The Journal of Cell Biology
The Journal of Cell Biology, Volume 161, Number 6, June 23, 2003 1191–1203
The Rockefeller University Press, 0021-9525/2003/06/1191/13 $8.00
Adhesion-independent mechanism for suppression of
tumor cell invasion by E-cadherin
Alice S.T. Wong and Barry M. Gumbiner
Department of Cell Biology, University of Virginia, Charlottesville, VA 22908
oss of E-cadherin expression or function in tumors
leads to a more invasive phenotype. In this study, we
investigated whether the invasion suppressor activity
of E-cadherin is mediated directly by tighter physical cell
adhesion, indirectly by sequestering
-catenin/T cell factor (TCF) signaling, or by
other signaling pathways. To distinguish mechanisms, we
expressed wild-type E-cadherin and various E-cadherin
mutants in invasive E-cadherin–negative human breast
(MDA-MB-231) and prostate (TSU-Pr1) epithelial carcinoma
cell lines using a tetracycline-inducible system. Our data
-catenin and thus
confirm that E-cadherin inhibits human mammary and
prostate tumor cell invasion. We find that adhesion is neither
necessary nor sufficient for suppressing cancer invasion.
Rather, the invasion suppressor signal is mediated through
-catenin–binding domain of the E-cadherin cytoplasmic
tail but not through the p120
depletion also results in invasion suppression. However,
alteration in the
target genes is not required for the invasion suppressor
activity of E-cadherin, suggesting the involvement of other
transcriptional regulation of
A disturbance in epithelial cell adhesion, which leads to a
more invasive and metastatic phenotype, is a hallmark of tumor
progression. E-cadherin, which has a widely acknowledged
role in cell–cell adhesion, also functions as an invasion/tumor
suppressor protein. Several immunohistochemical studies
have reported a strong correlation between E-cadherin loss
and the initiation and progression of tumors. This down-
regulation is generally due to transcriptional repression. Somatic
E-cadherin mutations have also been observed in a variety of
human epithelial cancers (Berx et al., 1998). A direct role for
E-cadherin in the suppression of tumor invasion has been
demonstrated by the reversion of undifferentiated, invasive
tumors to a differentiated phenotype after the transfection
of E-cadherin cDNA in cell culture models (Vleminckx et
al., 1991; Takeichi 1993). On the contrary, abrogation of
E-cadherin–mediated cell adhesion in the transfected cells by
function-perturbing antibodies and antisense RNA restores
invasion (Behrens et al., 1989; Frixen et al., 1991; Vleminckx
et al., 1991; Takeichi 1993). In a study using a transgenic
mouse model of pancreatic
onstrated that E-cadherin–mediated cell adhesion is crucial
in preventing the progression from well-differentiated adenoma
to invasive carcinoma (Perl et al., 1998). These data emphasize
the importance of E-cadherin in inhibiting tumor invasion and
metastasis, which occur at later stages of tumor development.
The cadherin-associated protein
potential to regulate cell motility or invasion. Although
-catenin was originally identified as an integral component
of the cadherin adhesion protein complex, it is also an essen-
tial intracellular mediator for the Wnt growth factor signal
transduction pathway through its interaction with the leuko-
cyte enhancer factor (LEF)*/T cell factor (TCF) family to
regulate transcription of target genes (for review see Peifer
and Polakis 2000). The matrix metalloproteinase matrilysin
and fibronectin have been identified as target genes of the
-catenin/TCF signaling pathway (Crawford et al., 1999;
Gradl et al., 1999), and
-catenin could regulate invasion
through these or other invasion-related genes.
Although the role of E-cadherin in the regulation of tumor
invasion is well established, the exact mechanism of its invasion
suppressor activity is less well defined. One possible mechanism
is that the adhesive function of E-cadherin simply prevents
cells from dissociating from one another and migrating into
tumorigenesis, it has been dem-
-catenin also has the
Address correspondence to Barry M. Gumbiner, Dept. of Cell Biology,
School of Medicine, University of Virginia, PO Box 800732, Charlottesville,
VA 22903. Tel.: (434) 243-9290. Fax: (434) 924-2794.
Alice Wong’s present address is Department of Zoology, University of
Hong Kong, 4S-14 Kadoorie Biological Sciences Bldg., Pokfulam Rd.,
Key words: E-cadherin; b-catenin; invasion; adhesion; signaling
*Abbreviations used in this paper: LEF, leukocyte enhancer factor; NS,
nonspecific; rtTA, reverse tetracycline-responsive transcriptional activator;
siRNA, small interfering RNA;
TCF, T cell factor.
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1192 The Journal of Cell Biology
Volume 161, Number 6, 2003
adjacent tissue. In favor of this hypothesis, it has been shown
that adhesion-blocking E-cadherin antibodies increase the
invasive behavior of cells (Behrens et al., 1989; Frixen et al.,
1991). Alternatively, the interaction between E-cadherin
-catenin at the adherens junction raises the intriguing
possibility that the loss of E-cadherin function may actively
regulate the levels of free
-catenin, thereby altering its abil-
ity to regulate target genes that support tumor invasion.
This hypothesis is supported by previous findings demon-
strating that cadherin expression strongly antagonizes
-catenin signaling in Drosophila
binding and sequestering it in cadherin–catenin complexes
at the plasma membrane (Heasman et al., 1994; Funayama
et al., 1995; Fagotto et al., 1996; Sanson et al., 1996). It is
also possible that E-cadherin could control invasion proper-
ties by altering cytoskeletal and junctional organization. One
possible mechanism could be through p120
on Rho GTPases (Noren et al., 2000; Grosheva et al.,
also enters the nucleus and interacts with the
putative transcription factor Kaiso (Daniel and Reynolds
1999), and although its function is not yet known, it could
potentially mediate effects of cadherins. E-cadherin could
and Xenopus embryos by
via its activity
also control invasion by facilitating juxtacrine signaling via
other receptor systems, since E-cadherin is fundamental for
the establishment and maintenance of numerous other kinds
of cell–cell interactions, for example, gap junctions, tight
junctions, and juxtacrine ligand–receptor interactions. Thus,
there are multiple ways that a reduction in E-cadherin ex-
pression could lead to enhanced tumor cell invasion.
