Fabrice Journe
email: fabrice.journe@bordet.be

Virginie Durbecq

Carole Chaboteaux

Ghizlane Rouas

Guy Laurent

Denis Nonclercq

Christos Sotiriou

Jean-Jacques Body

Denis Larsimont

Fabrice Journe Ae

A E Carole

Chaboteaux Ae

A E Guy

Laurent Ae

A E Christos

Sotiriou Ae

Jean-Jacques Body

Á V Durbecq

Á C Chaboteaux

Á G Rouas

Á C Sotiriou

J.-J Body

Á D Larsimont

Á D Nonclercq

Association between farnesoid X receptor expression and cell proliferation in estrogen receptor-positive luminal-like breast cancer from postmenopausal patients

Keywords: NR1H4, Breast cancer, Estrogen receptor, Ki-67, Immunohistochemistry, Bile acids

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Introduction

The farnesoid X receptor (FXR, NR1H4) was initially isolated in 1995 as an orphan nuclear receptor activated by farnesol, a metabolic intermediate of the mevalonate pathway [START_REF] Forman | Identification of a nuclear receptor that is activated by farnesol metabolites[END_REF]. In subsequent studies, FXR was deorphanized by the identification of bile acids (in particular chenodeoxycholic acid, CDCA) as cognate ligands [START_REF] Wang | Endogenous bile acids are ligands for the nuclear receptor FXR/ BAR[END_REF][START_REF] Makishima | Identification of a nuclear receptor for bile acids[END_REF][START_REF] Parks | Bile acids: natural ligands for an orphan nuclear receptor[END_REF]. Presently, FXR is considered as a member of the metabolic nuclear receptor family [START_REF] Modica | Nuclear bile acid receptor FXR as pharmacological target: are we there yet?[END_REF]. Like other nuclear receptors, it functions as a ligand-activated transcription factor. Thus it contains a DNA binding domain capable of interacting with specific cis-DNA sequences (FXR response elements, FXRE's) associated with the promoters of target genes. It also exhibits a ligand binding domain accepting small lipophilic molecules [START_REF] Lee | FXR, a multipurpose nuclear receptor[END_REF]. Upon ligand-induced activation, FXR generally heterodimerizes with the retinoid X receptor (RXR) and binds to inverted repeat-1 motifs (IR-1) or other motifs present in FXRE's [START_REF] Edwards | BAREing it all: the adoption of LXR and FXR and their roles in lipid homeostasis[END_REF].

In early studies, FXR was shown to exhibit a rather narrow tissue distribution, its expression being restricted to the liver, the intestine, the kidney and the adrenal gland [START_REF] Forman | Identification of a nuclear receptor that is activated by farnesol metabolites[END_REF]. In the liver and the gastrointestinal tract, it functions as a bile acid sensor and regulates bile acid homeostasis, lipid and glucose metabolism, and hepatic regeneration [START_REF] Lee | FXR, a multipurpose nuclear receptor[END_REF]. In hepatocytes, it indirectly inhibits the cytochrome p450 enzyme cholesterol 7-a-hydroxylase (CYP7A1) involved in the conversion of cholesterol into bile acids, and it induces the synthesis of a variety of proteins implicated in the metabolism and transport of lipids [START_REF] Edwards | BAREing it all: the adoption of LXR and FXR and their roles in lipid homeostasis[END_REF][START_REF] Kalaany | LXRS and FXR: the yin and yang of cholesterol and fat metabolism[END_REF]. Thus, FXR has emerged as a new therapeutic target for the management of lipid disorders [START_REF] Claudel | The farnesoid X receptor: a novel drug target?[END_REF][START_REF] Westin | FXR, a therapeutic target for bile acid and lipid disorders[END_REF].

In addition, FXR has recently been detected in vascular smooth muscle cells [START_REF] Bishop-Bailey | Expression and activation of the farnesoid X receptor in the vasculature[END_REF] and endothelial cells [START_REF] He | Downregulation of endothelin-1 by farnesoid X receptor in vascular endothelial cells[END_REF]. Surprisingly, we and others have also found FXR expression in breast cancer tissue and breast cancer cell lines [START_REF] Silva | Lipids isolated from bone induce the migration of human breast cancer cells[END_REF][START_REF] Swales | The farnesoid X receptor is expressed in breast cancer and regulates apoptosis and aromatase expression[END_REF][START_REF] Journe | Farnesol, a mevalonate pathway intermediate, stimulates MCF-7 breast cancer cell growth through farnesoid-X-receptor-mediated estrogen receptor activation[END_REF]. With regard to breast cancer development, the presence of high plasma levels of deoxycholic acid in postmenopausal breast cancer patients, as compared to healthy controls matched for age and body mass index, has been documented, suggesting that this bile acid might be involved in mammary gland carcinogenesis [START_REF] Costarelli | Plasma deoxycholic acid concentration is elevated in postmenopausal women with newly diagnosed breast cancer[END_REF]. Moreover, accumulation of bile acids from serum has been reported in breast cyst fluid and has been discussed as a potential risk factor for developing breast cancer [START_REF] Raju | Bile acids in human breast cyst fluid: the identification of lithocholic acid[END_REF][START_REF] Javitt | Breast-gut connection: origin of chenodeoxycholic acid in breast cyst fluid[END_REF][START_REF] Costarelli | Plasma bile acids and risk of breast cancer[END_REF]. Strikingly, recent data from our group reveal a positive crosstalk between FXR and estrogen receptor alpha (henceforth abbreviated as ER), accounting for FXR-mediated ER activation in absence of estrogens and resulting in a mitogenic response of breast carcinoma cells to farnesol [START_REF] Journe | Farnesol, a mevalonate pathway intermediate, stimulates MCF-7 breast cancer cell growth through farnesoid-X-receptor-mediated estrogen receptor activation[END_REF].

In this retrospective study, we assessed by immunohistochemistry FXR expression in breast cancer tissue specimens and we analyzed its correlation with classical breast cancer biomarkers in order to evaluate the FXR prognostic significance in breast cancer. Furthermore, we determined the effects of FXR activation by bile acid in breast cancer cell lines grown in steroid-free medium supplemented or not with estrogen.

Materials and methods

Breast cancer tissue sampling

Formalin-fixed, paraffin-embedded primary breast cancer samples collected from 204 patients at the Institut Jules Bordet were used for the evaluation of FXR expression. Patients with primary invasive breast cancer were included in the study, while patients with bilateral breast cancer as well as previous or concomitant cancer other than breast cancer were excluded. All marker analyses were performed on breast cancer tissue specimens collected in patients who did not receive neoadjuvant therapy. A subpopulation of 120 untreated breast cancer patients was selected to evaluate the ''pure'' prognostic value of FXR. The study was performed according to the REMARK recommendations from the National Cancer Institute-European Organisation for Research and Treatment Cancer [START_REF] Mcshane | REporting recommendations for tumor MAR-Ker prognostic studies (REMARK)[END_REF].

The clinico-pathological characteristics of the population are described in Table 1. Objective assessment of FXR and other biomarkers was performed by immunohistochemical analysis of tissue microarrays (TMA). Thus, representative core needle specimens (0.6 mm diameter) of invasive cancer tissue (identified as such by histological examination of hematoxylin/eosin-stained sections) were punched from donor tissue blocks and transferred to recipient blocks using a specific arraying device (Beecher Instruments Silver Spring, MD, USA). A minimum of 4 tissue cores was taken per donor tissue block to minimize the problem of tumor heterogeneity. The ethics committee of the Institute approved the use of the tissue material for this study.

Immunohistochemical evaluation of FXR expression

TMA blocks were cut and tissue sections were mounted on poly-L-Lysine-coated glass slides. Immunohistochemical staining was performed with an antibody raised against human FXR (mouse monoclonal anti-human FXR/NR1H4 antibody, clone A9033A, R&D Systems, Minneapolis, MN, USA). Prior to immunostaining, antigen retrieval was achieved by microwave pretreatment (2 9 10 min at a power of 650 W) in citrate buffer pH 6.0. Thereafter, tissue sections were incubated for 30 min at 37°C in presence of the primary antibody diluted 1:25. FXR antigen-antibody reaction was visualized using Ventana automated system with the highly sensitive Nexes reagents (Enhanced Nexes reagent, Ventana Medical Systems, Tucson, AZ, USA). Nuclear staining was defined as positivity. FXR expression was scored from 0 to 8 by adding a score reflecting the proportion of positively stained cells (none: 0, \1/100: 1, 1/100 to 1/10: 2, 1/10 to 1/3: 3, 1/3 to 2/3: 4 and [2/3: 5) and a score reflecting the staining intensity (none: 0, weak: 1, intermediate: 2 and strong: 3), as defined by Allred et al. [START_REF] Allred | Prognostic and predictive factors in breast cancer by immunohistochemical analysis[END_REF]. The semi-quantitative analysis was performed in a single-blind fashion by an experienced pathologist (D.L.).

