Loredana Mauro

Michele Pellegrino

Rosamaria Lappano

Adele Vivacqua

Francesca Giordano

Maria Grazia Palma

Sebastiano Andò
email: sebastiano.ando@unical.it

Marcello Maggiolini

Prof Sebastiano Andò

E-cadherin mediates the aggregation of breast cancer cells induced by tamoxifen and epidermal growth factor

Keywords: Tamoxifen, EGF, E-cadherin, Three-dimensional cultures, Breast cancer

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Breast cancer is the most common malignancy in women worldwide with over one million cases diagnosed each year [START_REF] Smigal | Trends in breast cancer by race and ethnicity: update 2006[END_REF][START_REF] Bray | The changing global patterns of female breast cancer incidence and mortality[END_REF][START_REF] Jemal | Cancer statistics, 2002[END_REF]. Estrogen is required for normal proliferation and differentiation of breast epithelial cells and is implicated in the development and progression of breast cancer [START_REF] Ishii | Tamoxifen stimulates the growth of cyclin D1overexpressing breast cancer cells by promoting the activation of signal transducer and activator of transcription 3[END_REF]. The classical mechanism of estrogen action mainly involves two members of the nuclear receptor superfamily, the estrogen receptor (ER) α and ERß [START_REF] Giguere | Estrogen receptor beta: re-evaluation of estrogen and antiestrogen signaling[END_REF]. Steroid receptors, including ERα, have two main transcriptional activation functions named AF1 and AF2 which are associated with the N-terminal and the hormone binding domains, respectively. Agonists of ERα induce a conformational change in the hormone binding pocket that allows the recruitment of coactivators, which in turn maximize the transcriptional response to ligands [START_REF] Mc Kenna | Combinatorial control of gene expression by nuclear receptors and coregulators[END_REF]. Antagonists of ERs bind to the hormone binding pocket, however they block the AF2 in an inactive conformation. In addition, ERα can be activated by a host of extracellular signals, including growth factors, in the absence of the cognate ligand 17ß-estradiol (E 2 ) [START_REF] Cenni | Ligand-independent activation of steroid receptors: new roles for old players[END_REF][START_REF] Weigel | Ligand-independent activation of steroid hormone receptors[END_REF][START_REF] Bunone | Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation[END_REF]. Of interest, partial antagonists of ERα, such as 4-hydroxytamoxifen (OHT), may display cell-and promoter-specific agonist effects that are thought to be due, at least in part, to AF1 activity and/or to the ability to recruit cofactors [START_REF] Feng | Potentiation of estrogen receptor activation function 1 (AF-1) by Src/JNK through a serine 118-independent pathway[END_REF][START_REF] Lavinsky | Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes[END_REF][START_REF] Lee | MEKK1 activation of human estrogen receptor alpha and stimulation of the agonistic activity of 4-hydroxytamoxifen in endometrial and ovarian cancer cells[END_REF]. OHT was the first antiestrogen approved for the clinical management of ER-positive breast cancers and has been recommended as first-line treatment for the past three decades [START_REF] Ishii | Tamoxifen stimulates the growth of cyclin D1overexpressing breast cancer cells by promoting the activation of signal transducer and activator of transcription 3[END_REF]. Despite the beneficial effects of OHT for patients at all stages of ER-positive breast cancer [START_REF] Jaiyesimi | Use of tamoxifen for breast cancer: twentyeight years later[END_REF], the major obstacle to its use is the development of tamoxifen-resistance which either occurs de novo or in patients showing initial benefits [START_REF] Massarweh | Tamoxifen resistance in breast tumors is driven by growth factor receptor signaling with repression of classic estrogen receptor genomic function[END_REF]. Growth factors, including EGF, are involved in breast cancer progression [START_REF] Lichtner | Estrogen/EGF receptor interactions in breast cancer: rationale for new therapeutic combination strategies[END_REF][START_REF] Kim | The role of the epidermal growth factor receptor family in mammary tumorigenesis and metastasis[END_REF]. Binding of EGF to the cognate receptor EGFR initiates a transduction cascade with sequential phosphorylation and activation of a series of intracellular signaling molecules. Among the downstream pathways activated by EGF, it is important to mention the cell survival cascade mediated by PI3-kinase (PI3-K) and AKT/PKB [START_REF] Vanhaesebroeck | PI3K-PDK1 connection: more than just a road to PKB[END_REF][START_REF] Cantley | The phosphoinositide 3-kinase pathway[END_REF], the cell proliferation pathway mediated by the mitogen activated protein kinases (MAPK) ERK1/2 [START_REF] Pearson | Mitogen-activated protein (MAP)kinase pathways: regulation and physiological functions[END_REF][START_REF] Peyssonnaux | The Raf/MEK/ERK pathway: new concepts of activation[END_REF] and the stress-induced pathways mediated by the stress activated protein kinase/JNK and p38 MAPKs [START_REF] Kyriakis | Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation[END_REF]. On the other hand, rapid E 2 stimulation through ERα also activates these transduction pathways which synergistically trigger cell survival and proliferation [START_REF] Schiff | Breast cancer endocrine resistance: how growth factor signaling and estrogen receptor coregulators modulate response[END_REF][START_REF] Santen | The role of mitogen-activated protein (MAP)kinase in breast cancer[END_REF]. In addition, ERα can be promptly activated in a ligand-independent manner by EGF which mimics E 2 in inducing agonist effects including breast cancer cell growth [START_REF] Vignon | Antiestrogens inhibit the mitogenic effect of growth factors on breast cancer cells in the total absence of estrogens[END_REF][START_REF] Ignar-Trowbridge | Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor[END_REF][START_REF] Hewitt | Lessons in estrogen biology from knockout and transgenic animals[END_REF].

