See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/6908024

Altered expression of -catenin/E-cadherin in

meningiomas

Article in Histopathology September 2006

DOI: 10.1111/j.1365-2559.2006.02440.x Source: PubMed

CITATIONS READS

27 94

5 authors, including:

Bernd Romeike Martin Jung

Universittsklinikum Jena Universitt des Saarlandes
141 PUBLICATIONS 1,863 CITATIONS 68 PUBLICATIONS 946 CITATIONS

SEE PROFILE SEE PROFILE

Eckart Meese
Universitt des Saarlandes
372 PUBLICATIONS 7,988 CITATIONS

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Antibody Design View project

Translational brain tumor research View project

All content following this page was uploaded by Martin Jung on 02 April 2017.

The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document
and are linked to publications on ResearchGate, letting you access and read them immediately.
Histopathology 2006, 49, 178187. DOI: 10.1111/j.1365-2559.2006.02440.x
Altered expression of b-catenin/E-cadherin in meningiomas
E C Brunner, B F M Romeike,1 M Jung,2 N Comtesse & E Meese
Institute of Human Genetics, 1Institute of Neuropathology and 2Institute of Biochemistry and Molecular Biology, Medical
School, University of Saarland, Homburg Saar, Germany
Date of submission 24 August 2005
Accepted for publication 22 September 2005
Brunner E C, Romeike B F M, Jung M, Comtesse N & Meese E
(2006) Histopathology 49, 178187
Altered expression of b-catenin/E-cadherin in meningiomas
Aims: Meningiomas are generally slow-growing benign
tumours representing approximately 20% of all pri-
mary intracranial tumours. The hallmark of tumori-
genesis of meningiomas is the loss of chromosome 22,
including loss of heterozygosity of the neurofibromato-
sis type 2 (NF2) gene. The NF2 encoded protein merlin
appears to function as a tumour suppressor gene by
controlling cadherin-mediated cellcell adhesion. The
E-cadherin cell adhesion system includes b-catenin that
indirectly connects cadherin to actin filaments. The
aim of this study was to analyse the expression and the
subcellular location of E-cadherin and b-catenin in
human meningiomas, including meningiomas of dif-
ferent histomorphological subtypes and different World
Health Organization (WHO) grades.
Methods and results: Immunohistochemical analysis
revealed lack of E-cadherin expression at the cell
Keywords: b-catenin, cellcell contact, E-cadherin, meningiomas
Abbreviations: ER GIC, endoplasmic reticulum Golgi intermediate compartment, NF2, neurofibromatosis
Introduction
Meningiomas are neoplasms of the leptomeninges
covering the brain and the spinal cord. They are
generally slow-growing benign tumours corresponding
to grade I in the World Health Organization (WHO)
classification (MI). Atypical meningiomas (WHO
grade II, MII), which are characterized by increased
cellularity and increased mitotic activity, account for
approximately 8% of this tumour type. Anaplastic
meningiomas (WHO grade III, MIII) account for
approximately 2% of this tumour type. Meningiomas
Address for correspondence: Eckart Meese PhD, Institute of Human
Genetics, Medical School, University of Saarland, Building 60, 66421
Homburg Saar, Germany. e-mail: hgemee@uniklinik-saarland.de
! 2006 The Authors. Journal compilation ! 2006 Blackwell Publishing Limited.
membrane in 34% of meningiomas independent of
their WHO grade. Loss of membranous b-catenin
occurred in 79% of meningiomas. An intense peri-
nuclear granular immunoreactivity of b-catenin with-
out nuclear location was detected in the majority of
meningiomas. Both immunofluorescence and Western
blot analysis of fractionated meningioma cells located
b-catenin mostly on the Golgi apparatus and ER Golgi
intermediate compartment (ERGIC). Cytogenetic ana-
lysis of meningiomas showed no correlation between
NF2 loss and the loss of the proper location of
b-catenin.
Conclusions: The lack of membranous b-catenin and or
membranous E-cadherin in meningiomas may indicate
an altered interaction between meningioma cells inde-
pendent of loss of NF2 and independent of the tumour
grade.
have a wide range of histological appearances. Of the
various subtypes, arachnoidal (meningothelial), fibro-
blastic and transitional meningiomas are the most
common. Meningiomas represent approximately 20%
of all primary intracranial tumours and are among
the most common tumours of the human nervous
system.1,2
Loss of chromosome 22, including loss of hetero-
zygosity (LOH) of NF2, is a consistent chromosomal
alteration in this tumour.35 Mutational inactivation of
the second allele of NF2 occurs in up to 60% of all
meningiomas. Recent studies indicate that the NF2
encoded protein merlin functions as a tumour suppres-
sor by controlling cadherin-mediated cellcell contact.6
Cadherins are transmembrane glycoproteins locali-
zed in the cell plasma membrane. The cytoplasmic