E-cadherin expression was found to suppress the rate of
tumor cell growth by inhibiting
naling in the noninvasive human SW480 colorectal tumor
cell line (Gottardi et al., 2001). To explore the invasion sup-
pressor activity of E-cadherin, we express wild-type E-cadherin
and various E-cadherin chimeras in invasive E-cadherin–
negative human breast and prostate epithelial carcinoma cell
lines using a tetracycline-inducible system.
-catenin/TCF nuclear sig-
Inducible expression of E-cadherin in
human cancer cell lines
We first screened for human tumor cell lines derived from
various tissues that express little or no endogenous E-cad-
Analysis of E-cadherin expression and in vitro behaviors of different human epithelial cancers
Cancer cell linesType of tumor
Soft agar growth
Yes (APC mutation)
The cell lines were from human carcinomas as described in American Type Culture Collection.
E-cadherin expression was determined by Western blot analysis.
In vitro invasion assay on Matrigel filters was performed as described in Materials and methods.
Anchorage-independent assay on soft agar.
expression system. (A) MDA-MB-231
and (B) TSU-Pr1 cells. Equivalent
micrograms of lysates from stable clones
expressing the empty vector control,
E-cadherin, E-cadherin–?-catenin, IL2R–
cytoplasmic tail, E-cadherin?p120, and
E-cadherin??-catenin; each untreated (?)
or treated (?) with 1 ?g/ml doxycycline
were analyzed by Western blot using a
mouse anti–human E-cadherin mAb
(Transduction Laboratory). The blot was
reprobed with an antiactin antibody as a
A tet-on inducible E-cadherin
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E-cadherin suppression of tumor cell invasion |
Wong and Gumbiner 1193
herin and have an invasive phenotype (Table I). The MDA-
MB-231 breast cancer line and TSU-Pr1 prostate cancer line,
which lack E-cadherin and are very invasive as measured by
an in vitro Matrigel invasion assay (Table I), were selected.
Because high levels of E-cadherin are sometimes inhibitory to
cell growth (St. Croix et al., 1998; Sasaki et al., 2000; Got-
tardi et al., 2001; Stockinger et al., 2001), we chose to restore
E-cadherin expression in a tetracycline-inducible (tet-on) ex-
pression system. This allowed us to select and grow the clones
with no or minimal levels of E-cadherin expression.
We first expressed wild-type E-cadherin in MDA-MB-
231 (Fig. 1 A) and TSU-Pr1 (Fig. 1 B) cell lines and ob-
tained at least three independent clones for each construct.
Parental and empty vector controls did not express E-cad-
herin (Fig. 1). Transfected clones expressed high levels of
wild-type E-cadherin after doxycycline induction but had
only a minimal basal level of expression as determined by
Western blot analysis with an anti–E-cadherin antibody
(Fig. 1). It is important to note that levels of E-cadherin in
these stable cell lines were less than those detected in E-cad-
herin–expressing human epithelial cell lines, including
MCF-7, indicating that expression was within normal levels.
Fluorescence microscopy revealed that the E-cadherin pro-
tein was localized to areas of cell to cell contacts (see Fig. 9).
We then expressed other mutant E-cadherin constructs
(e.g., the E-cadherin–
-catenin fusion, the IL2R–E-cad-
herin cytoplasmic tail chimera, the E-cadherin
tant, and the E-cadherin
-catenin) (Fig. 2) in MDA-MB-
231 (Fig. 1 A) and TSU-Pr1 (Fig. 1 B) cells and obtained
multiple clones for each construct. Western blot analysis re-
vealed high levels of expression of these constructs after doxy-
cycline induction and little or no expression when unin-
E-cadherin induction. In vitro Matrigel
invasion of tetracycline-regulated
MDA-MB-231 and TSU-Pr1 cells was
determined as a function of increasing
cell numbers and concentration of
doxycycline. (A) An increasing number of
cells was added to the top compartment
of Matrigel filters in the absence (white
bars) or presence (black bars) of 1 ?g/ml
doxycycline. (B) 2 ? 105 MDA-MB-231
cells and 105 TUS-Pr1 cells were added
onto Matrigel filters in the presence of
varying amount of doxycycline (indicated).
Values shown are the mean number of
cells counted in four fields for replicate
transwells from one experiment. Bars
Inhibition of invasion by
E-cadherin constructs used in this study
(adopted from Gottardi et al. 
with some modifications). Wild-type
E-cadherin is shown at the top. The
E-cadherin–?-catenin fusion chimera was
designed to mediate adhesion without
interacting with ?-catenin. Conversely,
the IL2R–cytoplasmic tail chimera binds
?-catenin but is defective in adhesion.
The E-cadherin?p120 mutant contains
point mutations (EED ? AAA) that abolish
p120ctn binding to E-cadherin and abolish
adhesion. The E-cadherin??-catenin
mutant has the ?-catenin–binding region
truncated, it lacks the ability to bind
?-catenin, and exhibits little or no
Schematic diagram of
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1194 The Journal of Cell Biology
Volume 161, Number 6, 2003
duced (Fig. 1). Although we did not obtain stable
transfection of TSU-Pr1 cells expressing the E-cadherin–
catenin chimera and the E-cadherin
were able to analyze the effect of these constructs and several
others in a transient transfection and invasion assay system
(see Fig. 4).
-catenin mutant, we
Tetracycline-regulated E-cadherin expression
suppresses the invasive phenotype
To investigate the invasion suppression effect of E-cadherin,
we performed an in vitro invasion assay on Matrigel filters,
which is hypothesized to mimic the three-step process of in-
vasion; adhesion, proteolytic dissolution of the extracellular
matrix, and migration (Albini, 1998). The number of cells
that transverse the filters reached a maximum at 5 to 10
10 cells; so we chose to use 2 and 1
MB-231 and TSU-Pr1, respectively, in all future invasion
assays (Fig. 3 A).
