Alternative Splicing of CHEK2
and Codeletion with NF2
Instability in Meningioma1
Hong Wei Yang*, Tae-Min Kim†, Sydney S. Song*,
Nihal Shrinath*, Richard Park†, Michel Kalamarides‡,§,
Peter J. Park†, Peter M. Black*, Rona S. Carroll*
and Mark D. Johnson*
*Department of Neurosurgery, Brigham and Women’s
Hospital and Harvard Medical School, Boston, MA, USA;
†Center for Biomedical Informatics, Harvard Medical
School, Boston, MA, USA;‡Inserm, U674, Paris, France;
§Université Paris 7 - Denis Diderot, Institut Universitaire
d’Hématologie, Paris, France
Mutations of the NF2 gene on chromosome 22q are thought to initiate tumorigenesis in nearly 50% of meningiomas,
and 22q deletion is the earliest and most frequent large-scale chromosomal abnormality observed in these tumors. In
aggressive meningiomas, 22q deletions are generally accompanied by the presence of large-scale segmental abnor-
malities involving other chromosomes, but the reasons for this association are unknown. We find that large-scale
chromosomal alterations accumulate during meningioma progression primarily in tumors harboring 22q deletions,
suggesting 22q-associated chromosomal instability. Here we show frequent codeletion of the DNA repair and tumor
suppressor gene, CHEK2, in combination with NF2 on chromosome 22q in a majority of aggressive meningiomas. In
addition, tumor-specific splicing of CHEK2 in meningioma leads to decreased functional Chk2 protein expression. We
show that enforced Chk2 knockdown in meningioma cells decreases DNA repair. Furthermore, Chk2 depletion in-
creases centrosome amplification, thereby promoting chromosomal instability. Taken together, these data indicate
that alternative splicing and frequent codeletion of CHEK2 and NF2 contribute to the genomic instability and associ-
ated development of aggressive biologic behavior in meningiomas.
Neoplasia (2012) 14, 20–28
Although most meningiomas grow slowly, 10% to 15% of these
tumors are WHO grade 2 or 3 lesions that display increased growth
and recurrence rates. Mutation or deletion of the NF2 gene on chro-
mosome 22q is observed in nearly half of all sporadic meningiomas ,
and germ line mutations of NF2 (as observed in neurofibromatosis
type 2) lead to the development of meningiomas in humans and in
mice . Monosomy 22q often occurs in the context of NF2 mutation
and is the earliest and most frequent chromosomal alteration observed
in meningiomas [1,3,4]. We and others have shown that meningiomas
that display deletions of chromosome 22q are more likely to display
other large-scale chromosomal alterations . The presence of frequent
large-scale chromosomal alterations correlates with increasing histologic
grade and the risk of recurrence in meningiomas [5–7].
Based on the association between chromosome 22q deletions and
we hypothesized that 22q loss leads to defects in the DNA homologous
recombination (HR) or nonhomologousendjoining pathways, thereby
tumor progressioninmeningiomas.CHEK2isatumor suppressorgene
a kinase (Chk2) that is involved in the HR and nonhomologous end
joining DNA repair pathways . Here we show that alternative splicing
Address all correspondence to: Mark D. Johnson, MD, PhD, Department of Neuro-
surgery, Brigham and Women’s Hospital, Boston, MA 02115. E-mail: mjohnson27@
1This work was supported by a grant from the Brain Science Foundation to M.D.J.
and R.S.C. and by a National Institutes of Health Director’s New Innovator Award
(DP2OD002319) and R01 NS062219 from the National Institute of Neurological
Disorders and Stroke to M.D.J. The authors have no conflicts of interest or competing
Received 11 November 2011; Revised 23 December 2011; Accepted 3 January 2012
Copyright © 2012 Neoplasia Press, Inc. All rights reserved 1522-8002/12/$25.00
Volume 14 Number 1January 2012pp. 20–28
and frequent codeletion of CHEK2 with NF2 in meningiomas har-
boring chromosome 22q deletions impair DNA repair and increase
chromosomal instability, thereby promoting meningioma progression.