undercoat proteins for cadherins are catenins, inclu-
ding b-catenin, which interact with cadherins through
their cytoplasmic domains. There is mounting evidence
that the E-cadherin cell adhesion system plays a
significant role during multistage human tumorigen-
esis. Altered expression of b-catenin occurs in several
human cancers including gastric cancer, colon cancer
and hepatocellular and oesophageal tumours.7,8
Studies on meningiomas have also shown that
E-cadherin plays a role in the morphogenesis of this
tumour.9 Recent studies have failed to provide evidence
for E-cadherin expression in malignant meningioma
while showing E-cadherin expression in benign men-
ingiomas, regardless of their histological subtype.10
Other studies have shown expression of E-cadherin at
the cell borders in syncytial and transitional subtypes
of meningioma but have failed to show E-cadherin
expression in fibroblastic subtypes.11
Our aim in this study was to analyse the expres-
sion and the subcellular location of E-cadherin and
b-catenin in human meningiomas. We analysed 38
meningiomas of different histological subtypes and
different WHO grades by immunohistochemistry. Anti-
body reactivity was confirmed by Western blot analysis
of tissue lysates. b-Catenin and E-cadherin protein
expression was analysed by Western blot analysis of
fractionated primary cell cultures from meningiomas.
Materials and methods
t u mo u r s a m p l e s
The tumour tissues were used with consent from the
patients. The 38 patients (35 with primary meningi-
oma and three with relapse) underwent surgery in the
Institute of Neurosurgery, Medical Faculty, University
of Saarland. The 24 females and 13 males had a
median age of 58 years (range 3186 years) at the
time of surgery. Tumours were histologically classified
at the Institute of Neuropathology. Grading is given in
Table 1. Two adenocarcinomas (brain metastases of
colorectal carcinomas) from one female and one male
patient served as positive controls for the immuno-
histochemical analysis. For immunohistochemistry tis-
sue blocks from the tumours were embedded in paraffin
after fixation in buffered formalin. Tissue samples for
Western blot analysis were frozen in liquid nitrogen
immediately after surgery and stored at ) 70"C.
cell culture
The meningioma primary cell cultures were derived
from fresh tumour tissues. Cells were kept in Dulbeccos
! 2006 The Authors. Journal compilation ! 2006 Blackwell Publishing Ltd, Histopathology, 49, 178187.
b-Catenin E-cadherin in meningiomas 179
modified Eagles medium (DMEM; BioWhittaker, Walk-
ersville, MD, USA) supplemented with 10% fetal calf
serum (FCS) and 1% penicillin streptomycin. Cells
were grown in 5% CO2 atmosphere.
karyotyping
Chromosomal analysis of meningioma primary cell
cultures was performed according to standard proto-
cols.12 Karyotypes were prepared by Giemsa-trypsin
(GTG)-banding of chromosomes according to the
International System for Human Cytogenetic Nomen-
clature.13
i mm u no h i sto c h e mi s t r y
Expression of beta-catenin and E-cadherin was ana-
lysed in paraffin-embedded sections of 38 meningiomas
and two adenocarcinomas (brain metastases of colo-
rectal carcinomas) and normal non-tumorous arach-
noid tissue as controls.
The sections were deparaffinized in xylene and rehy-
drated in ethanol. Endogenous peroxidase activity was
quenched by incubation in 1.0% H2O2 for 10 min. The
sections were microwaved for 20 min at 700 W in
citrate buffer. Incubation with the primary antibody was
done for 30 min at 37"C in a humid atmosphere. The
affinity-purified monoclonal mouse anti-b-catenin anti-
body (Zymed Laboratories, South San Francisco, CA,
USA) was used at a dilution of 1 : 100 (concentration
5 lg ml). The monoclonal mouse anti-E-cadherin anti-
body (clone G-10; Santa Cruz Biotechnology, Santa
Cruz, CA, USA) was used at a dilution of 1 : 50
(concentration 4 lg ml). The specificity of the antibod-
ies was confirmed by Western blot analysis. The secon-
dary antibody was a biotin-labelled rabbit anti-mouse
IgG (Dako Cytomation, Glostrup, Denmark). The incu-
bation was carried out for 20 min at 37"C in a humid
atmosphere at a concentration of 1 : 300. The slides
were treated with streptavidin (1 : 300; Dako Cyto-
mation) for 20 min at 37"C in a humid atmosphere
and incubated with the chromogen diaminobenzidine
(1 : 50; Dako Cytomation) for about 5 min at room
temperature. The slides were counterstained with haem-
atoxylin, dehydrated with ethanol and xylene and
mounted with Entellan (Merck, Darmstadt, Germany).
Sections were examined by light microscopy and inter-
preted by two persons (E.C.B., B.F.M.R.).
w es t e r n bl ot a n a ly s i s
Expression of b-catenin and E-cadherin was analysed
by Western blotting of lysates from meningioma
180 E C Brunner et al.

