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Molecular Cancer Research 1:765-775 (2003)
© 2003 American Association for Cancer Research

Signaling and Regulation

Heregulin Targets {gamma}-Catenin to the Nucleolus by a Mechanism Dependent on the DF3/MUC1 Oncoprotein1

Yongqing Li1,2, Wei-hsuan Yu1, Jian Ren1, Wen Chen1, Lei Huang1, Surender Kharbanda1,2, Massimo Loda1 and Donald Kufe1

1 Dana-Farber Cancer Institute, Harvard Medical School and 2 ILEX Products, Inc., Boston, MA

Requests for reprints: Donald Kufe, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115. Phone: (617) 632-3141; Fax: (617) 632-2934. E-mail: donald_kufe{at}dfci.harvard.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The DF3/MUC1 transmembrane oncoprotein is aberrantly overexpressed in most human breast carcinomas and interacts with the Wnt effector {gamma}-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 {gamma}-catenin and targeting of the MUC1-{gamma}-catenin complex to the nucleolus. Significantly, nucleolar localization of {gamma}-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 {gamma}-catenin. In concert with these results, we show nucleolar localization of MUC1 and {gamma}-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 {gamma}-catenin.

Key Words: MUC1 • ErbB2 • heregulin • {gamma}-catenin • nucleolus • breast cancer


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The 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 heterodimerization (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/neuregulin-1 (HRG) binds to the ErbB3 and ErbB4 receptors and activates ErbB2 through heterodimerization and transphosphorylation (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 activation 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 disruption of epithelial cell polarity and initiation of proliferation (11, 12). In normal polarized glandular epithelial cells, effectors of the Wnt signaling pathway, ß- and {gamma}-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 glycoprotein, which is overexpressed by breast and other carcinomas (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 ~25-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 ß-catenin (19). The SAGNGGSSL motif also serves as a binding site for {gamma}-catenin (plakoglobin) (19). Glycogen synthase kinase 3ß (GSK3ß) phosphorylates MUC1 on serine in a SPY site adjacent to that for ß/{gamma}-catenin binding and decreases the interaction between MUC1 and ß-catenin (20). Conversely, EGFR- or c-Src-mediated phosphorylation of MUC1 on tyrosine in the SPY site up-regulates the formation of MUC1-ß-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 ß-catenin (20) has supported a role for MUC1 in regulating adherens junction function. Other studies have demonstrated that MUC1 also colocalizes with ß-catenin in the nucleus (16, 24). Less is known about the regulation of binding between MUC1 and {gamma}-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 {gamma}-catenin. The functional significance of this signaling pathway is supported by the finding that HRG targets {gamma}-catenin to the nucleolus by a MUC1- dependent mechanism and that a RRK motif in MUC1-CD is required for this response.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
HRG 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 immunoprecipitates 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.



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  		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. Lysates were subjected to immunoprecipitation (IP) with anti-MUC1 (DF3) N-ter (left panel) or anti-ErbB2 (right panel). Mouse IgG was used as a control. The immunoprecipitates were analyzed by immunoblotting (IB) with anti-ErbB2 and anti-MUC1 N-ter. Intensity of the signals was determined by densitometric scanning and compared with that obtained for untreated cells. B. Lysates from control and HRG-treated ZR-75-1 cells were subjected to immunoprecipitation with anti-ErbB3 (left panel) or anti-ErbB4 (right panel). The immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. 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) and anti-ErbB2 (red).

 
HRG Regulates Interaction of MUC1 and {gamma}-Catenin
To determine whether HRG affects the interaction between 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-ß-catenin demonstrated that HRG has little effect on binding of MUC1 and ß-catenin (Fig. 2A). By contrast, HRG treatment was associated with an increase in binding of MUC1 and {gamma}-catenin (Fig. 2A). For comparison, ZR-75-1 cells were stimulated with EGF. As shown previously, EGF induced binding of MUC1 and ß-catenin (22) (Fig. 2B). Conversely, EGF had little effect on the interaction of MUC1 with {gamma}-catenin (Fig. 2B). To extend these findings, we used human HCT116 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-{gamma}-catenin demonstrated that HRG induces binding of MUC1 and {gamma}-catenin (Fig. 2D). These findings indicate that HRG stimulates the formation of MUC1-{gamma}-catenin complexes.



