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J. Pathol. 186: 8?16 (1998)
COMPARISON OF INTEGRIN, CADHERIN, AND
CATENIN EXPRESSION IN SQUAMOUS CELL
CARCINOMAS OF THE ORAL CAVITY
??????? ???????1, ???? ?. ???????2 ??? ????? ?. ????1*
Keratinocyte Laboratory, Imperial Cancer Research Fund, Lincoln?s Inn Fields, London WC2A 3PX, U.K.
2
Department of Oral Pathology, Eastman Dental Institute, Gray?s Inn Road, London WC1X 8LD, U.K.
1
SUMMARY
In addition to their role in maintenance of tissue integrity, cell adhesion molecules regulate the growth and differentiation of stratified
squamous epithelia. Reduced expression of E-cadherin and the ,  and  integrins is already reported to correlate with poor
histological differentiation in oral squamous cell carcinomas. However, it is not clear how closely cadherin and integrin loss are related
in any given tumour, nor whether cadherin loss is correlated with changes in expression of the cytoplasmic regulatory proteins known
as catenins. Double-label immunofluorescence has been used to stain a panel of 22 oral squamous cell carcinomas with antibodies to ten
proteins, including E- and P-cadherin, the major keratinocyte integrin subunits, and �-, �- and �-catenin. Overall, E-cadherin expression
and integrin expression correlated well with tumour grade, while P-cadherin staining was more variable. All tumours, regardless of
differentiation status, showed reduced staining for at least two of the catenins, implying that the adhesive function of E- and P-cadherin
could be impaired even when cadherin expression is normal. It is concluded that in all squamous cell carcinomas, regardless of degree
of histological differentiation, there is some perturbed expression of cell adhesion molecules and that integrin and E-cadherin loss are
closely related. 1998 John Wiley & Sons, Ltd.
KEY WORDS?cadherin;
integrin; catenin; squamous cell carcinoma; SCC; oral cancer
INTRODUCTION
Cell adhesion molecules are essential for maintaining
the structural integrity and organization of stratified
squamous epithelia. Within these tissues, receptors of
the integrin family mediate adhesion to the underlying
basement membrane, while cadherins mediate calciumdependent intercellular adhesion.1?3 The most abundant
integrins expressed by normal keratinocytes are , a
collagen receptor, and  and , which are laminin
receptors,  being a component of hemidesmosomes.4
Cadherins are subdivided into the classical and desmosomal types; the classical cadherins expressed by keratinocytes are E- and P-cadherin.2,3 The adhesive
function of cadherins is dependent on their association
with cytoplasmic regulatory proteins, the best characterized of which are �-catenin, �-catenin and �-catenin (also
known as plakoglobin).5 The integrins and P-cadherin
are predominantly expressed in the basal layer of
stratified squamous epithelia, while E-cadherin and the
catenins are expressed in all the living cell layers.1,6
Cell adhesion molecules play more than a purely
structural role within stratified squamous epithelia.
Integrin?extracellular matrix interactions regulate the
initiation of terminal differentiation1 and epidermal
stem cells express higher levels of the �integrins than
keratinocytes that have lower proliferative potential.7
Expression of a dominant-negative classical cadherin
mutant inhibits proliferation and stimulates terminal
differentiation of keratinocytes in culture,8 while
*Correspondence to: Dr Fiona M. Watt, Keratinocyte Laboratory,
Imperial Cancer Research Fund, 44 Lincoln?s Inn Fields, London
WC2A 3PX, U.K.
CCC 0022?3417/98/010008?09 $17.50
1998 John Wiley & Sons, Ltd.
expression of a mutant desmosomal cadherin in transgenic mouse epidermis has been shown to stimulate
proliferation.9
In view of the structural and regulatory functions of
integrins and cadherins in normal stratified squamous
epithelia, there has been considerable interest in whether
expression of these molecules is altered in squamous cell
carcinoma (SCC). In the case of E-cadherin, there is a
strong correlation between reduced expression and
decreased differentiation or increased invasiveness.10?12
There is also good evidence for reduced expression of
P-cadherin in SCCs.13 Down-regulation of expression of
desmosomal cadherins is also associated with invasion
and metastasis14 (and refs cited therein). Expression of
the catenins has not been studied as extensively, but
there is evidence that the absence of a particular catenin,
or failure to form cadherin/catenin complexes, results in
increased invasiveness in in vitro models.15,16
Integrin expression is also altered in SCCs but the
expression patterns are variable, both between tumours
and in different regions of the same tumour; furthermore, within a given tumour different integrins may
have different expression patterns.17?19 Three patterns of
integrin expression have been described: ?normal? (confined to the layer of neoplastic keratinocytes adjacent to
the tumour stroma); overexpression (throughout the
tumour, not confined to a single cell layer); and loss of
expression (usually focal, but occasionally extensive).18
The clinical significance of these changes is not entirely
clear. There is compelling evidence that overexpression
of  is correlated with poor prognosis in tumours of
the oral cavity;17 however, in a smaller study of the same
type of tumour, focal loss of expression of  or the �Received 25 November 1997
Revised 3 February 1998
Accepted 26 May 1998
9
CELL ADHESION MOLECULES IN SCC
Table I?Details of the SCC cases used in this study
Case No.