Induction of E-cadherin expression caused a significant
reduction in invasive activity for both cell lines compared
cells for MDA-
with their uninduced controls without doxycycline (Fig. 3
A). Suppression occurred over the entire range of cell densi-
ties, suggesting the invasion suppressor activity of E-cad-
herin is not strongly dependent on the density of cell con-
tacts (Fig. 3 A). Parental and empty vector controls
exhibited no difference in invasive activity in the absence or
presence of doxycycline, indicating that transfection and ex-
pression of the tetracycline regulator had no significant ef-
fects on the behavior of these cells (Fig. 3 A). The data also
suggest that the low levels of E-cadherin in the uninduced
state were not sufficient to alter invasiveness. In general,
there is no consistent difference in the invasiveness of E-cad-
herin–transfected cell lines in the uninduced state (Fig. 4 A,
E-cad) compared with mock transfectants (Fig. 4 A, Mock)
for both cell lines.
E-cadherin expression resulted in a dose-dependent de-
crease in tumor invasion (Fig. 3 B). At concentrations (0.1–
g/ml of doxycycline) over a range of increasing E-cad-
herin expression, there was a corresponding decrease in inva-
siveness, and after the maximum level of E-cadherin was at-
of the E-cadherin cytoplasmic tail but not increased
cell adhesion. (A) Induction of construct expression
in stable lines by tet-on system. Stable clones
expressing the empty vector control, E-cadherin,
E-cadherin–?-catenin, IL2R–cytoplasmic tail,
E-cadherin?p120, or E-cadherin??-catenin were
added to the top compartment of Matrigel filters in
the absence (white bars) or presence (black bars) of
1 ?g/ml doxycycline. (B) Cells were transiently
transfected with constructs along with GFP vector
to sort expressing cells by flow cytometry. 105
sorted cells were then employed for in vitro Matrigel
invasion assays. Values shown are the mean number
of cells counted in four fields for replicate transwells
from one experiment. Bars represent SD.
*P ? 0.05, which is considered statistically
different from uninduced (white bars) cells.
Inhibition of invasion due to expression
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E-cadherin suppression of tumor cell invasion |
Wong and Gumbiner 1195
tained at 2
on invasion (Fig. 3 B). Therefore, we selected 1
doxycycline for all future experiments. Together, these data
confirm that expression of E-cadherin at near normal level
can function as an invasion suppressor in the cases of human
breast (MDA-MB-231) and prostate (TSU-Pr1) carcinoma
g/ml of doxycycline, there was no further effect
Invasion suppressor activity is independent of adhesion
and maps to the cytoplasmic domain of E-cadherin
We asked whether the invasion suppressor activity of E-cad-
herin results from increases in physical cell adhesion or from
-catenin nuclear signaling because both of
these factors have been strongly implicated in tumorigenesis.
Additional mechanisms are also possible, such as the p120-
cadherin interaction, which has been implicated in cell mo-
tility, or other potential signal pathways. To address these
possibilities, various mutant E-cadherin constructs were ex-
pressed in cells (Fig. 2). The cytoplasmic domain of E-cad-
herin fused to the extracellular domain of the interleukin-2
subunit (IL2R–E-cadherin cytoplasmic tail chi-
-catenin and p120
signaling molecules but is defective in adhesion (Gottardi et
al., 2001). The E-cadherin
to AAA substitution and is incapable of binding p120
although it still interacts with other catenins, it is defective
in cell adhesion (Thoreson et al., 2000; unpublished data;
Fig. 5). E-cadherin fused directly to
-catenin fusion chimera) lacks the
gion but still mediates strong homophilic adhesive activity
and contains the juxtamembrane region for p120
and potentially other
construct has an EED
-catenin, and it has been found to exhibit little or no adhe-
sive activity in some cell systems (Stappert and Kemler,
1994; Lickert et al., 2000; Kaplan et al., 2001) but mediates
adhesion in others (Yap et al., 1998).
Stable transfection of the IL2R–E-cadherin cytoplasmic
tail chimera and the E-cadherin
cantly suppressed invasion, indicating that the adhesive
function of E-cadherin is not necessary for its invasion sup-
pressor effect in tumor cells (Fig. 4 A). Rather, the cytoplas-
mic tail of cadherin is more important. E-cadherin–
nin fusion chimera designed to rescue adhesion failed to
suppress invasion in MDA-MB-231 cells (Fig. 4 A), indicat-
ing that increased adhesion is not sufficient to suppress in-
vasion. We were unable to generate stable transfectants
of TSU-Pr1 cells expressing the E-cadherin–
sion and the E-cadherin
-catenin mutant. Therefore, we
turned to a transient transfection and invasion assay system
which entails enriching transfected cells by sorting for coex-
pressed GFP (Fig. 4 B). This was a valid measure of the ef-
fect of E-cadherin on invasion, since the transient transfec-
tion assay yielded the same result for E-cadherin and the
mutant constructs that were expressed stably (Fig. 4, A and
B). Expression of the E-cadherin–
transient assay did not suppress invasion in TSU-Pr1 cells
(Fig. 4 B). Nor did the expression of E-cadherin
mutant suppress invasion in the TSU-Pr1 carcinoma cell
line (Fig. 4 B). These data suggest that increased cell adhe-
sion does not account for the invasion suppressor activity of
E-cadherin; rather, it is likely occurs as a result of signaling
through the cytoplasmic tail.
-catenin mutant lacks the ability to bind
-catenin fusion in this
cadherin-expressing cell lines.
(A and B) Use of a laminar flow
adhesion assay and (C and D) use of a cell
aggregation assay. (A) MDA-MB-231 and
(B) TSU-Pr1 cells expressing E-cadherin
and mutant E-cadherin constructs.
Micrographs of (C) MDA-MB-231 and
(D) TSU-Pr1 cells expressing various
constructs in aggregation assays. All
cadherin cell lines were induced with
1 ?g/ml doxycycline to express a
transgene, and the mock control cells
were treated with the same level of
doxycycline. Bar, 20 ?m.