In the epithelium and in epithelium-derived tumors, cell-cell adhesion and tumor mass mostly rely on functions of E-cadherin, which is an intercellular adhesion molecule generally considered as a tumor suppressor [START_REF] Takeichi | Cadherin cell adhesion receptors as a morphogenetic regulator[END_REF][START_REF] Takeichi | Morphogenetic roles of classic cadherins[END_REF]. Alteration in the activity of E-cadherin and the cadherin-catenin complex has been implicated in cancer progression [START_REF] Perl | A causal role for E-cadherin in the transition from adenoma to carcinoma[END_REF], invasion [START_REF] Behrens | Dissecting tumor cell invasion: epithelial cells acquire invasive properties after the loss of uvomorulin mediated cell-cell adhesion[END_REF][START_REF] Frixen | E-cadherin mediated cell-cell adhesion prevents invasiveness of human carcinoma cells[END_REF][START_REF] Vleminckx | Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role[END_REF], and metastasis [START_REF] Dorudi | E-cadherin expression in colorectal cancer. An immunocytochemical and in situ hybridization study[END_REF][START_REF] Mbalaviele | E-cadherin expression in human breast cancer cells suppresses the development of osteolytic bone metastases in an experimental metastasis model[END_REF]. Considering that in our recent study ERα-mediated homotypic tumor cell adhesion was induced by E 2 through an increase of E-cadherin expression [START_REF] Mauro | Evidences that leptin upregulates E-cadherin expression in breast cancer: effects on tumor growth and progression[END_REF], in the present investigation we evaluated whether the mixed ER agonist/antagonist OHT and the well-known ER activator EGF could mimic the aforementioned action of E 2 in MCF7 breast cancer cells.

Materials and Methods

Plasmids. The plasmid containing the human E-cadherin promoter (-p1008/+49) was obtained from Dr. Y. S. Chang (Chang-Gung University, Republic of China) [START_REF] Tsai | The Epstein-Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases[END_REF].

Cell lines and culture conditions. MCF7 cells were grown in DMEM/F-12 containing 10% FBS supplemented with 1% L-glutamine and 1% penicillin/streptomycin (Sigma, Milan, Italy). Cells were switched to medium without serum 48h before RT-PCR, immunoblots, and evaluation of ERK1/2 phosphorylation.