PD1
PD2
PD6
PD11
PD16
PD20
MD3
MD4
MD5
MD7
MD9
MD10
MD13
MD14
MD15
MD17
MD18
MD22
WD8
WD12
WD19
WD21
Age
(years)
Gender
Site
Grade
TNM
Stage
56
82
55
53
37
82
61
80
71
79
58
82
53
54
74
57
44
54
42
59
45
47
M
F
M
M
F
F
M
F
M
M
M
F
F
M
M
M
F
F
F
M
F
M
Oropharynx
Max alveolus
Tongue
Mand alveolus
Mand alveolus
Tongue
Floor of mouth
Mand alveolus
Max alveolus
Tongue
Mand alveolus
Mand alveolus
Pillar of fauces
Tongue
Max alveolus
Mand alveolus
Mand alveolus
Tongue
Gum
Mand alveolus
Mand alveolus
Tongue
PD
PD
PD
PD
PD
PD
MD
MD
MD
MD
MD
MD
MD
MD
MD
MD
MD
MD
WD
WD
WD
WD
T4N0M0
T2N0M0
T2N1M0
T3N1M0
T4N2M0
T1N0M0
T3N0M0
T2N0M0
T2N0M0
T3N0M0
T2N0M0
T4N0M0
T2N0M0
T1N0M0
T1N0M0
T4N1M0
T2N0M0
T3N0M0
T1N0M0
T3N0M0
T2N0M0
T2N0M0
IV
II
III
III
IV
I
III
II
II
III
II
IV
II
I
I
IV
II
III
I
III
II
II
PD: poorly differentiated; MD: moderately differentiated; WD: well differentiated; Max: maxilla; Mand: mandible.
integrins was a feature of the most poorly differentiated
tumours.18,19 In experiments in which cell lines derived
from SCCs are ?repaired? by transfection of the integrin
subunits that they lack, there is one report of suppression of abnormal growth and stimulation of differentiation,20 but another in which there was no effect.21 To
complicate the situation further, there is increasing
evidence for cross-talk between molecules mediating
cell?cell and cell?extracellular matrix adhesion: in cultured keratinocytes, inhibition of cadherin-mediated
adhesion results in decreased integrin expression
by proliferating cells and de novo expression by
differentiating cells.8,22
Given these observations, the purpose of this study was
to investigate the distribution of integrins, cadherins, and
catenins within a single cohort of SCCs in order to compare changes in expression of the different proteins.
MATERIALS AND METHODS
Materials
Details of the tumours are provided in Table I.
Twenty-two oral SCCs were examined. There were 12
males and 10 females with an average age of 60�years
(range 37?82 years). Three tumours were recurrences
(MD14, MD18, WD12), but the remaining 19 were
primary lesions. In this retrospective study, limited
clinical details were available and it was not possible to
determine smoking or alcohol histories for all patients.
Only two cases (MD7 and MD9), however, were positively identified as being non-smokers. All samples were
from resection specimens and were representative of the
1998 John Wiley & Sons, Ltd.
body of the lesions, including the deep or invasive
margins, which were the areas used for grading and
immunohistochemical analysis.
Samples were embedded in OCT, snap-frozen in
liquid nitrogen, and stored at 70C until use. Haematoxylin and eosin-stained sections adjacent to those used
for immunohistochemistry were evaluated independently by two pathologists and were graded as well,
moderately, or poorly differentiated, according to the
method first described by Broders.23 Each pathologist
agreed independently on 20 cases and the remaining two
were agreed by consensus.
Tumours were staged according to the pathological
?tumour, node, metastasis? status (TNM) as recommended by the IUCC and WHO.23 The primary tumour
(T) was graded 1?4 according to size and infiltration, T1
being a tumour of less than 2 cm. N1 corresponds to a
single affected lymph node, while N2 indicates one large
node or bilateral nodes. M0 indicates no metastases. The
stage was assigned on the basis of the TNM status, stage
I being a simple tumour of less than 2 cm with no
metastases and stage IV being the most severe disease.
As a positive control and to confirm the specificity of
each antibody, frozen sections of normal buccal mucosa
were prepared and included in each staining run. The
distribution of integrins and cadherins in normal mucosa
is known and has been reported previously.18,19,24
Antibodies
Table II lists all the primary antibodies used.