Materials and Methods
Tumor Specimens and Cell Lines
All studies were performed with written informed consent and
under the auspices of a human subjects institutional review board
protocol approved by the Partners Human Research Committee. A
total of 47 primary human meningioma specimens were used for
whole genome analysis. Among these were 18 initial and recurrent
pairs involving meningiomas that progressed to a higher histologic
grade (17 patients with two specimens and 1 patient with three speci-
mens) . Four established human meningioma cell lines were used
in this study (IOMM-Lee, CH157-MN, F5, and Me3TSC) [9–13].
CH157-MN was obtained from Dr DH Gutmann, Washington
University School of Medicine, St. Louis, MO. All cell lines were
cultured in Dulbecco modified Eagle medium supplemented with
10% fetal bovine serum for less than six passages and were maintained
at 37°C in a 5% CO2atmosphere.
Reverse Transcription–Polymerase Chain Reaction, Cloning,
and Direct Sequencing of CHEK2 Transcripts
Total RNA was extracted from primary meningioma specimens, and
CHEK2 was performed. The primers used were 5′-ATGTCTCGG-
GAGTCGGATG (sense) and 5′-ACCACGGAGTTCACAACACAG
(antisense). The PCR products were separated on a 1.2% agarose gel to
visualize alternatively spliced transcripts of CHEK2. The PCR products
were then cloned into a pGEMT-Easy vector (Promega, Madison, WI),
and 10 colonies were randomly selected for direct sequencing.
Lentivirus Production and Generation of Chk2 Knockdown
Meningioma Cell Lines
Lentiviral small hairpin RNA (shRNA) vectors for CHEK2 (clones
V2LHS_1932 and V2LHS_196805) as well as a pGIPZ control
vector were purchased from Open Biosystems (Rockford, IL). The
lentiviral vectors were packaged in 293FT cells using the ViraPower
LentiviralExpression System(Invitrogen,GrandIsland,NY) according
to the manufacturer’s protocol. Human IOMM-Lee and CH157-MN
meningioma cells were transduced with the appropriate lentiviruses,
and stable cell lines were selected using puromycin.
500K SNP and Genome-wide Array Comparative Genomic
500K single nucleotide polymorphism (SNP) analysis of DNA
extracted from primary human meningioma specimens was per-
formed as described previously . Briefly, the tumor cell content
of each meningioma specimen was first evaluated histologically using
hematoxylin and eosin, and only specimens with a tumor cell content
greater than 90% were used for analysis. Genomic DNA was isolated
using a commercially available kit (Qiagen, Valencia, CA). The DNA
was then labeled and analyzed using Affymetrix 500K single nucle-
otide polymorphism (SNP; Santa Clara, CA) arrays according to the
manufacturer’s protocol. The data were normalized and analyzed
using the Affymetrix GTS software package.
For the array comparative genomic hybridization (CGH) analysis,
cultured IOMM-Lee cells stably expressing control or shChk2 vectors
were exposed to a low, sublethal dose of UV irradiation (50 J/m2,
1 minute) and then serially cultured for 10 passages. Meningioma cell
genomic DNA was then extracted. Genomic test and reference DNA
were independently labeled with fluorescent dyes, cohybridized to
a NimbleGen Human CGH 385K Whole-Genome Tiling array
(NimbleGen, Madison, WI) and scanned using a 5-μm scanner.
Log2ratio values of the probe signal intensities (Cy3/Cy5) were cal-
culated and plotted versus genomic position using Roche-NimbleGen
NimbleScan software (NimbleGen) according to the manufacturer’s
protocol. The data were displayed using the Roche-NimbleGen
SignalMap software (NimbleGen).
Cell Cycle Analysis
Established human CH157 meningioma cells were plated in 10-cm
culture dishesfor 24hours.The cells werethen labeledwith propidium
iodide, and flow cytometry cell cycle analysis was performed using a
FACScan Flow Cytometer (BD Biosciences, Bedford, MA).