Transfections and Luciferase Assays. MCF7 cells were plated into 24-well dishes with 500µl regular growth medium/well the day before transfection. The medium was replaced with that lacking serum on the day of transfection, which was performed using FuGene6 reagent as recommended by the manufacturer (Roche Diagnostics, Milan, Italy) with a mixture containing 0.5µg reporter plasmid and 2ng pRL-thymidine kinase. After 24h, we renewed the serum-free medium containing the indicated treatment, and then cells were incubated for approximately 16h. Luciferase activity was measured with the dual luciferase kit (Promega Italia, Milan, Italy) according to the manufacturer's recommendations.

Firefly luciferase values were normalized to the internal transfection control provided by the Renilla luciferase activity. The data are reported as normalized relative light unit (RLU) values.

RT-PCR.

The evaluation of gene expression was performed by semiquantitative RT-PCR as we have previously described [START_REF] Maggiolini | A non-radioactive method for inexpensive quantitative RT-PCR[END_REF]. For E-cadherin, pS2 and the acidic ribosomal phosphoprotein P0 (36B4), which was used as a control gene, the primers were, respectively, 5'-GGAATCCAAGCAGAATTGC-3' (E-cadherin forward), and 5'-TATGTGGCAATGCGTTCTCTATCCA-3' (E-cadherin reverse), 5'-TTCTATCCTAATACCATCGACG-3' (pS2 forward) and 5'-TTTGAGTAGTCAAAGTCAGAGC-3' (pS2 reverse), 5'-CTCAACATCTCCCCCTTCTC-3' (36B4 forward), and 5'-CAAATCCCATATCCTCGTCC-3' (36B4 reverse), to yield products of 285, 210 and 408 bp, respectively, with 20, 15 and 15 PCR cycles.

Western Blotting. Equal amounts of whole protein extract were resolved on a 8-10% SDSpolyacrylamide gel, transferred to a nitrocellulose membrane (GE Healthcare, Milan, Italy), probed overnight at 4°C with the antibodies against E-cadherin, EGFR, ß-actin (Santa Cruz Biotechnology, Milan, Italy), pERK1/2 and total ERK (Cell Signaling Technology and Celbio, Milan, Italy) and then revealed using the enhanced chemiluminescence system (GE Healthcare).

ERα, EGFR and Sp1 silencing experiments. Cells were plated onto 10-cm dishes, maintained in antibiotic-free medium for 24h and then transfected for additional 24h before treatments with a mixture containing Opti-MEM, 8µl/well LipofectAMINE 2000 (Invitrogen, Milan, Italy) and 0.5µg/well control siRNA or ERα siRNA (Sigma), vector or shEGFR purchased from SuperArray (Bioscience Corporation, Frederick, MD), 200nM/well of control siRNA or Sp1 siRNA (Applied Biosystems, Milan, Italy).

Chromatin immunoprecipitation (ChIP) assay. MCF7 cells were treated with 1µM OHT or 100ng/ml EGF for 1h. Thereafter, cells were cross-linked with 1% formaldehyde and sonicated.

Supernatants were immunocleared with salmon DNA/protein A agarose (Upstate Biotechnology, Lake Placid, NY) and immunoprecipitated with anti-ERα antibody or with anti-RNA Pol II (Santa Cruz Biotechnology). Pellets were washed as reported [START_REF] Morelli | Nuclear insulin receptor substrate 1 interacts with estrogen receptor a at ERE promoters[END_REF], eluted with elution buffer and digested with proteinase K. DNA was obtained by phenol/chloroform extraction and precipitated with ethanol; 3µl of each sample were used for PCR with Sp1 primers: 5'-TAGCAACTCCAGGCTAGAGG-3' and 5'-AACTGACTTCCGCAAGCTCACA-3'. The PCR conditions were 94°C for 1 min, 56°C for 2 min, and 72°C for 2 min. The amplification products obtained in 30 cycles were analysed in a 2% agarose gel and visualized by ethidium bromide staining.