Table 1. Immunohistochemical analysis of 38 meningioma tumour samples of different tumour grades using antibodies against
beta-catenin and E-cadherin
IHC-staining with
IHC staining with b-catenin antibody E-cadherin-antibody

Granular
Identification Cytoplasm clusters Cytoplasm
no of Grading without perinuclear without Cytogenetic
meningioma regarding Cytoplasmic cytoplasmic and in Cytoplasmic cytoplasmic aberrations
tumours typing membrane membrane cytoplasm membrane membrane chr. 22 Invasion

H355 MI ara. + + NA

H362 MI ara. +* + N

H372 MI ara. + + +* NA

H374 MI ara. + + + N

H375 MI ara. +* + +* N

H403 MI ara. + + +* N

H413 (relapse) MI ara. + + NA

H446 MI ara. + + + NA

H448 MI ara. + +* N

H392 MI fibro. +* ) 22

H395 MI fibro. + + NA

H398 MI fibro. +* (+) del22,(q11) Osseous invasion

or ) 22

H445 MI secr. + + +* ) 22 Dura invasion

H497 MI secr. +* + NA

H521 MI secr. +* + N

H376 MI trans. + + +* ) 22

H480 MI trans. + + N

H514 MI trans. + + del22,(q13)

H350 MII + + ) 22

H365 MII +* +* +* ) 22 Osseous invasion

H416 MII + +* NA Dura invasion

H419 MII + +* N

H421 MII + + +* ) 22 Osseous and soft

tissue invasion

H427 (relapse) MII +* + + ) 22

H429 MII + + + ) 22

H444 MII + + ) 22

! 2006 The Authors. Journal compilation ! 2006 Blackwell Publishing Ltd, Histopathology, 49, 178187.
 b-Catenin E-cadherin in meningiomas 181

Table 1. (Continued)
IHC-staining with
IHC staining with b-catenin antibody E-cadherin-antibody

Granular
Identification Cytoplasm clusters Cytoplasm
no of Grading without perinuclear without Cytogenetic
meningioma regarding Cytoplasmic cytoplasmic and in Cytoplasmic cytoplasmic aberrations
tumours typing membrane membrane cytoplasm membrane membrane chr. 22 Invasion

H449 MII + + +* NA Dura invasion

H457 MII +* + ) 22

H469 MII + + N Dura invasion

H490 MII + +* ) 22

H492 MII + + +* N

H500 MII + + + N Dura invasion

H526 MII + + ) 22

H533 MII + + + ) 22

H386 MIII + + NA Dura invasion

H386 (relapse) MIII + + +* N Brain invasion

H451 MIII + + + ) 22 Brain invasion

H588 MIII +* +* +* ) 22 Brain invasion

Cytogenetic analysis regarding chromosome 22 aberrations and analysis of the invasion pattern. +, Positive immunoreactivity;
, negative immunoreactivity; (+), faint staining; *focal stained areas of cells in tissue slides; ara., arachnoidal; fibro.,
fibroblastic; secr., secretory; trans., transitional; N, normal; NA, not analysed; del, deletion; , no invasion.