DNA Repair and Centrosome Duplication Assays
Cultured human meningioma cells were exposed to UV irradiation
(50 J/m2for 5 minutes) to induce DNA double-strand breaks (DSBs).
The cells were then fixed at various time points, and immunofluores-
cence was used to detect the presence of phospho-histone γ-H2AX foci
as a marker of DSBs. Immunostaining was performed using an Alexa
Fluor 488–conjugated phospho-histone γ-H2AX (Ser139) antibody
(20E3; Cell Signaling Technology, Beverly, MA). The percentage of
nuclei displaying γ-H2AX-immunoreactive foci was determined by
Cultured meningioma cells or 293T cells expressing an shCHEK2
vector or a scrambled shRNA control vector were stained using an
antipericentrin antibody (ab4448; Abcam, Cambridge, MA) to detect
centrosomes. The number of centrosomes in each cell was determined
by direct visualization under fluorescence microscopy. Centrosome
number in at least 680 cells was determined for each condition. Cells
containing three or more centrosomes were identified as abnormal.
Statistical significance was determined using the proportion test.
Total protein was extracted using RIPA buffer supplemented with
proteinase inhibitors and phosphatase inhibitor cocktail II (Boston
Bioproducts, Ashland, MA). Protein extracts were then separated by
gel electrophoresis using 10% SDS-PAGE–Tris-HCl gels. The pro-
tein was transferred to nitrocellulose membranes, washed, and sub-
sequently probed using specific antibodies. Antibodies used included
anti-Chk2 and anti-Cdc25A antibodies (Cell Signaling Technology),
anti-NF2 (Abcam), or anti–β-Actin (Sigma) as a loading reference.
After washing and incubating in the appropriate secondary antibody,
immunoreactive bands were visualized using the enhanced chemi-
luminescence system (Pierce, Rockford, IL).
MTT Growth Assay
Meningioma cells (CH157-MN, IOMM-Lee, F5 or Me3TSC)
were plated in 96 well plates (1 × 104cells/well) and maintained in
growth medium supplemented with serum. MTT fluorometric assays
were performed according to the manufacturer’s protocol as described
previously . Six wells were used for each condition. Statistical
significance was determined using the t test.
Neoplasia Vol. 14, No. 1, 2012CHEK2 in Meningioma ProgressionYang et al.
Alternative Splicing and Deletion of CHEK2 in Meningioma
To further investigate the relationship between 22q deletions, the
accumulation of large-scale chromosomal abnormalities, and tumor
progression, we performed a 500K SNP analysis of DNA from 18 ini-
tial (I) and recurrent (R) paired meningioma specimens where the re-
current tumor progressed to a higher histologic grade. As seen in
Figure 1, tumors that lacked 22q deletions generally progressed to a
higher grade without accumulating additional large segmental chromo-
somal changes. In one case, progression to a higher grade was accom-
panied by the new appearance of a 22q deletion concurrent with other
segmental chromosomal changes (arrow, Figure 1A). Each of the re-
maining nine tumors harbored 22q deletions and displayed an increase
in segmental chromosomal deletions during progression. The overall
pattern of chromosomal changes that developed was similar to that re-
ported in previous genomic studies of nonrecurrent meningiomas and
included frequent losses of 22q, 1p, and 14q [4–7]. Thus, the accu-
mulation of frequent segmental chromosomal changes was observed
almost exclusively in tumors harboring deletions of chromosome 22q
(Figure 1A). Tumors lacking segmental chromosome 22q deletions
progressed without accumulating such changes. The accumulation of
segmental chromosomal changes thus accompanies tumor progression
and is closely associated with chromosome 22q loss in a majority of
The observed association between 22q deletion and the accumu-
lation of segmental chromosomal abnormalities in meningiomas sug-
gested the presence of a defect in pathways regulating chromosomal
stability. We reasoned that this defect was likely to be present at the
time of the earliest large-scale chromosomal changes and that it might
Figure 1. Chromosomal instability occurs in the context of 22q deletion during meningioma progression. (A) 500K SNP analyses for 12
initial (I) and recurrent (R) paired primary meningioma specimens illustrating accumulation of large-scale chromosomal changes at the
time of recurrence. Note the association between 22q deletion and the presence of numerous segmental chromosomal abnormalities.