Three-dimensional spheroid cultures. MCF7 cells were plated in single-cell suspension in 2%-agarcoated plates and left untreated or treated with 1µM OHT or 100ng/ml EGF for 48h. To block Ecadherin function, the medium was supplemented with E-cadherin antibody (1:100 dilution; Chemicon International, Temecula, CA) or EGTA to a final concentration of 4mM. To generate three-dimensional spheroids, the plates were rotated for 4h at 37°C. The three-dimensional cultures were photographed using a phase-contrast microscope (Olympus, Sun Valley, CA). The extent of aggregation was scored by measuring the spheroids with an ocular micrometer. The spheroids between 25 and 50, 50 and 100, and >100µm (in the smallest cross-section) were counted in 10 different fields under ×10 magnification.

Statistical Analysis. Statistical analysis was performed using ANOVA, followed by Newman-Keuls testing to determine differences in means.

Results

OHT and EGF transactivate the E-cadherin promoter and induce E-cadherin mRNA and protein expression.

On the basis of our previous study demonstrating that E-cadherin expression is induced by E 2 [START_REF] Mauro | Evidences that leptin upregulates E-cadherin expression in breast cancer: effects on tumor growth and progression[END_REF], and considering the potential of OHT and EGF to exert agonist activity through ERα, we evaluated whether these compounds could regulate E-cadherin expression like E 2 . An expression vector containing the human E-cadherin promoter was transactivated by OHT and EGF in MCF7 cells (Fig. 1A). treating cells with OHT in combination with EGF the transcriptional response was similar using each compound alone (Fig. 1A). In line with these findings, OHT and EGF induced E-cadherin mRNA expression at the same extent treating cells with both compounds (Fig. 1B,C). Then, we explored whether the agonistic effect displayed by OHT in upregulating E-cadherin expression was also evident in the modulation of the classical estrogen target gene pS2. Hence, MCF7 cells were treated with E 2 , OHT, the combination of both compounds and EGF. The up-regulation of pS2 by E 2 was abolished by OHT (Fig. 2A,B), confirming that OHT can act as a mixed agonist-antagonist depending on the gene response evaluated. Moreover, also EGF was able to upregulate pS2 mRNA expression (Fig. 2A,B).

Thereafter, we asked whether OHT and EGF could up-regulate E-cadherin expression at the protein level. Upon exposure to each compound or a combination of both, we observed an increase of Ecadherin protein levels which was prevented by the ER antagonist ICI, the Src and ERK inhibitors PP2 and PD, respectively (Fig. 3A-D). The latter findings showing the involvement of the MAPK pathway was also confirmed by the ability of OHT and EGF to trigger a rapid ERK1/2 phosphorylation similar to that observed upon E 2 exposure (Fig. 4). Taken together these results suggest that both OHT and EGF involve ERα as well as Src and MAPK signaling in the regulation of E-cadherin expression.

ERα and EGFR silencing abrogates the E-cadherin up-regulation by OHT and EGF.

The results obtained prompted us to define the role exerted by ERα and EGFR in mediating E-cadherin stimulation by both OHT and EGF. Interestingly, after knocking-down ERα expression neither OHT nor EGF was able to up-regulate E-cadherin expression (Fig. 5A,B) confirming the data obtained using the ER antagonist ICI. Moreover, EGFR silencing also resulted in the abrogation of the E-cadherin increase upon exposure to each compound (Fig. 5C,D). The role of EGFR signaling was further ascertained observing an upregulation of EGFR protein expression particularly treating MCF7 cells with OHT (Fig. 5E). These results suggest that a functional cross-talk between ERα and EGFR triggers E-cadherin expression by the agents used.

OHT and EGF induce the recruitment of ERα to the Sp1 site located within the E-cadherin promoter sequence.

The interaction of ERα with the E-cadherin promoter sequence was investigated by ChIP assay. To this end, the MCF7 chromatin was immunoprecipitated with anti-ERα or anti-RNA-Pol II antibodies. PCR was used to determine the recruitment of ERα to the E-cadherin region containing the Sp1 site (-144/-132nt). Interestingly, the weak ERα recruitment to the E-cadherin promoter observed in cells treated with vehicle increased upon OHT and EGF treatment (Fig. 6A). In addition, both compounds induced the recruitment of RNA-Pol II indicating the ability to stimulate E-cadherin transcription (Fig. 6A).