Other antibodies used for immunohistochemistry were raised against estrogen receptor (ER, clone 6F11), progesterone receptor (PgR, clone AB), HER-2/neu (clone CB11), p27 (clone 1B4), c-myc (clone 9E11), mucin-1 (muc-1, clone Ma552) and cytokeratins 8/18 (CK8/18, clone 5D3) (from Novocastra, Newcastle upon Tyne, UK); Ki-67 (clone MIB1), epidermal growth factor receptor (EGFR, kit), CK5/6 (clone D5/6B4) (from Dako Belgium, Heverlee, Belgium); GATA-3 (clone HG3-31) and cyclin E (clone He12) (from Santa Cruz Biotechnology, Santa Cruz, CA, USA); cyclin D (P2D11F11) (from Ventana Medical System); AIB-1 (clone 34) (from BD Biosciences, Erembodegem, Belgium) and topoisomerase II alpha (Topo II, clone KiS1) (from Chemicon, Temecula, CA, USA). All markers were scored from 0 to 8, except Ki-67 and Topo II for which the percentages of positively stained cells were reported, and HER-2/neu for which the expression was scored from 0 to 3 (scoring system recommended for the Dako Hercep Test, Carpinteria, CA, USA). Biomarker overexpression was defined as a Topo II score [10%, a HER-2/neu score of 3, and a scoring C2 for all other biomarkers. Samples with a Ki-67[25% were considered as highly proliferating tumors.

Breast cancer cell lines and culture conditions

The ER-positive MCF-7 breast cancer cell line (ATCC HTB-22) and the ER-negative MDA-MB-231 breast carcinoma cell line (ATCC HTB-26) were both obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured at 37°C in a humidified 95% air and 5% CO 2 atmosphere. For routine maintenance, cells were propagated in 75-cm 2 flasks containing Eagle's Minimum Essential Medium (MEM) with Phenol Red, supplemented with 10% heat-inactivated fetal bovine serum (FBS) and with L-glutamine, penicillin and streptomycin (Gibco BRL, Life Technologies, Merelbeke, Belgium) at standard concentrations. Cells were harvested by trypsinization (0.1% trypsin-0.02% EDTA) and subcultured twice weekly. For experiments, cells were plated in steroid-free medium (SFM) consisting of MEM without Phenol Red supplemented with 10% dextran-coated charcoal-treated FBS to suppress potential influence of serumderived lipid compounds. One or two days after seeding, the culture medium was replaced by fresh SFM containing chenodeoxycholic acid (CDCA, Sigma, St Louis, MO, USA), 17b-estradiol (E 2 , Sigma), 4-hydroxytamoxifen (tamoxifen, Sigma), fulvestrant (ICI 182,780, Tocris, Bristol, UK), or vehicle (control). Cells were treated for 10-60 min or 1-3 days, with drugs alone or in combinations, as specified in ''Results''.

Western blot analysis

Cells were plated in 60-cm 2 Petri dishes (5 9 10 5 cells per dish) containing SFM. Two days after seeding, cells were cultured for 24 h in fresh SFM containing 50 lM CDCA, 1 nM 17b-estradiol, 100 nM 4-hydroxytamoxifen, 100 nM fulvestrant, or vehicle (control), alone or in combinations as specified in ''Results''. Then, cells were lysed using detergent cocktail, as previously described [START_REF] Journe | Farnesol, a mevalonate pathway intermediate, stimulates MCF-7 breast cancer cell growth through farnesoid-X-receptor-mediated estrogen receptor activation[END_REF]. Protein concentrations were determined by the BCA Protein Assay (Pierce, Rockford, IL, USA) using bovine serum albumin as standard. Equal amounts of cell proteins or 10 ng recombinant FXR protein (rFXR, Active Motif, Rixensart, Belgium) were subjected to Western blotting using a rabbit polyclonal anti-human FXR/NR1H4 antibody (Abcam, Cambridge, UK) diluted 1:1000, a mouse monoclonal antihuman ERa antibody (F-10, Santa Cruz Biotechnology) diluted 1:5,000, a mouse monoclonal anti-human PgR (A/B isoforms) antibody (NCL-PGR-AB, Novocastra) diluted 1:500, a mouse monoclonal anti-human cyclin D1 antibody (DCS6, Cell Signaling Technology, Beverly, MA, USA) diluted 1:2,000, or a rabbit polyclonal anti-mouse p27 antibody (Cell Signaling Technology) diluted 1:1000. b-actin was used as a loading control, and was demonstrated with a mouse monoclonal anti-human actin antibody (Chemicon) diluted 1:7,500. Of note, neutralization of the rabbit polyclonal anti-human FXR/NR1H4 antibody with excess rFXR (incubation for 2 h at room temperature) was used to document the specificity of the antibody. Peroxidase-labeled anti-rabbit IgG antibody (1:10,000) or peroxidase-labeled anti-mouse IgG antibody (1:10,000) (Amersham Pharmacia Biotech, Roosendaal, The Netherlands) were used as secondary reagents to detect corresponding primary antibodies. Bound peroxidase activity was revealed using the SuperSignal Ò West Pico Chemiluminescent Substrate (Pierce). Immunostaining signals were digitalized with a PC-driven LAS-3000 CCD camera (Fujifilm, Tokyo, Japan), using a software specifically designed for image acquisition (Image Reader, Raytest Ò , Straubenhardt, Germany). Immunoreactive band intensities were quantified using the software AIDA Ò Image Analyser 3.45 (Raytest Ò ).

Receptor co-immunoprecipitation

The formation of FXR/ER complexes in cells exposed to CDCA was determined by co-immunoprecipitation experiments, using first an anti-FXR antibody to immunoprecipitate FXR, followed by an anti-ER antibody to demonstrate by Western blotting the presence of ER in the immunoprecipitates. MCF-7 cells were plated in 60cm 2 Petri dishes (10 6 cells per dish) in SFM and cultured for 24 h. Control cells were not incubated further, while treated cells were exposed to 50 lM CDCA for 10, 20, 30 and 60 min as indicated in ''Results''. Cell monolayers were rinsed, harvested and lysed using detergent cocktail, as described in ''Western blot analysis'' section. Clarified supernatants containing equivalent amounts of proteins (1 mg), as determined by the BCA Protein Assay, were diluted with lysis buffer up to 500 ll; aliquots were saved for total FXR and ER expression determinations (see ''Western blot analysis''). In order to remove proteins that may otherwise cross react at the time of immunoprecipitation, supernatants were incubated with 100 ll of anti-rabbit IgG antibody-agarose (Sigma) for 2 h under agitation, and then centrifuged. Supernatants were therefore incubated overnight with the rabbit polyclonal anti-human FXR/NR1H4 antibody (Abcam) diluted 1:100. FXR-antibody complexes were precipitated with 100 ll of anti-rabbit IgG antibodyagarose for 2 h under agitation, and collected by centrifugation. Pellets were washed four times with the lysis buffer (see ''Western blot analysis''), suspended in 60 ll electrophoresis sample buffer, and boiled for 5 min.

Samples were finally subjected to Western blotting to assess ER levels, using the mouse monoclonal antihuman ERa antibody F-10, as described above. Non specific interactions were evaluated by omitting the anti-FXR antibody during the immunoprecipitation process.

Immunofluorescence microscopy

Cells were plated in SFM on sterile round glass coverslips in 12-well dishes, and cultured for 3 days without any treatment or cultured for 2 days and exposed to 50 lM CDCA or vehicle for additional 24 h. Cell monolayers were fixed with paraformaldehyde (PAF) and processed for immunofluorescence staining as described previously [START_REF] Journe | Farnesol, a mevalonate pathway intermediate, stimulates MCF-7 breast cancer cell growth through farnesoid-X-receptor-mediated estrogen receptor activation[END_REF]. Mouse monoclonal anti-FXR/NR1H4 antibody (clone A9033A, R&D Systems) or rabbit polyclonal anti-ER antibody (HC-20, Santa Cruz Biotechnology) were used as primary reagents (dilution 1:50). The cell preparations were subsequently, and in succession, exposed to a dextran polymer coated with both peroxidase and antibodies raised against mouse or rabbit immunoglobulins (EnVision TM , Dako Belgium), rabbit anti-peroxidase antiserum (Laboratory of Hormonology, Marloie, Belgium), biotinylated swine anti-rabbit immunoglobulins antibodies (Dako Belgium) and Texas Red-conjugated streptavidin (Vector Laboratories, Burlingame, CA, USA). The coverslips were mounted on glass slides using commercial anti-fading medium (Vectashield Ò , Vector Laboratories). The cell preparations were examined on a Leitz Orthoplan microscope equipped with a Ploem system for epi-illumination. Excitation wavelength of 560 nm and emission wavelength of 590 nm were used for the observation of Texas Red fluorescence. The appearance of immunostained cell preparations was documented by using a PC-driven digital camera (Leica DC 300F, Leica Microsystems AG, Heerbrugg, Switzerland). Microscopic fields were digitalized and stored thanks to a software specifically designed for image acquisition (Leica IM 50, Leica Microsystems AG).

Crystal violet growth assay

Cell number was assessed indirectly by staining with crystal violet dye as previously described [START_REF] Journe | Farnesol, a mevalonate pathway intermediate, stimulates MCF-7 breast cancer cell growth through farnesoid-X-receptor-mediated estrogen receptor activation[END_REF]. Briefly, cells were seeded in 96-well plates (10 3 cells per well) in SFM supplemented or not with 1 nM 17b-estradiol, and cultured for 24 h. Cells were then exposed to 1-200 lM CDCA or vehicle (control) for 3 days. After medium removal, cells were gently washed with PBS, fixed with glutaraldehyde/PBS and stained with crystal violet. Cells were destained under running tap water and subsequently lysed with Triton X-100. The absorbance was measured at 550 nm using a Microplate Autoreader EL309 (BIO-TEK Instruments, Winooski, VT, USA). Blank wells were used for background subtraction and untreated cell cultures were run in parallel as controls.

