Home | Molecular Cancer Research - Heregulin Targets ......Surender Kharbanda,1,2 Massimo Loda,1 and...
Transcript of Home | Molecular Cancer Research - Heregulin Targets ......Surender Kharbanda,1,2 Massimo Loda,1 and...
-
Heregulin Targets ;-Catenin to the Nucleolusby a Mechanism Dependent on theDF3/MUC1 Oncoprotein
Yongqing Li,1,2 Wei-hsuan Yu,1 Jian Ren,1 Wen Chen,1 Lei Huang,1
Surender Kharbanda,1,2 Massimo Loda,1 and Donald Kufe1
1Dana-Farber Cancer Institute, Harvard Medical School and 2ILEX Products, Inc., Boston, MA
AbstractThe DF3/MUC1 transmembrane oncoprotein is
aberrantly overexpressed in most human breast
carcinomas and interacts with the Wnt effector
;-catenin. Here, we demonstrate that MUC1 associates
constitutively with ErbB2 in human breast cancer cells
and that treatment with heregulin/neuregulin-1 (HRG)
increases the formation of MUC1-ErbB2 complexes.
The importance of the MUC1-ErbB2 interaction is
supported by the demonstration that HRG induces
binding of MUC1 and ;-catenin and targeting of the
MUC1-;-catenin complex to the nucleolus. Significantly,
nucleolar localization of ;-catenin in response to HRG is
dependent on MUC1 expression. Moreover, mutation of a
RRK motif in the MUC1 cytoplasmic domain abrogates
HRG-induced nucleolar localization of MUC1 and
;-catenin. In concert with these results, we show
nucleolar localization of MUC1 and ;-catenin in human
breast carcinomas but not in normal mammary ductal
epithelium. These findings demonstrate that MUC1
functions in cross talk between ErbB2 and Wnt pathways
by acting as a shuttle for HRG-induced nucleolar
targeting of ;-catenin.
IntroductionThe ErbB family of receptor tyrosine kinases includes
ErbB1/epidermal growth factor receptor (EGFR), ErbB2/neu,
ErbB3, and ErbB4. Activation of ErbB1, ErbB3, and ErbB4 is
conferred by direct binding of at least 10 different growth
factors that induce receptor homodimerization and hetero-
dimerization (1). The ErbB2 receptor, which has no known
ligand, is transactivated through heterodimerization with the
other ErbB family members (2, 3). Stimulation of EGFR with
the epidermal growth factor (EGF) induces the formation of
EGFR-ErbB2 heterodimers (4). Similarly, heregulin/neuregu-
lin-1 (HRG) binds to the ErbB3 and ErbB4 receptors and
activates ErbB2 through heterodimerization and transphos-
phorylation (5). ErbB2 may thus function as a coreceptor
that potentiates signaling of the other ErbB family members
(6–8). Dimerization of the ErbB receptors results in activa-
tion of the intrinsic kinase function and phosphorylation of
tyrosine residues that serve as binding sites for proteins that
contain Src homology 2 or phosphotyrosine binding domains
(9, 10). Activation of ErbB2 is also associated with disrup-
tion of epithelial cell polarity and initiation of proliferation
(11, 12). In normal polarized glandular epithelial cells,
effectors of the Wnt signaling pathway, h- and g-catenin,are localized to the adherens junction where they function
with E-cadherin in cell-cell interactions (13). Loss of polarity
as found with ErbB2 activation (11), however, is associated
with catenin translocation from the adherens junction to the
cytoplasm and nucleus (14). A functional relationship between
ErbB2 signaling and Wnt regulation of catenins is unknown,
although both ErbB2 and Wnt have been linked to the
development of breast carcinomas.
Human DF3/MUC1 is a mucin-like transmembrane glyco-
protein, which is overexpressed by breast and other carcino-
mas (15). MUC1 expression is restricted to the apical borders
of normal secretory epithelial cells and is aberrantly expressed
by breast carcinoma cells at high levels over the entire
cell surface (15). Importantly, overexpression of MUC1 is
sufficient to induce transformation (16). The MUC1 protein
consists of a NH2-terminal (N-ter) ectodomain with variable
numbers of conserved 20-amino acid tandem repeats that are
modified by O-glycosylation (17, 18). The f25-kd COOH-terminal (C-ter) subunit includes a transmembrane domain and
a 72-amino acid cytoplasmic domain (CD). The extracellular
>250-kd ectodomain associates with the C-ter subunit as a
heterodimer. A SAGNGGSSL motif in the MUC1-CD
functions as a binding site for h-catenin (19). The SAGNG-GSSL motif also serves as a binding site for g-catenin(plakoglobin) (19). Glycogen synthase kinase 3h (GSK3h)phosphorylates MUC1 on serine in a SPY site adjacent to that
for h/g-catenin binding and decreases the interaction betweenMUC1 and h-catenin (20). Conversely, EGFR- or c-Src-mediated phosphorylation of MUC1 on tyrosine in the SPY
site up-regulates the formation of MUC1-h-catenin complexes(21, 22). The demonstration that MUC1 and E-cadherin, a
transmembrane protein that functions in Ca2+-dependent
epithelial cell-cell interactions (23), compete for binding to
h-catenin (20) has supported a role for MUC1 in regulating
Received 3/3/03; revised 6/20/03; accepted 6/24/03.The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Grant support: National Cancer Institute grant CA97098. Note: Y.L. and W.-h.Y.contributed equally to this work.Requests for reprints: Donald Kufe, Dana-Farber Cancer Institute, HarvardMedical School, Boston, MA 02115. Phone: (617) 632-3141; Fax: (617) 632-2934.E-mail: [email protected] D 2003 American Association for Cancer Research.
Vol. 1, 765–775, August 2003 Molecular Cancer Research 765
on April 5, 2021. © 2003 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
adherens junction function. Other studies have demonstrated
that MUC1 also colocalizes with h-catenin in the nucleus(16, 24). Less is known about the regulation of binding
between MUC1 and g-catenin.The present studies demonstrate that MUC1 interacts
with ErbB2 and that HRG stimulation of human breast
carcinoma cells is associated with increased binding of
MUC1 and g-catenin. The functional significance of thissignaling pathway is supported by the finding that HRG targets
g-catenin to the nucleolus by a MUC1-dependent mechanismand that a RRK motif in MUC1-CD is required for this
response.
