Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin

doi:10.1016/j.isci.2020.101683

. 2020 Oct 15;23(11):101683.

doi: 10.1016/j.isci.2020.101683. eCollection 2020 Nov 20.

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin

Philip Bischoff^{1

2}, Marja Kornhuber^{1

3}, Sebastian Dunst¹, Jakob Zell^{1

2}, Beatrix Fauler⁴, Thorsten Mielke⁴, Anna V Taubenberger⁵, Jochen Guck⁶, Michael Oelgeschläger¹, Gilbert Schönfelder^{1

2}

Affiliations

¹ German Federal Institute for Risk Assessment (BfR), German Centre for the Protection of Laboratory Animals (Bf3R), Max-Dohrn-Straße 8-10, 10589 Berlin, Germany.
² Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 10117 Berlin, Germany.
³ Freie Universität Berlin, 14195 Berlin, Germany.
⁴ Max Planck Institute for Molecular Genetics, Microscopy and Cryo-Electron Microscopy Service Group, 14195 Berlin, Germany.
⁵ Biotechnology Center, Technische Universität Dresden, 01307 Dresden, Germany.
⁶ Max Planck Institute for the Science of Light, Max-Planck-Zentrum für Physik und Medizin, 91058 Erlangen, Germany.

PMID: 33163938
PMCID: PMC7607435
DOI: 10.1016/j.isci.2020.101683

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin

Philip Bischoff et al. iScience. 2020.

. 2020 Oct 15;23(11):101683.

doi: 10.1016/j.isci.2020.101683. eCollection 2020 Nov 20.

Authors

Affiliations

¹ German Federal Institute for Risk Assessment (BfR), German Centre for the Protection of Laboratory Animals (Bf3R), Max-Dohrn-Straße 8-10, 10589 Berlin, Germany.
² Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 10117 Berlin, Germany.
³ Freie Universität Berlin, 14195 Berlin, Germany.
⁴ Max Planck Institute for Molecular Genetics, Microscopy and Cryo-Electron Microscopy Service Group, 14195 Berlin, Germany.
⁵ Biotechnology Center, Technische Universität Dresden, 01307 Dresden, Germany.
⁶ Max Planck Institute for the Science of Light, Max-Planck-Zentrum für Physik und Medizin, 91058 Erlangen, Germany.

PMID: 33163938
PMCID: PMC7607435
DOI: 10.1016/j.isci.2020.101683

Abstract

Estrogens play an important role in the development and progression of human cancers, particularly in breast cancer. Breast cancer progression depends on the malignant destabilization of adherens junctions (AJs) and disruption of tissue integrity. We found that estrogen receptor alpha (ERα) inhibition led to a striking spatial reorganization of AJs and microclustering of E-Cadherin (E-Cad) in the cell membrane of breast cancer cells. This resulted in increased stability of AJs and cell stiffness and a reduction of cell motility. These effects were actomyosin-dependent and reversible by estrogens. Detailed investigations showed that the ERα target gene and epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) essentially regulates AJ reorganization and E-Cad microclustering. Our results not only describe a biological mechanism for the organization of AJs and the modulation of mechanical properties of cells but also provide a new perspective on how estrogens and anti-estrogens might influence the formation of breast tumors.

Keywords: Cancer; Cell Biology.

PubMed Disclaimer

Conflict of interest statement

A European (EP 3517967 A1) and international (PCT) (WO 2019145517 A1) patent application for the use of the here-described endpoint (estrogen-dependent reorganization of adherens junctions) to screen substances for estrogenic or anti-estrogenic activity has been filed at the European Patent Office by the employer (German Federal Institute for Risk Assessment [BfR]) of the following authors: P.B., M.K., S.D., M.O., and G.S.. The German Federal Institute for Risk Assessment (BfR) is a scientifically independent institution within the portfolio of the Federal Ministry of Food and Agriculture (BMEL) in Germany. The authors' freedom to design, conduct, interpret, and publish research is explicitly not compromised. All remaining authors do not have any competing financial interests.

