The cyclin D1 gene is a target of the β-catenin/LEF-1 pathway

Abstract

beta-Catenin plays a dual role in the cell: one in linking the cytoplasmic side of cadherin-mediated cell-cell contacts to the actin cytoskeleton and an additional role in signaling that involves transactivation in complex with transcription factors of the lymphoid enhancing factor (LEF-1) family. Elevated beta-catenin levels in colorectal cancer caused by mutations in beta-catenin or by the adenomatous polyposis coli molecule, which regulates beta-catenin degradation, result in the binding of beta-catenin to LEF-1 and increased transcriptional activation of mostly unknown target genes. Here, we show that the cyclin D1 gene is a direct target for transactivation by the beta-catenin/LEF-1 pathway through a LEF-1 binding site in the cyclin D1 promoter. Inhibitors of beta-catenin activation, wild-type adenomatous polyposis coli, axin, and the cytoplasmic tail of cadherin suppressed cyclin D1 promoter activity in colon cancer cells. Cyclin D1 protein levels were induced by beta-catenin overexpression and reduced in cells overexpressing the cadherin cytoplasmic domain. Increased beta-catenin levels may thus promote neoplastic conversion by triggering cyclin D1 gene expression and, consequently, uncontrolled progression into the cell cycle.

Full text

Proc. Natl. Acad. Sci . USA
Vol. 96, pp. 5522–5527, May 1999
Cell Biology
The cyclin D1 gene is a target of the
b
-cateninyLEF-1 pathway
MICHAEL SHTUTMAN*, JACOB ZHURINSKY*, INBAL SIMCHA*, CHRIS ALBANESE†,MARK D’AMICO†,
RICHARD PESTELL†,AND AVRI BEN-ZE’EV*‡
*Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel; and †Department of Medicine and Developmental and
Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461
Communicated by Elizabeth D. Hay, Harvard Medical School, Boston, M A, March 15, 1999 (received for review December 30, 1998)
ABSTR ACT
b
-Catenin plays a dual role in the cell: one in
linking the cytoplasmic side of cadherin-mediated cell–cell
contacts to the actin cytoskeleton and an additional role in
signaling that involves transactivation in complex with tran-
scription factors of the lymphoid enhancing factor (LEF-1)
family. Elevated
b
-catenin levels in colorectal cancer caused
by mutations in
b
-catenin or by the adenomatous polyposis
coli molecule, which regulates
b
-catenin degradation, result in
the binding of
b
-catenin to LEF-1 and increased transcrip-
tional activation of mostly unknown target genes. Here, we
show that the cyclin D1 gene is a direct target for transacti-
vation by the
b
-cateninyLEF-1 pathway through a LEF-1
binding site in the cyclin D1 promoter. Inhibitors of
b
-catenin
activation, wild-type adenomatous polyposis coli, axin, and
the cytoplasmic tail of cadherin suppressed cyclin D1 pro-
moter activity in colon cancer cells. Cyclin D1 protein levels
were induced by
b
-catenin overexpression and reduced in cells
overexpressing the cadherin cytoplasmic domain. Increased
b
-catenin levels may thus promote neoplastic conversion by
triggering cyclin D1 gene expression and, consequently, un-
controlled progression into the cell cycle.
b
-Catenin is a major component of adherens junctions linking
the actin cytoskeleton to members of the cadherin family of
transmembrane cell–cell adhesion receptors (1, 2). In addition,
b
-catenin can translocate into the nucleus (3–7), where it can
complex with transcription factors of the LEF-1 family and
regulate the expression of specific genes (8, 9). By playing such
a dual role—a structural role in cell–cell junctions and a
regulatory role in the nucleus—
b
-catenin can transduce
changes in cell adhesion and junction formation to control
transmembrane signaling and gene expression (1, 10–12).