ResultsHRG Induces the Association of MUC1 and ErbB2
Previous studies have demonstrated that human ZR-75-1
breast cancer cells express MUC1 and the four ErbB family
members (EGFR and ErbB2–4) (20, 22, 25). To determine
whether MUC1 associates with ErbB2, anti-MUC1 (DF3)
N-ter immunoprecipitates from lysates of human ZR-75-1
cells were analyzed by immunoblotting with anti-ErbB2. The
results demonstrate that ErbB2 coprecipitates with MUC1
(Fig. 1A). Whereas HRG stimulates ErbB2 activity, lysates
were prepared from ZR-75-1 cells treated with HRG for
5 min. Immunoblot analysis of anti-MUC1 immunoprecipi-
tates with anti-ErbB2 demonstrated that HRG stimulates the
formation of complexes containing MUC1 and ErbB2
(Fig. 1A). In the reciprocal experiment, immunoblot analysis
of anti-ErbB2 immunoprecipitates with anti-MUC1 confirmed
that HRG increases the basal association of MUC1 and
ErbB2 (Fig. 1A). Treatment of ZR-75-1 cells with EGF had
little (if any) effect on binding of MUC1 and EGFR (22). As
a control and in contrast to the effects of HRG, treatment
with EGF also had no apparent effect on binding of MUC1
and ErbB2 (data not shown). HRG binds to ErbB3 and ErbB4
and induces their heterodimerization with ErbB2 (3). To
determine whether MUC1 associates with ErbB3 or ErbB4,
immunoprecipitates prepared with antibodies against these
receptors were subjected to immunoblotting with anti-MUC1.
The results show that MUC1 associates with ErbB3 and
ErbB4 (Fig. 1B). Moreover, HRG stimulated the association
of MUC1 with ErbB3 and ErbB4, but to a much lesser extent
than that found for MUC1 and ErbB2 (Fig. 1B). To define
the subcellular localization of MUC1 and ErbB2, confocal
microscopy was performed with mouse anti-MUC1 and rabbit
anti-ErbB2. In control ZR-75-1 cells, MUC1 was distributed
uniformly over the cell membrane (Fig. 1C, left). A similar
pattern was obtained for the distribution of ErbB2 (Fig. 1C,
second panel). Overlay of the signals supported some
colocalization (red + green ! yellow) (Fig. 1C, right).Following HRG stimulation for 5 min, MUC1 was clustered
in patches on the cell surface (Fig. 1D, left). Staining for
ErbB2 revealed a similar pattern (Fig. 1D, second panel), and
overlay of the signals showed increased colocalization of
MUC1 and ErbB2 in clusters at the cell membrane (Fig. 1D,
right). There was no apparent HRG-induced localization of
MUC1 N-ter to the nucleus (Fig. 1D). Moreover, as a control,
there was no increased colocalization of MUC1 and ErbB2
in cells stimulated with EGF (Fig. 1E). These findings
demonstrate that colocalization of MUC1 and ErbB2 at the
cell membrane is regulated by HRG stimulation.
HRG Regulates Interaction of MUC1 and c-CateninTo determine whether HRG affects the interaction be-
tween MUC1 and catenins, lysates from control and HRG-
treated ZR-75-1 cells were subjected to immunoprecipitation
with anti-MUC1. Immunoblot analysis of the precipitates with
anti-h-catenin demonstrated that HRG has little effect onbinding of MUC1 and h-catenin (Fig. 2A). By contrast, HRGtreatment was associated with an increase in binding of MUC1
and g-catenin (Fig. 2A). For comparison, ZR-75-1 cells werestimulated with EGF. As shown previously, EGF induced
binding of MUC1 and h-catenin (22) (Fig. 2B). Conversely,EGF had little effect on the interaction of MUC1 with
g-catenin (Fig. 2B). To extend these findings, we used humanHCT116 carcinoma cells that are MUC1 negative as
determined by immunoblotting with anti-MUC1 antibodies
and by reverse transcription-PCR for sequences encoding the
C-ter [(26) and data not shown]. Moreover, flow cytometric
analysis of HCT116 cells demonstrated that all four ErbB
family members are expressed at the cell membrane and that
ErbB2 is detectable at somewhat higher levels than these
found for EGFR, ErbB3, and ErbB4 (Fig. 2C). HCT116 cells
that stably express an empty vector or MUC1 were treated
with HRG. In concert with the findings in ZR-75-1 cells,
immunoblot analysis of anti-MUC1 immunoprecipitates with
anti-g-catenin demonstrated that HRG induces binding ofMUC1 and g-catenin (Fig. 2D). These findings indicate thatHRG stimulates the formation of MUC1-g-catenin complexes.
Nucleolar Localization of MUC1-c-Catenin ComplexesTo define the subcellular localization of MUC1-g-catenin
complexes, ZR-75-1 cells were analyzed by confocal
microscopy after incubation with antibodies against MUC1
C-ter and g-catenin. The results show colocalization of MUC1C-ter and g-catenin at the cell membrane (Fig. 3A). Bycontrast, HRG stimulation for 20 min was associated with
localization of MUC1 C-ter in the nucleus (Fig. 3B). A similar
pattern was observed for g-catenin, and overlay demonstratedcolocalization with MUC1 C-ter (Fig. 3B). The well-circum-
scribed colocalization of MUC1 and g-catenin in the nucleussuggested a nucleolar pattern (Fig. 3B). Indeed, staining with
an anti-nucleolin antibody confirmed HRG-induced redistri-
bution of MUC1 C-ter to the nucleolus (Fig. 3C). A similar
pattern of nucleolar colocalization for MUC1 C-ter with
g-catenin was observed in the ErbB2-positive MCF-7 breastcancer cells (data not shown). Notably, stimulation of ZR-75-1
cells with EGF was associated with localization of MUC1
C-ter in a diffuse pattern throughout the nucleus (Fig. 3D).