Gene silencing with small interfering RNA (siRNA)

The siRNA targeting the human FXR with the cDNA sequence 5 0 -GAGGAUGCCUCAGGAAAUA-3 0 was synthesized and annealed by Eurogentec (Seraing, Belgium). The siRNA duplex negative control (scramble; cDNA sequence 5 0 -AAAGCGUCUGGAAAAGUCG-3 0 ) from Eurogentec was used to evaluate the non-specific effects on transfection on gene expression. MCF-7 cells (10 [50 years (=menopausal status), and node negative vs. node positive. Distant metastasis-free survival was defined as the interval elapsed between the time of surgery and the diagnostic of distant metastasis. P-values \ 0.05 were considered as statistically significant. In vitro data are reported as means ± SD and statistical analysis was performed by analysis of variance (ANOVA). Dunnett post hoc test was used to compare treated conditions to the untreated condition (control) and Tukey post hoc test was performed for multiple comparisons between groups. For in vitro studies, the level of statistical significance was arbitrarily set at 0.01.

Results

Evaluation of FXR expression in breast cancer cell lines and tissue specimens

In preliminary experiments, primary antibodies used in this study for FXR detection by Western blotting (rabbit polyclonal anti-FXR antibody) and immunofluorescence/ immunohistochemistry (mouse monoclonal anti-FXR antibody, clone A9033A) were tested with regard to immunoreactivity and specificity. Results are illustrated in Fig. 1 and2.

Immunoblot analysis of human recombinant FXR (rFXR) with the polyclonal antibody detected an immunoreactive band at the expected molecular mass for FXR (60 kDa) (Fig. 1a). Furthermore, a drastic reduction in band density was observed when the antibody was preincubated with the rFXR protein. Immunoblot analysis of lysates from ER-positive MCF-7 and ER-negative MDA-MB-231 breast carcinoma cells revealed the presence of similar 60 kDa immunoreactive bands (Fig. 1a), which disappeared when the neutralized anti-FXR antibody was used. The rabbit polyclonal anti-FXR antibody did not give satisfactory results when used for immunofluorescence microscopy and immunohistochemistry (data not shown). By contrast, mouse monoclonal anti-FXR antibody demonstrated the presence of FXR immunoreactivity in nuclei of both MCF-7 and MDA-MB-231 cells (Fig. 1b). Thus, the latter antibody was used for FXR immunostaining in clinical breast tumor specimens (Fig. 2).

FXR expression in ER-positive and ER-negative breast cancer tissue specimens FXR expression was assessed by immunohistochemistry in 204 breast cancer samples, along with a panel of biomarkers routinely used for the immunohistochemical characterization of breast carcinoma [START_REF] Colozza | Proliferative markers as prognostic and predictive tools in early breast cancer: where are we now?[END_REF]. These samples were representative of a typical breast cancer population, Fig. 2 Expression of FXR and biomarkers in human breast cancer tissue specimens. Cancer samples were subjected to immunohistochemical analysis with different antibodies as described in ''Materials and Methods''. Representative data of low/moderate FXR expression (sample 1) and of high FXR expression (sample 2) in ER-positive breast cancer tissue. FXR expression clearly correlates with that of ER, Ki-67, cyclin D and p27. Magnification bar: 50 lm insofar as ER expression was present in 2/3 of cases, HER-2/neu receptor expression was present in nearly 15% of cases, histological grade 1 and grade 3 samples represented each one 1/4 of the population, and 80% of the samples originated from node negative patients (Table 1).

Representative microscopic fields in Fig. 2 illustrate FXR immunostaining in tissue specimens from two breast tumor cases with low/moderate (sample 1, left column) and high (sample 2, right column) expressions of the FXR protein. In the whole population, FXR was detected (median score = 5) in 82.4% of the samples (Table 2). Interestingly, a significantly higher FXR expression was detected in the ER-positive subgroup (median score = 6) than in the ER-negative one (median score = 4). These data are in agreement with the observations on breast cancer cell lines showing that FXR is expressed at a higher level in ER-positive MCF-7 cells, as compared to ER-negative MDA-MB-231 cells (Fig. 1).

In the total population, significant correlations (Spearman's rho) were observed between the expression of FXR and that of steroid hormone receptors (ER and PgR), and between FXR and the proliferation marker Ki-67 (Table 2) (typical cases illustrated in Fig. 2). FXR expression also correlated with Ki-67 and the nodal status in the ER-positive subgroup while no significant correlation between FXR and other biomarkers was detected in the ER-negative subgroup (Table 2). Nevertheless, in the ER-positive subgroup, no relationship was observed between FXR and the tumor histological grade.

FXR expression in ER-positive breast cancers according to menopausal status of patients

In the ER-positive population, we found a weak correlation between FXR expression and the patients' age at diagnosis in the premenopausal but not in postmenopausal patients (Table 3). These data suggest that FXR expression increases with age to reach a maximum in tumors from postmenopausal patients.

Moreover, an analysis of the ER-positive population revealed that the statistically significant associations between FXR expression and Ki-67 expression (P = 0.017), and between FXR expression and the patients' nodal status (P = 0.029) were documented only in the postmenopausal women (Table 3). These data suggest that the lack of estrogens related to menopause may disclose the correlation between FXR expression and cellular proliferation in the ERpositive tumors.

Prognostic importance of FXR expression in ER-positive tumors

To assess the prognostic value of FXR with regard to distant metastasis-free survival, FXR evaluation was performed on 120 node-negative breast cancer patients from Institut Jules Bordet. We selected patients who underwent breast cancer surgery between 1987 and 1989 and did not receive systemic adjuvant therapy, and for whom a median follow-up of 10 years (range 2-16 years) was available (Table 4).

FXR expression was significantly correlated with ER and PgR, the transcription factor GATA-3 and the ER coactivator AIB-1, indicating a close association between FXR expression and the hormone-responsive phenotype. FXR expression was also correlated with CK8/18 and muc-1, supporting further association with the ER-positive luminal-like breast cancer subtype (Table 4).

As previously reported for the total patient population described in Table 1, FXR expression was significantly 4). In agreement with the fact that FXR expression was similar in the tumors of good and poor prognosis patients, no association was observed between FXR expression and the risk of cancer recurrence in the total population or in the ER-positive subgroup (data not shown).

Effect of FXR activation on cell proliferation and biomarker expression: in vitro data

The findings outlined above prompted us to examine the effect of the primary bile acid chenodeoxycholic acid (CDCA), the most potent endogenous ligand/activator of FXR [START_REF] Parks | Bile acids: natural ligands for an orphan nuclear receptor[END_REF], on the proliferation of MCF-7 and MDA-MB-231 cells cultured in steroid-free medium (SFM) supplemented or not with estrogen (Fig. 3a). In estrogen-supplemented medium, CDCA had no effect, while it stimulated the proliferation of the ER-positive MCF-7 cells in SFM. In agreement with the lack of correlation between FXR and Ki-67 observed in ER-negative tumor specimens, CDCA had no mitogenic action on the ER-negative MDA-MB-231 cells, regardless of the culture conditions. Of note, as reported by another group [START_REF] Swales | The farnesoid X receptor is expressed in breast cancer and regulates apoptosis and aromatase expression[END_REF], bile acid concentrations over 100 lM were toxic for breast cancer cells. The specific involvement of FXR in the proliferative response of MCF-7 cells to CDCA was checked by silencing the expression of the receptor with an anti-FXR siRNA. As shown in Fig. 3b, FXR expression silencing by the siRNA partly suppressed the proliferative response of MCF-7 cells to CDCA, while a scramble RNA was inactive. Of note, siRNA against FXR did not affect ER expression in MCF-7 cells, confirming the specificity of the siRNA.

Previous work of our group has disclosed the existence of a crosstalk between FXR and ER in breast carcinoma cells [START_REF] Journe | Farnesol, a mevalonate pathway intermediate, stimulates MCF-7 breast cancer cell growth through farnesoid-X-receptor-mediated estrogen receptor activation[END_REF]. Thus, as illustrated in Fig. 3c, the mitogenic action of CDCA on MCF-7 cells was abrogated by the antiestrogens 4-hydroxytamoxifen and fulvestrant (ICI 182,780). As expected, antiestrogens did not alter FXR expression in MCF-7 cells. In addition, co-immunoprecipitation experiments revealed that FXR interacts with ER when activated by CDCA (Fig. 4). As a consequence of such interactions, FXR activation by CDCA results in typical estrogenic responses, i.e. ER downregulation and induction of the progesterone receptor (PgR) (Fig. 5).

In addition, as illustrated in Fig. 5a, CDCA and E 2 exerted opposite effects on the cdk inhibitor p27, whereas these drugs induced an increase of the cell cycle mediator cyclin D1, suggesting that they somehow differ in their action on G1 phase progression. Results obtained here with E 2 are consistent with previous work showing that MCF-7 cell exposure to estrogen leads to a decrease of p27 [START_REF] Prall | Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2[END_REF] due to enhanced proteasomal degradation [START_REF] Foster | Multifaceted regulation of cell cycle progression by estrogen: regulation of Cdk inhibitors and Cdc25A independent of cyclin D1-Cdk4 function[END_REF], and a concomitant activation of cyclin E-cdk2. On the other hand, the fact that CDCA induces both a proliferative response (Fig. 3) and an increase of p27 (Fig. 5) was in agreement with in vivo data and suggests that the latter cdk inhibitor accumulates in an inactive form or is prevented from exerting an inhibitory effect on cyclin E-cdk2. Cyclin D1-cdk4 complexes might titrate p27 away from cyclin E-cdk2 complexes, allowing these complexes to become active [START_REF] Sherr | Inhibitors of mammalian G1 cyclindependent kinases[END_REF].