b
-Catenin-mediated signaling depends on its accumulation
and subsequent translocation into the nucleus. The level of
b
-catenin in the cell is regulated by its association with the
tumor suppressor molecule adenomatous polyposis coli (APC;
refs. 13 and 14), axin (15, 16), and glycogen synthase kinase 3
b
(GSK-3
b
; ref. 17). Phosphorylation of
b
-catenin by the APC–
axin–GSK-3
b
complex (18, 19) leads to its degradation by the
ubiquitin–proteasome system (6, 20). The failure of this deg-
radation in cells expressing mutant APC or
b
-catenin leads to
the accumulation of
b
-catenin and is common in human colon
cancer and melanoma (21–23). Elevated
b
-catenin levels in
such tumors are suggested to confer uncontrolled activation of
gene transcription by the
b
-cateninyLEF-1 complex that may
contribute to tumor progression (1, 24, 25). The nature of the
target genes of the
b
-cateninyLEF complex is, however, largely
unknown, except for the recently discovered c-MYC that was
shown to contain LEF-1-binding sequences in its promoter (26).
Cyclin D1 is a major regulator of the progression of cells into
the proliferative stage of the cell cycle (27). Although the
cyclin D1 gene is not amplified in human colon cancer, the
expression of cyclin D1 is elevated in about 30% of human
adenocarcinomas and in adenomatous polyps of the colon (28,
29), and expression of anti-sense cyclin D1 cDNA abolished
the growth of SW480 colon cancer cells in nude mice, indi-
cating a critical role for cyclin D1 in tumorigenesis (30).
In this study, we investigated the possibility that the cyclin
D1 gene is a target for the
b
-cateninyLEF-1 complex and show
that the cyclin D1 promoter contains a LEF-1 binding se-
quence that is activated in human colon cancer cells. We
further show that transcriptional activation of the cyclin D1
gene in such cells can be inhibited by enhancing the degrada-
tion of
b
-catenin with wild-type APC and axin or by its binding
to the cadherin cytoplasmic tail. Unscheduled activation of
cyclin D1 transcription by the
b
-cateninyLEF-1 complex in
colon cancer cells may result in uncontrolled cell proliferation
and thus contribute to tumor progression in these cells.
MATERIALS AND METHODS
Plasmid Constructions. Plasmids containing the luciferase
reporter under the cyclin D1 promoter and the deletion
constructions derived from it have been described (31). The
mutated LEF-1 binding site at nucleotides 275 and 274 from
AT to GC (2163mtLefCD1LUC) and the deletion of the
LEF-1 binding site from 281 to 273 (2163DLefCD1LUC)
were constructed by PCR-based site directed mutagenesis by
using the 2163CD1LUC plasmid. Reporter plasmids contain-
ing luciferase under a multimeric consensus- or mutant inac-
tive-LEF-1 binding site (TOPFLASH and FOPFLASH; ref. 9)
and vectors expressing various forms of
b
-catenin (7), LEF-1
(5), axin, APC (15), and the N-cadherin tail (32) have been
described.
Cell Lines and Transfections. SW480, 293T, and Neuro 2A
cell lines were maintained in DMEM with 10% (volyvol) calf
serum. Transient transfections were performed by the calcium
phosphate precipitation method with 293T and Neuro 2A cell
lines and by Lipofectamine with SW480 cells. A
b
-galactosi-
dase-expressing plasmid (0.5
m
g) was included in each trans-
fection to monitor the transfection efficiency. We found that
b
-galactosidase expression was not affected significantly by
either LEF-1 or
b
-catenin cotransfection. After 48 h, the cells
were lysed, and luciferase and
b
-galactosidase activities were
determined by enzyme assay kits from Promega. Luciferase
activity was normalized to
b
-galactosidase activity as an in-
ternal transfection control. N-cadherin-tail-expressing cell
lines were generated by stably transfecting the pECE-N-
cadherin tail and pSV-hygro expression vectors into SW480
cells (7, 32). Individual clones were selected for resistance to
100
m
gyml hygromycin. The green fluorescent protein (GFP)–
DN-
b
-catenin and the mutant
b
-catenin constructs (
b
-cat Y33)
were HA-tagged (7).
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
PNAS is available online at www.pnas.org.