Moreover, the lack of colocalization with nucleolin indicated
that EGF induces nuclear targeting of MUC1 C-ter to
nonnucleolar sites (Fig. 3D). Following EGF stimulation,
nuclear MUC1 C-ter colocalizes with h-catenin and notg-catenin (unpublished data).
Nucleolar Targeting of g-Catenin by MUC1766
on April 5, 2021. © 2003 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
Role of MUC1 in the Subcellular Distribution of c-CateninTo assess the functional role of MUC1 in g-catenin
signaling, HCT116/vector and HCT116/MUC1 cells were
analyzed for localization of g-catenin following HRG stimu-lation. The confocal images show that g-catenin localizes to thecell membrane of HCT116/vector cells (Fig. 4A). Moreover,
treatment of the HCT116/vector cells with HRG for 20 min had
no apparent effect on the distribution of g-catenin (Fig. 4A).In HCT116/MUC1 cells, MUC1 C-ter and g-catenin werepredominantly detectable at the cell membrane (Fig. 4B). By
contrast, HRG treatment of HCT116/MUC1 cells for 20 min
was associated with colocalization of MUC1 C-ter and g-cateninin discrete nuclear structures (Fig. 4B). As found in ZR-75-1
cells, colocalization of MUC1 C-ter and nucleolin indicated
that MUC1 C-ter and g-catenin are targeted to the nucleolus(data not shown).
Whereas a RRK motif in MUC1-CD may contribute to
nuclear localization, similar studies were performed on
HCT116 cells stably expressing a MUC1(RRK ! AAA)mutant. Coimmunoprecipitation studies demonstrated that
binding of MUC1 to g-catenin is not affected by the RRK! AAA mutation (data not shown). In contrast to HCT116/
FIGURE 1. HRG stimulates interaction of MUC1 and ErbB2. ZR-75-1 cells were left untreated or stimulated with 20-ng/ml HRG for 5 min. A. Lysateswere subjected to immunoprecipitation (IP ) with anti-MUC1 (DF3) N-ter (left panel ) or anti-ErbB2 (right panel ). Mouse IgG was used as a control. Theimmunoprecipitates were analyzed by immunoblotting (IB ) with anti-ErbB2 and anti-MUC1 N-ter. Intensity of the signals was determined bydensitometric scanning and compared with that obtained for untreated cells. B. Lysates from control and HRG-treated ZR-75-1 cells were subjected toimmunoprecipitation with anti-ErbB3 (left panel ) or anti-ErbB4 (right panel ). The immunoprecipitates were analyzed by immunoblotting with the indicatedantibodies. ZR-75-1 cells were grown to 60% confluence and incubated in medium with 0.1% serum for 24 h. The cells were left untreated (C),stimulated with 20-ng/ml HRG for 5 min (D), or stimulated with 10-ng/ml EGF for 5 min (E), fixed, and double stained with anti-MUC1 N-ter (green ) andanti-ErbB2 (red ).
Molecular Cancer Research 767
on April 5, 2021. © 2003 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
vector cells (Fig. 5A), MUC1 C-ter staining was intense over
the cell membrane of HCT116/MUC1(RRK ! AAA) cells(Fig. 5B). Similar patterns were observed for g-catenin in bothHCT116/vector and HCT116/MUC1(RRK ! AAA) cells (Fig.5, A and B). However, in contrast to HCT116/vector cells
(Fig. 5A), stimulation of HCT116/MUC1(RRK ! AAA) cellswith HRG for 20 min was associated with redistribution of
both MUC1 C-ter and g-catenin to the cytoplasm (Fig. 5B).Moreover, there was no detectable HRG-induced targeting of
MUC1 C-ter and g-catenin to the nucleolus (Fig. 5B).To extend these observations, the localization of MUC1
C-ter and g-catenin was assessed by subcellular fractionationof control and HRG-treated cells. Immunoblot analysis of the
nuclear fractions demonstrated that MUC1 C-ter is detectable
in the nuclei of HCT116/MUC1 cells but not of HCT116/
vector or HCT116/MUC1(RRK ! AAA) cells (Fig. 6). Theresults also demonstrate that HRG increases nuclear targeting
of MUC1 C-ter in the HCT116/MUC1 cells (Fig. 6). More-
over, HRG treatment of HCT116/MUC1, but not HCT116/vector
or HCT116/MUC1(RRK ! AAA), was associated with anincrease in nuclear g-catenin (Fig. 6). Equal loading of thenuclear fractions was confirmed by immunoblotting for lamin B
(Fig. 6). Moreover, purity of the nuclear preparations was
demonstrated with antibodies against the cytosolic InBa, themembrane-associatedMUC1N-ter subunit, and the endoplasmic
reticulum protein, calreticulin (Fig. 6). These findings collec-
tively indicate that the RRK motif is important for nucleolar
localization of MUC1 C-ter and g-catenin in the response toHRG stimulation.
Confocal Microscopy of Human Breast CarcinomasTo define the localization of MUC1 C-ter and g-catenin in
mammary tissues, confocal microscopy was first performed on
normal ductal epithelium. The results show localization of
MUC1 C-ter along the apical borders of the epithelial cells
lining the ducts (Fig. 7A). g-Catenin colocalized with MUC1C-ter at the apical borders and was expressed at lateral borders
of the ductal epithelium (Fig. 7A). Little (if any) MUC1 C-ter
or g-catenin was detectable in the nucleus (Fig. 7A).Significantly, sections from ErbB2-positive breast carcinomas
showed immunoflourescence staining of MUC1 C-ter and
g-catenin as discrete nuclear clusters (Fig. 7B). Sections werealso stained with anti-MUC1 C-ter and antinucleolin. The
results demonstrate prominent colocalization of MUC1 C-ter
FIGURE 2. HRG stimulates the interaction between MUC1and g-catenin. A. Lysates from ZR-75-1 cells left untreated orstimulated with HRG for 5 min were subjected to immuno-precipitation with anti-MUC1 N-ter or, as a control, IgG. Theimmunoprecipitates were analyzed by immunoblotting withthe indicated antibodies. B. Lysates from ZR-75-1 cells leftuntreated or stimulated with 10-ng/ml EGF for 5 min weresubjected to immunoprecipitation with anti-MUC1 or IgG. Theimmunoprecipitates were analyzed by immunoblotting withthe indicated antibodies. C. HCT116 cells were incubatedwith antibodies against the indicated ErbB family members(open patterns ) or control mouse IgG (solid patterns ) andanalyzed by flow cytometry. Similar results were obtainedfor HCT116/MUC1 cells. D. HCT116/vector (HCT116/V ) andHCT116/MUC1 cells were left untreated or stimulated withHRG. Anti-MUC1 N-ter immunoprecipitates were subjectedto immunoblotting with anti-g-catenin or anti-MUC1 N-ter.