Several of the antibodies were directly conjugated
to FITC; FITC-CD29 was purchased from Janssen
J. Pathol. 186: 8?16 (1998)
10
C. BAGUTTI ET AL.
Table II?Primary antibodies
Specificity
Antibody
Species
E-cadherin
P-cadherin
P-cadherin
������-catenin
�-catenin
�-catenin
HECD-1
NCC-CAD299
6A9
CD29
3E1
HAS6
DH4
GoH3
VB1
VB2
VB3
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Rabbit
Rat
Rabbit
Rabbit
Rabbit
Table III?Examples of Fisher?s exact test: (a) Correlation
between grade of differentiation and E-cadherin expression; (b)
correlation between E-cadherin expression and � � �integrin expression. The numbers in each column represent the
number of tumours in each category. Scores are given in
parentheses. For explanation of P values, see Results.
E-cadherin expression
(a) Differentiation
PD
MD/WD
Substantial loss
(0, 1)
Patchy/normal
(2, 3)
3
1
3
15
P<0�.
Sources of antibodies are described in refs 6, 7, 21, 26 and 27.
Pharmaceuticals and HAS6, GoH3, HECD-1 and
6A9 were conjugated according to the method of
Rinderknecht,25 using celite-bound FITC (Sigma
Chem.). The secondary antibodies were FITCconjugated rabbit anti-mouse, donkey anti-rabbit,
goat anti-rat IgG (all from Sigma Chem.); Texas
Red-conjugated donkey anti-mouse, donkey antirabbit, goat anti-rat IgG (all from Jackson Lab.); and
biotin-conjugated anti-mouse antibody (Sigma Chem.).
Staining procedures
Serial 6 靘 frozen sections were prepared on a cryostat (Slee), air-dried at room temperature, fixed in 100%
acetone for 10 min, and then washed in PBS containing
1 m? calcium chloride and 1 m? magnesium chloride
(PBSABC).
For immunoperoxidase labelling of E- and
P-cadherin, the sections were incubated with the
primary antibody for 1 h, with a biotin-conjugated antimouse antibody for 30 min, and with an avidin?biotin?
horseradish peroxidase complex (Dakopatts) for 30 min.
The substrate 3,3-diaminobenzidene tetrahydrochloride
in 1 per cent hydrogen peroxide was then applied for
approximately 10 min. After each step, the sections were
washed in PBSABC for 10 min. After the last wash, the
slides were rinsed in water for 2 min, counterstained with
Mayer?s haematoxylin, and mounted in colophonium
resin. The staining intensity for E- or P-cadherin was
evaluated and scored independently by two observers.
For double-label immunofluorescent staining, sections
were incubated sequentially with each primary antibody
for 1 h and then with a mixture of both secondary
antibodies for 45 min. Slides were washed in PBSABC
after each incubation. Double-labelling of E- or
P-cadherin (both mouse monoclonal antibodies) and the
�integrin subunit or �-, �- or �-catenin (all rabbit
antibodies) was detected with FITC-labelled anti-mouse
and Texas Red-labelled anti-rabbit secondary antibodies. In experiments in which E- or P-cadherin labelling was compared with the � � and �integrin
subunits, Texas Red-labelled anti-mouse secondary antibodies and FITC-conjugated HAS6 and GoH3 or CD29
were used. For double-labelling of the �integrin sub 1998 John Wiley & Sons, Ltd.
� � �integrin expression
(b) E-cadherin expression
Reduced (0, 1, 2)
Normal (3)
Substantial loss
(0, 1)
Patchy/normal
(2, 3)
19
2
2
16
P<0�1.
unit and E- or P-cadherin, HECD-1 and 6A9 were
conjugated to FITC and �was localized with a Texas
Red-labelled anti-mouse antibody.
As a negative control, primary antibodies were omitted and the sections were incubated with the secondary
antibodies alone. All fluorescently labelled specimens
were embedded in Gelvatol and examined and photographed using a Zeiss Axiophot microscope (Carl Zeiss
Ltd.).
Immunohistochemical staining was evaluated and
scored by two individuals, one of whom did not know
the pathological grading beforehand. The pattern and
intensity of staining were scored on a four-point semiquantitative scale: 3=strong staining in a normal distribution; 2=patchy or heterogeneous staining; 1=weak
and fragmented staining; 0=loss of staining. In addition,
a score of 4 was used to indicate increased suprabasal
expression where this was not normally seen. In those
lesions in which the expression pattern of any of the
proteins was very variable from region to region, we
gave a double score.
Statistical analysis
Correlations between expression of two adhesion molecules and between expression of E- or P-cadherin and
differentiation status were tested using Fisher?s exact test
(illustrated on two examples in Table III). For analysis,
scores of 0 and 1, and of 2 and 3 were grouped together.
