Estrogens promote cell–cell adhesion of normal and malignant mammary cells through increased desmosome formation

Molecular and Cellular Endocrinology 364 (2012) 126–133

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Estrogens promote cell–cell adhesion of normal and malignant mammary cells through increased desmosome formation Marie Maynadier 1, Monique Chambon 1, Ilaria Basile, Michel Gleizes, Philippe Nirde, Magali Gary-Bobo, Marcel Garcia ⇑ Institut des Biomolécules Max Mousseron, UMR 5247 CNRS, Université Montpellier 1, Université Montpellier 2, 15 Av. Charles Flahault, BP 14491, 34093 Montpellier, Cedex 5, France

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Article history: Received 8 March 2012 Received in revised form 24 August 2012 Accepted 27 August 2012 Available online 3 September 2012 Keywords: Breast Cancer Estrogen Adhesion Desmosome

a b s t r a c t The association of estrogen receptor alpha (ERa) expression with differentiated breast tumors presenting a lower metastasis risk could be explained by the estrogen modulation of cell adhesion, motility and invasiveness. Since desmosomes play a crucial role in cell–cell adhesion and may interfere in tumor progression, we studied their regulation by estrogens in human breast cancer and normal mammary cells. Estrogens increased the formation of desmosomes in normal and malignant cells. Furthermore, four desmosomal proteins (desmocollin, c-catenin, plakophilin and desmoplakin) appeared significantly upregulated by estrogens in three ERa-expressing cancer cell lines and this effect was reversed by a pure antiestrogen. Finally, silencing of ERa or desmoplakin expression by specific siRNA revealed that estrogen-modulated desmosomal proteins are essential for the estrogenic control of intercellular adhesion. This estrogen modulation of desmosome formation could contribute to the lower invasiveness of ERapositive tumors and to the integrity of epithelial layers in estrogen target tissues. Ó 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Estrogens are involved in the development of the mammary gland and in the etiology of human breast cancer (Medina, 2005). Most of their effects are mediated by two specific nuclear receptors, ERa and ERb (Dahlman-Wright et al., 2006; Osborne and Schiff, 2011; Thomas and Gustafsson, 2011). ERa assessment in primary tumors has been established as a way to predict the efficacy of endocrine therapies based on anti-estrogens or aromatase inhibitors, which are widely used as first-line adjuvant therapy (Fisher et al., 2008). It has been demonstrated that ERa mediate an estrogen mitogenic action in human breast cancer cells (Schiff et al., 2005) but, paradoxically, the presence of ERa is associated with more differentiated and less invasive tumors that have, therefore, a more favorable clinical prognosis (Fisher et al., 2008; Hanrahan et al., 2006; Mirza et al., 2002). However, the role

Abbreviations: ERa, estrogen receptor alpha; E2, estrogen; FBS-DCC, dextrancoated charcoal stripped fetal bovine serum; SEM and TEM, scanning and transmission electron microscopy; HRP, horse radish peroxydase; PVDF, polyvinylidene fluoride; ICI182780, fulvestrant; siRNA, small-interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SD, standard deviation. ⇑ Corresponding author. Address: Equipe ‘Glyco et nanovecteurs pour le ciblage thérapeutique’, IBMM, 15 Av. Charles Flahault, BP 14491, 34093 Montpellier, Cedex 5, France. Tel.: +33 411 759 617; fax: +33 411 759 641. E-mail address: [email protected] (M. Garcia). 1 These authors contributed equally to this work. 0303-7207/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2012.08.016

of these hormones in cancer progression, including invasion and metastasis, is more difficult to define given the various pathways involved. Several studies described the in vitro regulation of breast cancer cell invasion by estrogens and antiestrogens. ERa-positive cell lines are less invasive and do not metastasize in the same way as ERa-negative cells (Platet et al., 2004; Rochefort et al., 1998). The unliganded and estradiol-activated estrogen receptors decrease cancer cell invasion in vitro through distinct mechanisms corresponding to protein–protein interaction with still uncharacterized partner(s) and to a mechanism involving the functional receptor domains required for transcriptional activation of target genes respectively (Platet et al., 2000, 2004; Rochefort et al., 1998; Sisci et al., 2004). Cell-matrix and the cell–cell adhesion constitute initial elements of resistance to the release of cancer cells from primary tumor and subsequent cell migration (Mareel and Leroy, 2003; Zhang et al., 2010). Several studies have already demonstrated that breast cancer cell adhesion is estrogen dependent, notably, by involving adherens junctions and particularly of proteins such as E-cadherin (Behrens et al., 1992; Fujita et al., 2003; Hartsock and Nelson, 2008; Van Roy and Berx, 2008; Vleminckx et al., 1991). In the present study, ultrastructural and biochemical approaches were used to study the effects of estrogens on other structures known for their importance in intercellular adhesion: the desmosomes. We have analyzed the effects of prolonged estrogen treatment on cell–cell adhesion of both breast and