Overall, observations performed in vitro match in vivo findings since they confirm the relationship between FXR expression/activation, ER expression/activation and enhanced cell proliferation.

Discussion

The nuclear receptor FXR has been recently demonstrated in breast cancer as well as in normal mammary tissue [START_REF] Silva | Lipids isolated from bone induce the migration of human breast cancer cells[END_REF][START_REF] Swales | The farnesoid X receptor is expressed in breast cancer and regulates apoptosis and aromatase expression[END_REF][START_REF] Journe | Farnesol, a mevalonate pathway intermediate, stimulates MCF-7 breast cancer cell growth through farnesoid-X-receptor-mediated estrogen receptor activation[END_REF]. The present study reveals a higher FXR expression in ER-positive luminal-like tumors, as compared to ER-negative breast tumors. These data are consistent with the semi-quantitative analysis of FXR in the ER-positive cell line MCF-7 and the ER-negative cell line MDA-MB-231 and support the hypothesis of a lower FXR expression in less differentiated tumor cells (such as ER-negative breast carcinoma cells). Similar observations have been made in other malignancies. Indeed, FXR mRNA levels decrease in colon adenomas and even more in colon carcinomas, and become undetectable in undifferentiated colon adenocarcinoma SW480 cells and in metastasis-derived SW620 cells. On the other hand, FXR gene expression increases with the degree of differentiation in the Caco-2 and HT-29 cell lines [START_REF] De Gottardi | The bile acid nuclear receptor FXR and the bile acid binding protein IBABP are differently expressed in colon cancer[END_REF]. Thus, lesser expression of FXR seems to be associated with a more undifferentiated phenotype in breast and colon carcinomas.

In the whole group of breast cancers, significant correlations were observed between the expression of FXR and those of proliferation markers, including cyclin D and c-myc. These results are in agreement with a previous study which reports that FXR is highly expressed in a Barrett's esophagus-derived cell line and may influence cell survival [START_REF] De Gottardi | Expression of the bile acid receptor FXR in Barrett's esophagus and enhancement of apoptosis by guggulsterone in vitro[END_REF]. Indeed, the FXR antagonist guggulsterone induces cell apoptosis through an effect attributed to the downregulation of cyclin D and c-myc [START_REF] Shishodia | Guggulsterone inhibits NF-kappaB and IkappaBalpha kinase activation, suppresses expression of anti-apoptotic gene products, and enhances apoptosis[END_REF]. Moreover, in the ER-positive subgroup of breast tumors, FXR expression was highly correlated with cyclin D and p27 protein expression. This result is in line with the fact that the expressions of both cyclin D1 and p27 are closely related [START_REF] Fredersdorf | High level expression of p27(kip1) and cyclin D1 in some human breast cancer cells: inverse correlation between the expression of p27(kip1) and degree of malignancy in human breast and colorectal cancers[END_REF][START_REF] Sweeney | Lack of relationship between CDK activity and G1 cyclin expression in breast cancer cells[END_REF] and correlate with estrogen receptor positivity in primary breast cancer [START_REF] Hui | Cyclin D1 and estrogen receptor messenger RNA levels are positively correlated in primary breast cancer[END_REF][START_REF] Catzavelos | Decreased levels of the cell-cycle inhibitor p27Kip1 protein: prognostic implications in primary breast cancer[END_REF]. Therefore, insofar as mitogen-dependent progression through G 1 phase is mediated by induction of the cyclin D family [START_REF] Sherr | Cancer cell cycles[END_REF][START_REF] Sherr | CDK inhibitors: positive and negative regulators of G1-phase progression[END_REF], our study suggests that FXR expression is associated with cell proliferation.

On the other hand, no association was observed between FXR expression and neoplasm recurrence in the total population or in the ER-positive subgroup (data not shown). Interestingly, in good prognosis patients (no recurrence during 10 years of follow-up), FXR expression was correlated with several proliferation factors (cyclin D and p27), whereas in the poor outcome patients (distant metastasis), it was exclusively correlated with c-myc. Of note, c-myc and cyclin D1 participate in the activation of the cyclin E-cdk2 complexes by overriding the cell cycle arrest imposed by p27 overexpression [START_REF] Mateyak | c-Myc regulates cyclin D-Cdk4 and -Cdk6 activity but affects cell cycle progression at multiple independent points[END_REF]. No definite conclusion can, however, be drawn since the number of patients with poor prognosis was low. Nevertheless, these data suggest that FXR expression could be associated with proliferation in ER-positive tumors through different mechanisms contributing to tumor aggressiveness. Interestingly, the association between FXR expression and cell proliferation (Ki-67) was only noted in As previously demonstrated for the FXR activator farnesol [START_REF] Journe | Farnesol, a mevalonate pathway intermediate, stimulates MCF-7 breast cancer cell growth through farnesoid-X-receptor-mediated estrogen receptor activation[END_REF], CDCA might promote a positive crosstalk between FXR and ER, accounting for FXR-mediated ER activation in absence of estrogen stimulation and mitogenic response of ER-positive cells to this FXR agonist. It must be noted that the concentration of CDCA used in the current study (50 lM) is clinically relevant since it is in the range of those observed in human breast cyst fluid (42-94 lM) [START_REF] Javitt | Breast-gut connection: origin of chenodeoxycholic acid in breast cyst fluid[END_REF].

The correlation between FXR expression and tumor cell proliferation in postmenopausal patients could be extended to the clinical context of breast cancer patients treated with aromatase inhibitors. Indeed, the latter drugs markedly decrease plasma estrogen level by inhibiting or inactivating aromatase enzymes responsible for the synthesis of estrogens from androgenic substrates [START_REF] Brodie | Aromatase inhibitors and their application in breast cancer treatment*[END_REF], and also strongly inhibit intratumoral aromatase activity present in the majority of breast cancers [START_REF] Blankenstein | Intratumoral levels of estrogens in breast cancer[END_REF]. Thus, aromatase inhibitor treatments could disclose a possible crosstalk between FXR and ER, which may induce proliferative responses and lead to some forms of resistance to hormone therapy.

Bile acids, which are endogenous ligands/activators of FXR, have been implicated in the development of colorectal cancers [START_REF] Nagengast | Role of bile acids in colorectal carcinogenesis[END_REF]. Moreover, the involvement of FXR ligands in breast carcinogenesis has been previously documented. Indeed, high concentrations of plasma deoxycholate acid (DCA) are found in human breast cyst fluid from patients with fibrocystic disease of the breast and have been discussed as risk factors for developing breast cancer [START_REF] Raju | Bile acids in human breast cyst fluid: the identification of lithocholic acid[END_REF][START_REF] Javitt | Breast-gut connection: origin of chenodeoxycholic acid in breast cyst fluid[END_REF]. More recently, plasma bile acid concentrations were reported to be higher in postmenopausal women newly diagnosed with breast cancer, as compared to healthy controls matched for age and body mass index [START_REF] Costarelli | Plasma deoxycholic acid concentration is elevated in postmenopausal women with newly diagnosed breast cancer[END_REF]. In addition, the current study reveals that chenodeoxycholic acid (CDCA) promotes the growth of MCF-7 cells. Altogether, these data further support the hypothesis that bile acids might play a role in the etiology of breast carcinoma [START_REF] Lewis | The metabolic consequences of slow colonic transit[END_REF].

Obesity in postmenopausal women increases the risk of developing breast cancer [START_REF] Carmichael | Obesity as a risk factor for development and poor prognosis of breast cancer[END_REF], and results in increased bile acid output [START_REF] Halmy | High serum bile acid level in obesity: its decrease during and after total fasting[END_REF]. Breast cancer incidence is epidemiologically linked to high-fat diets [START_REF] Cho | Premenopausal fat intake and risk of breast cancer[END_REF] which increase the amount of bile acids in the body [START_REF] Costarelli | Acute effects of dietary fat composition on postprandial plasma bile acid and cholecystokinin concentrations in healthy premenopausal women[END_REF]. Moreover, some studies indicate that high-fat diets are associated with increased risk for ER-positive breast tumors [START_REF] Harlan | Estrogen receptor status and dietary intakes in breast cancer patients[END_REF][START_REF] Kushi | Dietary fat and risk of breast cancer according to hormone receptor status[END_REF][START_REF] Jain | Tumor characteristics and survival of breast cancer patients in relation to premorbid diet and body size[END_REF]. All of these data suggest that FXR activation by higher bile acid levels associated with obesity or high-fat diets might contribute to breast carcinogenesis.

Finally, a recent study indicates that the bile acid salt sodium deoxycholate (DC) may promote the survival and the migration of the metastatic MDA-MB-231 cell line through the induction of urokinase-type plasminogen activator, a critical factor for the dissemination of breast cancer cells [START_REF] Silva | Lipids isolated from bone induce the migration of human breast cancer cells[END_REF]. These data suggest that, in addition to the involvement of FXR in cell survival/proliferation, this nuclear receptor could be involved in tumor invasiveness/ metastasis, as further supported by the significant correlation between FXR expression and nodal status in our population. In this regard, we have found recently that FXR expression in primitive breast tumors appeared associated with the propensity for bone metastasis [START_REF] Journe | Farnesoid X receptor in primary breast carcinoma: a new marker to predict bone metastasis?[END_REF].

In conclusion, the current study suggests a close association between FXR and ER expression. Moreover, FXR expression was correlated with tumor proliferation only in ER-positive breast cancers in postmenopausal women. Therefore, FXR may prove to be a valuable biomarker to further characterize high and low proliferating tumors in ER-positive breast cancers, especially in estrogen deprived patients (postmenopause, treatment with aromatase inhibitors).