Abbreviations: APC, adenomatous polyposis coli; GSK-3
b
, glycogen
synthase kinase 3
b
; GFP, green fluorescent protein.
‡To whom reprint requests should be addressed. e-mail: lgbenzev@
weizmann.weizmann.ac.il.
5522

Immunoblotting. Protein levels were determined by immu-
noblotting by using a monoclonal antibody against cyclin D1
(DCS-6; Neomarkers, Freemont, CA), a polyclonal antiserum
against
b
-catenin, and monoclonal antibodies against pan
cadherin (CH-19), and vinculin (h-VIN 1) as described (ref. 7;
all from Sigma). Anti-LEF-1 rabbit antiserum was a gift from
R. Grosschedl (University of California, San Francisco).
In Vitro Translation and DNA Binding Analysis. In vitro
translated proteins were prepared by using a coupled transcrip-
tion and translation kit (Promega).
b
-Catenin and LEF-1 were
expressed from pCIneo constructs, and the efficiency of trans-
lation was determined by a reaction containing [
35
S]methionine,
followed by SDSyPAGE and Western blotting. For protein–DNA
interaction, in vitro translated LEF-1 and
b
-catenin were incu-
bated with the following
32
P-labeled duplex oligonucleotide
probes: CD1 (59-CTCTGCCGGGCTTTGATCTTTGCTTAA-
CAACA-39), CD1TOP (59-CTCTGCCGGCCTTTGATCTTT-
GCTTAACAACA-39), and CD1FOP (59-CTCTGCCGGGCT-
TTGGCCTTTGCTTAACAACA-39). The wild-type and mu-
tant LEF-1 binding sequences are underlined. The binding
reaction contained '40,000 cpm of
32
P-labeled DNA that was
incubated for 30 min with the in vitro translated proteins in 20 mM
Hepes, pH 7.9y75 mM NaCly1 mM dithiotreitoly2 mM MgCl
2
y
10% (vol/vol) glyceroly0.1 mg of BSAy10
m
g/ml salmon sperm
DNA. DNA–protein complexes were electrophoresed in 4%
native acrylamide gels and visualized by autoradiography. Bind-
ing reactions also were carried out with nuclear extracts from
SW480 cells (3
m
g protein per reaction mixture) that were
prepared as described (33).
RESULTS
Activation of the Cyclin D1 Promoter and Elevation of
Cyclin D1 in Cells Transfected with
b
-Catenin. We assessed
the effect of
b
-catenin overexpression on cyclin D1 abundance
in human 293T cells, previously shown to respond by activation
of
b
-cateninyLEF-1-driven transcription (7, 21, 22). Cells were
transfected with expression vectors encoding mutant N-
terminal
b
-catenin constructs (GFP–DN-
b
-catenin and
b
-cat
Y33) that render it stable against degradation (7, 22, 23).
Cyclin D1 protein levels were induced 2-to 3-fold after trans-
fection with these
b
-catenin constructs, as compared with
transfection with a construct encoding the GFP that served as
control (Fig. 1A). To examine whether the cyclin D1 promoter
was a direct transcriptional target for activation by the
b
-cate-
ninyLEF-1 complex, an HA-tagged DN-
b
-catenin expression
plasmid (pHA-DN-
b
-catenin) was cotransfected with the hu-
man cyclin D1 promoter linked upstream to a luciferase
reporter (Fig. 2A). The cyclin D1 promoter was induced
12-fold by this
b
-catenin (Fig. 1B) and was not affected by
either the empty expression plasmid (Fig. 1B; pA3) or the
construct encoding GFP. The cyclin D1 promoter was also
induced 10- to 15-fold by
b
-catenin overexpression in the
human Neuro 2A neuroblastoma cell line (see below; Fig. 3).