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ovarian cancer cell lines and on primary cultures of normal mammary cells. 2. Materials and methods 2.1. Materials Stock solutions of the anti-estrogen ICI182,780 (gift of A. Wakeling, AstraZeneca, Cheshire, UK) and 17b-estradiol (E2) (Sigma– Aldrich Chimie, St. Quentin Fallavier, France) were prepared in ethanol. Desmocollin 2/3 (7G6), plakophilin 3 (651114), glyceraldehyde 3-phosphate dehydrogenase (abbreviated as GAPDH) (ab8245), b-catenin (610153) and c-catenin (15F11) and cytokeratin-18 monoclonal antibodies were respectively purchased from Zymed (Clinisciences, Montrouge, France), Tebu-bio (Le Perray en Yvelines, France), Abcam (Paris, France), and BD Bioscience (Le Pont de Claix, France) for the two catenins and Beckman Coulter (France) for the last one. Rabbit polyclonal antibodies against desmoplakin I/ II (sc-33555) and E-cadherin (sc-7870) were obtained from Santa Cruz (Heidelberg, Germany). Mouse monoclonal ERa (SRA-1000) was obtained from Stressgen (Ann Arbor, USA). For immunofluorescence staining, Alexa Fluor 488 anti-rabbit and/or Alexa Fluor 568 anti-mouse (Fisher Bioblock scientific, Illkirch, France) were used as secondary antibodies. A non-relevant IgG1 mouse monoclonal antibody (MOPC21), used as control, was purchased from Letton Bionetics Inc. (Kensington, CA, USA) or a purified rabbit IgG1 (Sigma Chemical Co., St. Louis, MO) were used as negative controls for antibodies. Mouse and rabbit peroxydase-conjugated secondary antibodies for Western blotting were purchased from GE Healthcare (Orsay, France) and Jackson Immunoresearch (Cambridge, UK). 2.2. Cell cultures Two breast cancer cell lines (MCF7 and T47D) and one ovarian cell line (BG1) were maintained in monolayer cultures in phenol red-free Dulbecco’s modified Eagle’s/Ham’s F-12 medium (DMEM) supplemented with 10% of dextran-coated charcoal stripped fetal bovine serum (FBS-DCC) and 50 lg/ml gentamycin. For collagen I outgrowth, MCF7 cells were embedded in collagen I gel at 4 °C (0.3 ml, 0.2 mg/ml) and then added on a pre-set collagen I layer in 24-well plates. For steroid stripping, cells were grown for 5 days in phenol red-free DMEM supplemented with 10% FBS-DCC. The initial seeding concentration was corrected as a function of the respective hormone dependent effects on cell growth in order to obtain the same number of cells after 5 days of culture (1.5 106 cells per 6-well). The cell seeding ratios were 0.5 for E2-treated cells, 1 for control cells and 1.5 for ICI182,780-treated cells. By this way, growth effect was avoided. However, we assume that these corrected seeding could decrease the observed differences between the treatments since a high number of cells could interact for a longer period in control and ICI182,780 treatment. For primary cultures, epithelial cell clusters with polarized ductal and acinar structures (organoids) were isolated from reduction mammoplasties and maintained in 3D collagen I scaffold as described (Chambon et al., 1984). All patients gave their written informed consent and this study was reviewed and approved by our Institutional Review Board. For scanning (SEM) and transmission (TEM) electron microscopy studies, floating collagen membranes were used as supports. Organoids were first attached to collagen I (from Flow SA, UK) layer and then harvested to obtain floating collagen membranes that retracted after 2 days of culture. Organoids were then grown for 4 days in the presence of 10% FBSDCC for estrogen withdrawal and finally treated or not with 1 nM estradiol (E2) for 7–12 days.