Fig. 1 FXR

 1 Fig. 1 FXR expression in breast cancer cell lines. (a) FXR was assessed in MCF-7 and MDA-MB-231 cells by Western blotting with a rabbit polyclonal anti-FXR/NR1H4 antibody. The specificity of the signal was proved by the immunodetection of a recombinant FXR protein (rFXR), and the significant reduction of the signal by using rFXR-neutralized antibody. b-actin was used as a loading control. Quantitative data were obtained from densitometric analyses (n = 2) and are presented as mean percentages. (b) Demonstration of FXR in MCF-7 and MDA-MB-231 cell lines by immunofluorescence microscopy with a mouse monoclonal anti-FXR/NR1H4 antibody. Texas Red labeling. Magnification bar: 10 lm

Fig. 3

 3 Fig. 3 Effects of CDCA on the proliferation of MCF-7 and MDA-MB-231 cells. (a) Breast cancer cells were treated for 72 h with 1-200 lM CDCA or vehicle in steroid-free medium (SFM) (filled symbols) or E 2 -supplemented SFM (open symbol). Cell proliferation was determined by crystal violet staining assay. Data are presented as percentages of control values (mean ± SD). Mean of results pooled from 2 experiments (n = 12). * ANOVA, P \ 0.05 vs. untreated cells. (b) Effect of FXR gene silencing on the mitogenic effect of CDCA in MCF-7 cells. Cancer cells were transfected for 6 h with 50 nM siRNA duplex against FXR, corresponding scramble (negative control), or vehicle (mock). Transfected cells were exposed to 50 lM CDCA or vehicle for 3 days before assessment of cell proliferation as

Fig. 4 Fig. 5

 45 Fig.4Western blotting of ER in immunoprecipitated FXR preparations. MCF-7 cells were incubated in SFM with 50 lM CDCA or vehicle (control) for 10, 20, 30 or 60 min. Solubilized protein preparations were submitted to FXR immunoprecipitation, as described in ''Material and Methods''. ER expression was assayed in the immunoprecipitates by Western blotting. The second control ''no FXR antibody'' means that no primary antibody was used for immunoprecipitation. The broken line refers to the non specific signal level. Quantitative data were obtained from densitometric analyses (n = 3) and are presented as percentages of control values (mean ± SD). * ANOVA, P \ 0.05 vs. control. Total ER and FXR levels were determined by Western blotting in cell extracts before immunoprecipitation

Table 1

 1 Clinico-pathological characteristics of the study population

	Total population	ER+ population	ER-population
	n = 204	n = 109	n = 59

Table 2

 2 Association of FXR expression with pathological markers according to ER status

		FXR score	FXR score	FXR score
		Total population	ER+ subgroup	ER-subgroup
	FXR
	Median score	5	6*	4*
	Positivity	168/204 (82.4%)	94/109 (86.2%)**	44/59 (74.6%)**
	Age at diagnosis	0.009 a ; ns b (204) c	0.004; ns (109)	-0.135; ns (59)
	Histological grade	-0.059; ns (185)	-0.007; ns (108)	0.004; ns (59)
	Menopausal status (\50 vs. [50)	0.043; ns (204)	0.019; ns (109)	0.091; ns (59)
	Metastasis	-0.090; ns (166)	-0.028; ns (79)	-0.014; ns (53)
	ER score	0.228; 0.009 (129)
	PgR score	0.202; 0.021 (129)	0.206; ns (77)	0.070; ns (52)
	Ki67%	0.288; \0.001 (164)	0.385; \0.001 (106)	0.181; ns (58)
	Tumor size	-0.159; ns (173)	-0.142; ns (102)	0.072; ns (50)
	Nodal status	0.054; ns (182)	0.228; 0.020 (103)	0.097; ns (59)
				significant
	associations

* Mann-Whitney test, P \ 0.001; ** Kappa test, P = 0.060 a Correlation coefficient (Spearman's rho); b Significance; ns = No significant; c Number of cases. Bold text indicates statistically

Table 3

 3

	Association of FXR expression with pathological markers in ER-positive tumors		FXR score B50 Years old, ER+ subgroup	FXR score [50 Years old, ER+ subgroup
	according to menopausal status	FXR
		Median score	6*	6*
		Positivity	24/26 (92.3%)**	70/83 (84.3%)**
	* Mann-Whitney test,	Age at diagnosis	0.396 a ; 0.044 b (26) c	-0.053; ns (83)
	P = 0.843; ** Kappa test,	Histological grade	-0.079; ns (26)	0.027; ns (82)
	P = 0.303 a Correlation coefficient (Spearman's rho);	Metastasis PgR score	-0.326; ns (18) 0.403; ns (18)	0.057; ns (61) 0.192; ns (59)
	b Significance; ns = No	Ki67%	0.237; ns (26)	0.454; \0.001 (80)
	significant; c Number of cases.	Tumor size	0.086; ns (26)	-0.226; ns (76)
	Bold text indicates statistically significant associations	Nodal status	0.201; ns (26)	0.251; 0.028 (77)

Table 4

 4 Association of FXR expression with pathological markers in ER-positive population according to prognosis

		FXR score	FXR score	FXR score
		Total population	ER+ population, good	ER+ population, poor
			prognosis (no metastasis in	prognosis (distant
			10 years of follow-up)	metastasis)
	FXR
	Median score	5	5.5*	5.25*
	Positivity	93/120 (77.5%)	49/60 (81.7%)**	11/14 (78.6%)**
	Hormone-responsive markers
	ER score	0.289 a ; 0.001 b (120) c		0.245; ns (13)
	GATA-3 score	0.514; \0.001 (118)	0.393; 0.002 (59)	-0.178; ns (12)
	AIB-1 score	0.408; \0.001 (115)	0.399; 0.002 (59)	0.333; ns (14)
	PgR score	0.247; 0.006 (120)	0.213; ns (60)
	Luminal subtype markers
	CK8/18	0.252; 0.005 (120)	0.205; ns (60)	0.039; ns (14)
	CK5/6	-0.064; ns (120)	-0.103; ns (60)	no data
	Muc-1	0.207; 0.023 (119)	0.170; ns (60)	-0.370; ns (14)
	Proliferative markers
	Ki67 %	0.245; 0.007 (118)	0.249; ns (58)	0.468; ns (14)
	Topo II %	0.208; 0.022 (120)	0.267; 0.038 (60)	0.338; ns (14)
	Cyclin D score	0.498; \0.001 (118)	0.513; \0.001 (58)	0.493; ns (14)
	Cyclin E score	0.004; ns (120)	0.259; 0.045 (60)	-0.018; ns (14)
	c-myc Score	0.278; 0.002 (119)	0.254; 0.049 (60)	0.653; 0.015 (13)
	p27 Score	0.553; \0.001 (119)	0.467; \0.001 (60)	0.469; ns (14)
	neu Score	-0.059; ns (120)	0.007; ns (60)	no data
	EGFR score	-0.082; ns (120)	no data	-0.069; ns (14)
	* Mann-Whitney test, P = 0.799; ** Kappa test, P = 0.800
	a Correlation coefficient (Spearman's rho); b Significance; ns = No significant; c Number of cases. Bold text indicates statistically significant
	associations

Breast Cancer Res Treat (2009) 115:523-535

Acknowledgements This study received financial support from the ''Fondation Medic'', from the ''Fondation contre le Cancer'', from the Belgian Fund for Medical Scientific Research (grants no. 3.4512.03 and 3.4501.08), from the ''Fondation Lambeau-Marteaux'', and from ''Les Amis de l'Institut Bordet''. Virginie Durbecq, Christos Sotiriou and Guy Laurent are respectively Scientific Research Worker, Research Associate and Senior Research Associate of the National Fund for Scientific Research (Belgium).


Doris Auer

Daniel Reimer

Verena Porto

Martina Fleischer

Julia Roessler

Annemarie Wiedemair

Christian Marth

Elisabeth Müller-Holzner

Günter Daxenbichler

Alain G Zeimet
email: alain.zeimet@i-med.ac.at

A E Daniel

Reimer Ae

Verena Porto Ae

A E Julia

Roessler Ae

A E Christian

Marth Ae

Elisabeth Mu ¨ller-Holzner

A E Gu

Daxenbichler Ae

Á D Reimer

Á M Fleischer

Á J Roessler

Á A Wiedemair

Á C Marth

Á E Mu ¨ller-Holzner

Á A G Zeimet

Expression of coxsackie-adenovirus receptor is related to estrogen sensitivity in breast cancer

Keywords: Breast cancer, Coxsackie-adenovirus receptor, Estrogen receptor, HDAC inhibitor, Hormone sensitivity, Tamoxifen

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The coxsackie-adenovirus receptor (CAR) is considered essential for cell attachment and cell entry of the coxsackie B and adenovirus. During the past decade CAR was extensively studied in the context of adenovirus-based gene therapy. Moreover, in a number of tumor entities CAR expression has been found to correlate inversely with the biologic aggressiveness of tumors. In bladder cancer, for example, the loss of CAR expression is associated with established markers of biologically aggressive bladder transitional cell carcinoma such as lymph node metastases, stage and high pathologic grade [START_REF] Matsumoto | Loss of coxsackie and adenovirus receptor expression is associated with features of aggressive bladder cancer[END_REF].

CAR has also been shown to be physically associated with epithelial tight junctions and involved in cell-cell adhesion. In polarized epithelial cells, CAR is co-localized with the tight junctions, where it associates with ZO-1 and contributes to the barrier functions in the circulation of macromolecules and ions [START_REF] Cohen | The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction[END_REF].