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  		FIGURE 2. HRG stimulates the interaction between MUC1 and {gamma}-catenin. A. Lysates from ZR-75-1 cells left untreated or stimulated with HRG for 5 min were subjected to immunoprecipitation with anti-MUC1 N-ter or, as a control, IgG. The immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. B. Lysates from ZR-75-1 cells left untreated or stimulated with 10-ng/ml EGF for 5 min were subjected to immunoprecipitation with anti-MUC1 or IgG. The immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. C. HCT116 cells were incubated with antibodies against the indicated ErbB family members (open patterns) or control mouse IgG (solid patterns) and analyzed by flow cytometry. Similar results were obtained for HCT116/MUC1 cells. D. HCT116/vector (HCT116/V) and HCT116/MUC1 cells were left untreated or stimulated with HRG. Anti-MUC1 NH2-terminal immunoprecipitates were subjected to immunoblotting with anti-{gamma}-catenin or anti-MUC1 N-ter.

 
Nucleolar Localization of MUC1-{gamma}-Catenin Complexes
To define the subcellular localization of MUC1-{gamma}-catenin complexes, ZR-75-1 cells were analyzed by confocal microscopy after incubation with antibodies against MUC1 C-ter and {gamma}-catenin. The results show colocalization of MUC1 C-ter and {gamma}-catenin at the cell membrane (Fig. 3A). By contrast, HRG stimulation for 20 min was associated with localization of MUC1 C-ter in the nucleus (Fig. 3B). A similar pattern was observed for {gamma}-catenin, and overlay demonstrated colocalization with MUC1 C-ter (Fig. 3B). The well-circumscribed colocalization of MUC1 and {gamma}-catenin in the nucleus suggested a nucleolar pattern (Fig. 3B). Indeed, staining with an antinucleolin antibody confirmed HRG-induced redistribution of MUC1 C-ter to the nucleolus (Fig. 3C). A similar pattern of nucleolar colocalization for MUC1 C-ter with {gamma}-catenin was observed in the ErbB2-positive MCF-7 breast cancer 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 ß-catenin and not {gamma}-catenin (unpublished data).



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  		FIGURE 3. HRG induces nucleolar colocalization of MUC1 C-ter and {gamma}-catenin. 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 (A) or stimulated with 20-ng/ml HRG for 20 min (B), fixed, and double stained with anti-MUC1 C-ter (green) and anti-{gamma}-catenin (red). Nuclei were stained with SYNTOX blue. High (x100) (upper panels) and low (x63) (lower panels) magnifications are shown. ZR-75-1 cells were stimulated with 20-ng/ml HRG for 20 min (C) or with 10-ng/ml EGF for 20 min (D), fixed, and stained with anti-MUC1 C-ter and anti-nucleolin.

 
Role of MUC1 in the Subcellular Distribution of {gamma}-Catenin
To assess the functional role of MUC1 in {gamma}-catenin signaling, HCT116/vector and HCT116/MUC1 cells were analyzed for localization of {gamma}-catenin following HRG stimulation. The confocal images show that {gamma}-catenin localizes to the cell 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 {gamma}-catenin (Fig. 4A). In HCT116/MUC1 cells, MUC1 C-ter and {gamma}-catenin were predominantly 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 {gamma}-catenin in 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 {gamma}-catenin are targeted to the nucleolus (data not shown).



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  		FIGURE 4. MUC1 is necessary for HRG-induced targeting of MUC1 C-ter and {gamma}-catenin to the nucleolus. HCT116/vector (A) and HCT116/MUC1 (B) cells were left untreated or stimulated with HRG for 20 min. The cells were assessed for reactivity with anti-MUC1 C-ter and anti-{gamma}-catenin. Nuclei were stained with SYNTOX blue.

 
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 {gamma}-catenin is not affected by the RRK -> AAA mutation (data not shown). In contrast to HCT116/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 {gamma}-catenin in both HCT116/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) cells with HRG for 20 min was associated with redistribution of both MUC1 C-ter and {gamma}-catenin to the cytoplasm (Fig. 5B). Moreover, there was no detectable HRG-induced targeting of MUC1 C-ter and {gamma}-catenin to the nucleolus (Fig. 5B).



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  		FIGURE 5. Nucleolar localization of MUC1 C-ter and {gamma}-catenin is conferred by the MUC1 RRK motif. HCT116/vector (A) and HCT116/MUC1(RRK -> AAA) (B) cells were left untreated or stimulated with HRG for 20 min. Cells were analyzed for staining with anti-MUC1 C-ter and anti-{gamma}-catenin. Morphology of the cells was visualized by bright-field microscopy.