Identification of a LEF-1 Binding Sequence in the Cyclin D1
Promoter. To identify the sequences in the cyclin D1 promoter
that can confer transactivation by
b
-catenin, a series of 59
promoter deletion constructions were used (Fig. 2 A). Such
mapping identified a region between nucleotides 2141 and
266 that was sufficient for transcriptional activation by
b
-cate-
nin (Fig. 2B). Analysis of the sequence between nucleotides
2141 and 266 indicated that nucleotides 281 to 273 repre-
sent a consensus LEF binding site (Fig. 2 A,Insert). To examine
the functional significance of this candidate LEF binding site,
we introduced (in the context of the 2163 bp reporter plasmid,
2163CD1LUC) a deletion (2163DLefCD1LUC) and point
mutations (163mtLefCD1LUC) that should render the LEF
binding site in the cyclin D1 promoter inactive (Fig. 3B).
b
-Catenin-driven transactivation of the cyclin D1 constructs
was inhibited when this putative LEF-1-binding domain was
either deleted or mutated (Fig. 3A). Mutations introduced
outside of this region and in the Sp1 binding site of this
construct (2163DSp1CD1LUC; ref. 34) had no effect on
b
-catenin-driven activation of the promoter (Fig. 3A). Trans-
activation of the cyclin D1 promoter by
b
-catenin also de-
pended on the level of cotransfected LEF-1. At low concen-
trations of the expression plasmid, LEF-1 elevated
b
-catenin-
mediated activation of cyclin D1 a further 2.5-fold (Fig. 3C) but
inhibited transcription at high plasmid concentrations, consis-
tent with its transcription repressor role when expressed in
excess over
b
-catenin (35, 36).
The
b
-CateninyLEF-1 Complex Binds in Vitro to the LEF-1
Consensus Sequence of the Cyclin D1 Promoter. To determine
whether the LEF-1 binding sequence in the cyclin D1 promoter
can bind a LEF-1y
b
-catenin heterodimer, we conducted elec-
trophoretic mobility-shift assays comparing the cyclin D1
LEF-1 site with a consensus LEF-1-binding site (CD1TOP; ref.
9) and a mutant LEF-1 binding site (CD1FOP). In vitro,
synthesized LEF-1 bound to the LEF-1 sequence in the cyclin
D1 promoter (Fig. 4A, CD1, lane 2), and the complex was
FIG. 1. Induction of cyclin D1 and
b
-catenin-responsive transac-
tivation of the cyclin D1 promoter. (A) Cells from the 293T line were
transfected with 4
m
g of GFP, GFP-linked DN-
b
-catenin (GFP-DN-
b
-cat), or mutant
b
-cat Y33. Cell lysates were analyzed by Western
blotting for levels of cyclin D1,
b
-catenin (
b
-cat), and vinculin. (B)
Cells from the 293T line were transfected with 0.8
m
g of a reporter
plasmid containing 1,745 bp of the cyclin D1 promoter
(21745CD1Luc) or with the empty pA3 plasmid, together with 4
m
g
of an HA-tagged DN-
b
-catenin-encoding plasmid (pHA-DN-
b
-cat), a
GFP expression construct, or pCIneo. The bars represent luciferase
activity in cells transfected with pHA-DN-
b
-catenin divided by the
activity in cells transfected with control plasmid (pCIneo). Each
transfection was carried out in duplicate plates. The means 6SD from
three separate transfections are shown.
Cell Biology: Shtutman et al.Proc. Natl. Acad. Sci. USA 96 (1999) 5523

supershifted with an LEF-1 antibody (Fig. 4 A, lanes 4 and 8).
The mutant sequence derived from the 2163mtLefCD1LUC
(CD1FOP), in contrast, failed to bind LEF-1 (Fig. 4A, CD1FOP,
lane 10).
b
-Catenin transcribed and translated in vitro did not bind
on its own, as expected, to this sequence in the cyclin D1 promoter
(Fig. 4A, lanes 1, 5, and 9). A supershift was introduced in the
electrophoretic mobility of this DNA fragment when
b
-catenin
and LEF-1 were added together (Fig. 4A, lanes 3 and 7),
implying the formation of a ternary complex consisting of the
DNA, LEF-1, and
b
-catenin. This binding of LEF-1 to the
cyclin D1 promoter was blocked by the addition of excess
unlabeled CD1 but not by the mutant CD1FOP (Fig. 4B).