Figures

**Figure 1**
Reorganization of Adherens Junctions (AJs) Upon Treatment with Anti-estrogens (A) Phase contrast and immunofluorescence images showing intercellular spacing and organization of AJs upon Fulv treatment for 48 h as compared with the solvent control. White boxes indicate enlarged cell membrane areas shown in A′-A″. Scale bars, 10 μm. (B) Optical sections from immunofluorescence images showing organization of tight junctions (ZO-1, magenta) and AJs (E-Cad, green) upon Fulv treatment for 48 h as compared with the solvent control. White dashed lines in xz sections indicate image planes of xy sections shown in B′-B‴’. Scale bars, 10 μm. (C) Immunofluorescence images showing AJ organization (E-Cad) upon Fulv treatment for 24, 48, and 144 h. Scale bar, 10 μm. (D) Immunofluorescence images showing AJ organization (E-Cad) upon short-term pre-treatment with Fulv for 24 h. Indicated time points depict time after removal of Fulv-containing medium. Scale bar, 10 μm. (E and F) Immunofluorescence images showing AJ organization (E-Cad, green) and ERα protein levels (magenta) upon treatment with the selective estrogen receptor degrader (SERD) Fulv for 48 h and treatment with the selective estrogen receptor modulator (SERM) 4-hydroxytamoxifen (4-OHT) for 72 h as compared with the solvent control. Scale bar, 10 μm. (G) Quantitative RT-PCR measurement of ERα (*ESR1*, magenta) and E-Cad (*CDH1*, green) mRNA expression levels upon treatment with Fulv and 4-hydroxytamoxifen (4-OHT) for 48 h. The mRNA expression levels of the ERα target gene *TFF1* serve as readout for ERα signaling activity. Relative mRNA expression levels are normalized to the solvent control (Ctrl). Biological replicates, n = 3. Error bars, mean ± SD. (H) Western blot analysis and quantification of ERα (magenta) and E-Cad (green) protein levels upon treatment with Fulv and 4-hydroxytamoxifen (4-OHT) for 48 h. Relative protein expression levels are normalized to the solvent control (Ctrl). Biological replicates, n = 3. Bars, mean of biological replicates. (H′) Representative western blots of quantification shown in (H). Loading control, Coomassie total protein staining. (I) Immunofluorescence images showing AJ organization (E-Cad, green) and ERα protein levels (magenta) of cells transfected with a pool of four different *ESR1* siRNAs compared with cells transfected with scrambled control siRNA for 72 h. Scale bar, 10 μm. (J) Quantitative RT-PCR measurement of ERα (*ESR1*, magenta) and E-Cad (*CDH1*, green) mRNA expression levels upon transfection of cells with a mix of four different *ESR1* siRNAs for 72 h. The mRNA expression levels of the ERα target gene *TFF1* serve as readout for ERα signaling activity. Relative mRNA expression levels are normalized to the scrambled control (Ctrl). Biological replicates, n = 3. Error bars, mean ± SD. (K) Western blot analysis and quantification of ERα (magenta) and E-Cad (green) protein levels upon transfection of cells with a mix of four different *ESR1* siRNAs for 72 h. Relative protein expression levels are normalized to the scrambled control (Ctrl). Biological replicates, n = 3. Bars, mean of biological replicates. (K′) Representative western blots of quantification shown in (K). Loading control, Coomassie total protein staining. See also Figure S1.