tissues. Frozen tissue samples were homogenized in
lysis buffer (125 mm TrisHCl (pH 6.8), 6% sodium do-
decyl sulphate (SDS), 10% glycerol, 10% b-mercapto-
ethanol and 10% complete protease inhibitor cocktail
(Roche, Mannheim, Germany). Samples were incuba-
ted at room temperature for 10 min and centrifuged at
1800 g for 10 min at 4"C. Supernatants were treated
with ultrasound for 10 s twice and centrifuged as
before. Supernatants were boiled for 5 min at 98"C and
centrifuged for 30 min at 20 375 g at 4"C. Super-
natants were used as samples and diluted with one
volume of loading buffer (Roti-load; ROTH, Karlsruhe,
Germany).
As a positive control for Western blotting, protein
lysates were made from HeLa cell culture. Cells were
washed twice with 1 PBS buffer, scraped and
resuspended in an appropriate volume of buffer. The
suspension was centrifuged for 5 min at 1006 g at
4"C. Pellets were resuspended in an appropriate
volume of lysis buffer (see above) and boiled for
! 2006 The Authors. Journal compilation ! 2006 Blackwell Publishing Ltd, Histopathology, 49, 178187.
5 min at 98"C. Samples were centrifuged for 30 min
at 20 375 g at 4"C. One volume loading buffer was
added to supernatants. All samples were stored at
)70"C.
Samples were denatured by boiling for 5 min at 98"C
and separated by electrophoresis in a 10% SDS-poly-
acrylamide gel. The proteins were transferred to a
polyvinylidene fluoride membrane (Hybond-P; Amer-
sham Biosciences, Freiburg, Germany). Membranes
were blocked with 5% non-fat dry milk PBS for 1 h
at room temperature. Membranes were incubated with
anti-b-catenin antibody (dilution 1 : 500, concentra-
tion 1 lg ml), respectively, with anti-E-cadherin anti-
body (dilution 1 : 100, concentration 2 lg ml) diluted
in 1% non-fat dry milk PBS for 2 h at room tempera-
ture. Blots were washed three times for 10 min with
1 PBS and treated with horseradish peroxidase-
conjugated antimouse IgG (Dianova, Hamburg, Ger-
many) in 5% non-fat dry milk PBS for 1 h at room
temperature. Blots were washed again with 1 PBS
182 E C Brunner et al.
and developed after 5 min incubation with enhanced
chemiluminescence reagent (ECL Plus; Amersham
Biosciences).
Membranes were stripped with buffer containing
0.5% SDS and 0.5% SSC, washed with 1 PBS and
blocked with 5% non-fat dry milk PBS for 1 h at
room temperature. Blots were washed three times for
10 min with 1 PBS and incubated with the
primary monoclonal antibody anti-a-tubulin (Sigma,
Munich, Germany) at a dilution of 1 : 10 000
for 1 h at room temperature. After further washing
steps membranes were incubated with the secon-
dary antibody sheep anti-mouse at a dilution of
1 : 30 000 in 5% non-fat dry milk PBS for 1 h at
room temperature. Membranes were washed again
and developed with ECL plus.
ce ll frac ti on at ion
Cells were washed twice with 1 PBS buffer, scraped
and centrifuged. The cell pellet was resuspended in lysis
buffer and homogenized. Homogenate was centrifuged
for 5 min at 2"C and 450 g. The pellet contained the
nuclei and larger cell fragments.
The postnuclear supernatant was centrifuged for
5 min at 18 500 g and 2"C. The pellet contained
mitochondria and lysosomes. The postmitochondrial
supernatant was applied on a 2.5-m sucrose bed and
centrifuged for 30 min at 950 300 g at 4"C. After
centrifugation the sucrose bed was separated into six
fractions.
The nuclei and cell membranes were separated by
a sucrose gradient with 2.52 m, 2.25 m and 2.15 m
sucrose layers. After centrifugation for 30 min at
950 300 g at 4"C, seven fractions were separated. After
sucrose gradient the samples were precipitated with
trichlorine acetic acid for 15 min at 11 200 g and 4"C
and washed with acetone. Pellets were dried and
dissolved in laemmli buffer. Western blot analysis was
performed with the following primary antibodies: anti-
E-cadherin (dilution 1 : 50), anti-b-catenin (dilution
1 : 500), anti-a-tubulin (dilution 1 : 10 000), anti-
Golgi-58K (dilution 1 : 1000; Sigma, Munich, Ger-
many), anti-680 ribosomal-(S3-protein) specific (rabbit
polyclonal, 5th blood, dilution 1 : 1000) and anti-
sec61a-endoplasmic reticulum (ER)-specific (rabbit
polyclonal, 4th blood, dilution 1 : 1000). After incu-
bation with the primary antibody, membranes were
washed with 1 PBS buffer and incubated with the
appropriate secondary antibody (sheep anti-mouse,
Dianova, 1 : 30 000; goat anti-rabbit, Dianova,
1 : 2500). Detection by ECL Plus was done as men-
tioned above.
! 2006 The Authors. Journal compilation ! 2006 Blackwell Publishing Ltd, Histopathology, 49, 178187.
i mmun o fl uore s cen ce
Expression of b-catenin was analysed by immunofluo-
rescence in methanol-fixed cells from meningioma cell
cultures and in HeLa cells. Cells were thawed from
N2-freezing, washed with 1 PBS buffer and dissem-
inated in DMEM-medium (BioWhittaker, supplemented
with 10% FCS and 1% penicillin streptomycin). Cells
were grown on glass coverslips and washed three times
with 1 PBS and fixed with 100% methanol for 5 min
at ) 20"C. Methanol was removed, coverslips were
washed again with 1 PBS and were air dried.
Coverslips were covered with 1 PBS and incubated
at 4"C. All steps in the immunofluorescence procedure
were carried out in a humid atmosphere. After PBS was
removed from the coverslips, cells were incubated with
the primary antibody for 1 h at room tempera-
ture. Primary antibodies were anti-b-catenin (dilution
1 : 100; concentration 5 lg ml) and the monoclonal
anti-Golgi-58K antibody (Sigma; dilution 1 : 50). Anti-
body was removed and coverslips were washed three
times with 1 PBS buffer. Residual buffer was removed
and coverslips were incubated with the secondary
antibody Alexa fluor 488 (1 : 400) or Alexa fluor 594
(1 : 1000; Molecular Probes, Karlsruhe, Germany) for
45 min at room temperature. Coverslips were washed
three times and the nuclei counterstained with 46-
diamidino-2-phenylindole (DAPI) at a 1 : 1000 dilution
(concentration 1 lg ml) for 5 min at room tempera-
ture. Coverslips were fixed on slides with antifade
reagent (Molecular Probes). Samples were analysed
using an epifluorescence microscope (Olympus AX70).
Results
We analysed the subcellular location of the cell
adhesion components E-cadherin and its associated
protein b-catenin in 38 meningiomas of different
tumour grades, including 18 common type (MI), 16
atypical (MII) and four anaplastic (MIII) meningiomas.
MI comprised nine meningiomas of the arachnoidal
subtype, three fibroblastic meningiomas, three trans-
itional and three secretory meningiomas (Table 1). As
positive controls we analysed two adenocarcinomas,
which were brain metastases of colorectal carcinomas.
In addition, we analysed the expression of b-catenin
and E-cadherin in non-tumorous arachnoid tissue
sections. Prior to immunohistochemical analysis we
showed that the b-catenin and E-cadherin antibodies
identified signals of the expected sizes in Western blot
experiments (Figure 1a,b).
The adenocarcinomas showed intense focal immuno-
reactivity of b-catenin at the cell membranes, in both
 b-Catenin E-cadherin in meningiomas 183