Well and moderately differentiated tumours were also
grouped for comparison with poorly differentiated
lesions. In those tumours with double scores, the lower
score was included in the statistical analysis.
J. Pathol. 186: 8?16 (1998)
11
CELL ADHESION MOLECULES IN SCC
RESULTS
Evaluation of E- and P-cadherin labelling
The variation in the pattern of staining and the
corresponding scores are illustrated in Fig. 1. As
observed in epidermis,6 E-cadherin was expressed in all
the living layers of normal epithelium (Fig. 1a). This
normal pattern of staining was observed in some of the
moderately or well-differentiated tumours and given a
score of 3 (Table IV). In other tumours, E-cadherin
staining was patchy and very heterogeneous (score 2): in
some areas staining was normal, while in other areas it
was fragmented (Fig. 1b). We also observed tumours in
which the staining was completely fragmented (score 1,
Fig. 1c) and one in which E-cadherin was virtually
absent (score 0, Fig. 1d). In some of the tumours, there
were regions where the cytoplasm was labelled with no
obvious membrane staining (e.g., Fig. 2c and Table IV).
There was a correlation between E-cadherin staining
and grade of differentiation of the tumour with the
poorly differentiated tumours showing the most pronounced reduction in E-cadherin (P<0�), (see Tables
III and IV).
In normal epithelium, P-cadherin staining was most
intense in the basal layer, as observed in the epidermis,6
with weak suprabasal staining (data not shown). As with
E-cadherin, some tumours showed normal P-cadherin
staining (score 3), while others had patchy (score 2) or
fragmented (score 1) staining (Table V). In the tumour
that lacked E-cadherin (PD1), P-cadherin was also
undetectable (score 0). There were tumours in which
strong P-cadherin staining extended above the layer of
keratinocytes in contact with the stroma (suprabasal
staining, score 4). Staining for P-cadherin was more
heterogeneous than for E-cadherin and this is indicated
by a double score in ten cases; for example, tumour
MD10 had regions of patchy P-cadherin staining and
regions of suprabasal expression (score 2/4). Cytoplasmic staining was observed in six tumours. In contrast to
E-cadherin, P-cadherin expression did not correlate with
grade of differentiation (P>0�).
Double-labelling for cadherins and integrins
Sections of the tumours were double-labelled with
anti-E-cadherin or anti-P-cadherin and antibodies to
different integrins (Tables IV and V and Fig. 2) in order
to analyse whether the reduced levels of each cadherin
observed coincided with altered expression of particular
integrins. Reduction in cadherin expression might be
expected to affect the expression of integrins, since we
have previously reported that abolition of cadherinmediated adhesion in cultured keratinocytes resulted in
a decrease in integrin expression in the proliferating
cells.8,22 In those tumours exhibiting heterogeneous
staining of either E-cadherin (e.g., tumour with patchy
staining, score 2) or P-cadherin (e.g., tumour with score
2 or tumour with partially suprabasal expression, score
1/4 or 2/4), areas staining most weakly for the cadherin
were examined for expression of each integrin. Integrin
staining was evaluated using the same scoring system as
that described for the cadherins.
1998 John Wiley & Sons, Ltd.
In those regions with reduced E- or P-cadherin staining (score 0, 1 or regions of fragmented staining in
tumours with a score of 2), expression of the �integrins
(total,  or ) was down-regulated, i.e. either lost
(score 0) or greatly reduced (score 1; classified as focal
loss in our previous study18). This was highly significant
for E-cadherin (P<0�1, Table III), but not for
P-cadherin. �integrin expression was normal or suprabasal in most of the tumours which had an E-cadherin
score of 3 (P<0�1; see Figs 2a and 2b). In one case
(MD3), showing low levels of E-cadherin and the �and
�integrin subunits, there was variable expression of �
with areas of suprabasal, cytoplasmic accumulation.
In the regions of tumours with reduced E-cadherin
staining, there was sometimes loss of �integrin expression (Table IV; see also Figs 2c and 2d) which, however,
was not statistically significant. Within the same region,
loss of �at the basement membrane was found associated with suprabasal expression of �in three cases
(MD3, MD5, MD9). Even though �only heterodimerizes with � �and �were not always co-expressed and
suprabasal staining for �was frequently seen in the
absence of �(e.g., MD3, MD5). This was previously
observed in oral SCCs and may indicate the formation
of .19,28 There was no correlation between reduced
P-cadherin staining and reduced levels of �and �(Table V).
Double-labelling for cadherins and catenins
Sections of the tumours were double-labelled with
anti-E-cadherin or anti-P-cadherin and antibodies to �-,
�- or �-catenin (Figs 2e?j, 3, and Tables IV and V).