Nucleolar Targeting of g-Catenin by MUC1768
on April 5, 2021. © 2003 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
and nucleolin in breast carcinoma cells (Fig. 7C). Similar
results were obtained for g-catenin and nucleolin (Fig. 7D). Theresults indicate that over 50% of the breast cancer cells within
invasive islands exhibit nucleolar localization of MUC1 C-ter
and g-catenin. These findings in tissues and those in culturedcells collectively demonstrate that MUC1-CD and g-catenin aretargeted to nucleolus.
DiscussionInteraction of MUC1 and ErbB2
The MUC1 mucin-like glycoprotein is expressed on the
apical borders of normal mammary epithelium and at
substantially increased levels over the entire cell surface of
breast carcinoma cells (15). Significantly, overexpression of
MUC1 is associated with transformation as evidenced by
anchorage-independent growth and tumorigenicity (16). The
shed MUC1 N-ter is believed to function in the generation of a
protective mucous barrier. The function of the C-ter, which
consists of an extracellular domain of f58 amino acids, atransmembrane domain, and a 72-amino acid cytoplasmic tail,
is largely unknown. The finding that MUC1-CD binds directly
to h- and g-catenin suggested that the C-ter might function intransducing signals from the cell surface to the interior of the
cell (19). Indeed, the demonstration that MUC1-CD functions
as a substrate for GSK3h (20) and c-Src (21) has indicated thatthe MUC1 C-ter may function in integrating signals from the
Wnt and growth factor receptor pathways. In this context,
activation of the EGFR is associated with tyrosine phospho-
rylation of MUC1-CD and regulation of the interaction between
MUC1 and h-catenin (22, 27).Recent studies have shown that MUC1 associates with EGFR
and ErbB2–4 in pregnant and lactating mouse mammary glands
(27). The present work has explored the interaction between
MUC1 and ErbB2–4 in human breast cancer cells. The results
FIGURE 3. HRG induces nu-cleolar colocalization of MUC1C-ter and g-catenin. ZR-75-1cells were grown to 60% conflu-ence and incubated in mediumwith 0.1% serum for 24 h. Thecells were left untreated (A) orstimulated with 20-ng/ml HRGfor 20 min (B), fixed, and doublestained with anti-MUC1 C-ter(green ) and anti-g-catenin (red).Nuclei were stained with SYN-TOX blue. High (�100) (upperpanels ) and low (�63) (lowerpanels ) magnifications areshown. ZR-75-1 cells were stim-ulated with 20-ng/ml HRG for20 min (C) or with 10-ng/mlEGF for 20 min (D), fixed, andstained with anti-MUC1 C-ter andanti-nucleolin.
Molecular Cancer Research 769
on April 5, 2021. © 2003 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
of coimmunoprecipitation studies demonstrate the association of
MUC1 with ErbB2–4. Significantly, treatment with HRG is
associated with increases in MUC1-ErbB2 complexes and
colocalization of these complexes in clusters at the cell
membrane (Fig. 8). Members of the ErbB family form both
homodimers and heterodimers in response to the diverse ligands
that stimulate these receptors (1, 28). The available evidence
suggests that ErbB2 functions as a coreceptor and is a preferred
heterodimerization partner among the ErbB family members
(1, 28). In addition, ErbB2 is overexpressed in in situ and
invasive ductal carcinomas of the breast (28). The finding that
HRG stimulates the association between ErbB2 and MUC1 may
therefore be of importance to ErbB2 signaling, particularly in
tumors that overexpress both of these proteins.
Interaction of MUC1 and c-Cateninh- and g-catenin bind directly to MUC1 at a SAGNGGSSL
motif in the CD (19). These vertebrate homologues of
Drosophila armadillo are found in the adherens junction
where they link E-cadherin to the actin cytoskeleton through
a-catenin (29). The finding that complexes between MUC1and h- or g-catenin contain little (if any) a-catenin hassupported a function distinct from their roles with E-cadherin
(19). In this regard, other studies have indicated that MUC1 and
E-cadherin compete for the same pool of h-catenin (20).Moreover, negative regulation of the MUC1-h-catenin interac-tion by GSK3h is associated with increased binding of h-catenin to E-cadherin (20). In this model, down-regulation of
GSK3h by Wnt signaling would subvert E-cadherin functionin homotypic cell-cell interactions by titrating binding of h-catenin to MUC1. MUC1 is expressed along the apical borders
of normal ductal epithelial cells that are devoid of cell-cell
interactions. By contrast, aberrant expression of MUC1 over
the entire surface of carcinoma cells may contribute to loss of
E-cadherin function by disrupting interactions with h- and/org-catenin.
The present results show that the MUC1-ErbB2 interaction
is associated with HRG-induced binding of MUC1 and g-catenin (Fig. 8). HRG stimulation had less of an effect on
the interaction between MUC1 and h-catenin. Conversely,EGFR signaling increases binding of MUC1 and h-catenin(22) but has little effect on the interaction between MUC1 and
g-catenin. EGFR signaling also increases phosphorylation ofMUC1 on tyrosine in the SPY site (22), while HRG
stimulation had no apparent effect on tyrosine phosphorylation
of MUC1-CD (data not shown). Activation of ErbB2, but not
FIGURE 4. MUC1 is neces-sary for HRG-induced targetingof MUC1 C-ter and g-catenin tothe nucleolus. HCT116/vector (A)and HCT116/MUC1 (B) cells wereleft untreated or stimulated withHRG for 20 min. The cells wereassessed for reactivity with anti-MUC1 C-ter and anti-g-catenin.Nuclei were stained with SYNTOXblue.