(a)
6

2
38
Figure 1. Western blot analysis of protein lysates from different

40

44

44

45

46

49
H

H
150 kDa meningiomas and controls using antibodies against E-cadherin and
E-cadherin b-catenin. a, The expression of E-cadherin (124 kDa) was demon-
124 kDa strated in meningiomas H403 (MI), H444, H449, H457, H469 and
100 kDa
H492 (MII). Meningioma H386 (MIII) showed no E-cadherin
expression. a-Tubulin was used as control. b, The expression of
b-catenin (92 kDa) was demonstrated in all of the meningiomas and
-Tubulin in HeLa cells. Lower bands are likely to represent degradation
50 kDa
55 kDa
products. a-Tubulin was used as control. c, Lack of E-cadherin
expression was found in meningiomas H374 and H392. HeLa cells
(b) showed expression of E-cadherin. a-Tubulin was used as control.
6

a
38

40

44

44

45

46

49

eL
d, Cell fractionation of protein lysates from a primary cell culture
H

derived from meningioma H392. Left panel, lane 1: whole protein

100 kDa
beta-catenin lysate; lane 2: postnuclear supernatant; lane 3: mitochondria,
92 kDa lysosomes; lane 4: postmitochondrial supernatant; lanes 511:
75 kDa nuclear and cellular membranes after fractionation by sucrose
gradient; right panel, lanes 16: postmitochondrial supernatant
fractions after sucrose bed separation. The cell fractions were
-Tubulin
analysed by antibodies against the following cell components: Golgi,
50 kDa endoplasmic reticulum (ER) and ribosome fractions. a-Tubulin was
55 kDa
used as control.

(c)
4

a
37

39

eL
H

150 kDa The typical staining along cellcell boundaries was

E-cadherin found in only eight out of 38 meningiomas (21%) and
100 kDa 124 kDa
was not homogeneous in all cells (Figure 2g). The
membranous b-catenin protein was detected in two out
of 18 MI meningiomas (11%), in five out of 16 MII
-Tubulin
50 kDa 55 kDa
meningiomas (31%) and in one out of four MIII
meningiomas (Table 2).
The remaining meningiomas showed granular cyto-
(d) lane 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6
plasmic b-catenin staining without membranous
100 kDa beta-catenin
92 kDa
accentuation but with an intense perinuclear granular
75 kDa
immunoreactivity in most of the cases (Figure 2e and
50 kDa Golgi 58K Table 2). These perinuclear clusters might represent
58 kDa accumulated protein in cytoplasmic compartments.
-Tubulin
Primary cell cultures were grown from eight menin-
50 kDa
55 kDa giomas including four MI meningiomas, four MII
meningiomas and HeLa cells as positive controls. The
17 kDa ER 61 alpha cell cultures were analysed by immunofluorescence to
37 kDa
study whether cytoplasmic accumulated b-catenin was
mostly localized on the Golgi vesicles. Staining with the
17 kDa
Ribosomes 680 b-catenin antibody and an anti-Golgi antibody showed
25 kDa 30 kDa immunoreactivity on the membranes of the Golgi
vesicles (Figure 3). These results were confirmed by
double-staining experiments with both antibodies. In
cytoplasm and nuclei (Figure 2a). Non-tumorous arach- addition, the subcellular location of b-catenin was
noid cells showed b-catenin labelling at the cell mem- analysed by Western blot analysis of fractionated
brane (Figure 2c). Membranous location of b-catenin is meningioma primary cell cultures. b-Catenin was
common in cells with intact cell adhesion15. Nuclear found in cell fractions containing the ER and the Golgi
immunoreactivity indicates cells with translocation apparatus, presumably representing the ER Golgi
of b-catenin into the nucleus15. Immunohistochemical intermediate compartment (ERGIC) (Figure 1d).
analysis of meningiomas showed b-catenin expression in b-Catenin usually binds to the inner cytoplas-
all meningioma samples (38 38). Analysing the sub- mic domain of the integral membrane glycoprotein
cellular location, we found no nuclear immunoreactivity E-cadherin15. We analysed the expression of E-cadherin
in any of the analysed meningiomas (Table 1). in adenocarcinomas, in normal arachnoid tissue and in
! 2006 The Authors. Journal compilation ! 2006 Blackwell Publishing Ltd, Histopathology, 49, 178187.
184 E C Brunner et al.