**Figure 2**
Quantification of Adherens Junction (AJ) Reorganization (A) Main steps of a CellProfiler/CellProfiler Analyst (CP/CPA)-based image analysis pipeline including segmentation and classification of immunofluorescence images (E-Cad staining) to categorize cells into “Continuous AJs” (blue circles) and “Discontinuous AJs” (orange squares) based on their AJ organization. (A′) Representative results of a CP/CPA-based analysis of the AJ organization in cells treated with Fulv for 48 h or solvent control-treated cells. The plot shows the fraction of cells classified as Continuous AJs (Morphology Index, MI) for each condition. The reorganization of AJs upon Fulv treatment results in an increase of Discontinuous AJs and therefore a decrease of the MI. Each data point represents an average MI from three individual images per treatment condition. Biological replicates, n = 3. Bars, mean of biological replicates. (B) Application of the CP/CPA-based image analysis pipeline on images from ERα signaling inhibition (Fulv titration) and restoration (10 nM Fulv in combination with 17β-Estradiol titration) experiments. The derived MI is normalized to the negative control (solvent, Ctrl = 1) and the positive control (10 nM Fulv, Fulv = 0). Each data point represents an average MI from three individual images per treatment condition. Biological replicates, n = 3. Bars, mean of biological replicates. (C) Quantification of AJ organization upon transfection with four different *ESR1* siRNAs individually and in combination (Mix) compared with controls with scrambled siRNA (Scr) or transfection reagent only (Rea). The MI is normalized to the negative control (solvent, Ctrl = 1) and positive control (10 nM Fulv, Fulv = 0). Biological replicates, n = 3. Bars, mean of biological replicates. (D) Time-resolved quantification of AJ reorganization in MCF-7/E-Cad-GFP cells (stably expressing an *E-Cad::GFP* plasmid) upon Fulv treatment. Cells were imaged every 2 h by live-cell fluorescence microscopy over the course of 24 h after pre-treatment with Fulv for 24 h. Image analysis was performed using the commercial Harmony software (PerkinElmer) following the principle as described in (A). Biological replicates, n = 2. Error bars, mean ± SD of three technical replicates. See also Videos S1 and S2.

**Figure 3**
ERα Signaling Activity under Anti-estrogenic and Estrogenic Conditions Quantitative RT-PCR measurement of mRNA expression levels of typical ERα target genes (*BCL2L1*; *PGR*; *TFF1*) and E-Cad (*CDH1*, green) upon ERα signaling inhibition (Fulv titration), restoration (10 nM Fulv in combination with 17β-Estradiol titration), and stimulation (17β-Estradiol titration) for 48 h. Relative mRNA expression levels for each treatment condition are normalized to the respective solvent control (Ctrl). Biological replicates, n = 3. Error bars, mean ± SD.

**Figure 4**
Clustering of E-Cad at Cell Membranes under Anti-estrogenic Conditions (A) High-resolution immunofluorescence images showing AJ organization (E-Cad) and formation of E-Cad microclusters upon Fulv treatment for 48 h as compared with the solvent control. White boxes indicate enlarged cell membrane areas shown in A′-A⁗. Scale bars, 10 and 1 μm. (B) Transmission electron microscopy (TEM) images showing the organization of cell-cell contact zones upon Fulv treatment for 48 h as compared with the solvent control. Black boxes indicate enlarged cell membrane areas shown in B′-B⁗. Arrowheads indicate cell membranes. Scale bars, 10 and 1 μm. (C) Transmission electron microscopy images showing localization of immuno-gold-labeled E-Cad molecules (Immuno-TEM) upon Fulv treatment for 48 h as compared with the solvent control. Black boxes indicate enlarged cell membrane areas shown in C′-C⁗. Black arrowheads indicate individual gold particles along cell membranes. White arrowheads indicate gold particle clusters along remaining cell-cell contact zones. Scale bars, 1 and 0.1 μm. (D) Fluorescence images showing AJ organization (GFP) and formation of E-Cad microclusters in Fulv-treated cells expressing an *E-Cad::GFP* plasmid (pE-Cad::GFP) for 48 h as compared with the solvent control. Transfected cells are visualized by GFP expression. White boxes indicate enlarged cell membrane areas shown in D′-D⁗. Scale bars, 10 and 1 μm. (E) Immunofluorescence images showing Fulv-treated and solvent control-treated cells upon siRNA-mediated E-Cad (*CDH1*) knockdown for 48 h. Cells transfected with a pool of four different siRNAs (siCDH1) show reduced E-Cad protein levels (green). F-Actin staining (magenta) visualizes the cortical F-Actin network and serves as a proxy for AJ organization in knockdown cells (asterisks). Scale bar, 10 μm. (E′) Representative western blot of experiment shown in (E). Loading control, Coomassie total protein staining. See also Figure S2.