Adhesive properties of
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1196 The Journal of Cell Biology
Volume 161, Number 6, 2003
The cytoplasmic domain of the cadherin can bind directly
to p120ctn and ?-catenin, and either protein could play a
role in invasion suppression. The construct that is defective
in p120ctn binding, the E-cadherin?p120 mutant, was still
able to suppress invasion. However, constructs with dele-
tions of the ?-catenin–binding domain, the E-cadherin–?-
catenin chimera and the E-cadherin??-catenin mutant, did
not suppress invasion (Fig. 4, A and B). Thus, the inhibition
of invasiveness by E-cadherin is associated with the ?-cate-
nin–binding domain but not the p120ctn binding domain.
Our interpretation of the data that adhesion is neither
necessary nor sufficient for suppressing cancer invasion de-
pends on the use of various E-cadherin constructs with pre-
viously tested adhesion activities. However, these cadherin
constructs have not been tested for adhesive function in
MDA-MB-231 and TSU-Pr1 cells. In particular, the
E-cadherin?p120 mutant that lacks p120ctn binding should
be examined, since the role of p120ctn or the binding site in
adhesion has been variable depending on the system (Ozawa
and Kemler, 1998; Yap et al., 1998; Aono et al., 1999;
Thoreson et al., 2000; Ireton et al., 2002; Myster et al.,
2003; Pacquelet et al., 2003). Therefore, we examined their
relative adhesive activities using an adhesion flow assay
to measure the strength of cell attachment to purified
ectodomain of E-cadherin (Brieher et al., 1996; Gottardi et
al., 2001) and an aggregation assay (Shimoyama et al.,
1992). All cells were treated with 1 ?g/ml doxycycline to in-
duce transgene expression; the mock cells were treated with
the same level of doxycycline. The E-cadherin–?-catenin
chimera construct adhered strongly to cadherin-coated sub-
strates and exhibited adhesive activity almost as good as the
wild-type E-cadherin (Fig. 5 A). By contrast, cells expressing
the IL2R–E-cadherin cytoplasmic tail and the E-cad-
herin?p120 construct exhibited only background adhesive
activity, which was indistinguishable from E-cadherin–nega-
tive mock-transfected MDA-MB-231 and TSU-Pr1 con-
trols (Fig. 5, A and B). The E-cadherin??-catenin construct
had little or no adhesive activity, presumably due to the lack
of interactions with the cytoskeleton which are important
for strong cell–cell adhesion (Fig. 5 A). These results were
consistent with those of aggregation assays; prominent ag-
gregates were observed exclusively in cells expressing wild-
type E-cadherin and E-cadherin–?-catenin but not other
cadherin constructs (Fig. 5, C and D). We did not perform
adhesion and aggregation assays on E-cadherin–?-catenin
chimera fusion and E-cadherin??-catenin mutant constructs
in TSU-Pr1 cells because we did not obtain stable transfec-
tants for these two constructs.
We also examined whether an adhesion blocking anti–E-
cadherin antibody, HECD-1, could revert the suppression
of tumor cell invasion in the MDA-MB-231 and TSU-Pr1
cell lines. HECD-1 did not revert the invasion suppression
mediated by E-cadherin expression in either cell line (Fig.
6 A). For a positive control, HECD-1 caused cells expressing
E-cadherin, but not in E-cadherin–negative mock-trans-
fected cells, to dissociate and scatter (Fig. 6 B). Thus, treat-
ment with an antibody that disrupts the adhesive function of
E-cadherin did not abrogate the invasion suppression activ-
ity of E-cadherin.
Suppression of invasiveness involves ?-catenin but is
independent of TCF-mediated transcription
Since these data suggest roles for the ?-catenin–binding do-
main, and by implication ?-catenin, in the invasion suppres-
HECD-1 does not revert E-cadherin–mediated
invasion suppression. (A) Doxycycline-untreated
(white bars) and treated (black bars). Mock-
transfected control cells and cells stably expressing
E-cadherin were plated on Matrigel filters for invasion
assays in the presence (hatched bars) or absence
(black bars) of HECD-1 antibody (50 ?g/ml). After
24 h, the number of cells traversing the filter was
determined. Data shown are the mean number of
cells counted in four fields for replicate transwells
from one experiment. Bars represent SD. (B)
Corresponding control cell dissociation experiment
(phase–contrast micrographs) showing inhibition
of cell to cell adhesion by HECD-1 (50 ?g/ml).
Bar, 50 ?m.
Treatment with anti–E-cadherin mAb
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E-cadherin suppression of tumor cell invasion | Wong and Gumbiner 1197
sor function of E-cadherin we examined whether reduction
or loss of ?-catenin could suppress invasion in MDA-MB-
231 and TSU-Pr1 cells. To do so, we chose to deplete
?-catenin using small interfering RNA (siRNA) with a spe-
cific siRNA shown previously to deplete ?-catenin (Deng et
al., 2002). Treatment of cells with the ?-catenin–siRNA,
but not nonspecific (NS)-siRNA, resulted in a significant de-
crease in ?-catenin levels (Fig. 7 A). Importantly, treatment
suppresses invasion. (A) Western blot of cell lysates
from equal protein of corresponding samples with
antibodies as indicated. (B) MDA-MB-231 and TSU-Pr1
cells were transfected with ?-catenin-siRNA (black bars)
or a NS-siRNA (hatched bars) for 1 d and then harvested
for invasion assays. Data are expressed as the mean
number of cells counted in four fields for replicate
transwells from one experiment. Bars represent SD.
*P ? 0.05, which is considered statistically different
from untreated (white bars) cells.
siRNA-mediated depletion of ?-catenin
regulation in invasive activity of MDA-MB-231
and TSU-Pr1 cells. (A) No detectable changes in
?-catenin/TCF signaling by E-cadherin. Analysis of
TCF reporter gene activation in various cell lines.