CAR is a 46-kDa integral membrane protein with a typical transmembrane domain, a long cytoplasmic domain, and an extracellular domain composed of two immunoglobulin-like domains [START_REF] Cohen | The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction[END_REF] and belongs to the immunoglobulin superfamily [START_REF] Williams | The immunoglobulin superfamily-domains for cell surface recognition[END_REF]. The human CAR gene is located on chromosome 21q11.2 and consists of at least eight exons distributed over an area of 54 kb. Beside the transmembrane CAR isoform (hCAR), the skipping of exons III-VI in CAR 2/7, exons IV-VI in CAR 3/7, and exons V-VI in CAR 4/7 results in at least three soluble CAR splice variants (CAR 2/7, CAR 3/7, CAR 4/7) that contain the leading sequence encoded by exon I, different portions of the immunoglobulin domains, but lack the transmembrane domain [START_REF] Chen | Structure and chromosomal localization of the murine coxsackievirus and adenovirus receptor gene[END_REF].

The role of membranous CAR in tumor progression and metastasis seems to differ among various tumor entities and remains the subject of debate. Several studies performed in human tumors, including prostate cancer, bladder cancer, and glioblastoma multiform, indicate a significant reduction in CAR expression during tumor progression [START_REF] Okegawa | The dual impact of coxsackie and adenovirus receptor expression on human prostate cancer gene therapy[END_REF][START_REF] Okegawa | The mechanism of the growth-inhibitory effect of coxsackie and adenovirus receptor (CAR) on human bladder cancer: a functional analysis of car protein structure[END_REF][START_REF] Sachs | Integrin alpha(v) and coxsackie adenovirus receptor expression in clinical bladder cancer[END_REF][START_REF] Kim | The coxsackievirus and adenovirus receptor acts as a tumour suppressor in malignant glioma cells[END_REF]. In bladder cancer cell lines CAR was shown to be involved in modulating cell cycle regulators, such as p21 CIP and Rb, suggesting a possible role of CAR as a tumor suppressor [START_REF] Okegawa | The mechanism of the growth-inhibitory effect of coxsackie and adenovirus receptor (CAR) on human bladder cancer: a functional analysis of car protein structure[END_REF]. Furthermore, it has been shown that the retroviral transfection of CAR into the primarily CAR-deficient human bladder cancer cell line T24 causes to growth inhibition [START_REF] Zhang | Overexpression of coxsackie and adenovirus receptor inhibit growth of human bladder cancer cell in vitro and in vivo[END_REF]. The same effect was observed in CAR-deficient human prostate cancer and malignant glioma cells [START_REF] Okegawa | The dual impact of coxsackie and adenovirus receptor expression on human prostate cancer gene therapy[END_REF][START_REF] Kim | The coxsackievirus and adenovirus receptor acts as a tumour suppressor in malignant glioma cells[END_REF]. However, in breast cancer, Martin et al. [START_REF] Martin | The coxsackie-adenovirus receptor has elevated expression in human breast cancer[END_REF] reported that CAR expression was found to be higher in metastatic tumors than in early breast cancers. Furthermore, these authors also found that tumors with nodal involvement were associated with higher levels of CAR. On the other hand, tumors from patients with local recurrences exhibited reduced CAR expression, even in comparison with those cases that remained disease-free over the follow-up period. Moreover, CAR expression increased with higher grade of malignancy, but did not reach statistical significance. In survival analysis, high CAR expression was revealed to be significantly related to poor overall survival in breast cancer patients [START_REF] Martin | The coxsackie-adenovirus receptor has elevated expression in human breast cancer[END_REF].

In ovarian cancer, however, no prognostic relevance was pointed out for the membranous form of CAR, but expression of the soluble CAR isoforms CAR 3/7 and 4/7 was shown to be an independent prognosticator predicting unfavorable progression-free (CAR 4/7) and overall survival (CAR 3/7 and CAR 4/7) [START_REF] Reimer | Soluble isoforms but not the transmembrane form of coxsackie-adenovirus receptor are of clinical relevance in epithelial ovarian cancer[END_REF].

Interestingly, in human ovarian cancer cell lines (PEO4, PEO1), CAR expression was enhanced and transduction efficacy by adenoviral vectors was increased by 17bestradiol treatment [START_REF] You | Coxsackievirus-adenovirus receptor expression in ovarian cancer cell lines is associated with increased adenovirus transduction efficiency and transgene expression[END_REF]. For that reason and because of the major role of steroid hormones in the biology of breast cancer, we examined whether a relationship exists between CAR expression and hormone sensitivity in cancers of the breast. In addition, the expression of membranous CAR (hCAR) and its soluble isoforms (CAR 2/7, CAR 3/7, and CAR 4/7) was investigated for prognostic significance in a set of 95 breast cancer patients. In order to investigate a possible functional relationship between steroid hormones and the family of CAR isoforms, in vitro assays of CAR inducibility by 17b-estradiol and 4-hydroxy-tamoxifen were performed in hormone receptor-positive and -negative breast cancer cell lines.

Materials and methods

Patients

Tissue samples from patients with breast cancer were collected during primary surgery at the Department of Obstetrics and Gynecology in Innsbruck (n = 95). Breast tissue samples obtained from patients who underwent esthetic surgery for other than inflammatory or malignant conditions served as control (n = 11). All included patients gave written consent for tissue use in research, and the study was approved by the local Institutional Ethics Review Board. Tissues were immediately frozen and stored at -80°C.

All patients (n = 95) were monitored within the outpatient follow-up program of the Department of Obstetrics and Gynecology, Innsbruck Medical University, and the median observation period of the included patients was 92 (52-130) months. Clinicopathologic characteristics are summarized in Table 1.

Estrogen and progesterone receptor levels were obtained from patient charts and were routinely determined by immunohistochemistry using the commercially available ERICA and PRICA kits (Abbott, Wiesbaden, Germany). Receptor status was recorded as percentage of positive staining cells (0-100%) and as staining intensity (1-3) for both ER and PR. Samples with a percentage C10 were considered receptor-positive. HER2/neu status was analyzed immunohistochemically using the HercepTest (DAKO, Denmark A/S, Glostrup, Denmark). Membrane immunoreactivity and membrane staining patterns were evaluated and scored using the 0 to 3+ scoring system according to the manufacturer's protocol. Tumors with a HER-2/neu score of 2+ in immunohistochemical analysis were also tested for HER-2/neu gene amplification using fluorescence in situ hybridization (FISH) and the PathVysion kit (Vysis, Downers Grove, IL). According to the HER-2/neu testing recommendations of the American Society of Clinical Oncology and the College of American Pathologists, tumors were classified as positive when the immunohistochemical score was 3+ or gene amplification was detected by means of in situ hybridization [START_REF] Wolff | American Society of Clinical Oncology/ College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer[END_REF].

Cell lines

Established breast cancer cell lines MCF-7, T-47D, MDA-MB-361, Zr75-1, 734-B, BT-20 and MDA-MB-231 obtained from ATCC (American Type Culture Collection, Maryland, USA) were cultured in appropriate medium for 24 h. The original medium was displaced by MEM without phenol red (Gibco) supplemented with 10% charcoal/dextran-treated FBS (HyClone). Each of the cell lines examined was treated with 10 -10 M 17b-estradiol (estradiol) alone, 10 -6 M 4-hydroxy-tamoxifen (tamoxifen) alone, 10 -10 M 17b-estradiol plus 10 -6 M 4-hydroxy-tamoxifen, transforming growth factor (TGF) -alpha (10 ng/ml), TGFbeta (10 ng/ml), and epidermal growth factor (EGF) (10 ng/ ml) for 72 h. Furthermore, cells were treated with trichostatin A (TSA) (100 ng/ml) for 24 h alone, 2.5 lM 5-aza-2 0 deoxycytidine (5-AZA) for 48 h alone, 10 -10 M 17b-estradiol alone or in combination with these agents.

RNA extraction and RT reaction

Total RNA was isolated from tissue samples or cultured cells using the guanidium thiocyanate-phenol-chloroform method according to the manufacturer's protocol (RNAgents Ò Total RNA Isolation System, Promega, Madison, WI). Integrity was evaluated by assessing the 18S-and 28-S-ribosomal RNA bands in 1% ethidium bromide-stained agarose gels. DNAse treatment and reverse transcription were performed as described recently [START_REF] Reimer | Soluble isoforms but not the transmembrane form of coxsackie-adenovirus receptor are of clinical relevance in epithelial ovarian cancer[END_REF].

Primers and probes

Specific primers and probes for hCAR, its soluble splice variants CAR 2/7, CAR 3/7, CAR 4/7 and for TBP (TATA box-binding protein; a component of the DNA-binding protein complex TFIID as an endogenous RNA control) were determined with the computer program ''Primer Express'' (Applied Biosystems, Foster City, CA). To prevent amplification of contaminating genomic DNA, the forward primers were placed at a junction between two exons. The various splice variants were distinguished by placing the forward primer between exon 2 and exon 7 for CAR 2/7, exon 3 and exon 7 for CAR 3/7, exon 4 and exon 7 for CAR 4/7 and between exon 6 and exon 7 for hCAR. The reverse primer (CAR Ex7 AS) and the probe (CAR Ex7 Taq) used for all splice variants were placed in exon 7. Sequences of primers and probes are given in Table 2.