Next, the E-cadherin protein increase stimulated by OHT and EGF was no longer evident silencing Sp1 in MCF7 cells (Fig. 6B). A similar result was obtained using mithramycin, an inhibitor of Sp1-DNA interactions (Fig. 6C).These data confirm the role exerted by the Sp1 site in mediating the upregulation of E-cadherin upon OHT and EGF treatments.

OHT and EGF induce MCF7 cell-cell adhesion in three-dimensional cultures.

On the basis of our previous study showing that the increase of E-cadherin expression by E 2 is responsible for enhanced MCF7 cell aggregation [START_REF] Mauro | Evidences that leptin upregulates E-cadherin expression in breast cancer: effects on tumor growth and progression[END_REF], we evaluated whether OHT and EGF could induce the same effect. Considering that multicellular spheroid cultures can closely reproduce some biological features of tumors and improve the relevance of results obtained by in vitro studies [START_REF] Dangles | Two-and threedimensional cell structures govern epidermal growth factor survival function in human bladder carcinoma cell lines[END_REF][START_REF] Kunz-Schughart | Multicellular spheroids: a three-dimensional in vitro culture system to study tumour biology[END_REF][START_REF] Santini | Three-dimensional spheroid model in tumor biology[END_REF],

we used three-dimensional MCF7 cultures as an experimental model. MCF7 cell-cell adhesion was enhanced following a 48h exposure to OHT and EGF (Fig. 7), which both induced the formation of spheroids prevalently with a diameter greater than 50µm (Table 1). Moreover, supplementing the culture medium with function-blocking E-cadherin antibody or the calcium-chelating agent EGTA we verified the direct involvement of E-cadherin in the enhanced cell-cell adhesion upon OHT and EGF treatments. In the presence of the E-cadherin antibody, the MCF7 cells showed the formation of small aggregates and limited intercellular contact, whereas in the presence of EGTA MCF7 cells remained rounded and separately suspended (Fig. 7).

Discussion

Estrogens play a major role in the development of breast cancer. Nearly 70% of breast tumors express the ERα which allows for the use of antiestrogen therapy as a reliable therapeutic approach for all stages of the disease [START_REF] Massarweh | Resistance to endocrine therapy in breast cancer: exploiting estrogen receptor/growth factor signaling crosstalk[END_REF]. The ER antagonist tamoxifen has been shown to improve survival in early breast cancer [START_REF]Tamoxifen for early breast cancer: an overview of the randomised trials[END_REF] as well as the quality of life in patients with metastatic disease [START_REF] Jaiyesimi | Use of tamoxifen for breast cancer: twentyeight years later[END_REF]. Despite the documented benefits of ER-targeted therapy in breast cancer, approximately 50% of patients with advanced disease do not respond to first-line treatment with tamoxifen due to de novo or intrinsic resistance. Furthermore, almost all patients with metastatic disease and many who receive tamoxifen as adjuvant therapy eventually experience tumor relapse (acquired resistance). Thus, tamoxifen resistance occurs frequently in breast cancer patients and seriously limits treatment efficacy [START_REF] Massarweh | Resistance to endocrine therapy in breast cancer: exploiting estrogen receptor/growth factor signaling crosstalk[END_REF][START_REF] Arpino | Cross-talk between the estrogen receptor and the HER tyrosine kinase receptor family: molecular mechanism and clinical implications for endocrine therapy resistance[END_REF]. Multiple mechanisms have been proposed to contribute to the failure of tamoxifen therapy, including changes in the uptake or metabolism of tamoxifen, presence of ER mutants and ligand-independent ER activation, loss of expression of ER and/or cofactors and modification of the estrogen response element [START_REF] Massarweh | Resistance to endocrine therapy in breast cancer: exploiting estrogen receptor/growth factor signaling crosstalk[END_REF][START_REF] Herynk | Estrogen receptor mutations in human disease[END_REF].