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2.3. Cell–cell adhesion A first dissociation assay involves subconfluent cells subjected to a moderate incubation with trypsin–EDTA (Gibco). MCF7 cells deprived of steroids for 5 days were differently seeded in order to obtain the same confluence 5 days later, according to treatment effect. After attachment, cells were treated for 5 days with 1 nM E2 or 1 nM E2 + 1 lM ICI182,780. For the release of the entire cell monolayer as a single cell suspension, MCF7 cells were usually incubated for 15 min with a solution of 0.5 mg/ml trypsin–EDTA diluted in Dulbelcco’s phosphate buffer saline without calcium and magnesium (DPBS) (Gibco). Here, to investigate the adhesive strength in cells pretreated with hormones, adherent cells were simply washed with DPBS and the 0.5 mg/ml trypsin treatment was shortened to only 7 min. Thereby cells were released as aggregates of several cells or as single cells whose number was counted by two investigators using Malassez’s chambers. A SD < 5% was observed between duplicates. The second dissociation assay was performed with dispase as described previously (Kimura et al., 2007) to analyze the presence of calcium-independent desmosomes on confluent cells. MCF7 cells were deprived of steroid and treated for 5 days with E2 or E2 + ICI182,780 to obtain the same confluence (98%) as described above. Cell sheets were detached from wells by 45 min incubation with dispase (2.4 U/ml; Gibco) diluted in DPBS. At the end of the treatment, dispase was inactivated by dilution with low calcium medium. The cell suspension was incubated for 90 min in this low calcium medium and then subjected to 20 rotation cycles. Then, single cells and fragments were counted under an inverted microscope. 2.4. Immunohistochemistry For immunoperoxydase detection, cells were fixed with 4% paraformaldehyde (PFA) in phosphate saline buffer (PBS) for 1 h. They were then permeabilized by 25 min incubation with 0.05% Saponin in 0.05% Tween-PBS (PBS-Ts) and blocked with 2.5% horse serum in PBS-Ts. Cells were incubated for 1 h with anti-cytokeratin-18 (1 lg/ml), desmocollin 2/3 (12 lg/ml) or c-catenin (1:2000) antibodies. Cells were extensively washed and incubated for 30 min with a biotinconjugated horse anti-IgG antibody, then washed again and incubated with streptavidin–biotin peroxydase complex (Vectastain ABC kit) as described by the manufacturer (Vector Laboratories, Burlingame, CA, USA). All incubations with antibodies were performed in PBS supplemented with 0.05% Saponin and 1% bovine c-globulin and all washes were done in PBS-Ts. A non-relevant mouse monoclonal antibody MOPC21 was used as negative control. For immunofluorescence, cells grown on coverslips were fixed with 4% PFA for 12 min, cold methanol for 4 min, cold acetone for 2 min, and then saturated overnight at 4 °C with 2.5% goat serum in phosphate saline buffer (PBS) containing 4% bovine c-globulin. For immunostaining, cells were incubated with desmoplakin I/II (2 lg/ml) antibody for 2 h. Cells were extensively washed and incubated for 1 h with Alexa fluor antibody, then washed again and incubated with DAPI (0.5 lg/ml, Sigma–Aldrich Chimie) for nuclear staining. All incubations were performed in PBS supplemented with 1% bovine c-globulin and all washes were done with PBS. Negative controls were performed with a purified rabbit IgG1. 2.5. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) MCF7 cells grown on glass coverslips and organoids of normal cells grown on floating collagen membranes were fixed with glutaraldehyde (3.75%) for 2 h and post-fixed with 1% osmium tetroxide for 1 h in Millonig buffer at pH 7.3. Coverslips and floating collagen membranes were rinsed in Millonig buffer and dehydrated through a graded series of alcohol and isoamyl acetate