 
To extend these observations, the localization of MUC1 C-ter and {gamma}-catenin was assessed by subcellular fractionation of 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). The results also demonstrate that HRG increases nuclear targeting of MUC1 C-ter in the HCT116/MUC1 cells (Fig. 6). Moreover, HRG treatment of HCT116/MUC1, but not HCT116/vector or HCT116/MUC1(RRK -> AAA), was associated with an increase in nuclear {gamma}-catenin (Fig. 6). Equal loading of the nuclear fractions was confirmed by immunoblotting for lamin B (Fig. 6). Moreover, purity of the nuclear preparations was demonstrated with antibodies against the cytosolic I{kappa}B{alpha}, the membrane-associated MUC1 NH2-terminal subunit, and the endoplasmic reticulum protein, calreticulin (Fig. 6). These findings collectively indicate that the RRK motif is important for nucleolar localization of MUC1 C-ter and {gamma}-catenin in the response to HRG stimulation.



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  		FIGURE 6. HRG-induced nuclear localization of MUC1 and {gamma}-catenin. Nuclear fractions were analyzed by immunoblotting with the indicated antibodies. Whole cell lysates (WCL) were used as a positive control.

 
Confocal Microscopy of Human Breast Carcinomas
To define the localization of MUC1 C-ter and {gamma}-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). {gamma}-Catenin colocalized with MUC1 C-ter at the apical borders and was expressed at lateral borders of the ductal epithelium (Fig. 7A). Little (if any) MUC1 C-ter or {gamma}-catenin was detectable in the nucleus (Fig. 7A). Significantly, sections from ErbB2-positive breast carcinomas showed immunoflourescence staining of MUC1 C-ter and {gamma}-catenin as discrete nuclear clusters (Fig. 7B). Sections were also stained with anti-MUC1 C-ter and antinucleolin. The results demonstrate prominent colocalization of MUC1 C-ter and nucleolin in breast carcinoma cells (Fig. 7C). Similar results were obtained for {gamma}-catenin and nucleolin (Fig. 7D). The results indicate that over 50% of the breast cancer cells within invasive islands exhibit nucleolar localization of MUC1 C-ter and {gamma}-catenin. These findings in tissues and those in cultured cells collectively demonstrate that MUC1-CD and {gamma}-catenin are targeted to nucleolus.



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  		FIGURE 7. Colocalization of MUC1 C-ter and {gamma}-catenin to the nucleolus of human breast carcinoma cells. Sections of normal mammary ductal epithelium (A) and two anti-HER2/ErbB2-positive primary invasive ductal breast carcinomas (B, upper and lower panels) were assessed for reactivity with anti-MUC1 C-ter and anti-{gamma}-catenin. Morphology was visualized at high and low (inset) power by H&E staining. Breast carcinoma cells were stained with anti-MUC1 C-ter (C) or anti-{gamma}-catenin (D) and anti-nucleolin.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
Interaction 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 ~58 amino acids, a transmembrane domain, and a 72-amino acid cytoplasmic tail, is largely unknown. The finding that MUC1-CD binds directly to ß- and {gamma}-catenin suggested that the C-ter might function in transducing signals from the cell surface to the interior of the cell (19). Indeed, the demonstration that MUC1-CD functions as a substrate for GSK3ß (20) and c-Src (21) has indicated that the 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 phosphorylation of MUC1-CD and regulation of the interaction between MUC1 and ß-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 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.



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  		FIGURE 8. Schematic representation of the involvement of MUC1 in HRG-induced targeting of {gamma}-catenin to the nucleolus.

 
Interaction of MUC1 and {gamma}-Catenin
ß- and {gamma}-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 {alpha}-catenin (29). The finding that complexes between MUC1 and ß- or {gamma}-catenin contain little (if any) {alpha}-catenin has supported 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 ß-catenin (20). Moreover, negative regulation of the MUC1-ß-catenin interaction by GSK3ß is associated with increased binding of ß-catenin to E-cadherin (20). In this model, down-regulation of GSK3ß by Wnt signaling would subvert E-cadherin function in homotypic cell-cell interactions by titrating binding of ß-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 ß- and/or {gamma}-catenin.

The present results show that the MUC1-ErbB2 interaction is associated with HRG-induced binding of MUC1 and {gamma}-catenin (Fig. 8). HRG stimulation had less of an effect on the interaction between MUC1 and ß-catenin. Conversely, EGFR signaling increases binding of MUC1 and ß-catenin (22) but has little effect on the interaction between MUC1 and {gamma}-catenin. EGFR signaling also increases phosphorylation of MUC1 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 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 epithelial 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 {gamma}-catenin signaling as another potential mechanism for increasing invasiveness. Thus, HRG-induced increases in binding of {gamma}-catenin to MUC1 could decrease the availability of {gamma}-catenin for linking E-cadherin to the actin cytoskeleton and thereby disrupt homotypic cell-cell signaling.