Transactivation of the Cyclin D1 Promoter in Colon Cancer
Cells and Its Inhibition by APC, Axin, and the Cadherin
Cytoplasmic Domain. To analyze effects on transcription of
the cyclin D1 promoter in a cellular system where endogenous
b
-catenin is elevated, we employed SW480 colon carcinoma
cells in which the level of
b
-catenin is increased because of
inactivating mutations in APC (37). Nuclear extracts from
these cells could specifically bind to and shift the electro-
phoretic mobility of the LEF-1 binding sequence in the cyclin
D1 promoter (Fig. 5A, lane 3), similar to in vitro translated
LEF-1 (Fig. 5A, lane 1). An additional shifted band (Fig. 5A,
marked X) bound nonspecifically; it also was observed when
the mutant LEF-1 binding site (CD1FOP) was used (Fig. 5A,
lanes 6– 8).
When the cyclin D1 promoter, in the context of the
2163CD1LUC reporter plasmid, was transfected into SW480
cells, transcription from this plasmid was active without co-
transfection of exogenous
b
-catenin (Fig. 5B). This transcrip-
tion was reduced by cotransfection of wild-type APC or a
mixture of APC and axin (Fig. 5B), which act together to
decrease levels of
b
-catenin by enhancing its degradation (15,
16). In contrast, APC and axin did not affect the basal
transcription observed in the deletion mutant of this cyclin D1
promoter construct (2163DLefCD1LUC; Fig. 5B). We have
FIG. 2. Identification of the LEF-1 binding sequence in the cyclin
D1 promoter. (A) Schematic representation of reporter constructs
from the cyclin D1 promoter, deletion constructs, and the LEF-1-
binding sequence between nucleotides 281 and 273 of the promoter
(Insert). (B) The promoter deletion constructs of the cyclin D1
promoter (0.8
m
g) shown in Awere transfected into 293T cells as
described in Fig. 1B.
FIG. 3. The LEF-1-binding sequence in the cyclin D1 promoter is
required for
b
-catenin-mediated transactivation. (A) Cells from the
293T line were transfected with the indicated reporter plasmid (0.8
m
g), together with 4
m
gofDN-
b
-catenin or the control plasmid
(pCIneo). The promoter activity is presented as in Fig. 1B.(B)
Schematic representation of mutations in the 2163 cyclin D1 pro-
moter construct, including an AT to GC change at nucleotides 275 and
274 (2163mtLefCD1LUC) and deletion of the LEF-1 binding site
(2163DLefCD1LUC) between nucleotides 281 to 273. (C) Neuro 2A
cells were transfected with 0.8
m
gof21745CD1Luc, 2
m
gofDN-
b
-
catenin, or a control plasmid (pCIneo), along with increasing amounts
of a LEF-1 expression plasmid. DNA concentrations were kept
constant with empty vector DNA. Transfections were carried out in
triplicate and the means 6SD are presented.
5524 Cell Biology: Shtutman et al.Proc. Natl. Acad. Sci. USA 96 (1999)

shown previously that either full-length N-cadherin or its
cytoplasmic domain can block the constitutive
b
-catenin-
mediated transactivation in SW480 cells by competing with
LEF-1 for
b
-catenin binding (7, 32). We therefore analyzed the
effect of a stably overexpressed N-cadherin cytoplasmic tail in
SW480 cells on the transcriptional activity of the cyclin D1
promoter and the level of the cyclin D1 protein. In SW480 cells
stably expressing high levels of the cadherin cytoplasmic tail
(Fig. 6B, clone A8) but not in clones expressing low levels of
the cadherin tail (Fig. 6B, clone A20),
b
-catenin-mediated
transactivation of the cyclin D1 promoter was inhibited (Fig.
6A; compare clones A8 and A20), and the level of cyclin D1
protein was reduced specifically (Fig. 6B; compare clones A8
and A20).