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solutions before critical-point drying. After mounting on aluminum support, samples were coated with gold and later examined under a JEOL Model JSM-35 scanning electron microscope at 15 kV. For TEM experiments, MCF7 cells were grown for 4 days in phenol red-free medium containing 10% FBS-DCC then grown during the indicated times with or without 1 nM E2. Cells plated in 24-well plates or on collagen I gel were fixed in a freshly prepared mixture of 3% paraformaldehyde and 0.05% glutaraldehyde in PBS, and postfixed with 1.33% osmium tetroxide in collidine buffer, dehydrated and embedded in Epon. Ultra-thin sections were stained with uranyl acetate solution and examined with a Philips EM 300 transmission electron microscope (Eindhoven, Netherlands) at 60 kV. Organoids were cut out of the collagen gel, fixed with 3.75% glutaraldehyde and post-fixed with 1% osmium tetroxide in Millonig buffer, dehydrated and embedded in Araldite. Ultra-thin sections were stained with uranyl acetate and lead citrate before examination. 2.6. Immunoblotting and RNA-interference For Western blot analysis, 50–200 lg of soluble proteins were resolved by 12% or 7.5% SDS–PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk powder for 1 h, washed and probed overnight at 4 °C with a 1:500 dilution of desmosomal proteins and ERa antibodies or a 1:1000 dilution of GAPDH antibody. After washing, membranes were incubated for 1 h at room temperature with a goat anti-rabbit or a sheep antimouse HRP-labeled secondary antibody purchased, respectively, from Jackson Immunoresearch (Suffolk, UK) and Amersham (Piscataway, NJ, USA). Hence, protein bands were detected with ECL detection reagents (GE Healthcare) according to the manufacturer’s instructions. Molecular weights were determined with Precision Plus Protein All Blue Standards (Bio-Rad Laboratories). RNA-interference experiments were performed by transfection of a pool of three 19-nt sense oligomers, 50 -GCA TCC AGC TTC AGA CAA A-30 , 50 -ACA CCA ACA TCG CTC AGA A-30 and 50 -GTG CAG AAC TTG GTA AAC A-30 , which recognize human desmoplakin I/II mRNAs (Zhou et al., 2004). The best concentrations of siRNAs for an effective gene silencing were determined by incubating cells with a dose range of 0–100 nM, followed by the analysis of desmoplakin I/II expression by Western blotting. Specific oligomers of ERa siRNA have been previously described (Maynadier et al., 2008). A 18-nt oligomer siRNA specific for luciferase (50 -AAC GTA CGC GGA ATA CTT CGA -30 , sense) was used as a negative control to check non specific effects of transfection. For desmoplakin I/II silencing, cells were plated 24 h before transfection, while for ERa silencing, cells were plated on six-well plates after transfection. After 4 days of transfection completion, cells were lysed for protein extraction and immunoblot analysis. 2.7. Statistical analysis Quantitative analysis of Western blots was performed with the PC-Bas 2.0 densitometric software (Fuji, Stanford, CT, USA). Statistical analysis was performed using the Student’s t test to compare paired groups of data. A P value of <0.05 was considered statistically significant. The Mann–Whitney–Wilcoxon test was used to compare distributions of aggregate sizes in different treated samples. 3. Results 3.1. Estrogens increase cell–cell adhesion The estrogen-dependency of intercellular adhesion of MCF7 breast cancer cells was studied in cell monolayers or after partial