Real-time PCR amplification

RT-PCRs were performed using an ABI Prism 7900 Detection System (Applied Biosystems, Foster City, CA) in a total volume of 25 ll reaction mixture containing 5 ll of each appropriately diluted RT sample (standard curve points and test samples), 12.5 ll TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA), 900 nm of each primer and 250 nm of the probe. Cycling conditions were an initial step at 50°C for 2 min, a denaturing step at 95°C for 10 min, and 45 cycles at 95°C for 15 s and 65°C for 1 min. Real-time PCR efficiencies were determined by amplifying serially diluted cDNA isolated from the breast cancer cell line SKBR-3. Real-time PCR assays were conducted in duplicate and the mean value was used for calculation. Only PCR experiments with a

Immunohistochemical demonstration of membranous hCAR

Immunohistochemistry was performed on paraffin-embedded 4 lm sections using the UltraView Universal DAB Detection Kit (Ventana, Tucson, AZ, USA) based on a mouse anti-rabbit Ventana BenchMark automated slide staining system. Antigen-retrieval was performed with cell conditioning solution 1 (standard); slides were incubated for 1 h at 37°C with the rabbit polyclonal anti-CAR H-300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) used as primary antibody (dilution 1:50). Counterstaining was attained using Nexes Hematoxylin, followed by Nexes Bluing Reagent. Negative controls were obtained by omitting the primary antibody.

Western blot analyzes

Extracts were prepared by lysing cell pellets (5 Blots were exposed to ECL plus Western blotting detection reagents (Amersham/GE Healthcare) and detected by photo imager.

Statistics

Differences between expression levels of CAR transcripts in cell culture experiments were assessed with the Student's t-test. For analysis between patient subgroups regarding, for example, hormone receptor, lymph node status or grade of malignancy we used either the Kruskal-Wallis or the Mann-Whitney U non-parametric test. Survival analyses for disease-free survival and overall survival were conducted with the Kaplan-Meier method and differences between groups were determined with the logrank test.

Results

mRNA expression of CAR isoforms related to clinicopathologic parameters

Expression levels of hCAR mRNA and that of its soluble splice variants CAR 2/7, CAR 3/7, and CAR 4/7 were assessed in breast cancer samples of 95 patients and 11 normal breast tissue samples. Soluble CAR 2/7 and CAR 3/ 7 isoforms, were not detectable in four and three cancers, respectively.

mRNA of CAR 2/7, CAR 3/7, and CAR 4/7 isoforms was expressed to a higher extent in controls than in tumor samples (Table 3). Between controls and tumors no significant difference in hCAR expression was observed. Expression of the soluble CAR isoforms CAR 2/7, 3/7, and Between patients with and without lymph node involvement no significant difference in CAR expression (membrane and soluble isoforms) was observed, and no reliable association between tumor size and CAR expression was observed. Furthermore, survival analyses did not reveal a prognostic significance for expression of the various CAR isoforms in terms of either disease-free or overall survival. mRNA levels of CAR isoforms related to the hormone receptor status Estrogen receptor (ER)-positive tumors showed increased hCAR transcript levels [median value 1.67 (Q 1 0.80 and Q 3 3.49)] in comparison to ER-negative tumors [median value 1.05 (Q 1 0.61 and Q 3 1.69)] (P = 0.011) (Fig. 1a). A closer look at the immunohistochemical results obtained for steroid receptors revealed that especially ER-positive tumors with a high staining intensity were found to be strongly associated with increased hCAR expression (P \ 0.0001) (Fig. 1b). PR-positive tumors showed a tendency to higher hCAR expression [median value 1.31 (Q 1 0.85 and Q 3 2.49)] than did PR-negative tumors [median value 1.02 (Q 1 0.53 and Q 3 2.16)], but these differences did not reach statistical significance (P = 0.065). When the subgroup of ER-positive tumors was compared to ER-negative/PRpositive and ER-negative/PR-negative tumor subgroups, we observed that elevated hCAR expression was strictly associated with ER expression and was independent of tumor PR expression (Table 4). However, no consistent association between the expression of the mRNA of any soluble CAR isoform and steroid hormone receptivity was observed. As depicted in Fig. 2a andb, the close relationship between the membranous hCAR and ER was corroborated when both molecules were demonstrated by immunohistochemistry.

Inducibility of CAR isoforms by estradiol and tamoxifen

In order to explore whether the revealed correlation between CAR and ER expression in the breast cancer samples analysed, simply reflects the grade of tumor differentiation or whether this correlation is based on a functional link between both molecules, we evaluated the inducibility of hCAR and its soluble isoforms by estradiol or tamoxifen in vitro. When hormone receptor-positive and -negative cell lines were compared, a significantly higher constitutive expression of hCAR and its soluble isoforms was observed in the hormonesensitive cell lines (P \ 0.03).

In the hormone receptor-positive cell line T47-D, treatment with estradiol significantly induced the expression of membranous and soluble CAR isoforms (P \ 0.05). Significant inducibility of all CAR isoforms was observed to a lesser extent when cells were treated with tamoxifen or the combination of tamoxifen and estradiol (Fig. 3). These effects were also found in the other hormone receptorpositive cell lines analysed, but in those cell lines the differences did not reach statistical significance. The revealed effects of steroid and tamoxifen treatment were not found to be confined to the mRNA level but were also evidenced at the protein level by means of Western Blot analyzes (Fig. 4). Of note is the fact that in the hormone receptorpositive cell line MCF-7 no constitutive expression of any CAR isoform was detectable and CAR expression was not inducible with estradiol or tamoxifen treatment.

Mechanisms possibly involved in the estrogen and tamoxifen induction of CAR isoforms

As no responsive element for ER was found in the promotor of CAR using the transcription factor-binding profile database JASPAR (htpp://jaspar.cgb.ki.se), we wanted to explore whether CAR expression is induced in an auto-or paracrine manner by factors known to be up-regulated by estrogens or tamoxifen in hormone-sensitive breast cancer cells. However, treatment of T47D cells with TGF-beta, TGF-alpha or EGF virtually did not affect the expression of hCAR and tended to decrease the transcripts of the soluble isoforms (data not shown).

To explore whether epigenetic phenomenons are potentially involved in the ER-dependent induction of the expression of the various CAR isoforms, cell lines were furthermore treated with the histone deacetylase (HDAC) inhibitor TSA and the DNA methyltransferase inhibitor 5-AZA. In six of the seven cell lines tested, the combination of TSA and 5-AZA led to a highly significant 2.3-to 3.5fold up-regulation of hCAR-specific transcripts (P \ 0.01) (Fig. 5). This effect was less prominent when cells were treated with TSA alone (1.8-to 2.5-fold) (P \ 0.05) and was completely missing when cells were treated with 5-AZA alone. Interestingly, sensitivity to TSA was significantly higher in receptor-positive as compared to receptornegative cell lines (P \ 0.03). Inducibility of the soluble isoforms paralleled that observed for membranous CAR and was of the same magnitude. It is worth to note that neither TSA, 5-AZA, nor a combination of both agents, was able to induce demonstrable CAR expression in MCF-7 cells that consequently should be regarded as a CARdeficient breast cancer cell line.

Discussion

Steroid hormone receptors play a critical role in the development and progression of breast cancer. Most importantly, the expression of ER and/or PR by tumor cells provides important information that is critical for the choice of treatment [START_REF] Cordera | Steroid receptors and their role in the biology and control of breast cancer growth[END_REF]. In addition, the expression of steroid hormone receptors correlates with well-differentiated tumors [START_REF] Ikeda | Estrogen receptors and their downstream targets in cancer[END_REF]. CAR, which was intensively studied during the last decade in the context of adenoviral-based gene therapy, has also been identified as a molecule associated with low malignant phenotype in urogenital cancers [START_REF] Matsumoto | Loss of coxsackie and adenovirus receptor expression is associated with features of aggressive bladder cancer[END_REF]. Even more, membranous CAR was implicated in cell cycle control and therefore considered to be a molecule with tumor-suppressing properties [START_REF] Okegawa | The mechanism of the growth-inhibitory effect of coxsackie and adenovirus receptor (CAR) on human bladder cancer: a functional analysis of car protein structure[END_REF].

Indeed, in our series of breast cancers, hCAR correlated with the grade of malignancy. Moreover, HER2/neu-positive tumors tended to show decreased hCAR expression as compared to HER2/neu-negative tumors. Although these findings were not true for the soluble CAR isoforms, it seems that high hCAR expression is associated with welldifferentiated tumors. However, no association with lymph node status or tumor size was observed, and neither for membranous nor for soluble CAR isoforms was a prognostic significance in terms of disease-free or overall survival revealed. In this regard our findings disagree with those reported by Martin et al. [START_REF] Martin | The coxsackie-adenovirus receptor has elevated expression in human breast cancer[END_REF], who found poor overall survival for breast cancer patients with elevated transmembrane CAR levels.