?-catenin/TCF–mediated transcriptional activation
was determined by transient transfection of
TOPFLASH or FOPFLASH reporter constructs, which
contain wild-type or mutant TCF-binding motifs
upstream of the firefly luciferase cDNA. APC mutant
colorectal cancer cell line SW480 served as a
positive control. Stable clones expressing the empty
vector control and E-cadherin in the absence
(white bars) or presence (black bars) of 1 ?g/ml
doxycycline were assessed for their ability to
modulate TCF-dependent transcription. TCF-
mediated transcriptional activity was defined as
the ratio of TOPFLASH:FOPFLASH luciferase
activities, each corrected internally for Renilla
luciferase activities and where no transactivation
equals 1. (B) Inhibition of ?-catenin/TCF signaling
pathway does not suppress invasion. Cells were
transiently transfected with GFP alone, an empty
vector, a ?-catenin/engrailed repressor, and a
dominant-negative (dn) TCF and were assessed for
in vitro invasion. (C) Constitutively active form of
TCF (VP16?TCF) does not rescue E-cadherin–
mediated invasion suppression. Values shown are
the mean number of cells counted in four fields for
replicate transwells from one experiment. Bars
No role for ?-catenin/TCF transcriptional
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1198 The Journal of Cell Biology | Volume 161, Number 6, 2003
of parental MDA-MB-231 and TSU-Pr1 cells with ?-cate-
nin–siRNA suppressed invasion, but no inhibition was ob-
served for a NS-siRNA (Fig. 7 B). These results indicate that
?-catenin contributes to the invasive behavior of MDA-MB-
231 and TSU-Pr1 cells and suggest that the invasion sup-
pression activity of E-cadherin could be mediated through
the sequestration of ?-catenin.
Since the invasion suppressor activity of E-cadherin is as-
sociated with ?-catenin, it could occur through changes in
?-catenin signaling in the nucleus in association with LEF/
TCF to regulate transcription. The ?-catenin/TCF–depen-
dent transcription was assayed using a TCF-responsive
reporter, TOPFLASH (Korinek et al., 1997). Both MDA-
MB-231 and TSU-Pr1 cell lines displayed low TCF-depen-
dent transcriptional activities, with mean relative TCF activ-
ities between three and six (Fig. 8 A). As a positive control,
the SW480 colorectal cancer cell line, which harbors muta-
tions in the APC tumor suppressor protein, had a 137-fold
enhanced transcriptional activity. In no case did E-cadherin
induction alter TCF-dependent transcription activity com-
pared with their uninduced controls or the mock transfec-
tants (Fig. 8 A). Thus, the invasion inhibition activity of
E-cadherin does not seem to be attributable to changes in
?-catenin/TCF–dependent nuclear signaling.
To further test the possibility that the suppression of cell
invasiveness may be mediated through a ?-catenin/TCF–
dependent signaling pathway, we asked whether expression
of constructs known to inhibit ?-catenin/TCF nuclear sig-
naling at the level of target genes could inhibit cell invasion
in these cell lines. ?-catenin fused to the engrailed repressor
domain, and a dominant-negative TCF-3 construct lacking
a ?-catenin–binding site in its NH2 terminus were expressed
transiently together with GFP, and cells were sorted by
FACS® analysis and tested in the invasion assay. Neither the
?-catenin/engrailed repressor nor the dominant-negative
TCF construct caused a reduction in invasion through
Matrigel filters (Fig. 8 B). Conversely, we asked whether in-
vasiveness that was suppressed by E-cadherin could be res-
cued by expression of a downstream activator of the ?-cate-
nin signaling pathway, a constitutively active form of TCF.
Suppression of invasion by E-cadherin expression was not
reversed by VP16?TCF, a TCF-3 construct has its NH2-
terminal ?-catenin–binding region replaced with the potent
herpes simplex virus VP16 transactivation domain (Fig. 8
C). Together, these observations indicate that modulation of
the ?-catenin/TCF signaling pathway does not contribute
significantly to the invasion suppression activity of E-cad-
herin in these cells.
TSU-Pr1 cells. (A–D) Phase microscopy and indirect immunofluorescence staining of (E–H) E-cadherin (CY3), (I–L) ?-catenin (FITC), and
(Q–T) paxillin (FITC) in mock and E-cadherin stable cell lines. E-cadherin–transfected cells were treated with 1 ?g/ml doxycycline to induce
E-cadherin expression, and the control cells were treated with the same level of doxycycline. (M–P) F-actin was visualized after the binding of
Texas red–conjugated phalloidin. (U–X) Effect of E-cadherin expression on cell migration. Confluent monolayers of MDA-MB-231 and TSU-Pr1
cultures of cells treated with 1 ?g/ml doxycycline to induce expression of E-cadherin were scraped with a pipet tip to create a wound. The
mock controls were treated with the same level of doxycycline. Cells were given fresh medium containing doxycycline in the presence of
roscovitine (20 ?M, to reduce cell growth), cultured for 24 h, and photographed. Bar, 10 ?m.
Expression of E-cadherin has no obvious effect on morphology, cytoskeletal organization, or migration of MDA-MB-231 and
The Journal of Cell Biology
E-cadherin suppression of tumor cell invasion | Wong and Gumbiner 1199
Roles of cell morphology, cytoskeletal organization,
cell motility, and N-cadherin in invasion suppression
Previous studies showed that expression of E-cadherin can
alter cell morphology, cytoskeletal organization, and motil-
ity of some tumor cell types (Frixen et al., 1991; Luo et al.,
1999). In the case of MDA-MB-231 and TSU-Pr1 cells,
however, stable expression of E-cadherin did not signifi-
cantly alter cell morphology compared with the mock-
transfected controls (Fig. 9, A–D). Immunofluorescence
microscopy revealed E-cadherin staining was enriched at
the intercellular boundaries of E-cadherin transfectants only
(Fig. 9, E–H). Even without expression of E-cadherin,
?-catenin was present at low levels at cell contacts, probably
due to its association with endogenous N-cadherin or other
cadherins such as cadherin-11 (Fig. 10; Bussemakers et al.,
1994; Wang et al., 2002). Expression of E-cadherin re-
sulted in increased recruitment of ?-catenin at the cell–cell
contacts (Fig. 9, I–L). F-actin organization, visualized by
Texas red–labeled phalloidin, appeared not to be signifi-
cantly altered in E-cadherin–expressing MDA-MB-231 and
TSU-Pr1 cells (Fig. 9, M–P). Similarly, E-cadherin–trans-
fected cells and mock-transfected cells displayed similar
staining patterns for the focal adhesion protein paxillin
(Fig. 9, Q–T). We also examined whether E-cadherin al-
tered cell motility using a classical wound-healing assay
(Fig. 9, U–X). E-cadherin had no obvious effect on the mo-
tility of these cell monolayers.