**Figure 5**
Analysis of Breast Tumor Tissue Samples from METAcancer Project and ADAPT Trial (A and B) Immunofluorescence images showing AJ organization (E-Cad, green) and ERα localization (magenta) in representative breast tumor tissue samples from patients with diagnosed invasive ductal carcinoma (IDC). An asterisk indicates sections with clustered appearance of E-Cad along cell membranes. Samples obtained from (A) METAcancer project and (B) ADAPT trial (results summarized in Table 1 and Table 2). The numbers of histological sections were anonymized. Scale bar, 10 μm. See also Figures S1 and S2.

**Figure 6**
Role of the Actomyosin Cytoskeleton for Adherens Junction Reorganization and Biomechanical Properties of Cells (A) Immunofluorescence images showing AJ organization (E-Cad) in Fulv-treated cells and solvent control-treated cells after pre-treatment for 48 h and after application of Latrunculin A-containing medium (1 μM Latrunculin A for 30 min) to depolymerize the actin cytoskeleton. Indicated time points depict time after removal of Latrunculin A-containing medium. Scale bar, 10 μm. (B) Immunofluorescence images showing AJ organization (E-Cad) in Fulv-treated cells and solvent control-treated cells after pre-treatment for 48 h and after application of Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor-containing medium (10 μM Y-27632 for 10 h) to reduce the Myosin-II motor protein activity. Indicated time points depict time after removal of Y-27632-containing medium. Scale bar, 10 μm. (C) Quantification of cell stiffness (Apparent elastic [Young's] modulus) by atomic force microscopy (AFM) indentation measurements on cells treated with Fulv for 48 h (red datasets, E_Repl.1 = 0.71 ± 0.12 kPa; E_Repl.2 = 0.65 ± 0.14 kPa; E_Repl.3 = 0.59 ± 0.16 kPa; mean ± SD) as compared with solvent control-treated cells (black datasets, E_Repl.1 = 0.48 ± 0.12 kPa; E_Repl.2 = 0.41 ± 0.15 kPa; E_Repl.3 = 0.47 ± 0.12 kPa). Biological replicates, n = 3. Boxes, 25th, 50th (median), and 75th percentiles. Whiskers, 10th and 90th percentiles. Cross, mean for each group. (D) Apparent elastic (Young's) modulus distribution maps of 50 × 50 μm regions of cells treated with Fulv for 48 h as compared with solvent control-treated cells at a spatial resolution of 1 μm. Scale bar, 10 μm. (E) Immunofluorescence images showing AJ organization (E-Cad) of cells grown on extracellular matrix constituents (fibronectin, laminin) upon Fulv treatment for 48 h as compared with the solvent control. Scale bar, 10 μm. See also Figure S3.

**Figure 7**
Adherens Junction (AJ) Stability and Cell Motility under Anti-estrogenic Conditions (A) Representative western blots showing full-length E-Cad (120 kDa) protein and E-Cad fragment (90 kDa, 69 kDa) bands upon application of trypsin-containing medium for 3 min to disrupt AJs. Loading control, Coomassie total protein staining. (A′) Western blot analysis and quantification of full-length E-Cad protein levels of experiment shown in (A) normalized to solvent control (without trypsin treatment, Ctrl). Biological replicates, n = 3. Bars, mean of biological replicates. (A″) Western blot analysis and quantification of E-Cad fragment levels of experiment shown in (A) normalized to solvent control (trypsin-treated, Ctrl). Biological replicates, n = 3. Bars, mean of biological replicates. (B and B′) Representative fluorescence images from live cell imaging (CellTrace) of cells pre-treated with Fulv for 48 h and solvent control-treated cells. Indicated time points depict time after application of EGTA-containing medium (8 mM EGTA) to disrupt AJs. The plot shown in B’ displays the fraction of Fulv-treated (red dataset) and solvent control (black dataset) cells classified as “Rounded cells” over the course of 120 min. Three individual images were analyzed per time point and treatment condition using CellProfiler/CellProfiler Analyst. Biological replicates, n = 3. Error bars, mean of biological replicates +/− SD. Scale bar, 10 μm. (C) Quantification of cell motility (velocity) by manual tracking of cells treated with Fulv for 48 h (red datasets, V_Repl.1 = 0.0613 ± 0.0176 μm/min; V_Repl.2 = 0.0553 ± 0.0157 μm/min; V_Repl.3 = 0.051 ± 0.014 μm/min; mean ± SD) as compared with solvent control-treated cells (black datasets, V_Repl.1 = 0.113 ± 0.0237 μm/min; V_Repl.2 = 0.0757 ± 0.023 μm/min; V_Repl.3 = 0.0827 ± 0.0174 μm/min). For each treatment condition, a total of 30 cells from three individual areas (10 cells per area) were tracked every 10 min over the course of 16 h using Fiji plugins. Boxes, 25th, 50th (median), and 75th percentiles. Whiskers, 10th and 90th percentiles. Cross, mean for each group. (D) Cell path maps comprising trajectories of individual cells tracked in Figure 7C. See also Videos S3 and S4.