When regions of the tumour with reduced cadherin
expression were analysed, all tumours but one (MD7
when double-labelled with P-cadherin) had areas of
reduced expression of at least two of the three catenins,
but there was no correlation between the levels of
staining of individual catenins: for example, in MD10,
E-cadherin expression was normal while expression of
�-, �- or �-catenin was greatly reduced, whereas in PD6,
showing the same level of E-cadherin, the expression of
the catenins was only slightly altered (see Table IV).
Occasional cytoplasmic staining was observed for each
catenin (see Tables IV and V). Although �- and
�-catenin are known to localize to the nucleus under
certain conditions,29,30 we did not detect any specific
accumulation of �- and �-catenin in the nucleus, possibly as a result of the specimen preparation methods
used.
DISCUSSION
We have stained a panel of oral SCCs with ten
different antibodies, using double-label immunofluorescence to compare the expression of E- and P-cadherin
with five different integrin subunits and with �-, �-, and
�-catenin. Although there have been other studies of
cadherin and integrin expression in oral SCCs, we are
not aware of any previous attempts to compare the two
classes of adhesion molecules within the same tumour.10
J. Pathol. 186: 8?16 (1998)
12
C. BAGUTTI ET AL.
Fig. 1?E-cadherin expression in normal oral epithelium (a) and oral SCCs (b?d). Different levels of E-cadherin are indicated by scores of (a)
3, normal cell?cell border localization; (b) 2, patchy: areas of fragmented expression (arrow-head) and areas of normal expression (arrow);
(c) 1, fragmented; (d) 0, absent. (a)410; (b)160; (c, d)260?all magnifications reduced to 95% in printing
1998 John Wiley & Sons, Ltd.
J. Pathol. 186: 8?16 (1998)
13
CELL ADHESION MOLECULES IN SCC
Table IV?Comparison of E-cadherin with the levels of integrins and catenins. In tumours with heterogeneous E-cadherin
expression, expression of integrins and catenins was scored in the regions with the lowest level of E-cadherin
Integrin subunits
Tumour
PD1
PD2
PD6
PD11
PD16
PD20
MD3
MD4
MD5
MD7
MD9
MD10
MD13
MD14
MD15
MD17
MD18
MD22
WD8
WD12
WD19
WD21
Catenins
E-cadherin
�
�
�
�
�
�-
�-
�-
0
2c
3
2c
0/3c
0/1
2
1c
2c
3
3
3
3c
0/3
0c/3
3c
0/3
3
2c
2
2
3c
0
0/4
2
1
0/4
0
0
0
0
3/4
3/4
3/4
3
4c
0/3
3/4
0
3
3/4
0/4
2
3
0
1
3
n.d.
n.d.
n.d.
0
0
0
0/1
3
3
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
3
n.d.
n.d.
n.d.
0
1
3
n.d.
n.d.
n.d.
0/4c
0
0
4
3
4
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
3
n.d.
n.d.
n.d.
0
3
3
n.d.
n.d.
n.d.
2
0
0
3
3
4
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0/1
n.d.
n.d.
n.d.
0
3/4
2/4
2/4
3
1
0/4
0
0/4c
2/4
0/4
2/4
2
2/4
3
4c
3
2
2/4
3/4
3
3
0
2
2
2c
1
0/3
2
2/4
2/4
2c
2c
1
1c
1
1
2
1
2
2c
0c
2
2c
0
2
2
1
1
0/1
1
0
1c
2
2
2
0c
0/1
0c
2
0c
1
3
0/2
2
0c
0
0
3
3
1
0/3
1
1
2c
3/4
2
0/1
0/1
2
2
2
2
2
1c
2
2
3c
Tumours are identified by grade (PD, MD, WD) and a reference number. For scoring system see text.
c=cytoplasmic staining; n.d.=not determined.
In our panel, E-cadherin expression was correlated
with the differentiation status of the tumour, the most
poorly differentiated tumours having least expression.
This is in agreement with earlier studies10?12,31 and also
with an analysis of experimentally induced mouse skin
tumours carried out by Cano and colleagues32 (see also
reviews in refs 11 and 16). The correlation between
P-cadherin loss and tumour differentiation was not
significant and this is in agreement with the observation
that in mouse skin tumours, P-cadherin expression does
not correlate with degree of differentiation.32 Potential
reasons for reduced E-cadherin expression include suppression of the gene promoter, destabilization of the
protein through lack of catenin binding, or mutations
within the E-cadherin gene.5,11,16
As we previously reported,18 integrin loss was a
feature of the poorly differentiated tumours, although in
one of our panel (PD2) expression of the �and �subunits was normal or increased in some regions.