Figure 2. Immunohistochemical analysis of paraffin-embedded tissues using antibodies against b-catenin and E-cadherin. Immunoperoxidase
diaminobenzidine method with haematoxylin counterstain. a, b-Catenin expression in the nucleus, at the cell membrane and in the cytoplasm of
an adenocarcinoma (N955 02; metastasis of a colorectal carcinoma). b, E-cadherin expression at the cell membrane of the same tumour sample
as in (a). c, b-Catenin expression at the cell membrane of non-tumorous arachnoid cells. d, Lack of E-cadherin expression in non-tumorous
arachnoid cells. e, Cytoplasmic b-catenin expression with granular clusters in meningioma H374. f, Membranous expression of E-cadherin in
meningioma H374. g, Membranous and cytoplasmic expression of b-catenin in meningioma H469. h, Membranous expression of E-cadherin in
the same area of meningioma H469 as in (g).

those meningioma tissues that were also tested for at the cell membrane in 25 out of 38 meningi-
b-catenin expression. The adenocarcinomas showed omas (66%) (Figure 2f,h and Table 2). Membranous
focal membranous E-cadherin expression (Figure 2b), E-cadherin expression was not associated with a certain
whereas the non-tumorous arachnoid cells did not tumour grade or histological subtypes. Seven cases did
show specific E-cadherin staining (Figure 2d). Immuno- not show any staining and six revealed staining of
histochemical analysis revealed E-cadherin expression the cytoplasm without membranous accentuation.
! 2006 The Authors. Journal compilation ! 2006 Blackwell Publishing Ltd, Histopathology, 49, 178187.
 b-Catenin E-cadherin in meningiomas 185

a b c

Figure 3. Immunofluorescent staining of cell culture from meningioma H376 (MI) with the b-catenin antibody and an anti-Golgi antibody. The
same cells are double-stained with both antibodies but different secondary antibodies after methanol-fixation. a, Staining with b-catenin antibody
and a green fluorescent secondary antibody. b, Staining with anti-Golgi antibody and a red fluorescent secondary antibody. c, Staining with
b-catenin antibody and anti-Golgi antibody indicating colocalization.

Table 2. Immunohistochemical analysis of b-catenin and E-cadherin expression in meningiomas of different grades
b-Catenin expression E-cadherin expression

Positive at Positive in cytoplasm Positive at Positive in cytoplasm

cytoplasmic without cytoplasmic cytoplasmic without cytoplasmic
membrane membrane membrane membrane Negative

Percentage of the total number 21% 79% 66% 16% 18%

of analysed meningiomas (8 38) (30 38) (25 38) (6 38) (7 38)

Percentage of analysed MI 11% 89% 56% 16% 28%

(10 18) (3 18) (2 18) (16 18) (5 18)

Percentage of analysed MII 31% 69% 75% 12% 13%

(5 16) (11 16) (12 16) (2 16) (2 16)

Percentage of analysed MIII 25% 75% 75% 25% 0%

(1 4) (3 4) (3 4) (1 4) (0 4)

