expression were normalized to the average expression level in the lowest grade of tumor. NDRG2 expression in the ‘‘aggressive’’
meningiomas was significantly lower as compared with the ‘‘indolent’’ tumors (Mann-Whitney U test, P = 0.0379). C, relative NDRG2 mRNA expression in 45
meningiomas stratified by NDRG2 promoter methylation. NDRG2 mRNA levels and promoter methylation were determined by real-time reverse transcription-PCR and
sequencing of sodium bisulfite–treated DNA, respectively. Depending on each tumor’s promoter methylation score (see Materials and Methods), the cases were
subdivided into three groups, i.e., low/absent promoter methylation (n = 14 tumors), moderate promoter methylation (n = 17 tumors), and strong promoter methylation
(n = 14 tumors). Note significantly lower mean NDRG2 mRNA expression in the tumors with strong promoter methylation (P < 0.01, two-sided t test).
which completely lacked detectable mRNA expression in all five
anaplastic meningiomas examined by gene expression microarray
(Fig. 2). Other genes flanking NDRG2 on 14q11.2 (e.g., FLJ20859
and ZNF219) retained expression regardless of tumor grade,
suggesting that NDRG2 represents a specific target of transcriptional inactivation in anaplastic meningiomas.
To confirm differential expression of NDRG2, quantitative
reverse transcription-PCR analysis was done on an independent
set of 34 meningiomas, which included 15 benign, 11 atypical, and
8 anaplastic tumors. Although benign meningiomas showed
considerable variability in NDRG2 expression, anaplastic meningiomas consistently showed attenuated expression (P = 0.0015) with
a mean of 4.2-fold lower expression levels (Fig. 3A). It was not
possible to determine whether the ‘‘benign’’ tumors with attenuated NDRG2 expression were more aggressive, as the majority of
these tumors were only recently resected and there was insufficient
clinical follow-up. Therefore, to determine whether NDRG2
expression correlates with clinical behavior independent of tumor
grade, we examined an additional set of atypical (WHO grade II)
meningiomas. Total RNA was isolated from 16 atypical meningiomas, previously stratified by clinical behavior. Eight patients with
clinically ‘‘indolent’’ tumors were defined by a neurosurgeon (M.R.
Chicoine) as patients with no recurrence or death (median follow
up 9.9 years), whereas eight patients with ‘‘aggressive’’ disease had a
median time to tumor recurrence of 2.2 years and a median
survival of 8.2 years. Expression of NDRG2 was consistently
attenuated in the clinically aggressive subset and differed
significantly from the indolent subset (P = 0.0379), with an average
of 5.8-fold decreased expression (Fig. 3B).
Cancer Res 2005; 65: (16). August 15, 2005
Next, we examined corresponding NDRG2 protein expression by
immunohistochemistry in a cohort of 49 tumors selected based on
well-characterized biological behavior. This third independent
tumor set included 17 clinically stratified atypical meningiomas,
10 benign meningiomas with no recurrence after at least 10 years of
follow-up, 10 benign meningiomas with recurrence despite gross
total resection, and 12 anaplastic meningiomas. Representative
examples of protein expression are shown in Fig. 4. NDRG2 protein
expression was retained in all but one benign tumor, whereas
complete or partial loss of expression was seen in all 12 anaplastic
tumors. In intermediate grade tumors, loss of protein expression
was seen in only three of nine (33%) clinically indolent cases, as
compared with six of eight (75%) aggressive tumors. In total, 4 of 19
(21%) biologically benign meningiomas versus 23 of 30 (77%)
biologically aggressive meningiomas showed partial or complete
loss of protein expression (P < 0.001; Fisher’s exact test).
Finally, to investigate the mechanistic basis of NDRG2 gene
inactivation, we determined the methylation status of the NDRG2
promoter in 45 meningiomas (24 benign, 11 atypical, and 10
anaplastic meningiomas) using sequencing of sodium bisulfite–
modified DNA. These analyses revealed strong methylation,
preferentially involving CpG sites 1 to 12 and 16 (located between
nucleotides 20,564,169 and 20,564,309, Genbank accession no.
NC_000014) in 7 of 10 (70%) anaplastic meningiomas examined
(Fig. 5). In normal leptomeningeal tissue, these CpG sites were only
partially methylated or completely unmethylated. Among the
nonanaplastic meningiomas, strong NDRG2 promoter methylation
was detected in only 7 of 35 cases (20%; P < 0.01, m2 test). The
remaining tumors showed either low (14 tumors) or moderate
7124
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 2005 American Association for Cancer
Research.