A large body of clinical and experimental studies has suggested that alteration of ER function by growth factor pathways may be involved in tamoxifen resistance [START_REF] Massarweh | Tamoxifen resistance in breast tumors is driven by growth factor receptor signaling with repression of classic estrogen receptor genomic function[END_REF][START_REF] Massarweh | Resistance to endocrine therapy in breast cancer: exploiting estrogen receptor/growth factor signaling crosstalk[END_REF][START_REF] Long | Changes in epidermal growth factor receptor expression and response to ligand associated with acquired tamoxifen resistance or oestrogen independence in the ZR-75-1 human breast cancer cell line[END_REF][START_REF] Nicholson | Epidermal growth factor receptor expression in breast cancer: association with response to endocrine therapy[END_REF][START_REF] Osborne | Crosstalk between estrogen receptor and growth factor receptor pathways as a cause for endocrine therapy resistance in breast cancer[END_REF]. In this context, it has been reported that phosphorylation of ERα by EGFR-mediated signaling enhances the estrogeninduced transcriptional activity [START_REF] Joel | pp90rsk1 regulates estrogen receptor-mediated transcription through phosphorylation of Ser-167[END_REF][START_REF] Gee | Epidermal growth factor receptor/HER2/insulinlike growth factor receptor signalling and oestrogen receptor activity in clinical breast cancer[END_REF]. An increased recruitment of coactivators, such as AIB1 [START_REF] Shou | Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer[END_REF], and conformational changes [START_REF] Michalides | Tamoxifen resistance by a conformational arrest of the estrogen receptor a after PKA activation in breast cancer[END_REF] after receptor phosphorylation also contribute to enhanced transcriptional activity of ERα in tamoxifen-resistant breast cancer cells. In addition, an inverse relationship between the expression of ERα and EGFR or c-erbB2 was observed in primary breast cancer samples and was associated with decreased sensitivity to endocrine therapy and a poorer prognosis [START_REF] Nicholson | Epidermal growth factor receptor expression in breast cancer: association with response to endocrine therapy[END_REF][START_REF] Nicholson | Relationship between EGF-R, c-erbB2 protein expression and Ki67immunostaining in breast cancer and hormone sensitivity[END_REF]. Hormone-dependent breast cancer cells engineered to overexpress EGFR and c-erbB2 resulted in stimulated hormone-independent cell growth [START_REF] Van Agthoven | Ectopic expression of epidermal growth factor receptors induces hormone independence in ZR-75-1 human breast cancer cells[END_REF][START_REF] Benz | Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7cells transfected with HER2/neu[END_REF].

Our present data demonstrate that EGF enhances E-cadherin expression and promotes homotypic aggregation of breast cancer cells maintained as three-dimensional cultures.

E-cadherin is an intercellular adhesion molecule generally implicated as tumor suppressor in several

types of epithelial tumors, based on findings that the expression of this homotypic adhesion molecule is frequently lost in human epithelial cancers [START_REF] Perl | A causal role for E-cadherin in the transition from adenoma to carcinoma[END_REF][START_REF] Behrens | Dissecting tumor cell invasion: epithelial cells acquire invasive properties after the loss of uvomorulin mediated cell-cell adhesion[END_REF][START_REF] Frixen | E-cadherin mediated cell-cell adhesion prevents invasiveness of human carcinoma cells[END_REF]. However, it has well been shown in ovarian epithelial tumors that E-cadherin expression is much more elevated than normal ovaries, suggesting that E-cadherin can play a role in the development of ovarian carcinomas [START_REF] Reddy | Formation of E-cadherinmediated cell-cell adhesion activates AKT and mitogen activated protein kinase via phosphatidylinositol 3 kinase and ligand-independent activation of epidermal growth factor receptor in ovarian cancer cells[END_REF]. For instance, it is worth to mention that E-cadherin may serve not only as an intercellular adhesion molecule, but it may also trigger intracellular activation of proliferation and survival signals [START_REF] Steinberg | Cadherins and their connections: adhesion junctions have broader functions[END_REF][START_REF] Mauro | Evidences that leptin upregulates E-cadherin expression in breast cancer: effects on tumor growth and progression[END_REF]. Our recent findings provided evidence that E 2 may be involved in mammary tumorigenesis on the basis of its stimulatory effects on primary tumor mass either in vivo using MCF7 cell tumor xenograft or in vitro using MCF7 three-dimensional cultures [START_REF] Mauro | Evidences that leptin upregulates E-cadherin expression in breast cancer: effects on tumor growth and progression[END_REF]. These actions were elicited by E 2 through homotypic tumor cell adhesion consequent to an increase of E-cadherin expression [START_REF] Mauro | Evidences that leptin upregulates E-cadherin expression in breast cancer: effects on tumor growth and progression[END_REF].

