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.

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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



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



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.




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.




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.




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.




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.




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.




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.

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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

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