Arrow identifies a pair of specimens in which the initial specimen lacked 22q deletion, whereas the recurrent tumor displayed 22q
deletion and numerous additional large-scale chromosomal changes. (B) 500K SNP analyses of a portion of chromosome 22q in 47 human
meningioma specimens. The location of the CHEK2 and NF2 genes is as indicated. Arrowheads point to two tumors with interstitial 22q
deletions that involve NF2 and CHEK2. (C) Higher-resolution image of the SNP data shown in B illustrating frequent codeletion of NF2 and
CHEK2 in meningioma.
CHEK2 in Meningioma ProgressionYang et al. Neoplasia Vol. 14, No. 1, 2012
thus result from deletions involving chromosome 22q. To investigate
this possibility, we examined chromosome 22q for copy number altera-
tions involving genes that are involved in HR or DNA repair. We ob-
served that the CHEK2 tumor suppressor gene, which is located within
1.1 Mb of NF2, was codeleted with NF2 in all 30 specimens harboring
22q deletions. Although most of these specimens displayed monosomy
22q, two displayed interstitial deletionsthat involved both CHEK2 and
NF2 (Figure 1, B and C).
CHEK2 is a tumor suppressor gene that is involved in DNA repair
and genome stability [15–17]. Loss of CHEK2 has been associated
with the development of breast, prostate, and colon cancer
[8,18,19]. Thus, the high frequency of codeletion of CHEK2 with
NF2 raised the possibility that altered CHEK2 expression might con-
tribute to the genomic instability observed in meningiomas harboring
chromosome 22q deletions.
To examine the status of CHEK2 in meningiomas more closely,
we isolated mRNA from 20 primary human meningioma specimens
and performed RT-PCR for CHEK2. Thirteen of the 20 tumors
showed evidence for multiple splice variants of CHEK2. Importantly,
full-length CHEK2 mRNA was reduced or undetectable in 15 of 20
meningiomas (Figure 2A). To investigate this phenomenon further,
we cloned and sequenced CHEK2 transcripts from 10 primary me-
ningiomas. About 7 to 10 randomly selected CHEK2 clones were se-
quenced for each tumor to obtain an estimate of the relative abundance
of the different CHEK2 transcripts. Point mutations in the coding se-
quence of CHEK2 were not identified. However, several novel CHEK2
splice variants in which a frame shift introduced a stop codon and
eliminated the kinase domain were observed (Figure 2B). The ratio
of wild-type (WT) to alternative transcripts ranged from 10:0 to 1:8
(Figure 2C). Some tumors primarily expressed full-length CHEK2
transcripts, whereas others expressed truncated, nonfunctional CHEK2
splice variants lacking the kinase domain. Previous studies indicate
that proteins derived from such splice variants dimerize with WT
Chk2 and act as dominant negative proteins [20,21].
confirmed the presence of multiple isoforms of Chk2 protein in six
of eight tumors (Figure 2D). In half of the cases, these alternate
Chk2 isoforms were more abundant than full-length Chk2. Taken
together, these data indicate that heterozygous deletion and alternative
splicing of CHEK2 occurs frequently in primary meningiomas. These
genetic and posttranscriptional alterations are associated with decreased
expression of full-length Chk2 protein.