Notably, Western blot analysis did not show E-cadherin
expression in primary cell cultures derived from men-
ingiomas (Figure 1c). Likewise, immunofluorescent
staining of fixed cells from meningioma primary cell
cultures did not show specific E-cadherin immuno-
reactivity at the cell membranes but only in the
cytoplasm.
All seven cases without detectable E-cadherin expres-
sion showed no membranous b-catenin staining
including all meningiomas of the fibroblastic subtype.
Seven out of eight meningiomas that showed b-
catenin expression at the cell membrane also showed
E-cadherin at the cell membrane. In addition, there
were 17 meningiomas with membranous E-cadherin
but without b-catenin signals at the cell membrane.
Taken together, only seven of 38 (18%) meningiomas
! 2006 The Authors. Journal compilation ! 2006 Blackwell Publishing Ltd, Histopathology, 49, 178187.
showed a membranous location of both b-catenin and
E-cadherin, possibly indicating intact cellcell inter-
action. These meningiomas included two of 18 MI
meningiomas, four of 16 MII and one of four MIII
meningiomas.
Regarding invasion, brain invasion was present in
only three MIII meningiomas (Table 1), but there was
no correlation with a specific catenin cadherin stain-
ing pattern.
We performed cytogenetic analysis of primary cell
cultures in the majority of meningiomas to identify
meningiomas with loss of chromosome 22 (Table 1).
The karyotypes did not indicate a correlation between
the loss of chromosome 22 including loss of NF2 and
an altered location of b-catenin or E-cadherin in the
meningioma cells.
186 E C Brunner et al.
Discussion
E-cadherin and its associated intracellular catenins
play an important role in cellular integrity and mor-
phology. There is mounting evidence that impaired
cellcell adhesion also occurs in meningioma, which is
generally a benign human tumour. Our immunohisto-
chemical analysis revealed E-cadherin expression at the
cell membrane in 25 out of 38 meningiomas (66%).
Of the seven tumours that did not show E-cadherin
expression, there were five MI meningiomas and two
MII meningiomas. Several studies have described
reduced expression of E-cadherin in undifferentiated
cancers and elevated E-cadherin expression in more
differentiated tumours.9,10 Immunohistochemical ana-
lysis of non-tumorous arachnoid tissue showed no
specific E-cadherin staining. These data are in agree-
ment with those of Yamashima et al., who did not
identify E-cadherin at the cell membrane. However,
Yamashima et al. has shown E-cadherin in the cyto-
plasm of arachnoid villi.14
While there are conflicting results on the relation-
ship between E-cadherin expression and tumour grade
in meningiomas, our data argue against the assump-
tion that increased malignancy is associated with loss
of E-cadherin expression in meningiomas. In contrast,
our data lend support to previous studies that have
reported membranous E-cadherin to be independent of
the tumour grade or histological subtype.11
The cytoplasmic tail of E-cadherin binds b-catenin to
connect cadherin via a-catenin to actin filaments. This
complex formation is necessary to provide functional
cellcell adhesion. In our study we found membranous
b-catenin in only 21% of meningiomas with a slightly
decreased frequency in MI meningiomas compared
with MII meningiomas. We also detected intense
perinuclear granular immunoreactivity in most of the
meningiomas without nuclear location of b-catenin.
Our results indicate that loss of membranous b-catenin
is frequent in meningiomas. Immunofluorescent stud-
ies of primary meningioma cell cultures showed accu-
mulation of b-catenin in Golgi vesicles, but also in
other cytoplasmic compartments. This observation was
supported by the Western blot analysis of fractionated
cells showing b-catenin in cytoplasmic compartments,
specifically in the ER and the Golgi apparatus.
b-Catenin also plays a decisive role in the Wnt
signalling pathway, which has been implicated in the
pathogenesis of various human cancers.15 The activa-
ted pathway leads to the stabilization of b-catenin
and its translocation into the nucleus. Our analysis
of meningiomas did not show nuclear location of
b-catenin. However, the lack of membranous b-catenin
! 2006 The Authors. Journal compilation ! 2006 Blackwell Publishing Ltd, Histopathology, 49, 178187.
and E-cadherin in approximately 80% of all meningi-
omas provides evidence for an altered distribution of
these proteins in meningiomas. Meningiomas that lack
both membranous b-catenin and E-cadherin were found
in two of 18 MI meningiomas, four of 16 MII and one of
four MIII meningiomas. Notably, only one of the nine
arachnoidal MI meningiomas had membranous expres-
sion of both components. The latter case showed focal
membranous b-catenin expression with most of the cells
showing cytoplasmic staining. A normal interaction of
b-catenin and E-cadherin at the cell membranes appears
to be unlikely in these meningiomas.
Concerning brain invasion, which is not part of the
WHO grading system,2 two out of three MIII menin-
giomas lacked membranous b-catenin staining; how-
ever, there was no correlation with a specific staining
pattern.
The hallmark of tumorigenesis of meningiomas is the
loss of chromosome 22, including loss of heterozygosity
of the NF2 gene. Recent studies have shown that
merlin, encoded by NF2, acts as a tumour suppressor
by controlling cadherin-mediated cell contact.6 How-
ever, the frequency of NF2 mutations varies with the
histological subtype. While fibroblastic and transitional
meningiomas show mutations in 7080% of cases,
these mutations occur in only 25% of arachnoidal
meningiomas. This has led to the assumption that
arachnoidal meningiomas have a different genetic
origin.16 Moreover, reduced expression of the NF2
gene product is seen in only a minority of arachnoidal
meningiomas but is frequent in other histological
subtypes.17 We performed cytogenetic analysis in the
majority of meningiomas to identify loss of chromo-
some 22, including loss of one NF2 allele. Our
cytogenetic analysis of primary cell cultures from
meningiomas showed no correlation between chromo-
some 22 loss and the altered localization of b-catenin
and E-cadherin. These results make it unlikely that
loss of NF2 expression is associated with loss of the
proper localization of b-catenin and E-cadherin in
meningiomas.
Additional factors in the cell adhesion system have
also been reported to be involved in meningioma
development. The CD44 antigen is a transmembrane
glycoprotein that is involved in cell adhesion and
trafficking as well as in tumour motility and progres-
sion. CD44-mediated signal transduction leads to
activation of the GTPases Rac1 and Rho, resulting in
reorganization of the actin cytoskeleton.18 CD44 is also
a receptor for hyaluronic acid, which plays a role in the
extracellular matrix of the brain. As we have previ-
ously shown, the hyaluronidase MGEA519 causes a
humoral immune response in meningiomas, possibly