trypsin digestion. Firstly, in cell monolayer the deprivation of estrogen for 5 days induces scattering and decreased contacts between cells in contrast to estrogen-treatment that induced formation of tightly packed colonies with undetectable cell borders (Fig. 1a). The full reversion of this effect by an excess of a pure antiestrogen (ICI182,780) clearly identifies the implication of an ER, and probably ERa whose affinity for this antiestrogen is higher than ERb (Wakeling, 2000). This higher cell aggregation became visible after 2 days of estrogen treatment and was also found in monolayer cultured on collagen I or Matrigel (data not shown). In order to investigate more precisely the effect of E2 on ERa positive cells, we decided to compare the adhesiveness of cell sheets of breast cancer cells. Firstly, the resistance of a cell monolayer to dissociation into aggregates or single cells after a controlled mild trypsin treatment was studied (Fig. 1b). This treatment only released 24% of E2-treated cells as single cells in comparison to 62% of control cells. Moreover, 41% of E2-treated cells remained in aggregates containing more than 5 cells in comparison to only 1% of control cells and 9% of E2 + ICI182,780 treated cells. The second approach concerns confluent cultures dissociation with dispase as previously described (Kimura et al., 2007). Cell sheets were detached from their support with dispase and incubated in low calcium medium for 90 min. The E2-treated cell sheets showed an increased resistance to dissociation (Fig. 1c and d). The degree of dissociation was quantified by counting the number of fragments and of single cells released from the sheets (Fig. 1e and f). Whereas dispase dissociated the cell monolayer in single cells and aggregates in both control and in E2 + ICI182,780 treated cells, the monolayer remained nearly unaffected when cells were pretreated with E2. Thus, E2 appeared to significantly promote cell–cell adhesion and this effect was significantly reversed by antiestrogen addition. As desmosomes are the only type of junction that support calcium independency (Wallis et al., 2000), this result suggests an increase of desmosomes with E2 treatment. Using SEM analysis, we analyzed the effects of estrogen on the morphology of MCF-7 cells and normal breast epithelial cell maintained as primary cultures on floating collagen membrane (Fig. 1g). After a 7-day estrogen treatment, MCF7 cells were in clusters with gland-like structures and numerous microvilli whereas cells grown in estrogen-deprived medium appeared markedly flattened with rare microvilli. Similar morphological changes were also observed at the apical surface of normal mammary structures known as organoids. 3.2. Ultrastructural analysis revealed that estrogens increase desmosomes in normal and cancer mammary cells The desmosomes were then analyzed by TEM in cells cultured either on plastic or on collagen I gel. When MCF7 cells were grown as a monolayer on standard culture dishes, desmosomes (black arrows) were observed (Fig. 2a). However, when MCF7 cells were cultured on collagen I gel or when normal mammary cells were maintained in floating collagen gel, tight and adherens junctions (white arrows) were observed near lumens in addition to desmosomes (black arrows) along the adjacent plasma membranes. After E2 addition, cells showed a marked increase in the number of desmosomes between lateral membranes. At a higher magnification, the enlargement of the dense cytoplasmic plaques of the desmosomes was observed in normal cells with an apparent increase of intermediate filaments and to a lower extent in cancer cells (Fig. 2b). Desmosomal cadherins could be observed in the intercellular space although the well-described dense midline in keratinocytes desmosomes was absent in these cells. Desmosomes were then counted on electron microscopy photographs over a distance of 600 nm along the cell–cell border (Fig. 2c). A significant increase of 3 and 3.3-fold in the total number of desmosomes was observed

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SEM Fig. 1. Estrogen enhances cell–cell interactions. (a) Morphological structure of subconfluent MCF7 breast cancer cells treated for 5 days with 1 nM E2 (E2), 1 nM E2 + 1 lM ICI182,780 (E2 + ICI), or vehicle alone (control). Bars, 10 lm. (b) Adhesive strength was evaluated by a moderate trypsin dissociation. Monolayers of subconfluent adherent MCF7 cells were released as aggregates by moderate trypsin treatment as described in Materials and Methods. Data are means of 2 independent experiments ± SD. Differences of the overall cell distributions were significant (0.05) between Control and E2 and non significant between Control and E2 + ICI182,780 according to the Mann– Whitney–Wilcoxon test. (c–f) Monolayers of MCF7 confluent cells treated for 5 days as in (a) were detached with 2.4 U/ml dispase and incubated for 90 min in low calcium medium to destroy calcium-dependent cell–cell adhesion. The dissociation of the cell monolayer was photographed. (d) shows parts of (c) at higher magnification. Bars, 2 mm (c), 500 lm (d). The dissociation by LCM into single cells (e) and in monolayer fragments (f) was quantified by counting under microscope. Values represent mean ± SD, ⁄ P < 0.05 relative to control (Student’s t test). Data are representative of 3 independent experiments. (g) Scanning electron microscopy (SEM) of MCF7 cells or organoids of normal mammary cells treated (E2), or not (C) for 7 days. Bars, 5 lm. All data are representative of 3 experiments.

in MCF7 cells after 7 and 12 days of E2 treatment. A significant increase (1.8–3.4-fold) in desmosomes was also observed after 7 days treatment in three different primary cultures of normal mammary cells. 3.3. Estrogen promotes the expression of desmosomal proteins in three ERa-dependent cell lines To understand the mechanism by which estrogen enhances desmosomal formation, the expression of some major desmosomal proteins was studied. The desmosome is composed of transmem-

brane proteins of the broad cadherin family (desmogleins and desmocollins) that are linked to the intermediate filament cytoskeleton through plakophilin, c-catenin and desmoplakins (Bonne et al., 2003; Chidgey, 2002; Garrod and Chidgey, 2008). Desmocollin 2/3, c-catenin and cytokeratin-18 were first assessed by immunoperoxydase staining whereas desmoplakin I/II was detected by immunofluorescence in MCF7 cells (Fig. 3a). A clear increment in the expression of these desmosomal proteins was observed at the membranes of E2-treated cells in comparison to control or E2 + ICI182,780 treated cells. For desmocollin 2/3 and desmoplakin I/II, this difference was totally reversed by addition