Figure 2. Alternative splicing of CHEK2 in meningioma yields nonfunctional CHEK2 splice variants. (A) RT-PCR analysis of CHEK2 tran-
scripts using total RNA extracted from 20 primary meningioma specimens (labeled A through T). Arrows indicate location of splice variants
compared with the full-length CHEK2 mRNA (WT), M = marker. (B) Direct sequencing of CHEK2 clones illustrating the most commonly
identified splice variants lacking the kinase domain. (C) Frequency of full-length (wt) CHEK2 clones versus various splice variants in 10 pri-
mary meningioma specimens. (D) Western blot illustrating Chk2 and NF2 protein expression in eight primary meningioma specimens. Note
that in half of the specimens, alternate Chk2 isoforms were more abundant than full-length Chk2 (top band).
Neoplasia Vol. 14, No. 1, 2012CHEK2 in Meningioma Progression Yang et al.
Chk2 Depletion Decreases DNA Repair Capacity
in Meningioma Cells
We next examined the effect of decreased expression of full-length
Chk2 on DNA repair in meningioma cells. We performed SNP anal-
yses of four established meningioma cell lines (CH157-MN, F5,
IOMM-Lee, and Me3TSC). Two of these cell lines (CH157-MN
and Me3TSC) showed 22q deletions involving both NF2 and CHEK2
(Figure 3A). These two cell lines also displayed numerous segmental
chromosomal deletions, suggesting impaired DNA repair and chromo-
somal instability. To investigate this possibility, we exposed cells from
each of the four meningioma cell lines to UV irradiation (50 J/m2for
5 minutes) to induce DNA DSBs. The cells were then fixed at several
time points after irradiation and stained for phosphorylated gamma
H2AX (γ-H2AX), a sensitive marker for DNA double strand breaks
. The two cell lines with 22q deletions involving CHEK2
(CH157-MN and Me3TSC) showed significantly greater γ-H2AX
immunoreactivity 3 hours after UV irradiation when compared with
the cell lines in which CHEK2 was intact (F5 and IOMM-Lee), indi-
Chk2 is phosphorylated by ATM after DNA damage and facili-
tates DNA repair by phosphorylating and stabilizing p53, thereby
inducing G1arrest . Chk2 also cooperates with PML to mediate
p53-independent apoptosis after radiation exposure . In addi-
tion to its effects on p53, Chk2 phosphorylates the Cdc25A phos-
phatase to inhibit cell cycle progression . We therefore examined
the effect of Chk2 depletion on meningioma cell growth after DNA
damage. First, we overexpressed two different shRNAs directed
against CHEK2 mRNA to knockdown Chk2 expression in human
Figure 3. Impaired DNA repair capacity in meningioma cell lines with segmental 22q deletions. (A) 500K SNP analysis of genomic DNA
obtained from four established human meningioma cell lines (CH157-MN, F5, IOMM-Lee, and Me3TSC). Dark blue represents copy
number loss, whereas bright red represents gain. Each column contains data from a different cell line. Chromosome 22q is shown
at higher magnification to the right. The approximate locations of the NF2 and CHEK2 genes are as indicated. Note that 22q deletions
involving CHEK2 and NF2 occur in the CH157-MN and Me3TSC cell lines, but not the F5 and IOMM-Lee cell lines. (B) Cells from each of
four human meningioma cell lines were exposed to ultraviolet irradiation (50 J/m2) for 5 minutes. The cells were then fixed and stained
for γ-H2AX immunoreactivity (green) after 0, 1, 2, and 3 hours to detect DNA DSBs. Note the relative increase in nuclear γ-H2AX immuno-
reactivity in the CH157-MN and Me3TSC cell lines that harbor CHEK2 and NF2 deletions when compared with the cell lines that do not
have such deletions (F5 and IOMM-Lee). (C) Quantitation of data shown in B.A significant increase in γ-H2AX immunoreactivity wasnoted
after 3 hours in human meningioma cell lines harboring CHEK2 and NF2 deletions (CH157-MN and Me3TSC) when compared with me-
ningioma cell lines that do not have such deletions (F5 and IOMM-Lee). Data shown are mean ± SEM. *P < .05, t test.