Tumour PD1 was remarkable in lacking all the adhesion
molecules that we examined. A larger group of tumours
must now be studied to see whether PD tumours can be
subdivided into those with loss of E-cadherin and
integrins versus those with normal or suprabasal integrin
expression and loss of E-cadherin; if so, it will be
interesting to determine whether there is any difference
in prognosis.
In view of the evidence for cadherin/integrin crosstalk discussed in the Introduction, we might have
expected cadherin loss to correlate with increased or
suprabasal integrin expression, but this was not found.
1998 John Wiley & Sons, Ltd.
Suprabasal integrin expression did occur in some
tumours, as did suprabasal expression of P-cadherin; in
three of the six tumours with suprabasal P-cadherin
there was also suprabasal integrin expression and
there were cases of reduced P-cadherin labelling and
suprabasal �expression (e.g. MD9).
Reduced catenin staining was found in all tumours,
but there was no evidence for co-ordinated regulation of
expression of �-, �-, and �-catenin (see also ref. 10). The
consequence of reduced catenin expression is likely to be
impaired cadherin-mediated adhesion: �- and �-catenin
bind to the cadherin cytoplasmic domain and also
to �-catenin, which in turn links the entire adhesive
complex to the actin cytoskeleton.33 The functional
importance of �-catenin has been demonstrated by
transfection of tumour cells that lack �-catenin.34 Further aspects to be considered are modification of
�-catenin function by phosphorylation35 or by complex
formation with the adenomatous polyposis coli tumour
suppressor protein APC.36 �-Catenin has recently been
found to have signalling functions independent of cadherin binding and to form a complex with the transcription factor LEF-1 in the nucleus.30,37 �-Catenin binds to
desmosomal cadherins in addition to binding the classical cadherins, although with different stoichiometry,38
and thus alterations in the levels of desmosomal
cadherins may affect �-catenin association with E- and
P-cadherin.
In conclusion, reduced expression of cadherins and
integrins was a feature of poorly differentiated tumours,
while reduced catenin expression was seen in all
J. Pathol. 186: 8?16 (1998)
14
C. BAGUTTI ET AL.
Fig. 2?Double-label immunostaining of E-cadherin with integrins and catenins. (a, b) MD10: (a) E-cadherin,
score 3; (b) �integrin, score 3/4. (c, d) PD2: (c) E-cadherin, score 2c; (d) �integrin, score 3/4. (e, f) WD8: (e)
E-cadherin, score 2c; (f) �-catenin, score 2c. (g, h) MD5: (g) E-cadherin, score 2c; (h) �-catenin, score 1c. (i, j) WD8:
(i) E-cadherin, score 2c, (j) �-catenin, score 1c. (a, b, e, ,f, i, j)100; (c, d)41; (g, h)50?all magnifications
reduced to 77% in printing
1998 John Wiley & Sons, Ltd.
J. Pathol. 186: 8?16 (1998)
15
CELL ADHESION MOLECULES IN SCC
Table V?Comparison of P-cadherin with the levels of integrins and catenins. In tumours with heterogeneous P-cadherin expression,
expression of integrins and catenins was scored in the regions with the lowest level of P-cadherin
Integrin subunits
Tumour
PD1
PD2
PD6
PD11
PD16
PD20
MD3
MD4
MD5
MD7
MD9
MD10
MD13
MD14
MD15
MD17
MD18
MD22
WD8
WD12
WD19
WD21
Catenins
P-cadherin
�
�
�
�
�
�-
�-
�-
0
2/4
1
2
1/3
2c
1/4c
1
2/4
3/4
2
2/4
3c
1/3
0/3
3c
3c
2c
2/4
3
2
0/2
0
4
2
1
1
2
1
0
0
3
3
3
3
4c
2
3/4
0/3
2
3
0/3
2
3
0
1
3
n.d.
n.d.
n.d.
0
0
0
0/1
3
3
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
3
n.d.
n.d.
n.d.
0
3
3
n.d.
n.d.
n.d.
3
0
2
3/4
3
2/4
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
2
n.d.
n.d.
n.d.
0
1
1
n.d.
n.d.
n.d.
1
0
2
2
3
3
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
3
n.d.
n.d.
n.d.
0
3
2
3
3
0/1
0
2
1
3
3/4
2/4
3c
1/4
3
3/4
3c
2
2
3/4
3
2
0
1
1
2
1
0/1
1c
0
2
3
1
2
1c
1
0/1
1/3
1
1
2
2
2
2
0
2
0
0c
1c
0c
0
1
0
3
1
1
0
0c
0c
2c
0
1
0/1c
1
3
0
0
1
0
1c
0/1
0
0c
1
1c
2
2
1
0/1
1
1
2
0c
0/1
0
3
0c
1
Tumours are identified by grade (PD, MD, WD) and a reference number. For scoring system see text.
c=cytoplasmic staining; n.d.=not determined.