Nucleolar Targeting of g-Catenin by MUC1770
on April 5, 2021. © 2003 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
EGFR, in growth-arrested mammary acini results in reinitiation
of proliferation, disruption of tight junctions, loss of polarity,
and filled lumina (11). These results indicate that ErbB2
activation can selectively disrupt regulation of mammary epi-
thelial cell proliferation and organization. Other effectors, such
as Rac, Cdc42, and PI3K, which induce invasiveness of
mammary epithelial cells, may cooperate with ErbB2 in
disrupting polarized epithelia (30). One report has also
indicated that ErbB2 suppresses E-cadherin expression in
mammary epithelial cells (31), but such regulation was not
found in other studies (11). The present findings provide
evidence for the involvement of ErbB2 activation and the
regulation of g-catenin signaling as another potential mecha-nism for increasing invasiveness. Thus, HRG-induced increases
in binding of g-catenin to MUC1 could decrease the availabilityof g-catenin for linking E-cadherin to the actin cytoskeletonand thereby disrupt homotypic cell-cell signaling.
Nucleolar Localization of MUC1 C-Ter and c-CateninThe present results further indicate that HRG stimulation
is associated with nuclear targeting of MUC1 C-ter and g-catenin (Fig. 8). The well-circumscribed nuclear distribution
of the MUC1 C-ter signal and colocalization with anti-
nucleolin staining supported compartmentalization of MUC1
C-ter in the nucleolus. Similar results were obtained with g-catenin, supporting the likelihood that the MUC1-g-catenincomplex is targeted to the nucleolus in response to HRG
stimulation. In concert with these findings, MUC1 C-ter and
g-catenin are detectable in nucleoli of ErbB2-positive primarybreast carcinomas. The observation that over 50% of the
breast cancer cells exhibit nucleolar colocalization of MUC1
C-ter and g-catenin indicate that, as found in vitro , MUC1may interact with the ErbB2 signaling pathway in primary
breast carcinomas. The nucleolus is a membrane-free nuclear
subdomain in which rRNAs are transcribed and processed
into ribosome subunits (32). Additional functions that may be
attributable to the nucleolus include the processing of other
ribonucleoproteins (33, 34) and export of mRNAs and
tRNAs (35, 36). In addition, the nucleolus may function in
sequestering specific regulatory factors (37). For example,
Mdm2 is sequestered in the nucleolus by an ARF-dependent
mechanism (38–40). Disassembly of the nucleolus during
cell cycle progression can in turn release sequestered factors.
In the nucleus, g-catenin interacts with the T-cell factor/lymphoid enhancer factor transcription factors and functions
as a coactivator. Like h-catenin, g-catenin can contribute tocell transformation by a mechanism involving transactivation of
c-Myc expression (41).
FIGURE 5. Nucleolar locali-zation of MUC1 C-ter and g-catenin is conferred by theMUC1 RRK motif. HCT116/vec-tor (A) and HCT116/MUC1(RRK! AAA) (B) cells were left un-treated or stimulated with HRGfor 20 min. Cells were analyzedfor staining with anti-MUC1 C-terand anti-g-catenin. Morphology ofthe cells was visualized by bright-field microscopy.
Molecular Cancer Research 771
on April 5, 2021. © 2003 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
Activation of the Wnt signaling pathway is associated with
accumulation of h- and g-catenin in the nucleus. Themechanisms responsible for targeting h- and g-catenin to thenucleus are not clear. Neither protein has a definitive nuclear
localization signal; however, h-catenin is imported into thenucleus by binding directly to the nuclear pore machinery
(42). Moreover, binding to T-cell factor/lymphoid enhancer
factor transcription factors is probably not responsible for
nuclear localization of h-catenin (43). The adenomatouspolyposis coli protein can function as a h-catenin chaperonein nuclear export but apparently not in nuclear import (44, 45).
Recent studies have demonstrated that MUC1 colocalizes with
h-catenin in the nucleus and increases nuclear levels of h-
catenin (16, 24). These findings have indicated that MUC1
may function in the import and/or stabilization of nuclear
h-catenin. Importantly, the nuclear colocalization of MUC1-h-catenin complexes is found outside the nucleolus (16, 24,and unpublished data).
The present results in HCT116/vector and HCT116/MUC1
cells indicate that HRG-induced nucleolar localization of
g-catenin is dependent on MUC1 expression. The MUC1-CDcontains a RRK motif that may function as a monopartite
nuclear localization signal (46). Studies of the c-Myc nuclear
localization signal (PAAKRVKLD) have demonstrated the
functional role of neutral amino acids and the dipeptide LD in
nuclear targeting (47). The RRK basic cluster in the MUC1-CD
is also flanked by neutral amino acids and the LD dipeptide
(CQCRRKNYGQLD). Importantly, mutation of the MUC1
RRK motif to AAA abrogated HRG-induced nucleolar
localization of MUC1 C-ter. In addition, targeting of g-cateninto the nucleolus in response to HRG was not found in cells
expressing the MUC1(RRK ! AAA) mutant. These findingsprovide the first evidence that MUC1 functions in nuclear
signaling and that g-catenin is transported to the nucleolus by aMUC1-dependent mechanism.
Materials and MethodsCell Culture
Human ZR-75-1 and MCF-7 breast carcinoma cells
(American Type Culture Collection, Manassas, VA) were
cultured in RPMI 1640 high-glucose medium containing 10%
heat-inactivated fetal bovine serum (HI-FBS), 100-U/ml
penicillin, 100-Ag/ml streptomycin, and 2-mM L-glutamine.HCT116 colon carcinoma cells (American Type Culture
Collection) were grown in DMEM containing 10% HI-FBS
and antibiotics. Cells were maintained in medium with 0.1%
HI-FBS for 24 h and stimulated with 20-ng/ml HRG or 10-ng/
ml EGF (Calbiochem-Novabiochem, San Diego, CA) at 37jC.