Findings showing that expression of membranous CAR in ovarian cancer cell lines (PEO4, PEO1) can be induced with estradiol [START_REF] You | Coxsackievirus-adenovirus receptor expression in ovarian cancer cell lines is associated with increased adenovirus transduction efficiency and transgene expression[END_REF] prompted us to investigate whether an association exists between the expression of this membranous receptor or its soluble isoforms and hormone sensitivity in breast cancer. In the breast cancers analysed, we observed that high expression of transmembrane CAR is strongly related to estrogen but not to progesterone receptivity of tumors. Furthermore, hCAR expression was higher in receptor-positive breast cancer cell lines than in receptor-negative cell lines. These findings would primarily suggest that hCAR is associated with tumors exhibiting a highly differentiated phenotype, which are otherwise known to be highly sensitive for steroid hormones. However, the performed in vitro analyzes show that membranous as well as soluble CAR isoforms can be Fig. 4 Expression of hCAR and soluble CAR 4/7 isoform at the protein level after exposure of T47-D cells to E2, tamoxifen, or both Fig. 5 Expression of hCAR and its soluble isoforms in T47-D cells after treatment with DNA-methyltransferase (DNMT) inhibitor 5-AZA, HDAC inhibitor TSA either alone or in combination. TSA and 5-AZA treatment was performed with and without E2. *P \ 0.05; **P \ 0.01; ***P \ 0.001 as compared to the respective control induced in receptor-positive T-47D cells with either estradiol or tamoxifen. This was also the case, even to a lesser extent and with borderline statistical significance, for other receptor-positive cell lines, but was not observed in receptor-negative cell lines. Thus, theoretically it is possible that CAR expression is directly influenced by estradiol or by selective estrogen receptor modulators such as tamoxifen. However, screening of the putative CAR promotor region revealed no binding site for ER, but identified specific binding sites for SP-1 and the E2F transcription factor family. Earlier observations that expression of E2F transcription factors is increased following EGF stimulation [START_REF] Reimer | Heterogeneous cross-talk of E2F family members is crucially involved in growth modulatory effects of interferongamma and EGF[END_REF] prompted us to elucidate whether CAR up-regulation by estradiol could be driven in an autocrine or paracrine manner involving TGF-alpha, which is a ligand of the EGF receptor and has been shown to be released in enhanced amounts following estradiol stimulation [START_REF] Lippman | Growth regulation of human breast carcinoma occurs through regulated growth factor secretion[END_REF]. Moreover, in ER-positive breast cancer cells tamoxifen has been found to increase the release of TGF-beta [START_REF] Morena | Early nuclear alterations and immunohistochemical expression of Ki-67, Erb-B2, vascular endothelial growth factor (VEGF), transforming growth factor (TGF-beta1) and integrine-linked kinase (ILK) two days after tamoxifen in breast carcinoma[END_REF] which could be a further candidate with the potential to influence CAR expression. Yet, in our in vitro model hCAR expression remained unaffected and the transcripts of the soluble isoforms even tended to be decreased by EGF, TGF-alpha, and TGF-beta treatment.

As CAR expression is known to be under tight epigenetic control, particularly by histone acetylation, and the phytoestrogen genestein was shown to inhibit HDAC activity in T24 bladder cancer cells [START_REF] Pong | Mechanism of increased coxsackie and adenovirus receptor gene expression and adenovirus uptake by phytoestrogen and histone deacetylase inhibitor in human bladder cancer cells and the potential clinical application[END_REF], we were tempted to speculate that the stimulating effect of estradiol on the CAR gene could be mediated by epigenetic changes. We found a significant induction of hCAR transcripts when T-47D cells were treated with TSA alone or in combination with 5-AZA, but no effect was seen when cells were treated with 5-AZA alone. This corroborates the notion that the CAR gene is more likely regulated by histone acetylation than by methylation of its promotor CpG islands. Furthermore, the stimulatory effects of TSA on the expression of CAR were by far more pronounced than those observed for estradiol or tamoxifen treatment. In addition, a much lower sensitivity to TSA regarding CAR induction was revealed for the ER-negative versus ER-positive cell lines with the exception of the MCF-7 cell line, which must be considered CAR-deficient. Moreover, combining estradiol or tamoxifen with TSA did not result in an additive enhancement of CAR expression. This argues against there being two distinct mechanisms of action for up-regulating CAR by means of the investigated drugs and suggests that histone acetylation is influenced to a lesser extent by estradiol and tamoxifen than by the hyperacetylating agent TSA. Our data are consistent with the findings of Sun et al. [START_REF] Sun | Effect of estradiol on histone acetylation dynamics in human breast cancer cells[END_REF], who reported that estradiol increased the level of acetylated histones by reducing the rate of histone deacetylation in ER-positive but not negative breast cancer cell lines. The hypothesis that activated ER alters the balance of histone acetyltransferases and HDACs at the nuclear matrix as well as the dynamics of acetylation of histones associated with transcriptionally active and competent chromatin, could reliably explain the herein described association between ER positivity and high hCAR expression in breast cancers and the inducibility of hCAR expression in receptor-positive cell lines through ER activation.

At first glance, the fact that tamoxifen and also TSA induce expression of hCAR could represent a suitable approach ameliorating the transduction efficiency of adenoviral vectors in gene therapeutic strategies in ERpositive breast cancers. However, an additional important finding of our investigations is that the soluble isoforms of CAR, which are known to significantly interfere with cell entry of adenoviral particles [START_REF] Dorner | Alternatively spliced soluble coxsackie-adenovirus receptors inhibit coxsackievirus infection[END_REF], were also considerably up-regulated by tamoxifen or TSA treatment and might diminish the favorable effect of enhanced membranous CAR expression on therapeutic gene delivery.

In conclusion, we here describe an unknown association between estrogen receptivity and the expression of membranous CAR in breast cancer. As a proof of principle, inducibility of membranous CAR and its soluble isoforms by estradiol and tamoxifen was demonstrated in hormone receptor-positive breast cancer cell lines. As a consequence of these findings we suggest that the co-expression of ER and CAR in breast cancers is not only related to a more highly differentiated tumor phenotype, but also founded on a functional basis.

  FAM-CAC CTC CAA AGA GCC GTA CGT CCA CTG-TAMRA-3 0 FAM-CAG AGT CCG ACT CTC CCA CCT GCT AAG -TAMRA-3 0 Exon/exon boundaries are underlined. The reverse primer and probe are located in exon 7, whereas various splice variants are defined by specific forward primers 4/7 correlated with tumor histology as these isoforms were expressed to a significantly lower level in ductal and lobular carcinomas than in medullary/mucinous carcinomas (P \ 0.015, P \ 0.005 and P \ 0.015, respectively). Moreover, the membranous isoform was expressed to a higher extent in tumors with low-grade malignancy [median value 2.18 (Q 1 1.13 and Q 3 3.63)] than in tumors with a higher grade of malignancy [median value 1.05 (Q 1 0.68 and Q 3 1.91)] (P \ 0.004). HER2/neu-positive tumors showed a tendency toward decreased hCAR transcript levels [median value 1.00 (Q 1 0.64 and Q 3 2.36)] in comparison to HER2/neu-negative tumors [median value 1.45 (Q 1 1.00 and Q 3 2.94)]. In the subgroup of tumors qualified as steroid receptor-positive (n = 55) this difference was more prominent, in terms that HER2/neu negativity was associated with significantly higher hCAR expression [median value 1.72 (Q 1 0.20 and Q 3 7.68)] as compared with HER2/neu positivity [median value 0.87 (Q 1 0.26 and Q 3 3.90)]; (P \ 0.03).

Fig. 1

 1 Fig. 1 Expression of membranous hCAR in relationship to the ER status: (a) Breast cancers dichotomized in ER-positive and -negative tumors. (b) Subdivision on ER staining intensity

Fig. 2 Fig. 3

 23 Fig. 2 Immunohistochemical concordant expression of membranous hCAR and ER in a breast cancer sample: (a) high membranous immunostaining of hCAR. (b) High nuclear ER staining intensity

Table 1

 1 Clinicopathologic characteristics

		n	Median	Q 1 -Q 3
	Age (years)	95	60.3	48.6-72.0
	Median DFS (m)		84.0	37.0-131.0
	Median OAS (m)		92.0	53.8-130.3
		n	%
	Death during follow-up	49/95	51.6
	Relapse during follow-up
	Relapse	43/93	46.2
	Local relapse	14/93	15.1
	Systemic relapse	36/93	38.7
	pT
	pT-1		21.3
	pT-2		59.6
	pT-3		10.6
	pT-4		8.5
	pN
	pN-0		35.2
	pN-1		63.6
	pN-2		1.2
	Histology
	Ductal		82.1
	Lobular		12.6
	Medullary/mucinous		5.3
	Grade of malignancy
	1		29.0
	2		58.1
	3		12.9
	Estrogen receptor
	Negative		32.6
	Positive		67.4
	Progesterone receptor
	Negative		36.6
	Positive		63.4
	HER2/neu
	Negative		68.2
	Positive		31.8
	Menopausal state
	Pre		31.6
	Post		68.4
	M1
	M0		91.4
	M1		8.6

Table 2

 2 Primers and probes of real-time PCR

	Isoform	Exon	Oligonucleotide	Sequence
	CAR 2/7_S	2/7	Forward primer	5 0 -GGT GGA TCA AGT GGG AAG ATG T-3 0

Table 3

 3 CAR expression in breast cancer samples and healthy controls

	Breast cancer samples	Controls		P-
						value
		Median (Q 1 -Q 3 )		Median (Q 1 -Q 3 )
	hCAR	1.239	0.363-2.114 hCAR	1.856	1.456-2.256 0.17
	CAR2/7 0.496	0.046-0.945 CAR2/7 1.171	0.722-1.619 0.03
	CAR3/7 0.405	0.004-0.807 CAR3/7 0.937	0..527-1.347 0.07
	CAR4/7 0.672	0.289-1.054 CAR4/7 1.161	0.836-1.486 0.03

Table 4

 4 hCAR expression in ER-/PR-positive tumors

		ER-	ER-negative	ER-negative
		positive	PR-positive	PR-negative
	Median	1.672	1.185	0.999	P \ 0.04
	Q 1 -Q 3	0.796-3.486	0.751-1.448	0.582-1.847

Breast Cancer Res Treat (2009) 116:103-111