Unlike E-cadherin, N-cadherin has been found to pro-
mote cell motility and invasion (Islam et al., 1996; Hazan et
al., 2000). Nonetheless, we asked whether increased N-cad-
herin expression, which also binds ?-catenin strongly, had
any effect on cell invasion (Fig. 10). N-cadherin or E-cad-
herin was expressed transiently together with GFP and
sorted by FACS® before testing in invasion assays. As before,
expression of E-cadherin potently inhibited invasion (Fig.
10 A). In contrast, expression of N-cadherin did not result
in significant suppression of invasion even though it was ex-
pressed (Fig. 10 A), indicating the invasion suppressing ac-
tivity is unique to E-cadherin. In fact, N-cadherin expres-
sion promoted cell invasion when expressed in cells already
expressing E-cadherin (Fig. 10 B), consistent with its re-
ported invasion promoting activity (Islam et al., 1996; Hazan
et al., 2000).
Although the ability of E-cadherin to suppress tumor inva-
sion has been known for many years, the mechanisms re-
quired for this inhibition have been less well defined. We
have examined the mechanism by which E-cadherin expres-
sion suppresses tumor cell invasion in light of different as-
pects of cadherin function: the adhesive function and the
ability to bind ?-catenin and influence its signaling and
transcriptional activity in the nucleus. Additional mecha-
nisms are also possible, such as the p120-cadherin interac-
tion, which has been implicated in cell motility (Grosheva et
al., 2001; Noren et al., 2000), or even other potential signal
pathways (Carmeliet et al., 1999; Xu and Carpenter 1999;
Kaplan et al., 2001; Xu et al., 2002). We find that E-cad-
herin constructs that exhibit adhesive activity but do not
bind ?-catenin are unable to inhibit cell invasion. In con-
trast, using constructs that can bind ?-catenin but are defec-
tive in adhesion suppresses invasion. Thus, the homophilic
adhesive activity of E-cadherin is neither necessary nor suffi-
cient to mediate tumor cell invasion suppression in MDA-
(A) N-cadherin does not suppress invasion as well as
E-cadherin. Parental cells transiently transfected with
either N-cadherin or E-cadherin were compared with
GFP control cells in invasion assays. (B) N-cadherin
promotes invasion of E-cadherin–expressing cells.
Doxycycline-induced E-cadherin cells transiently
transfected with N-cadherin was compared with GFP
controls. Values shown are the mean number of cells
counted in four fields for replicate transwells from one
experiment. Bars represent SD. Western blot of cell
lysates from transient transfection samples before
analyzed by FACS® were examined for cadherin
expression with antibodies as indicated.
Effect of N-cadherin on invasion.
The Journal of Cell Biology
1200 The Journal of Cell Biology | Volume 161, Number 6, 2003
MB-231 breast and TSU-Pr1 prostate carcinoma cells. Since
the adhesive function of cadherin is not important in sup-
pressing tumor invasion, we also rule out the possibility that
E-cadherin controls invasion by facilitating juxtacrine sig-
naling via other receptor systems through the establishment
and maintenance of other kinds of cell–cell interactions.
Previous studies using adhesion-blocking E-cadherin anti-
bodies on MDCK (Behrens et al., 1985, 1989) and carci-
noma cells (Frixen et al., 1991) or by modulating cell densi-
ties (Chen and Obrink 1991) suggested that decreased
adhesion leads to increased invasion. In contrast, we did not
observe a density independence or an increase in cell inva-
siveness using the HECD-1 mAb that blocks the adhesive
function of human E-cadherin. Although it is not entirely
clear why our findings differ in this regard from the previous
reports, the role of E-cadherin may differ depending on the
type of tumor or cell context. For example, Sommers et al.
(1991) has found that treatment of E-cadherin–positive
MCF-7 breast cancer cells with an adhesion-blocking anti-
body caused the cells to detach from one another but did
not induce invasiveness in these cells. Furthermore, blocking
antibodies can potentially also change E-cadherin conforma-
tion and signaling. For the two human carcinoma lines in
our studies, the combined evidence from expression of mu-
tant constructs, antibody treatment, and density indepen-
dence lead us to conclude that E-cadherin suppresses inva-
sion in an adhesion-independent manner.
p120ctn has the potential to regulate cell motility and inva-
sion through its activity on Rho family GTPases and its in-
teraction with a putative transcription factor (Daniel and
Reynolds 1999). However, the mechanism by which E-cad-
herin suppresses tumor cell invasion is not likely mediated
through p120ctn, since mutation of the p120ctn-binding re-
gion has no effect. These findings may appear to differ
somewhat from those of Chen et al. (1997) who used an
E-cadherin construct that lacks the juxtamembrane region
(aa 595–617), which removes p120ctn binding (and perhaps
other functions), to show that this region is essential for sup-
pressing motility independent of adhesion. However, the
work by Chen et al. (1997) was done in L-cell fibroblasts
and astrocyte-like WC-5 cells, and the juxtamembrane do-
main might have a different role in those cell types com-
pared with the MDA-MB-231 breast and TSU-Pr1 prostate
cells. Furthermore, the roles of the p120ctn-binding domain
and p120ctn in regulating adhesion appear to differ signifi-
cantly among various cell types and organisms (for review
see Myster et al., 2003). Another possibility is that p120ctn
could be more important in regulating cytoskeletal organiza-
tion and cell motility in some cell types than invasion, since
p120ctn has been suggested to be a regulator of the actin cy-
toskeleton via Rho (Daniel and Reynolds 1999; Anastasiadis
et al., 2000; Noren et al., 2000; Grosheva et al., 2001). Our
findings indicate that E-cadherin functions as an invasion
suppressor in MDA-MB-231 breast and TSU-Pr1 prostate
carcinoma cells without any obvious effects on cytoskeletal
organization or cell motility. It is likely that E-cadherin sup-
presses invasion through a different mechanism involving
signaling through the cytoplasmic domain. For example,
E-cadherin has been proposed to suppress prostate cancer
invasiveness by modulating matrix metalloproteinase activity
(Luo et al., 1999), which could help tumor cells to invade
through basement membrane into surrounding tissue.