Cell TransfectionspIRESpuro2, pIRESpuro2-MUC1, and pIRESpuro2-
MUC1(RRK ! AAA) were transfected into HCT116 cells byLipofectAMINE. Stable transfectants were selected in the
presence of 0.4-Ag/ml puromycin (Calbiochem-Novabiochem).
Immunoprecipitation and ImmunoblottingLysates were prepared from subconfluent cells as described
(20). Equal amounts of cell lysate protein were incubated with
antibody DF3 (anti-MUC1) (15), anti-ErbB2 (Santa Cruz
Biotechnology, Santa Cruz, CA), anti-ErbB3 (Santa Cruz
Biotechnology), anti-ErbB4 (Santa Cruz Biotechnology), or
mouse IgG. The immune complexes were prepared as described
(20), separated by SDS-PAGE, and transferred to nitrocellulose
membranes. The immunoblots were probed with anti-MUC1,
anti-ErbB2, anti-ErbB3, anti-ErbB4, anti-h-catenin (Zymed, SanFrancisco, CA), or anti-g-catenin (Zymed). Reactivity wasdetected with horseradish peroxidase-conjugated second anti-
bodies and chemiluminescence (Perkin-Elmer Corp., Boston,
MA).
Immunoflourescence Confocal MicroscopyCultured cells were washed three times in PBS (containing
FIGURE 6. HRG-induced nuclear localization of MUC1 and g-catenin.Nuclear fractions were analyzed by immunoblotting with the indicatedantibodies. Whole cell lysates (WCL ) were used as a positive control.
Nucleolar Targeting of g-Catenin by MUC1772
on April 5, 2021. © 2003 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
Mg2+ and Ca2+), fixed with 3.7% formaldehyde in buffer A
(PBS containing 10-AM ZnCl2) for 10 min, permeabilized with0.25% Triton X-100/3.7% formaldehyde in buffer A for 5 min,
and postfixed with 3.7% formaldehyde in buffer A for 5 min.
The cells were then washed three times with PBS and
incubated with blocking buffer (PBS containing 4%
protease-free BSA and 5% normal goat serum). Incubation
with anti-MUC1, anti-ErbB2, anti-MUC1 C-ter (Neomarkers,
FIGURE 7. Colocalization of MUC1C-ter and g-catenin to the nucleolus ofhuman breast carcinoma cells. Sectionsof normal mammary ductal epithelium (A)and two anti-HER2/ErbB2-positive pri-mary invasive ductal breast carcinomas(B, upper and lower panels ) were as-sessed for reactivity with anti-MUC1 C-terand anti-g-catenin. Morphology was visu-alized at high and low (inset ) power byH&E staining. Breast carcinoma cells werestained with anti-MUC1 C-ter (C) or anti-g-catenin (D) and anti-nucleolin.
Molecular Cancer Research 773
on April 5, 2021. © 2003 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
Fremont, CA), anti-g-catenin, and anti-nucleolin (ResearchDiagnostics, Flanders, NJ) in blocking buffer was performed
overnight at 4jC. The cells were washed with PBS, incubatedovernight with secondary FITC- or Texas Red-conjugated goat
anti-hamster or anti-mouse IgG antibodies (Jackson Immuno-
Research Laboratories, West Grove, PA) at 4jC, washed withPBS, washed three times with buffer B (20-mM Tris, pH 7.5,
0.15-M NaCl), and stained with 0.2-AM of SYNTOX BlueNuclei C solution for 2 h. After washing again with buffer B, the
cells were mounted with Slowfade solution and analyzed by
confocal microscopy using an inverted Zeiss LSM510 scope
(Carl Zeiss, Inc., Thornwood, NY). Images were captured at
0.6-nm increments along the Z axis and converted to composites
by LSM510 software version 3.0.
Flow CytometryCells were incubated with anti-EGFR, anti-ErbB2, anti-
ErbB3, anti-ErbB4, or mouse IgG for 30 min, washed,
incubated with goat antimouse immunoglobulin-flourescein-
conjugated antibody (Santa Cruz Biotechnology), and fixed
in 1% formaldehyde/PBS. Reactivity was detected by immu-
noflourescence FACScan.
Subcellular FractionationPreparation of nuclear fractions was performed as described
(48). Purity of the fractionations was monitored by immunoblot
analysis with anti-lamin B (Oncogene Science, Cambridge,
MA), anti-calreticulin (Santa Cruz Biotechnology) and anti-
InBa (Santa Cruz Biotechnology) antibodies.
AcknowledgmentsThe authors acknowledge Kamal Chauhan for excellent technical support. D.K.has a financial interest in ILEX.
References1. Olayioye, M. A., Neve, R. M., Lane, H. A., and Hynes, N. E. The ErbBsignaling network: receptor heterodimerization in development and cancer.EMBO J., 19: 3159–3167, 2000.
2. Carraway, K. L., III and Cantley, L. C. A neu acquaintance for erbB3 anderbB4: a role for receptor heterodimerization in growth signaling. Cell, 78: 5– 8,1994.
3. Riese D. J., II and Stern, D. F. Specificity within the EGF family/ErbBreceptor family signaling network. Bioessays, 20: 41–48, 1998.
4. Wada, T., Qian, X. L., and Greene, M. I. Intermolecular association of thep185neu protein and EGF receptor modulates EGF receptor function. Cell, 61:1339– 1347, 1990.
5. Plowman, G. D., Culouscou, J. M., Whitney, G. S., Green, J. M., Carlton,G. W., Foy, L., Neubauer, M. G., and Shoyab, M. Ligand-specific activation ofHER4/p180erbB4, a fourth member of the epidermal growth factor receptorfamily. Proc. Natl. Acad. Sci. USA, 90: 1746– 1750, 1993.