CHEK2 in Meningioma ProgressionYang et al. Neoplasia Vol. 14, No. 1, 2012
CH157-MN meningioma cells. An 80% knockdown of Chk2 was
achieved using this approach (Figure 4A). Cell growth assays revealed
that Chk2 knockdown increased the growth of human CH157-MN
meningioma cells under control conditions (Figure 4B; P < .0001,
unpaired t test). This is consistent with the known role of Chk2 as
a regulator of the G2/M checkpoint . Next, we measured the
growth of these cells under control conditions or after UV irradia-
tion. As shown in Figure 4C, depletion of Chk2 significantly de-
creased meningioma cell growth after UV irradiation-induced
DNA damage (P < .01, unpaired t test). In addition, Chk2 depletion
significantly decreased cell growth after DNA damage caused by ex-
posure to the topoisomerase I inhibitor, camptothecin (Figure 4D;
P < .02, unpaired t test). Similar effects of Chk2 knockdown on
cell growth after UV irradiation or camptothecin-induced DNA
damage were obtained using the IOMM-Lee meningioma cell line
(data not shown).
To determine whether decreased expression of Chk2 alters cell cycle
progression in meningioma, we knocked down Chk2 in CH157-MN
impaired DNA repair, increased sensitivity to DNA damage, and de-
creased proliferation in meningioma cells after Chk2 depletion.
Chk2 Depletion Promotes Centrosome Amplification
and Chromosomal Instability
The striking accumulation of segmental chromosomal alterations
observed in meningiomas suggests the presence of an abnormality in
Figure 4. Chk2 depletion increases proliferation but decreases cell growth after DNA damage. (A) Western blot illustrating knockdown of
Chk2 protein in CH157-MN cells stably expressing two different shRNAs directed against human Chk2. Control cells were transfected
using an empty shRNA control vector. (B) MTT cell growth assay illustrating the effect of Chk2 depletion on the growth of human
CH157-MN meningioma cells under control conditions. Data shown are mean ± SEM. *P < .05, **P < .0001, unpaired t test. (C)
MTT cell growth assay illustrating effect of Chk2 depletion on the growth of CH157-MN cells after UV irradiation (50 J/m2for 5 minutes).
Data shown are mean ± SEM. *P < .05, t test. (D) MTT cell growth assay illustrating effect of Chk2 depletion on the growth of CH157-MN
cells after overnight exposure to increasing concentrations of camptothecin. Data shown are mean ± SEM. *P < .05, t test. (E) Flow
cytometry cell cycle analysis illustrating effect of Chk2 depletion on CH157-MN cell proliferation.
Neoplasia Vol. 14, No. 1, 2012CHEK2 in Meningioma ProgressionYang et al.
pathways governing chromosomal stability. Centrosome amplifica-
tion is a major contributor to chromosomal instability in cancer
. Previous studies have identified Cdc25A as a primary regulator
of centrosome number , and Cdc25A is a direct target of Chk2
. Chk2 phosphorylation of Cdc25A promotes its degradation
. We therefore investigated whether decreased Chk2 expression
leads to genomic instability through centrosome amplification in
Analysis of Chk2 and Cdc25A levels in a panel of 13 primary
human meningiomas revealed an inverse correlation between full-
length Chk2 expression and Cdc25A expression (Figure 5A). Addi-
tional Western blot analysis confirmed that enforced overexpression
of Chk2 decreased Cdc25A levels, whereas overexpression of two dif-
ferent Chk2 splice variants lacking the kinase domain (cloned from
primary meningioma specimens) failed to do so (Figure 5B). Immuno-
cytochemistry for the centrosome marker, pericentrin, in 293T cells
or CH157-MN meningioma cells revealed that Chk2 depletion in-
creased centrosome number (Figure 5C). Quantitation of this effect
indicatedthat thenumber of cells displayingthreeor morecentrosomes
increased two-fold after Chk2 knockdown (Figure 5D; P < .011,
To determine whether the centrosome amplification occurring after
Chk2 depletion was accompanied by an increased frequency of chro-
mosomal alterations in meningioma cells, we administered a sublethal
dose of UV irradiation (50 J/m2for 1 minute) to IOMM-Lee menin-
gioma cells expressing a Chk2 shRNA vector or a control vector and