NDRG2 in Aggressive Meningiomas
Figure 4. Immunohistochemical analysis
of NDRG2 protein expression. A, strong,
diffuse staining in both normal cortex and
overlying leptomeninges. B, benign
meningioma with strong and diffuse
immunoreactivity, consistent with retained
expression. C, anaplastic meningioma
scored as partial loss of expression based
on decreased staining intensity compared
with intratumoral blood vessels.
D, anaplastic meningioma with complete
loss of NDRG2 expression, but retained
immunoreactivity in adjacent blood vessel.
(17 tumors) levels of NDRG2 promoter methylation. Correlation of
the promoter methylation data with NDRG2 mRNA expression
revealed a significantly lower mean expression level in the 14
tumors with strong promoter methylation as compared with the
tumors with low/absent or moderate promoter methylation
(Fig. 3C). In total, these data suggest that the most frequent
mechanism of transcriptional down-regulation of NDRG2 in
aggressive meningiomas is NDRG2 promoter hypermethylation.
Nevertheless, the presence of low expression levels in some
meningiomas lacking promoter hypermethylation suggests that
other mechanisms of inactivation may also be taking place in a
subset of cases.
Figure 5. Demonstration of NDRG2 promoter methylation by
sequencing of sodium bisulfite–modified DNA from one anaplastic
meningioma (MN212; A), normal leptomeningeal tissue (B),
and in vitro methylated (SssI methylase-treated) normal
leptomeningeal tissue (C ). Note methylation of the six depicted
CpG sites (CpG sites 3-8) in the anaplastic meningioma (A) and
the positive control (C ). In contrast, normal leptomeningeal tissue
(B) either shows partial or no methylation at the depicted CpG
sites. Arrowheads, CpG sites in the depicted sequences
(noncoding strand sequences).
www.aacrjournals.org
7125
Cancer Res 2005; 65: (16). August 15, 2005
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 2005 American Association for Cancer
Research.
Cancer Research
N-myc downstream regulated gene 2 (NDRG2) is normally
expressed in brain, heart, and muscle and is one of four members of
the NDRG family (15). Although the NDRG1 gene is regulated by
N-myc, other members of this gene family are not similarly
regulated, with each member demonstrating distinct patterns of
expression during development (15, 16). The precise biological
function of this protein family is unknown, but an a/h hydrolase fold
domain of 220 amino acids suggests possible enzymatic function
(15). NDRG2 has been implicated in cell growth (17), differentiation
(18), apoptosis (19), and is rapidly responsive to mineralocorticoid
stimuli in the kidney (20). Most recently, NDRG2 has been found to
be up-regulated in the brains of Alzheimer’s patients (11), suggesting
roles in both cell growth and neurodegeneration.
The analytic approach described here, combining a priori
knowledge of tumor-associated cytogenetic alterations with transcriptome profiling, represents a potentially unique and powerful
method to identify clinically relevant molecular biomarkers in other
tumor types. Although our study clearly shows that NDRG2
inactivation is common in clinically aggressive meningioma, further
studies will be necessary to validate this biomarker using larger
cohorts of patients with more extensive follow-up data. In addition,
future genomic and functional studies will be required to define the
precise mechanism of NDRG2-associated growth regulation in
meningioma, which could potentially elucidate additional targets for
biologically based meningioma therapy.
Acknowledgments
Received 1/6/2005; revised 3/14/2005; accepted 5/4/2005.
Grant support: Doris Duke Charitable Foundation (E.A. Lusis), the James S.
McDonnell Foundation (D.H. Gutmann) and the Deutsche Krebshilfe (G. Reifenberger).
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.
We thank Dr. Niels A. Jensen from The Panum Institute 6.5, University of
Copenhagen N, Denmark for providing the NDRG2 antibody used in this study.
1. Perry A, Scheithauer BW, Stafford SL, Lohse CM,
Wollan PC. ‘‘Malignancy’’ in meningiomas: a clinicopathologic study of 116 patients, with grading implications. Cancer 1999;85:2046–56.
2. Perry A, Gutmann DH, Reifenberger G. Molecular
pathogenesis of meningiomas. J Neurooncol 2004;70:
183–202.
3. Louis DN, Scheithauer BW, Budka H, von Deimling A,
Kepes JJ. Meningiomas. In: Kleihues P, Cavenee WK,
editors. WHO classification of tumours. Pathology and
genetics of tumours of the nervous system. Lyon: IARC
Press; 2000. p. 176–84.
4. Gutmann DH, Donohoe J, Perry A, et al. Loss of DAL-1,
a protein 4.1-related tumor suppressor, is an important
early event in the pathogenesis of meningiomas. Hum
Mol Genet 2000;9:1495–500.