6. Tzahar, E., Waterman, H., Chen, X., Levkowitz, G., Karunagaran, D., Lavi, S.,Ratzkin, B. J., and Yarden, Y. A hierarchical network of interreceptor interactionsdetermines signal transduction by Neu differentiation factor/neuregulin andepidermal growth factor. Mol. Cell. Biol., 16: 5276–5287, 1996.
7. Graus-Porta, D., Beerli, R. R., Daly, J. M., and Hynes, N. E. ErbB-2, thepreferred heterodimerization partner of all ErbB receptors, is a mediator of lateralsignaling. EMBO J., 16: 1647–1655, 1997.
8. Olayioye, M. A., Graus-Porta, D., Beerli, R. R., Rohrer, J., Gay, B., andHynes, N. E. ErbB-1 and ErbB-2 acquire distinct signaling properties dependentupon their dimerization partner. Mol. Cell. Biol., 18: 5042– 5051, 1998.
9. Ricci, A., Lanfrancone, L., Chiari, R., Belardo, G., Pertica, C., Natali, P. G.,Pelicci, P. G., and Segatto, O. Analysis of protein-protein interactions involved inthe activation of the Shc/Grb-2 pathway by the ErbB-2 kinase. Oncogene, 11:1519– 1529, 1995.
10. Zrihan-Licht, S., Deng, B., Yarden, Y., McShan, G., Keydar, I., and Avraham,H. Csk homologous kinase, a novel signaling molecule, directly associates withthe activated ErbB-2 receptor in breast cancer cells and inhibits their proliferation.J. Biol. Chem., 273: 4065–4072, 1998.
11. Muthuswamy, S. K., Gilman, M., and Brugge, J. S. Controlled dimerizationof ErbB receptors provides evidence for differential signaling by homo- andheterodimers. Mol. Cell. Biol., 19: 6845–6857, 1999.
12. Janda, E., Litos, G., Grunert, S., Downward, J., and Beug, H. OncogenicRas/Her-2 mediate hyperproliferation of polarized epithelial cells in 3Dcultures and rapid tumor growth via the PI3K pathway. Oncogene, 21: 5148–5159, 2002.
13. Polakis, P. Wnt signaling and cancer. Genes Dev., 14: 1837–1851, 2000.
14. Lin, S. Y., Xia, W., Wang, J. C., Kwong, K. Y., Spohn, B., Wen, Y., Pestell,R. G., and Hung, M. C. h-catenin, a novel prognostic marker for breast cancer: itsroles in cyclin D1 expression and cancer progression. Proc. Natl. Acad. Sci. USA,97: 4262–4266, 2000.
15. Kufe, D., Inghirami, G., Abe, M., Hayes, D., Justi-Wheeler, H., andSchlom, J. Differential reactivity of a novel monoclonal antibody (DF3)with human malignant versus benign breast tumors. Hybridoma, 3: 223 –232,1984.
16. Li, Y., Liu, D., Chen, D., Kharbanda, S., and Kufe, D. Human DF3/MUC1carcinoma-associated protein functions as an oncogene. Oncogene, 22: 6107–6110, 2003.
17. Gendler, S., Taylor-Papadimitriou, J., Duhig, T., Rothbard, J., and Burchell,J. A. A highly immunogenic region of a human polymorphic epithelial mucinexpressed by carcinomas is made up of tandem repeats. J. Biol. Chem., 263:12820 –12823, 1988.
18. Siddiqui, J., Abe, M., Hayes, D., Shani, E., Yunis, E., and Kufe, D.Isolation and sequencing of a cDNA coding for the human DF3 breastcarcinoma-associated antigen. Proc. Natl. Acad. Sci. USA, 85: 2320– 2323,1988.
19. Yamamoto, M., Bharti, A., Li, Y., and Kufe, D. Interaction of the DF3/MUC1breast carcinoma-associated antigen and h-catenin in cell adhesion. J. Biol.Chem., 272: 12492 –12494, 1997.
20. Li, Y., Bharti, A., Chen, D., Gong, J., and Kufe, D. Interaction of glycogensynthase kinase 3h with the DF3/MUC1 carcinoma-associated antigen andh-catenin. Mol. Cell. Biol., 18: 7216–7224, 1998.
21. Li, Y., Kuwahara, H., Ren, J., Wen, G., and Kufe, D. The c-Src tyrosinekinase regulates signaling of the human DF3/MUC1 carcinoma-associatedantigen with GSK3h and h-catenin. J. Biol. Chem., 276: 6061–6064, 2001.
22. Li, Y., Ren, J., Yu, W.-H., Li, G., Kuwahara, H., Yin, L., Carraway, K. L.,and Kufe, D. The EGF receptor regulates interaction of the human DF3/MUC1
FIGURE 8. Schematic representation of the involvement of MUC1 inHRG-induced targeting of g-catenin to the nucleolus.
Nucleolar Targeting of g-Catenin by MUC1774
on April 5, 2021. © 2003 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
carcinoma antigen with c-Src and h-catenin. J. Biol. Chem., 276: 35239–35242, 2001.
23. Takeichi, M. Cadherins: a molecular family important in selective cell-celladhesion. Annu. Rev. Biochem., 59: 237– 252, 1990.
24. Li, Y., Chen, W., Ren, J., Yu, W., Li, Q., Yoshida, K., and Kufe, D. DF3/MUC1 signaling in multiple myeloma cells is regulated by interleukin-7. CancerBiol. Ther., 2: 187–193, 2003.
25. Shimizu, H., Koyama, N., Asada, M., and Yoshimatsu, K. Aberrantexpression of integrin and erbB subunits in breast cancer cell lines. Int. J.Oncol., 21: 1073–1079, 2002.
26. Ren, J., Li, Y., and Kufe, D. Protein kinase C y regulates function of the DF3/MUC1 carcinoma antigen in h-catenin signaling. J. Biol. Chem., 277: 17616–17622, 2002.
27. Schroeder, J., Thompson, M., Gardner, M., and Gendler, S. TransgenicMUC1 interacts with epidermal growth factor receptor and correlates withmitogen-activated protein kinase activation in the mouse mammary gland. J. Biol.Chem., 276: 13057 –13064, 2001.