maintained the cells for 10 passages in culture. Our previous SNP anal-
ysis indicated that IOMM-Lee cells did not harbor CHEK2 deletions
and were thuswell suitedfor investigating theeffects of Chk2depletion
on meningioma cells. After 10 passages, genomic DNA was extracted,
Figure 5. Chk2 depletion increases centrosome number and chromosomal instability in human meningioma cells. (A) Western blot
illustrating correlation between Chk2 and Cdc25A expression using protein lysates derived from 13 primary human meningioma speci-
mens. Arrows indicate samples with the lowest Chk2 expression. Densitometry measurements of the Chk2/Cdc25A ratio revealed a
relative increase in Cdc25A expression in meningiomas with the lowest Chk2 levels. (B) Western blot illustrating effect of overexpressed
Chk2 or Chk2 splice variants lacking the kinase domain on Cdc25A expression in 293T cells. Densitometry measurements revealed an
increase in the p-Cdc25A/Cdc25A ratio and a decrease in the overall expression of Cdc25A after Chk2 overexpression. (C) Light micro-
graphs illustrating effect of Chk2 depletion on centrosome number in IOMM-Lee meningioma cells. (D) Quantitative analysis of centro-
some duplication induced by Chk2 depletion in CH157-MN meningioma cells. Cells with three or more centrosomes were considered
abnormal. At least 680 cells were counted for each condition. P < .011, proportion test. (E) Array CGH analysis of DNA extracted from
IOMM-Lee meningioma cells after Chk2 depletion. Cells were exposed to a sublethal dose of UV irradiation (50 J/m2for 1 minute) and
were then passaged 10 times before harvesting. Data for chromosomes 6, 7, and 8 are shown. Note the increase of chromosomal
deletions in cells with shChk2 depletion.
CHEK2 in Meningioma ProgressionYang et al. Neoplasia Vol. 14, No. 1, 2012
and array comparative genomic hybridization analysis was performed.
As shown in Figure 5E, an increase in chromosomal alterations was
observed in meningioma cells overexpressing Chk2 shRNA, but not
in the parental meningioma cell line or in meningioma cells expressing
a scrambled control shRNA.
Based on these findings, we propose a model (Figure 6) in which a
monoallelic NF2 mutation prompts loss of the other NF2 allele
through 22q deletion. This step initiates meningioma growth. Be-
cause of the close physical proximity of CHEK2 to NF2, both genes
are frequently lost when 22q is deleted. In addition, CHEK2 is alter-
natively spliced in many meningiomas, leading to the production of
nonfunctional or dominant negative forms of the kinase. This dele-
tion and/or alternative splicing of CHEK2 leads to decreased func-
tional Chk2 protein, decreased DNA repair capacity, increased
centrosome number, increased chromosomal instability, and conse-
quent tumor progression.
Here we provide evidence that alternative splicing and deletion of the
tumor suppressor, CHEK2, contributes to chromosomal instability
in meningioma. Our finding that deletion and decreased expression
of functional Chk2 protein occurs in association with chromosome
22q deletions helps to explain the association between NF2 mutations,
22q loss, and the occurrence of large-scale chromosomal deletions in
meningioma. Monosomy 22q is found in 47% of meningiomas, and
it occurs in all three histologic grades . However, 22q deletions are
observed with increased frequency in grade 2 and 3 meningiomas. As
reported in other studies [1,4,6], we observed a general increase in the
frequency of chromosomal abnormalities among a majority of clinically
aggressive meningiomas, with a notable increase in the frequency of
22q, 1p, and 14q losses. Monosomy 22q is the earliest and most fre-
quently occurring chromosomal abnormality noted in meningiomas,
and its occurrence is driven by loss of heterozygosity of the NF2 gene
on 22q in most (but not all) of these tumors [1,28]. Importantly, the in-
with deletions involving 22q. In a few cases, these alterations were inter-
overall pattern suggests a model in which the accumulation of chromo-
somal changes accompanies tumor progression, and our analysis of initial
and recurrent meningioma pairs clearly establishes that this is the case.