Nucleolar Localization of MUC1 C-ter and {gamma}-Catenin
The present results further indicate that HRG stimulation is associated with nuclear targeting of MUC1 C-ter and {gamma}-catenin (Fig. 8). The well-circumscribed nuclear distribution of the MUC1 C-ter signal and colocalization with antinucleolin staining supported compartmentalization of MUC1 C-ter in the nucleolus. Similar results were obtained with {gamma}-catenin, supporting the likelihood that the MUC1-{gamma}-catenin complex is targeted to the nucleolus in response to HRG stimulation. In concert with these findings, MUC1 C-ter and {gamma}-catenin are detectable in nucleoli of ErbB2-positive primary breast carcinomas. The observation that over 50% of the breast cancer cells exhibit nucleolar colocalization of MUC1 C-ter and {gamma}-catenin indicate that, as found in vitro, MUC1 may 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, {gamma}-catenin interacts with the T-cell factor/lymphoid enhancer factor transcription factors and functions as a coactivator. Like ß-catenin, {gamma}-catenin can contribute to cell transformation by a mechanism involving transactivation of c-Myc expression (41).

Activation of the Wnt signaling pathway is associated with accumulation of ß- and {gamma}-catenin in the nucleus. The mechanisms responsible for targeting ß- and {gamma}-catenin to the nucleus are not clear. Neither protein has a definitive nuclear localization signal; however, ß-catenin is imported into the nucleus 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 ß-catenin (43). The adenomatous polyposis coli protein can function as a ß-catenin chaperone in nuclear export but apparently not in nuclear import (44, 45). Recent studies have demonstrated that MUC1 colocalizes with ß-catenin in the nucleus and increases nuclear levels of ß-catenin (16, 24). These findings have indicated that MUC1 may function in the import and/or stabilization of nuclear ß-catenin. Importantly, the nuclear colocalization of MUC1-ß-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 {gamma}-catenin is dependent on MUC1 expression. The MUC1-CD contains 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 {gamma}-catenin to the nucleolus in response to HRG was not found in cells expressing the MUC1(RRK -> AAA) mutant. These findings provide the first evidence that MUC1 functions in nuclear signaling and that {gamma}-catenin is transported to the nucleolus by a MUC1-dependent mechanism.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell 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-µg/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 37°C.

Cell Transfections
pIRESpuro2, pIRESpuro2-MUC1, and pIRESpuro2-MUC1(RRK -> AAA) were transfected into HCT116 cells by LipofectAMINE. Stable transfectants were selected in the presence of 0.4-µg/ml puromycin (Calbiochem-Novabiochem).

Immunoprecipitation and Immunoblotting
Lysates 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-ß-catenin (Zymed, San Francisco, CA), or anti-{gamma}-catenin (Zymed). Reactivity was detected with horseradish peroxidase-conjugated second antibodies and chemiluminescence (Perkin-Elmer Corp., Boston, MA).

Immunoflourescence Confocal Microscopy
Cultured cells were washed three times in PBS (containing Mg2+ and Ca2+), fixed with 3.7% formaldehyde in buffer A (PBS containing 10-µM ZnCl2) for 10 min, permeabilized with 0.25% 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, Fremont, CA), anti-{gamma}-catenin, and anti-nucleolin (Research Diagnostics, Flanders, NJ) in blocking buffer was performed overnight at 4°C. The cells were washed with PBS, incubated overnight with secondary FITC- or Texas Red-conjugated goat anti-hamster or anti-mouse IgG antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) at 4°C, washed with PBS, washed three times with buffer B (20-mM Tris, pH 7.5, 0.15-M NaCl), and stained with 0.2-µM of SYNTOX Blue Nuclei 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 Cytometry
Cells 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 immunoflourescence FACScan.

Subcellular Fractionation
Preparation 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), anti-I{kappa}B{alpha} (Santa Cruz Biotechnology) antibodies.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
The authors acknowledge Kamal Chauhan for excellent technical support. D.K. has financial interest in ILEX.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 National Cancer Institute grant CA97098. Note: Y.L. and W.-h.Y. contributed equally to this work. Back

Received March 3, 2003; revised June 20, 2003; accepted June 24, 2003.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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