We provide evidence that both the ?-catenin–binding do-
main of E-cadherin and ?-catenin levels play roles in cell in-
vasion. Nonetheless, on the basis of several different criteria,
we conclude that the invasion suppressor activity of E-cad-
herin is not related to a change in the well-studied pathway
involving the ?-catenin/TCF regulation of target gene
expression. E-cadherin expression is unable to change TCF-
dependent transcriptional activity as measured by TOP-
FLASH reporter assay. Reduction in the activity of ?-cate-
nin/TCF signaling using dominant-negative TCF and
?-catenin/engrailed repressor does not suppress invasion.
Nor does the expression of a constitutively active form of
TCF revert the invasion suppressor activity of E-cadherin.
These findings differ from those obtained with embryos and
cancer cells that have a strongly activated Wnt pathway in
which expression of E-cadherin antagonizes ?-catenin/TCF
signaling (Heasman et al., 1994; Funayama et al., 1995;
Fagotto et al., 1996; Sanson et al., 1996; Gottardi et al.,
2001). In breast and prostate carcinomas, the Wnt pathway
is not thought to be active, and components involved in me-
diating ?-catenin/TCF–dependent gene regulation may not
be expressed or active in MDA-MB-231 and TSU-Pr1 cells.
Although these cells exhibit low TCF-dependent reporter
gene activity, it is possible that the TOPFLASH reporter re-
sponds to other factors in these cells.
Our findings that the invasion suppressor activity of E-cad-
herin is mediated through the ?-catenin–binding region, but
not a ?-catenin/TCF–dependent nuclear signaling mecha-
nism, suggest the involvement of another signaling pathway.
Since ?-catenin depletion by siRNA treatment also sup-
presses invasion in both cell lines, the most likely explanation
is that E-cadherin binds and sequesters ?-catenin away from
a target protein other than TCF. For example, several pro-
teins that have the potential to regulate tumor invasion,
MUC1 (Li et al., 1998; Schroeder et al., 2003), NF-kB
(Deng et al., 2002), Nr-CAM (Conacci-Sorrell et al., 2002),
MITF (Widlund et al., 2002), and IQGAP (Kuroda et al.,
1998), have been shown to interact with ?-catenin, and it is
possible that E-cadherin expression titrates ?-catenin away
from one or more of them. We cannot rule out the possibil-
ity, however, that another signaling factor could also bind to
the ?-catenin–binding region. For example, Shc (Xu and
Carpenter 1999), G?12 (Kaplan et al., 2001), and PTP1B
(Xu et al., 2002) have reported to bind to a partially overlap-
ping region of the ?-catenin–binding domain, although the
functional significance of these interactions is not yet known.
Another formal possibility is that the association of ?-catenin
with the cytoplasmic tail of cadherin could recruit other sig-
naling molecules. For example, ?-catenin associated with
VE-cadherin recruits VEGF receptor-2/phosphatidylinosi-
tide 3-kinase, thus activating phosphatidylinositide 3-kinase
and AKT (Carmeliet et al., 1999), and ?-catenin has also
been found to mediate the interaction of E-cadherin with
EGF receptor (Hoschuetzky et al., 1994).
N-cadherin also binds ?-catenin but stimulates tumor cell
invasion (Islam et al., 1996; Hazan et al., 2000). Our data
suggest that the invasion suppressor activity is specific to
E-cadherin, despite the high homology between E- and
The Journal of Cell Biology
E-cadherin suppression of tumor cell invasion | Wong and Gumbiner 1201
N-cadherin sequences in the cytoplasmic region. This finding
is not really surprising; others have shown that N-cadherin has
a dominant effect over E-cadherin (Islam et al., 1996; Nie-
man et al., 1999; Hazan et al., 2000), which we have con-
firmed in our experiments. This could be due to the activa-
tion of other pathways that can overcome the suppressive
signals mediated by the ?-catenin–binding domain. Indeed,
there is considerable evidence that the invasion-promoting
activity of N-cadherin resides primarily in the extracellular
domain (Kim et al., 2000; Suyama et al., 2002), perhaps via
its interaction with the FGF receptor (Suyama et al., 2002).
Materials and methods
cDNAs for wild-type E-cadherin, an E-cadherin–?-catenin chimera, an
IL2R–E-cadherin cytoplasmic tail chimera, an E-cadherin?p120 (a gift
from A. Reynolds, Vanderbilt University, Nashville, TN) (Thoreson et al.,
2000), an E-cadherin??-catenin (a gift from D. Rimm, Yale University,
New Haven, CT) were excised from plasmid pcDNA3 (Fig. 2; Gottardi et
al., 2001) and subcloned into the XbaI site of the reverse tetracycline-
responsive transcriptional activator (rtTA)-responsive plasmid pUHD10.3,
a hygromycin-resistant pTRE vector (a gift from J. Massague, Memorial
Sloan-Kettering Cancer Center, New York, NY). All constructs were veri-
fied by restriction endonuclease digestion and DNA sequencing. The
?-catenin–engrailed repressor fusion construct was provided by U. Muel-
ler and P. McCrea (University of Texas, Houston, TX) (Montross et al.,
2000); dominant-negative Xenopus TCF-3 and activated TCF-3 were de-
scribed elsewhere (Vonica et al., 2000). Mouse N-cadherin (Miyatani et al.,
1992) was provided by R. Brackenbury (University of Cincinnati, Cincin-
nati, OH). pEGFP vector was purchased from CLONTECH Laboratories, Inc.