**Figure 8**
Role of Amphiregulin (AREG) and the Epidermal Growth Factor Receptor (EGFR) Pathway for AJ Reorganization (A) Time-resolved quantitative RT-PCR measurement of *AREG* (orange) and ERα (*ESR1*, magenta) mRNA expression levels upon Fulv treatment over the course of 48 h. The mRNA expression levels of the ERα target gene *TFF1* serve as readout for ERα signaling activity. Relative mRNA expression levels of each time point are normalized to the corresponding solvent control (Ctrl). Biological replicates, n = 3. Error bars, mean ± SD. (B) Quantitative RT-PCR measurement of *AREG* (orange) mRNA expression levels upon ERα signaling inhibition (Fulv titration) and restoration (10 nM Fulv in combination with 17β-Estradiol titration) for 48 h (samples from experiment shown in Figure 2A). Relative mRNA expression levels for each treatment condition are normalized to the solvent control (Ctrl). Biological replicates, n = 3. Error bars, mean ± SD. (C) Immunofluorescence images showing AJ organization (E-Cad) of cells transfected with a pool of four different *AREG* siRNAs in combination as compared with cells transfected with scrambled control siRNA for 72 h. Scale bar, 10 μm. (C′) Quantification of AJ organization of the experiment shown in (C). The MI is normalized to the negative control (solvent, Ctrl = 1) and positive control (10 nM Fulv, Fulv = 0). Biological replicates, n = 3. Bars, mean of biological replicates. (D) Quantitative RT-PCR measurement of *AREG* (orange) mRNA expression levels of cells transfected with a pool of four different siRNAs to knockdown ERα (siESR1) and AREG (siAREG) for 72 h. The mRNA expression levels of the ERα target gene *TFF1* serve as readout for ERα signaling activity. Relative mRNA expression levels are normalized to the scrambled control (Ctrl). Biological replicates, n = 3. Error bars, mean ± SD. (E) Western blot analysis and quantification of AREG protein levels upon Fulv treatment (normalized to solvent control, Ctrl), siESR1 knockdown, and siAREG knockdown (both normalized to scrambled control, Ctrl) for 72 h. Biological replicates, n = 3. Bars, mean of biological replicates. (F) Immunofluorescence images showing AJ organization (E-Cad, green) in Fulv-treated cells expressing an *AREG::GFP* plasmid (pAREG::GFP) for 48 h as compared with untransfected control cells. Transfected cells are visualized by GFP expression (magenta). Scale bar, 10 μm. (F′) Quantification of AJ organization of the experiment shown in (F). The MI is normalized to the negative control (solvent, Ctrl = 1) and positive control (10 nM Fulv, Fulv = 0). The trainings set was generated from reagent control images. Biological replicates, n = 3. Bars, mean of biological replicates. (G) Quantitative RT-PCR measurement of *AREG* (orange) mRNA expression levels of Fulv-treated cells expressing an *AREG::GFP* plasmid (pAREG) for 48 h as compared with untransfected control cells. The mRNA expression levels of the ERα target gene *TFF1* serve as readout for ERα signaling activity. Relative mRNA expression levels are normalized to the reagent control (Ctrl). Biological replicates, n = 1. (H) Immunofluorescence images showing AJ organization (E-Cad) of cells upon treatment with the EGFR inhibitor Gefitinib for 72 h as compared with solvent control-treated cells. Scale bar, 10 μm. (H′) Quantification of AJ organization of cells upon treatment with different concentrations of the EGFR inhibitor Gefitinib for 72 h as compared with solvent control-treated cells (Ctrl). The MI is normalized to the negative control (solvent, Ctrl = 1) and positive control (10 nM Fulv, Fulv = 0). Biological replicates, n = 3. Bars, mean of biological replicates. (I) Quantitative RT-PCR measurement of *AREG* (orange) and ERα (*ESR1*, magenta) mRNA expression levels upon Gefitinib treatment for 72 h. The mRNA expression levels of the ERα target gene *TFF1* serve as readout for ERα signaling activity. Relative mRNA expression levels for each treatment condition are normalized to the solvent control (Ctrl). Biological replicates, n = 3. Error bars, mean ± SD.