28. Harari, D. and Yarden, Y. Molecular mechanisms underlying ErbB2/HER2action in breast cancer. Oncogene, 19: 6102–6114, 2000.
29. Hulsken, J., Birchmeier, W., and Behrens, J. E-cadherin and APC competefor the interaction with h-catenin and the cytoskeleton. J. Cell Biol., 127: 2061–2069, 1994.
30. Keely, P. J., Westwick, J. K., Whitehead, I. P., Der, C. J., and Parise, L. V.Cdc42 and Rac1 induce integrin-mediated cell motility and invasiveness throughPI(3)K. Nature, 390: 632– 636, 1997.
31. D’Souza, B. and Taylor-Papadimitriou, J. Overexpression of ERBB2in human mammary epithelial cells signals inhibition of transcription of theE-cadherin gene. Proc. Natl. Acad. Sci. USA, 91: 7202–7206, 1994.
32. Shaw, P. J. and Jordan, E. G. The nucleolus. Annu. Rev. Cell Dev. Biol., 11:93–121, 1995.
33. Politz, J. C., Yarovoi, S., Kilroy, S. M., Gowda, K., Zwieb, C., and Pederson,T. Signal recognition particle components in the nucleolus. Proc. Natl. Acad. Sci.USA, 97: 55–60, 2000.
34. Lange, T. S. and Gerbi, S. A. Transient nucleolar localization of U6 smallnuclear RNA in Xenopus laevis oocytes. Mol. Biol. Cell, 11: 2419–2428, 2000.
35. Schneiter, R., Kadowaki, T., and Tartakoff, A. M. mRNA transport in yeast:time to reinvestigate the functions of the nucleolus. Mol. Biol. Cell, 6: 357 –370,1995.
36. Bertrand, E., Houser-Scott, F., Kendall, A., Singer, R. H., and Engelke, D. R.Nucleolar localization of early tRNA processing. Genes Dev., 12: 2463–2468,1998.
37. Visintin, R. and Amon, A. The nucleolus: the magician’s hat for cell cycletricks. Curr. Opin. Cell Biol., 12: 372 –377, 2000.
38. Zhang, Y. and Xiong, Y. Mutations in human ARF exon 2 disrupt itsnucleolar localization and impair its ability to block nuclear export of MDM2 andp53. Mol. Cell, 3: 579– 591, 1999.
39. Tao, W. and Levine, A. p19ARF stabilizes p53 by blocking nucleo-cytoplasmic shuttling of mdm2. Proc. Natl. Acad. Sci. USA, 96: 6937–6941,1999.
40. Lohrum, M. A., Ashcroft, M., Kubbutat, M. H., and Vousden, K. H.Identification of a cryptic nucleolar-localization signal in MDM2. Nat. Cell Biol.,2: 179– 181, 2000.
41. Kolligs, F. T., Kolligs, B., Hajra, K. M., Hu, G., Tani, M., Cho, K. R.,and Fearon, E. R. g-catenin is regulated by the APC tumor suppressor andits oncogenic activity is distinct from that of h-catenin. Genes Dev., 14:1319– 1331, 2000.
42. Fagotto, F., Gluck, U., and Gumbiner, B. M. Nuclear localization signal-independent and importin/karyopherin-independent nuclear import of h-catenin.Curr. Biol., 8: 181–190, 1998.
43. Prieve, M. G. and Waterman, M. L. Nuclear localization and formation ofh-catenin-lymphoid enhancer factor 1 complexes are not sufficient for activationof gene expression. Mol. Cell. Biol., 19: 4503–4515, 1999.
44. Henderson, B. R. Nuclear-cytoplasmic shuttling of APC regulates h-cateninsubcellular localization and turnover. Nat. Cell Biol., 2: 653 –660, 2000.
45. Neufeld, K. L., Zhang, F., Cullen, B. R., and White, R. L. APC-mediateddownregulation of h-catenin activity involves nuclear sequestration and nuclearexport. EMBO Rep., 1: 519 –523, 2000.
46. Dingwell, C. and Laskey, R. A. Nuclear targeting sequences—a consensus?Trends Biochem. Sci., 16: 478–482, 1991.
47. Makkerh, J. P., Dingwall, C., and Laskey, R. A. Comparative mutagenesis ofnuclear localization signals reveals the importance of neutral and acidic aminoacids. Curr. Biol., 6: 1025–1027, 1996.
48. Kharbanda, S., Saleem, A., Yuan, Z.-M., Kraeft, S., Weichselbaum, R., Chen,L. B., and Kufe, D. Nuclear signaling induced by ionizing radiation involvescolocalization of the activated p56/p53lyn tyrosine kinase with p34cdc2. CancerRes., 56: 3617–3621, 1996.
Molecular Cancer Research 775
on April 5, 2021. © 2003 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/
-
2003;1:765-775. Mol Cancer Res Yongqing Li, Wei-hsuan Yu, Jian Ren, et al. W.-h.Y. contributed equally to this work.National Cancer Institute grant CA97098. Note: Y.L. and
1 1Mechanism Dependent on the DF3/MUC1 Oncoprotein-Catenin to the Nucleolus by aγHeregulin Targets
Updated version
http://mcr.aacrjournals.org/content/1/10/765
Access the most recent version of this article at:
Cited articles
http://mcr.aacrjournals.org/content/1/10/765.full#ref-list-1
This article cites 48 articles, 28 of which you can access for free at:
Citing articles
http://mcr.aacrjournals.org/content/1/10/765.full#related-urls
This article has been cited by 21 HighWire-hosted articles. Access the articles at:
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's
.http://mcr.aacrjournals.org/content/1/10/765To request permission to re-use all or part of this article, use this link
on April 5, 2021. © 2003 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
http://mcr.aacrjournals.org/content/1/10/765http://mcr.aacrjournals.org/content/1/10/765.full#ref-list-1http://mcr.aacrjournals.org/content/1/10/765.full#related-urlshttp://mcr.aacrjournals.org/cgi/alertsmailto:[email protected]://mcr.aacrjournals.org/content/1/10/765http://mcr.aacrjournals.org/