Cell culture and stable transfections
The E-cadherin–negative MDA-MB-231 human breast cancer cell line (a
gift from N. Rosen, Memorial Sloan-Kettering Cancer Center, New York,
NY) was maintained in DME and the TSU-Pr1 human prostate cancer cell
line (a gift from P. Pandolfi, Memorial Sloan-Kettering Cancer Center, New
York, NY) in RPMI 1640; both media contain 10% FBS, 1% L-glutamine,
100 U/ml penicillin, and 100 ?g/ml streptomycin, and cells were grown in
a humidified atmosphere of 5% CO2 at 37?C. Cells were transfected with
Lipofectamine reagent (Invitrogen). Stable transfections were performed
first to establish clonal lines constitutively expressing rtTA encoded by the
pTet-ON regulator plasmid, which contains a neomycin resistance gene,
allowing cells to be selected in G418 (Gossen et al., 1995) (CLONTECH
Laboratories, Inc.). One of these rtTA clones was selected for secondary
stable transfection with rtTA-responsive pUHD10.3 hygromycin plasmids
containing a wild-type E-cadherin, an E-cadherin–?-catenin chimera, an
IL2R–E-cadherin cytoplasmic tail chimera, an E-cadherin?p120, an E-cad-
herin??-catenin (Fig. 2), or an empty vector alone, and colonies were se-
lected in 400 ?g/ml of G418 and 800 ?g/ml hygromycin B (Invitrogen).
Stable clones were screened for inducible expression by Western blotting
after 24 h induction with 1 ?g/ml of the doxycycline (CLONTECH Labora-
tories, Inc.). Positives were then subcloned one more time by limiting dilu-
tion and expanded into cell lines that were maintained in the selection me-
dium. At least three independent clones were selected per cadherin
construct based solely on expression of E-cadherin as determined by West-
ern blot analysis.
Transient transfection and flow cytometry cell sorting for
For some mutant constructs, we were unable to generate stable transfec-
tants with sufficient levels of expression. Therefore, these constructs were
transiently cotransfected along with pEGFP vector (as fluorescence marker)
using Fugene 6 (Boehringer) and subjected to FACS® 48 h posttransfection
(Vantage Sorter; Becton Dickinson). 105 sorted cells were immediately em-
ployed for Matrigel invasion assays as described below. Each experiment
was repeated at least three times.
Matrigel invasion assay
Cells were grown in the absence or presence of 1 ?g/ml of doxycycline for
48 h, harvested in 0.1% trypsin in PBS supplemented with 1 mM calcium
and 0.5 mM magnesium, washed, suspended in DME containing 0.1%
BSA in the presence and absence of doxycycline and counted. In some ex-
periments, anti–E-cadherin antibody HECD-1 (50 ?g/ml) (a gift from M.
Takeichi, Kyoto University) was added to the medium. 1–2 ? 105 cells
(unless otherwise specified) were seeded on the upper well of Biocoat
Matrigel chambers (8 ?m pore size; Becton Dickinson). Conditioned cell
culture medium of 3T3 fibroblasts containing 20 ?g/ml of human plasma
fibronectin (Invitrogen) in the absence or presence of 1 ?g/ml of doxycy-
cline filled the lower well as chemoattractant. The invasion assay was per-
formed at 37?C under 5% CO2/95% air atmosphere. After 24 h, medium
was aspirated, and cells on the upper side of the membrane were removed
with a cotton swab. The invading cells on the underside of the filters were
stained with DAPI, and four fields were counted for each of the replicate
membranes. We used SW480 colon cancer cell line as a negative control
for invasion (Gottardi et al., 2001). Each experiment was repeated at least
three times, although the results of one representative experiment were
shown. Data were expressed as mean ? SD. Statistical significance was
determined by unpaired Student’s t test, and differences between groups
were analyzed using the ANOVA; P ? 0.05 was considered significant.
siRNA duplex oligo (Dharmacon) (13.5 ?g/100 mm plate) targeting ?-cate-
nin mRNA (5?-AAGUCCUGUAUGAGUGGGAAC-3?) (Deng et al., 2002)
or a nonspecific RNA (5?-GGCTACGTCCAGGAGCGCACC-3?) as a nega-
tive control were transfected using Lipofectamine (Invitrogen). Matrigel in-
vasion assay was performed 1 d after transfection.
Adhesion flow and aggregation assays
Adhesion flow assays were performed as previously described by Brieher
et al. (1996) except glass capillary tubes were coated with a human E-cad-
herin–Fc recombinant fusion protein as an adhesive substrate. Aggregation
assays were done as described by Shimoyama et al. (1992) with only mi-
?-catenin–LEF/TCF reporter gene (TOPFLASH) assay
In 24-well plates, cells were transiently transfected with 0.5 ?g of the TOP-
LASH or FOPFLASH reporter plasmid (H. Clevers, University of Utrecht,
Utrecht, Netherlands) using Fugene-6 (Boehringer). Transfection efficien-
cies were determined by cotransfection of the pRL-TK reporter construct (a
gift from J. Massague, Memorial Sloan-Kettering Cancer Center, New York,
NY) that contained the Renilla luciferase cDNA. Activities of Firefly and
Renilla luciferases were measured sequentially from a single sample using
the Dual-Luciferase Reporter Assay System (Promega).
Cells were grown on glass coverslips, fixed in cold (?20?C) methanol, and
then incubated with antibodies. F-actin was visualized after the binding of
Texas red–conjugated phalloidin (Molecular Probes).
Monolayers of cells were wounded by scraping with a plastic pipet-tip,
rinsed several times with media to remove dislodged cells, and placed
back in medium with 10% FBS. Cells were given 20 ?M roscovitine (Cal-
biochem) to reduce cell growth, cultured for 24 h, stained by 0.1% crystal
violet, and photographed.
We thank Ellen Wong and Joanne Lannigan for technical assistance and
Cara Gottardi for advice throughout the project.
This work was supported by National Institute of Health grant
GM37432 to B.M. Gumbiner and a fellowship by the National Cancer In-
stitute of Canada to A.S.T. Wong.
Submitted: 3 December 2002
Revised: 8 May 2003
Accepted: 8 May 2003
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