5. Robb VA, Li W, Gascard P, Perry A, Mohandas N,
Gutmann DH. Identification of a third protein 4.1 tumor
suppressor, protein 4.1R, in meningioma pathogenesis.
Neurobiol Dis 2003;13:191–202.
6. Surace EI, Lusis E, Murakami Y, Scheithauer BW, Perry
A, Gutmann DH. Loss of tumor suppressor in lung
cancer-1 (TSLC1) expression in meningioma correlates
with increased malignancy grade and reduced patient
survival. J Neuropathol Exp Neurol 2004;63:1015–27.
7. Cai DX, Banerjee R, Scheithauer BW, Lohse CM,
Kleinschmidt-Demasters BK, Perry A. Chromosome 1p
and 14q FISH analysis in clinicopathologic subsets of
meningioma: diagnostic and prognostic implications.
J Neuropathol Exp Neurol 2001;60:628–36.
8. Weber RG, Bostrom J, Wolter M, et al. Analysis of
genomic alterations in benign, atypical, and anaplastic
meningiomas: toward a genetic model of meningioma
progression. Proc Natl Acad Sci U S A 1997;94:14719–24.
9. Gutmann DH, Donahoe J, Brown T, James CD, Perry A.
Loss of neurofibromatosis 1 (NF1) gene expression in
NF1-associated pilocytic astrocytomas. Neuropath Appl
Neurobiol 2000;26:361–7.
10. Livak KJ, Schmittgen TD. Analysis of relative gene
expression data using real-time quantitative PCR and
the 2(-DD C(T)) method. Methods 2001;25:402–8.
11. Mitchelmore C, Buchmann-Moller S, Rask L, West
MJ, Troncoso JC, Jensen NA. NDRG2: a novel Alzheimer’s
disease associated protein. Neurobiol Dis 2004;16:48–58.
12. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin
SB. Methylation-specific PCR: a novel PCR assay for
methylation status of CpG islands. Proc Natl Acad Sci
U S A 1996;93:9821–6.
13. Watson MA, Gutmann DH, Peterson K, et al.
Molecular characterization of human meningiomas by
gene expression profiling using high-density oligonucleotide microarrays. Am J Pathol 2002;161:665–72.
Cancer Res 2005; 65: (16). August 15, 2005
7126
References
14. Fathallah-Shaykh HM, He B, Zhao LJ, et al. Genomic
expression discovery predicts pathways and opposing
functions behind phenotypes. J Biol Chem 2003;278:
23830–3.
15. Qu X, Zhai Y, Wei H, et al. Characterization and
expression of three novel differentiation-related genes
belong to the human NDRG gene family. Mol Cell
Biochem 2002;229:35–44.
16. Shimono A, Okuda T, Kondoh H. N-mycdependent repression of ndr1, a gene identified by
direct subtraction of whole mouse embryo cDNAs
between wild type and N-myc mutant. Mech Dev 1999;
83:39–52.
17. Deng Y, Yao, Chau L, et al. N-Myc downstreamregulated gene 2 (NDRG2) inhibits glioblastoma cell
proliferation. Int J Cancer 2003;106:342–7.
18. Choi SC, Kim KD, Kim JT, et al. Expression and
regulation of NDRG2 (N-myc downstream regulated
gene 2) during the differentiation of dendritic cells.
FEBS Lett 2003;553:413–8.
19. Wu GQ, Liu XP, Wang LF, et al. Induction of
apoptosis of HepG2 cells by NDRG2. Xi Bao Yu Fen Zi
Mian Yi Xue Za Zhi 2003;19:357–60.
20. Boulkroun S, Fay M, Zennaro MC, et al. Characterization of rat NDRG2 (N-Myc downstream regulated
gene 2), a novel early mineralocorticoid-specific induced
gene. J Biol Chem 2002;277:31506–15.
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 2005 American Association for Cancer
Research.
Integrative Genomic Analysis Identifies NDRG2 as a
Candidate Tumor Suppressor Gene Frequently Inactivated in
Clinically Aggressive Meningioma
Eriks A. Lusis, Mark A. Watson, Michael R. Chicoine, et al.
Cancer Res 2005;65:7121-7126.
Updated version
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/65/16/7121
This article cites 19 articles, 7 of which you can access for free at:
http://cancerres.aacrjournals.org/content/65/16/7121.full#ref-list-1
This article has been cited by 24 HighWire-hosted articles. Access the articles at:
http://cancerres.aacrjournals.org/content/65/16/7121.full#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 2005 American Association for Cancer
Research.