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Fig. 2. Estrogen promotes desmosomes formation in MCF7 cancer cells and normal mammary cells. MCF7 cells and normal mammary cells were treated with 1 nM E2 (E2) or vehicle alone (C). (a) Tight and adherens junctions (white arrows) were observed by transmission electron microscopy (TEM) in MCF7 cells and normal mammary cells cultured 7 days in collagen gel, while desmosomes (black arrows) increased after E2 exposure (E2). Bar, 0.1 lm. (b) Higher magnification of desmosome sections. Bar, 0.05 lm. Data are representative of 3 experiments. (c) Desmosome quantification in MCF7 cells cultured for 7 days in monolayer cultures or for 12 days in collagen I gel in the presence (E2), or absence (C) of 1 nM E2, and normal mammary cells from 3 primary cultures obtained from different reduction mammoplasties after 7 days of E2 treatment. In each culture, the number of desmosomes was quantified on a total length of 600 lm of adjacent cell membranes analyzed in 3 separate areas. Data represent mean ± SD (bars) of the number of desmosomes per 100 lm of membrane. Electron microscopy photographs (final magnification of 22,500) were used for this quantification. ⁄P < 0.05 relative to control.

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Fig. 3. Estrogen treatment results in specific accumulation of desmosomal proteins. (a) Immunoperoxydase and immunofluorescent assessment of desmosomal proteins’ expression in MCF7 cells. MCF7 cells were untreated (C), or treated with 1 nM E2 (E2) or with E2 + 1 lM ICI 182,780 (E2 + ICI) for 5 days and then immunoperoxydase labeling observed with anti-desmocollin 2/3, anti-c-catenin, anti-cytokeratin18 and immunofluorescent staining with anti-desmoplakin I/II. Bar, 5 lm. (b) Western blot analysis of whole cell lysates of MCF7, T47D, and BG1 cells treated as described in (a). Gels were immunoblotted for desmoplakin I/II (Dsp I/II), desmocollin 2/3 (Dsc 2/3), b-catenin (b-cat), plakophilin 3 (Pkp 3), E-cadherin (Ecad) and c-catenin (c-cat) expressions. Equal amounts of proteins were loaded in each lane and blots were stripped and re-probed with the antibody for GAPDH, used as an equal loading control. The histogram represents E2/E2 + ICI ratio of densitometric values of protein signals. ⁄P < 0.05,⁄⁄P < 0.01 relative to respective E2-treated value. Data are mean of 3 independent experiments.

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of ICI182,780 whereas c-catenin and cytokeratin-18 levels were equivalent in ICI182,780 treated and control cells. Estradiol significantly increased the immunostaining of cytokeratin-18, a major component of intermediate filaments. This observation confirmed the increase of the fiber network observed by TEM. The regulation of four desmosomal proteins (desmoplakin I/II, desmocollin 2/3, plakophilin 3, c-catenin) was then analyzed in ERa-dependent cell lines from breast (MCF7, T47D) or ovarian (BG1) origin. Western blot analysis showed that all these proteins are significantly up-regulated by a 5-day E2 treatment in a range of 2–10-fold (Fig. 3b) as compared to E2 + ICI182,780 treatment. The

higher expression found in control cells versus E2 + ICI182,780 treated cells could reflect a partial activation of ERa due to residual E2 in control conditions. It should be noted that in these blots the internal control housekeeping protein, GAPDH was unaffected by these treatments. Moreover, among proteins involved in other junctions, b-catenin was increased by 1.2–2 depending of the cell type whereas E-cadherin was unaffected by these treatments. E-cadherin is required to specifically bind c-catenin in order to enhance desmosome formation, while its interaction with b-catenin cannot support desmosome formation (Lewis et al., 1997).