Fig. 3?Double-label immunostaining of P-cadherin and catenins in MD5. (a) P-cadherin, score 2/4; (b) �-catenin, score 2; (c) �-catenin, score
0; (d) �-catenin, score 1, partially cytoplasmic. (a, b, d)100; (c)50?all magnifications reduced to 95% in printing
1998 John Wiley & Sons, Ltd.
J. Pathol. 186: 8?16 (1998)
16
C. BAGUTTI ET AL.
tumours, regardless of differentiation status. It will be
important now to examine the consequences of reduced
catenin expression for keratinocyte adhesion, proliferation and differentiation, using available in vitro models.
ACKNOWLEDGEMENTS
We are grateful to everyone who provided advice and
reagents. CB was funded by fellowships from the Swiss
National Science Foundation, the HuggenbergerBischoff Foundation for Cancer Research, the CibaGeigy-Jubilee Foundation, the Eastman Dental
Institute, and ICRF. We thank Dr W. Barrett for
helping with the histological grading of the tumours.
REFERENCES
1. Watt FM, Hertle MD. Keratinocyte integrins. In: Leigh IM, Lane EB, Watt
FM, eds. The Keratinocyte Handbook. Cambridge: Cambridge University
Press, 1994; 153?164.
2. Takeichi M. Morphogenetic roles of classic cadherins. Curr Opin Cell Biol
1995; 7: 619?627.
3. Buxton RS, Magee AI. Structure and interactions of desmosomal and other
cadherins. Semin Cell Biol 1992; 3: 157?167.
4. Borradori L, Sonnenberg A. Hemidesmosomes: roles in adhesion, signalling
and human diseases. Curr Opin Cell Biol 1996; 8: 647?656.
5. H黮sken J, Behrens J, Birchmeier W. Tumor-suppressor gene products in
cell contacts: the cadherin?APC?armadillo connection. Curr Opin Cell Biol
1994; 6: 711?716.
6. Mol鑣 J-P, Watt FM. The epidermal stem cell compartment: variation in
expression levels of E-cadherin and catenins within the basal layer of human
epidermis. J Histochem Cytochem 1997; 45: 867?874.
7. Jones PH, Harper S, Watt FM. Stem cell patterning and fate in human
epidermis. Cell 1995; 80: 83?93.
8. Zhu AJ, Watt FM. Expression of a dominant negative cadherin mutant
inhibits proliferation and stimulates terminal differentiation of human
epidermal keratinocytes. J Cell Sci 1996; 109: 3013?3023 .
9. Allen E, Yu Q-C, Fuchs E. Mice expressing a mutant desmosomal cadherin
exhibit abnormalities in desmosomes, proliferation, and epidermal differentiation. J Cell Biol 1996; 133: 1367?1382.
10. Andrews NA, Jones AS, Helliwell TR, Kinsella AR. Expression of the
E-cadherin?catenin cell adhesion complex in primary squamous cell carcinomas of the head and neck and their nodal metastases. Br J Cancer 1997;
75: 1474?1480.
11. Birchmeier W, Behrens J. Cadherin expression in carcinomas: role in the
formation of cell junctions and the prevention of invasiveness. Biochim
Biophys Acta 1994; 1198: 11?26.
12. Mattijssen V, Peters H, Schalkwijk L. E-cadherin expression in head and
neck squamous cell carcinomas is associated with clinical outcome. Int J
Cancer 1993; 55: 580?585.
13. Sakaki T, Wato M, Kaji R, Mushimoto K, Shirasu R, Tanaka A.
Correlation of E- and P-cadherin expression with differentiation grade and
mode of invasion in gingival carcinoma. Pathol Int 1994; 44: 280?286.
14. Hiraki A, Shinohara M, Ikebe T, Nakamura S, Kurahara S, Garrod DR.
Immunohistochemical staining of desmosomal components in oral squamous cell carcinomas and its association with tumour behaviour. Br J
Cancer 1996; 73: 1491?1497.
15. Oyama T, Kanai Y, Ochiai A, et al. A truncated �-catenin disrupts the
interaction between E-cadherin and �-catenin: a cause of loss of intercellular
adhesiveness in human cancer cell lines. Cancer Res 1994; 54: 6282?6287.
16. Raftopoulos IS, Kouraklis G. Dysfunction of the E-cadherin/catenin cell
adhesion cascade in epithelial cancers. Oncol Rep 1996; 3: 793?803.
1998 John Wiley & Sons, Ltd.
17. Carey TE, Laurikainen L, Nair TS, et al. Regulation of expression and
phosphorylation of A9/alpha 6 beta 4 integrin in normal and neoplastic
keratinocytes. J Natl Cancer Inst Monogr 1992; 13: 75?86.