FIG. 4. Electrophoretic mobility-shift assays of the cyclin D1
promoter. (A) Duplex oligonucleotides containing the LEF-1 binding
sequences of the cyclin D1 promoter (CD1), a consensus LEF-1
binding sequences (CD1TOP) and a substitution of nucleotides 275
and 274 from AT to GC (CD1FOP) were ‘‘end labeled’’ with
[
32
P]dATP and incubated with in vitro translated LEF-1 andyor
b
-catenin or with anti-LEF-1 antibody. The protein–DNA complexes
were separated by electrophoresis and visualized by autoradiography.
(B)In vitro translated LEF-1 was incubated with the
32
P-labeled CD1
oligonucleotide, and increasing amounts of unlabeled CD1 or
CD1FOP oligonucleotides were used as competitors. The protein–
DNA complexes were analyzed as in Fig. 4A.
FIG. 5. Electrophoretic mobility-shift assays of the cyclin D1
promoter with SW480 nuclear lysates and regulation of cyclin D1
promoter transcriptional activity by APC and axin. (A) Oligonucleo-
tides labeled with [
32
P]dATP (CD1, lanes 1–5; CD1FOP, lanes 6–8)
were incubated with in vitro translated LEF-1 (lanes 1 and 2) or with
nuclear extracts from SW480 cells (lanes 3–8) in the presence of
100-fold excess of cold competitor oligonucleotides. (B) SW480 cells
were transiently transfected with 1
m
gof2163CD1LUC or
2163DLefCD1LUC and 2
m
g of either GFP or APC plus axin
expression vectors. The bars represent luciferase activity in the trans-
fected cells after normalizing for transfection efficiency with
b
-galac-
tosidase. Note that, in A, an additional, faster-migrating band was
obtained with CD1 and the SW480 nuclear extract (X). This band also
was seen with the mutant CD1FOP and therefore was considered
nonspecific.
Cell Biology: Shtutman et al.Proc. Natl. Acad. Sci. USA 96 (1999) 5525

DISCUSSION
Elevated
b
-catenin expression in colorectal cancer caused by
inactivating mutations in APC or in the GSK-3
b
phosphory-
lation sites of
b
-catenin results in the accumulation of
b
-cate-
nin in the nucleus and the uncontrolled activation of target
gene expression—a process believed to contribute to tumor
progression. The nature of these target genes is still largely
unknown, but their involvement in the control of cell prolif-
eration is suggested. In this study, we have identified the cyclin
D1 gene as a target for the
b
-cateninyLEF-1 complex. We have
shown that the LEF-1 binding consensus sequence in the cyclin
D1 gene promoter can bind to nuclear extracts from human
colon cancer cells and is transcriptionally active when linked to
reporter plasmids and introduced into human colon cancer
cells expressing elevated wild-type
b
-catenin. In such cells
expressing inactive mutant APC, cotransfection with wild-type
APC inhibited transactivation of the cyclin D1 gene. Based on
these results, we suggest that increased
b
-catenin levels in
colon cancer cells result in the activation of the cyclin D1 gene
promoter by the heterodimeric complex formed between
b
-catenin and LEF-1, which, in turn, results in the elevation of
cyclin D1 gene expression and protein level. This elevation, in
turn, may lead to uncontrolled stimulation of the cells into the
proliferative stage of the cell cycle. In the presence of wild-t ype
APC, the level of
b
-catenin is kept in check, and the formation
of the
b
-cateninyLEF-1 complex is prevented. The reduction
in the transcriptional activity of the cyclin D1 promoter,
resulting most probably from competition between the cyto-
plasmic tail of cadherin and LEF-1 for
b
-catenin binding (32),
and the concomitant decrease in cyclin D1 protein support this
view (Figs. 5 and 6). These results also show the potential
usefulness of the cadherin cytoplasmic domain (and its deriv-
atives) in the attempt to antagonize the possibly oncogenic
transcription driven by the
b
-cateninyLEF-1 complex (32).