indicating an involvement of this enzyme and its
substrate in meningioma development.
Acknowledgements
The authors thank Prof. Dr W. I. Steudel, Prof.
Dr W. Feiden, Dr I. Niedermayer and colleagues for
providing tumour samples and for histological analysis,
Prof. Dr R. Zimmermann and his staff for providing the
primary antibodies anti-680 ribosomal (S3)-specific
and anti-sec61a-endoplasmic reticulum-specific and
technical support, and Mrs S. Bernhardt for technical
help. This work was supported by a grant from the
Deutsche Krebshilfe (10-1966-Me).
References
1. Burger PC, Scheithauer BW, Vogel SW. Meningioma. Surgical
pathology of the nervous system and its coverings, 4th edn. New
York: Churchill Livingstone 2002; 4971.
2. Louis DN, Scheithauer BW, Budka H, von Deimling A, Kepes JJ.
Meningiomas. In Kleihues P, Cavenee WK eds. Pathology and
genetics of tumours of the nervous system. Lyon: IARC Press 2000;
176184.
3. Meese E, Blin N, Zang KD. Loss of heterosygosity and the origin
of meningioma. Hum. Genet. 1987; 77; 349351.
4. Dumanski JP, Rouleau GA, Nordenskjold M, Collins VP. Molecu-
lar genetic analysis of chromosome 22 in 81 cases of meningi-
oma. Cancer. Res. 1990; 50; 58635867.
5. Ruttledge MH, Sarrazin J, Rangaratnam S et al. Evidence for
the complete inactivation of the NF2 gene in the majority of
sporadic meningiomas. Nat. Genet. 1994; 6; 180184.
6. Lallemand D, Curto M, Saotome I, Giovannini M, McClatchey AI.
NF2 deficiency promotes tumourigenesis and metastasis by
destabilising adherens junctions. Genes Dev. 2003; 17; 1090
1100.
! 2006 The Authors. Journal compilation ! 2006 Blackwell Publishing Ltd, Histopathology, 49, 178187.
View publication stats
b-Catenin E-cadherin in meningiomas 187
7. Maruyama K, Ochiai A, Akimoto S et al. Cytoplasmic beta-
catenin accumulation as a predictor of hematogenous metastasis
in human colorectal cancer. Oncology 2000; 59; 302309.
8. Van Aken E, De Wever O, Correia da Rocha AS, Mareel M.
Defective E-cadherin catenin complexes in human cancer.
Virchows Arch. 2001; 439; 725751.
9. Figarella-Branger D, Pellissier JF, Bouillot P et al. Expression of
neural cell-adhesion molecule isoforms and epithelial cadherin
adhesion molecules in 47 human meningiomas: correlation with
clinical and morphological data. Mod. Pathol. 1994; 7; 752761.
10. Schwechheimer K, Zhou L, Birchmeier W. E-Cadherin in human
brain tumours: loss of immunoreactivity in malignant menin-
giomas. Virchows Arch. 1998; 432; 163167.
11. Tohma Y, Yamashima T, Yamashita J. Immunohistochemical
localisation of cell adhesion molecule epithelial cadherin in human
arachnoid villi and meningiomas. Cancer Res. 1992; 52; 1981
1987.
12. Limon J, Dal Cin P, Sandberg A. Application of long-term
collagenase disaggregation for the cytogenetic analysis of human
solid tumours. Cancer Genet. Cytogenet. 1986; 23; 305313.
13. Mitelman F. ISCN Guidelines for cancer cytogenetics. Supplement
to an International System for Cytogenetic Nomenclature. Basel:
S. Karger 1995.
14. Yamashima T, Tohma Y, Yamashita J. Expression of cell adhesion
molecule E-cadherin in human arachnoid villi. J. Neurosurg.
1992; 77; 749756.
15. Lustig B, Behrens J. The Wnt signaling pathway and its role in
tumour development. J. Cancer. Res. Clin. Oncol. 2003; 129; 199221.
16. Wellenreuther R, Kraus JA, Lenartz D et al. Analysis of the
neurofibromatosis 2 gene reveals molecular variants of menin-
gioma. Am. J. Pathol. 1995; 146; 827832.
17. Lee JH, Sundaram V, Stein DJ, Kinney SE, Stacey DW, Golubic M.
Reduced expression of schwannomin merlin in human sporadic
meningiomas. Neurosurgery 1997; 40; 578587.
18. Lee JY, Spicer AP. Hyaluronan: a multifunctional, megaDalton,
stealth molecule. Curr. Opin. Cell. Biol. 2000; 12; 581586.
19. Heckel D, Comtesse N, Brass N, Blin N, Zang KD, Meese E.
Novel immunogenic antigen homologous to hyaluronidase in
meningioma. Hum. Mol. Genet. 1998; 7; 18591872.