18. Jones J, Sugiyama M, Watt FM, Speight PM. Integrin expression in normal,
hyperplastic, dysplastic, and malignant oral epithelium. J Pathol 1993; 169:
235?243.
19. Downer CS, Watt FM, Speight PM. Loss of �and �integrin subunits
coincides with loss of basement membrane components in oral squamous
cell carcinomas. J Pathol 1993; 171: 183?190.
20. Jones J, Sugiyama M, Speight PM, Watt FM. Restoration of 醰�integrin
expression in neoplastic keratinocytes results in increased capacity for
terminal differentiation and suppression of anchorage-independent growth.
Oncogene 1996; 12: 119?126.
21. Jones J, Sugiyama M, Giancotti F, Speight PM, Watt FM. Transfection of
�integrin subunit into a neoplastic keratinocyte line fails to restore
terminal differentiation capacity or influence proliferation. Cell Adh Commun 1996; 4: 307?316.
22. Hodivala KJ, Watt FM. Evidence that cadherins play a role in the
downregulation of integrin expression that occurs during keratinocyte
terminal differentiation. J Cell Biol 1994; 124: 589?600.
23. Pindborg JJ, Reichart PA, Smith CJ, van der Waal I. Histological Typing of
Cancer and Precancer of the Oral Mucosa. 2nd edn. World Health
Organisation Histological Classification of Tumours. Berlin: SpringerVerlag, 1997.
24. Downer CS, Speight PM. E-cadherin expression in normal, hyperplastic and
malignant oral epithelium. Oral Oncol Eur J Cancer 1993; 29B: 303?305.
25. Rinderknecht H. Ultra-rapid fluorescent labelling of proteins. Nature 1962;
193: 167?168.
26. Bishop LA, Rahman D, Pappin DJC, Watt FM. Identification of an 80 kD
protein associated with  integrin as a proteolytic fragment of the �subunit: studies with human keratinocytes. Cell Adh Commun 1995; 3:
243?255.
27. Lewis JE, Jensen PJ, Wheelock MJ. Cadherin function is required for
human keratinocytes to assemble desmosomes and stratify in response to
calcium. J Invest Dermatol 1994; 102: 870?877.
28. Zhang K, Kim JP, Woodley DT, Waleh NS, Chen Y-Q, Kramer RH.
Restricted expression and function of laminin 1-binding integrins in normal
and malignant oral mucosal keratinocytes. Cell Adh Commun 1996; 4:
159?174.
29. Simcha I, Geiger B, Yehuda-Levenberg S, Salomon D, Ben-Ze?ev A.
Suppression of tumorigenicity by plakoglobin: an augmenting effect of
N-cadherin. J Cell Biol 1996; 133: 199?209.
30. Behrens J, von-Kries JP, Kuhl M, et al. Functional interaction of �-catenin
with the transcription factor LEF-1. Nature 1996; 382: 638?642.
31. Schipper JH, Frixen UH, Behrens J, Unger A, Birchmeier W. E-cadherin
expression in squamous cell carcinomas of head and neck: inverse correlation with tumor dedifferentiation and lymph node metastasis. Cancer Res
1991; 51: 6328?6337.
32. Navarro P, Gomez M, Pizarro A, Gamallo C, Quintanilla M, Cano A. A
role for the E-cadherin cell?cell adhesion molecule during tumor progression of mouse epidermal carcinogenesis. J Cell Biol 1991; 115: 517?533.
33. Cowin P, Burke B. Cytoskeleton?membrane interactions. Curr Opin Cell
Biol 1996; 8: 56?65.
34. Hirano S, Kimoto N, Shimoyama Y, Hirohashi S, Takeichi M. Identification of a neural �-catenin as a key regulator of cadherin function and
multicellular organization. Cell 1992; 70: 293?301.
35. Behrens J, Vakaet L, Friis R, et al. Loss of epithelial differentiation and gain
of invasiveness correlate with tyrosine phosphorylation of the E-cadherin/
�-catenin complex in cells transformed with a temperature-sensitive v-SRC
gene. J Cell Biol 1993; 120: 757?766.
36. Miller JR, Moon RT. Signal transduction through �-catenin and specification of cell fate during embryogenesis. Genes Dev 1996; 10: 2527?2539.
37. Fagotto F, Funayama N, Gluck U, Gumbiner BM. Binding to cadherins
antagonizes the signalling activity of beta-catenin during axis formation in
Xenopus. J Cell Biol 1996; 132: 1105?1114.
38. Kowalczyk AP, Borgwardt JE, Green KJ. Analysis of desmosomal
cadherin-adhesive function and stoichiometry of desmosomal cadherin?
plakoglobin complexes. J Invest Dermatol 1996; 107: 293?300.
J. Pathol. 186: 8?16 (1998)
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