**Figure 9**
Working Model Illustrating Mechanistic Events and Functional Consequences that are Involved in Estrogen-Dependent Adherens Junction (AJ) Reorganization Under estrogenic conditions, ERα monomers bind available estrogens, dimerize, and shuttle to the nucleus to activate target gene expression including *AREG*. Upon processing and secretion of AREG at the cell membrane, it can bind to the Epidermal growth factor receptor (EGFR) and induce further downstream signaling cascades that potentially cross talk with the ERα signaling pathway. Under anti-estrogenic conditions, anti-estrogens perturb ERα signaling activity through different modes of action. Whereas selective estrogen receptor modulators (SERMs) such as Tamoxifen (Tam) only compete with estrogens for receptor binding, selective estrogen receptor degraders (SERDs) like Fulvestrant (Fulv) additionally reduce available ERα protein levels. The inhibition of ERα signaling activity and the corresponding reduction of AREG expression levels eventually lead to the clustering of E-Cad and a striking reorganization of AJs involving the actomyosin cytoskeleton. This change in cell-cell contact architecture further correlates with an increase of cell stiffness and stability of AJs and decrease of cell motility. As these parameters represent functional readouts that are often associated with breast cancer progression and metastasis, the reorganization of AJs may provide a novel functional and clinically relevant endpoint to determine the activity of the estrogen signaling pathway.

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[1] Baluk P., Fuxe J., Hashizume H., Romano T., Lashnits E., Butz S., Vestweber D., Corada M., Molendini C., Dejana E. Functionally specialized junctions between endothelial cells of lymphatic vessels. J. Exp. Med. 2007;204:2349–2362. - PMC - PubMed

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[6] Britton D.J., Hutcheson I.R., Knowlden J.M., Barrow D., Giles M., McClelland R.A., Gee J.M., Nicholson R.I. Bidirectional cross talk between ERalpha and EGFR signalling pathways regulates tamoxifen-resistant growth. Breast Cancer Res. Treat. 2006;96:131–146. - PubMed

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[8] Busser B., Sancey L., Brambilla E., Coll J.L., Hurbin A. The multiple roles of amphiregulin in human cancer. Biochim. Biophys. Acta. 2011;1816:119–131. - PubMed

[9] Cardamone M.D., Bardella C., Gutierrez A., Di Croce L., Rosenfeld M.G., Di Renzo M.F., De Bortoli M. ERalpha as ligand-independent activator of CDH-1 regulates determination and maintenance of epithelial morphology in breast cancer cells. Proc. Natl. Acad. Sci. U S A. 2009;106:7420–7425. - PMC - PubMed

[10] Cardamone M.D., Bardella C., Gutierrez A., Di Croce L., Rosenfeld M.G., Di Renzo M.F., De Bortoli M. ERalpha as ligand-independent activator of CDH-1 regulates determination and maintenance of epithelial morphology in breast cancer cells. Proc. Natl. Acad. Sci. U S A. 2009;106:7420–7425. - PMC - PubMed

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Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin

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Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin

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