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Fig. 4. Down-regulation of desmoplakin I/II expression and cell–cell adhesion by ERa silencing. (a) Inhibition of ERa expression in MCF7 cells. Western blots of lysates from MCF7 cells transfected with ERa and control siRNAs. During 4 days cells were treated with 1 nM E2 or vehicle alone and cell lysates were collected 4 days after completion of transfection. Membranes were immunoblotted to quantitate ERa and desmoplakin I/II expression and reprobed for b-actin. Data are representative of 3 experiments. The histogram represents densitometric values of protein signals, with control cells considered as 100%. ⁄P < 0.05 relative to their respective control siRNA value. (b) Effect of different concentrations of ERa siRNAs on cell–cell adhesion evaluated by the counting of cell clusters (described in Fig. 1b). Data represent the mean percentage of cells in aggregates (P5 cells) from 3 independent experiments. Bars, SD. ⁄P < 0.05 relative to E2-treated cells transfected with control siRNA.

Fig. 5. Inhibition of estrogen-induced adhesion by desmoplakin I/II (Dsp) siRNAs. (a) Inhibition of desmoplakin I/II expression in MCF7 cells by specific siRNAs is shown by immunofluorescence, using a specific anti-desmoplakin I/II antibody, and revealed with Alexa fluor dyes conjugated with secondary antibody (time exposure and magnification are identical). From left to right: 100 nM control siRNA; 50 and 100 nM desmoplakin I/II siRNAs. Bar, 3 lm. (b) Western blot analysis of expression of desmoplakin I/II and GAPDH in cells treated with 100 nM control siRNA, 50 or 100 nM desmoplakin I/II siRNAs for 4 days The histogram represents densitometric values of protein signals, with control cells considered as 100%. ⁄P < 0.05 relative to their respective control siRNA value. (c) Effect of different concentrations of desmoplakin I/II siRNAs on cell–cell adhesion evaluated by the counting of cell cluster (method described in Fig. 1b). Data represent the mean percentage of cells in aggregates (P5 cells). Bars, SD. ⁄P < 0.05 relative to E2-treated cells transfected with control siRNA. All data are representative of 3 experiments.

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However, c-catenin displays a higher affinity for the desmosomal cadherin than for E-cadherin. Taken together, these data revealed the up-regulation of the major desmosomal proteins by estrogens in breast and ovarian cancer cells. 3.4. ERa silencing prevents estrogen-induced cell–cell adhesion and desmoplakin I/II expression in MCF7 cells To functionally evaluate the role of ERa in cell–cell adhesion specific siRNAs were used to knock-down ERa in MCF7 cells. Fig. 4a shows that siRNA treatment decreased ERa expression by 48% and this effect is sufficient to reduce the capacity of the cells to resist trypsin treatment. After E2 treatment, 42% of control cells resist as aggregates after trypsin treatment as compared to only 10% for ERasiRNA treated cells. In addition, the 2-fold increase in a key desmosomal protein, desmoplakin I/II, observed in presence of estrogens was totally blocked by the ERa silencing (Fig. 4b). 3.5. Desmoplakin I/II silencing prevents estradiol-induced adhesion in MCF7 cells To further specify the relevance of desmosomes in E2-induced adhesion, we used specific desmoplakin siRNAs to impair desmosomal formation (Vasioukhin et al., 2001). Desmoplakin I/II immunostaining presented a punctate distribution along adjacent cell membranes in control MCF7 cells, while, in cells transfected with desmoplakin I/II siRNAs, its expression decreased in a dose-dependent manner (Fig. 5a). In agreement with these observations, Western blot analysis of cellular extracts indicated a 60% and 80% decrease in desmoplakin I/II expression in cells transfected with 50 and 100 nM specific siRNAs, respectively (Fig. 5b). We then examined the ability of the treated cells to resist in the form of aggregates after mild trypsin treatment (Fig. 5c). In cells transfected with a non specific siRNA, E2 enhanced by 3.2-fold the number of aggregates resistant to trypsin but this effect appeared totally abolished by desmoplakin I/II silencing. This indicates a major role of desmoplakin I/II in estrogen-induced cell–cell adhesion.