Several lines of evidence indicate that CHEK2 is a bona fide tumor
suppressor gene in humans. Germ line mutations in the CHEK2 gene
have been identified in a subgroup of families with Li-Fraumeni-like
syndrome [15,29] and in patients with breast, prostate, or colon cancer
[18–20]. Moreover, alternative splicing of CHEK2 in breast or other
cancers leads to the production of dominant negative Chk2 variants
that can interfere with Chk2 function . Our own data also indicate
that severalofthealternative splice variants ofCHEK2found inmenin-
gioma lack the kinase domain and do not induce phosphorylation-
dependent degradation of Cdc25a. Thus, a combination of CHEK2
deletion and alternative splicing of CHEK2 transcripts leads to de-
creased functional Chk2 expression in meningiomas.
Themechanisms by whichloss of Chk2 promotes tumor formation
are still being unraveled. After DNA damage, Chk2 stabilizes p53 to
increase apoptosis  and promotes Cdc25A degradation to inhibit
cell cycle progression . Previous studies have suggested that Chk2
suppresses the oncogenic effect of DNA damage through these
mechanisms . It seems likely, however, that defects in DNA
replication also contribute to the large-scale chromosomal instability
observed in clinically aggressive meningiomas. Previous reports have
suggested that inhibition of Chk2 activity can lead to p53-independent
apoptosis through the induction of mitotic catastrophe . Here
we provide evidence that decreased Chk2 expression impairs DNA
repair and contributes to chromosomal instability by increasing centro-
some number. The loss of functional Chk2 protein increases centro-
some number by increasing Cdc25a expression, which, in turn,
regulates centrosome amplification . In addition, loss of Chk2-
dependent phosphorylation of BRCA1 may lead to abnormal mitotic
spindle assembly, lagging chromosomes, and genomic instability .
and chromosomal instability , a common feature of clinically
Figure 6. A proposed model by which a monoallelic NF2 mutation prompts loss of the other NF2 allele through 22q deletion. Monoallelic
mutation of NF2 leads to loss of the other NF2 allele through segmental 22q deletion. This results in frequent codeletion of CHEK2,
which is located close to the NF2 gene. Concurrently, alternative splicing of CHEK2 generates nonfunctional and dominant negative
forms of Chk2. The combined effect of these changes leads to decreased Chk2 function, increased centrosome amplification, and chro-
Neoplasia Vol. 14, No. 1, 2012CHEK2 in Meningioma Progression Yang et al.
Evidence from a mouse model of meningioma development suggests Download full-text
In patients who have neurofibromatosis II, most of the meningiomas
detected are also slowly growing lesions, although aggressive tumors
can develop. We have previously reported that recurrent alterations in-
volving the CDKN2A/CDKN2B locus accompany meningioma pro-
gression from grade 2 to 3, but not from grade 1 to 2. Moreover,
experiments in a mouse model of NF2-induced meningioma suggest
that alterations to the CDKN2A/CDKN2B locus alone do not alter
meningioma grade . Presumably, CDKN2A/CDKN2B deletions
cooperate with other accumulated chromosomal abnormalities to pro-
motemeningiomaprogression.Itis importanttonote thatwe andother
22q and no other large-scale chromosomal abnormalities [4,5,34]. Unlike
meningiomas with numerous large-scale chromosomal changes, tumors
with monosomy 22q alone generally exhibit benign clinical behavior.
Itwill beimportant to examine thestatusofCHEK2 splice variants and
overall Chk2 expression levels in this subset of meningiomas.
Taken together, our findings indicate that alternative splicing
of CHEK2 transcripts and codeletion of CHEK2 with NF2 im-
pair DNA repair and promote chromosomal instability in menin-
gioma. The resultant accumulation of large-scale chromosomal
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