Elevated transcription from the cyclin D1 gene induced by
the
b
-cateninyLEF-1 complex may also explain the notion
that, although alterations in the cyclin D1 gene were detected
in many types of human cancer (38–40), no mutations in the
cyclin D1 gene were identified in colorectal cancer, whereas
'30% of primary human colon tumors display increased
expression of cyclin D1 (28, 29). This increase in cyclin D1 in
only some colon cancers may result from the heterogeneity in
b
-catenin expression in the tumor and from the fact that many
colon cancer cell lines with mutant APC do not display
increased levels of
b
-catenin (41).
The involvement of cyclin D1 in human colon cancer also is
supported by experiments showing that expression of an
antisense cyclin D1 construct in SW480 colon cancer cells (also
used in this study) results in decreased cyclin D1, inhibition of
growth, and suppression of their tumorigenicity in nude
mice (30).
b
-Catenin and cyclin D1 apparently also share another
component of the Wnt pathway, GSK-3
b
. A recent study
showed that cyclin D1 is a target for GSK-3
b
, which, by
regulating cyclin D1 phosphorylation, may control the subcel-
lular localization and proteolysis of cyclin D1 by the ubiquitin–
proteasome system (42). This pathway may constitute another
means by which the Wnt signaling pathway can target cyclin D1
in colon cancer.
The link between
b
-catenin-mediated transactivation of the
cyclin D1 promoter and neoplastic transformation also may be
relevant to intestinal and mammary epithelial cells trans-
formed by the elevated expression of integrin-linked kinase
(ILK) (43). ILK-transfected cells were shown to become
neoplastic and to display high levels of cyclin D1 protein
constitutively (44). In addition, the level of extrajunctional
b
-catenin in such cells is increased, concomitant with nuclear
translocation of
b
-catenin and activation of
b
-catenin-
mediated LEF-1-directed transcription (45). Cyclin D1 eleva-
tion in these ILK-transfected cells may result from a direct
activation of cyclin D1 transcription by the
b
-cateninyLEF-1
complex.
In a recent study, the c-MYC promoter was identified also as
a target of the
b
-cateninyLEF-1 complex in human colorectal
cancer (26). In other types of tumors, overexpression of either
c-myc or cyclin D1 were shown to induce cell-cycle progression
in breast cancer cell lines (46), whereas, in fibroblasts, myc
induction of cell-cycle progression seems to be linked to the
induction of cyclin E, independently of cyclin D1 (47). To-
gether, these results are consistent with a model in which myc
and cyclin D1 can induce S phase entry in parallel pathways,
and the elevation of cyclin D1 in colorectal cancer, by inacti-
vation of APC or mutations in
b
-catenin, may promote
uncontrolled proliferation and thus contribute to the neoplas-
tic transformation of cells.
We are grateful to the following colleagues for sending reagents: S.
Sokol, R. Kemler, H. Clevers, M. van de Wetering, M. Wheelock, and
R. Grosschedl. These studies were supported by grants from the
USA–Israel Binational Foundation, the German–Israeli Foundation
for Scientific Research and Development, the Forchheimer Center for
Molecular Genetics, the Cooperation Program in Cancer Research
between the German Cancer Research Center and the Israel Ministry
of Science (to A.B.-Z.), and from the National Institute of Health (to
R.G.P.). A.B.-Z. holds the Lunenfeld–Kunin Chair in Cell Biology and
Genetics, and R.G.P. is a recipient of the Ira T. Hirschl Award and the
Susan G. Komen Breast Cancer Foundation Award.
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FIG. 6. Decreased transcription from the cyclin D1 promoter and
cyclin D1 protein in SW480 cells expressing the N-cadherin cytoplas-
mic domain. (A) Individual SW480 cell clones stably expressing
different levels of the N-cadherin cytoplasmic domain were transiently
transfected with 1
m
gof21745CD1LUC. The bars represent luciferase
activity in cells transfected with 21745CD1LUC divided by luciferase
activity in cells transfected with the empty pCIneo vector. (B) Total
cellular proteins from the cells described in Awere separated by
SDSyPAGE and analyzed by Western blotting with antibodies that
recognize the N-cadherin tail,
b
-catenin, and cyclin D1.
5526 Cell Biology: Shtutman et al.Proc. Natl. Acad. Sci. USA 96 (1999)

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