4. Discussion Previous studies have pointed out the crucial role of intercellular junctions in cancer spreading. Current literature highlights the role of tight and adherens junctions in cancer invasion but several evidences also evoked a preventing role of desmosomes in this process. Initial observations of Alroy et al. (1981) and Schindler et al. (1982) have shown an inverse correlation between the number of desmosomes and the aggressiveness of several carcinomas. The reduction of desmoplakin expression was then evidenced in different neoplasms, i.e., uterine adenocarcinoma, breast cancer, oral squamous carcinomas and oropharyngeal cancer (Hiraki et al., 1996; Nei et al., 1996). More recently, a combined modulation of adherens junctions and desmosomes was implicated in cancer invasion (Vasioukhin et al., 2001) and in epithelialmesenchymal transition of early tumors to invasive cancers (Dahlman-Wright et al., 2006). Besides the primary role of desmosomes in maintaining tissue integrity (Chidgey, 2002; Garrod and Chidgey, 2008), more and more studies indicate that desmosomes are also signaling centers and that loss or perturbation of their components contributes to cancer pathogenesis (Chidgey and Dawson, 2007; Dusek and Attardi, 2011). In recent studies the conditional knockout of desmosomal genes in mice more directly demonstrated a role of desmosomes in cancer suppression (Beaudry et al., 2010; Chun and Hanahan, 2010).

In this study, several complementary approaches indicate that estrogens induce cell adhesion by the formation of desmosomes. Firstly, TEM studies indicate the presence of a greater number of desmosomes after prolonged E2 treatment of normal or malignant mammary cells. Secondly, estrogens significantly increase key desmosomal proteins (desmoplakin I/II, c-catenin, desmocollin 2/3, plakophilin 3) detected by immunocytochemistry or immunoblotting analyses. The finding that ICI182,780, a pure anti-estrogen, clearly reversed these effects demonstrates the estrogen receptor-mediated action. Thirdly, partial inhibition of ERa expression causes a reduction of desmoplakin, an essential protein in the formation of desmosomes. Finally, the fact that siRNA silencing of desmoplakin expression was sufficient to prevent E2-dependent adhesion indicates that desmosomes play a major role in this pathway. We have demonstrated that cancer cells conserve estrogen responsiveness by increasing membrane contacts and desmosome number. These data are consistent with our previous results indicating that E2 prevented cancer cell invasion in vitro through a mechanism involving ERa functional domains (DNA binding domain and AF2 transcriptional activator domain) required for transcriptional activation (Platet et al., 2000). As a whole, our results suggest that estrogens could reduce invasion through at least two mechanisms: the initial ERa transactivation of specific genes that is sufficient to prevent Matrigel invasion at 24 h (Platet et al., 2000) and the activation of intercellular adhesion by desmosome formation after 4 days. These results could help to explain the favorable prognosis associated with the ERa status in primary breast cancers. The clinical observations that tumors of postmenopausal women who receive estrogen replacement therapy are less disseminated to distant sites than tumors of placebo-treated patients (Decker et al., 2003; Marsden and Blacks, 1997) is also consistent with an estrogen regulation of cell–cell adhesiveness. In addition to these effects that required long exposure to estrogen, rapid effects of ERa on actin cytoskeleton modulators have also revealed that cell membrane remodeling modulated cell motility (Giretti et al., 2008). In addition, TEM studies show that the estrogen-mediated increase of desmosomes is significant in primary cultures of normal mammary cells. These data raise the question of the physiological relevance of estrogen-induced desmosome formation in steroid target tissues. An attractive hypothesis for E2 function would be a balance between proliferation and modulation of desmosomes to maintain epithelium integrity. This proposal is argued by the observations of Wada-Hiraike et al. (2006) which demonstrated a reduction of desmosomes in colonic epithelial cells and a more frequent shedding of the epithelium after ERb inactivation in mice. However, more studies are required to understand the physiological impact of the ERa and ERb estrogen-regulation of desmosomes in the mammary gland and in other estrogen target tissues. In conclusion, our experiments provide novel insights into cellular adhesion of normal and malignant mammary cells containing estrogen receptors. A prolonged treatment with estrogen is associated with an increase of desmosomal proteins and the formation of desmosomes. We conclude that a functional interplay exists between estrogen receptors, desmosome formation and intercellular adhesion. Future investigations will define how estrogen-mediated desmosome formation alters the invasiveness or the endocrine response of hormone-dependent cancers. Acknowledgements We thank J.Y. Cance for art work. This work was supported in part by Grants of the ‘‘Ligue Régionale de Lutte contre le Cancer’’, the ‘‘Association pour la Recherche contre le Cancer’’ SFI20101201906 and BQR Grants from University of Montpellier 1.

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