A number of studies have evaluated the usefulness of E-cadherin expression with respect to survival of breast cancer patients. Several studies indicate that reduced E-cadherin expression is an independent indicator of poor survival, particularly in node-negative patients [START_REF] Siitonen | Reduced E-cadherin expression is associated with invasiveness and unfavorable prognosis in breast cancer[END_REF][START_REF] Charpin | Reduced Ecadherin immunohistochemical expression in node-negative breast carcinomas correlates with 10-year survival[END_REF][START_REF] Heimann | Separating favorable from unfavorable prognostic markers in breast cancer: The role of E-cadherin[END_REF][START_REF] Asgeirsson | Altered expression of E-cadherin in breast cancer: Patterns, mechanisms and clinical significance[END_REF]. However, other studies have found E-cadherin to be of little use in predicting clinical behavior of breast cancer patients [START_REF] Lipponen | Expression of E-cadherin (E-CD) as related to other prognostic factors and survival in breast cancer[END_REF]. Indeed, one study even noted that strong E-cadherin immunostaining correlated with poor survival [START_REF] Tan | The biological and prognostic significance of cell polarity and E-cadherin in grade I infiltrating ductal carcinoma of the breast[END_REF]. In short, there currently are conflicting opinions about the prognostic value of altered E-cadherin expression. Thus, the usefulness of E-cadherin as a predictor of clinical behavior for breast cancer patients is still under debate.

In the present study, we showed that a cross-talk between ERα and EGFR was involved in E-cadherin expression by both OHT and EGF as demonstrated using the pure ER antagonist ICI, the Src and ERK inhibitors PP2 and PD, respectively, and most importantly silencing either ERα or EGFR expression.

Moreover, OHT and EGF stimulated ERα recruitment to the Sp1 site within the E-cadherin promoter region, whereas blocking this site, either by silencing Sp1 or using mithramycin, ERα binding to the Ecadherin promoter region was no longer evident. Thus, in conclusion in this study, we provide further insights into the molecular mechanisms involved in the agonist activity exerted by tamoxifen in breast cancer, through the evidence that OHT per se is able to activate the promoter of E-cadherin, increasing E-cadherin expression at the mRNA and protein levels and thus may enhance homotypic aggregation of MCF7 breast cancer cells.

In addition, we have demonstrated that E-cadherin is a target of a cross-talk between EGF and OHT/ER signaling, since the upregulatory effect induced by OHT on E-cadherin expression was abrogated in the presence of either ERα or EGFR silencing. It is worth to note that in our experimental conditions tamoxifen induced an enhanced expression of EGFR protein levels, which may contribute to the failure of adjuvant endocrine therapy in breast cancer, as suggested by a growing body of data. The latter observations highlights how the agonistic effect of tamoxifen and EGF may sustains tumor mass growth through cell-cell aggregation. E-cadherin-positive MCF7 cells were seeded in 2% agar-coated plates and cultured as three dimensional spheroids. To block E-cadherin function, the medium was supplemented with E-cadherin antibody (1:100 dilution; a-E-cad) or EGTA (4 mM). Cells were untreated or treated with 1µM OHT or 100ng/ml EGF for 48h and then photographed under phase-contrast microscopy. Bar, 50 µm.

Legend for figures

Figure 1 .

 1 Figure 1. OHT and EGF transactivate the E-cadherin promoter and increase E-cadherin mRNA

Figure 2 .

 2 Figure 2. OHT acts as an agonist for E-cadherin mRNA expression but as an antagonist for pS2

Figure 3 .

 3 Figure 3. Immunoblots of E-cadherin from MCF7 cells.

Figure 4 .

 4 Figure 4. ERK1/2 phosphorylation in MCF7 cells.

Figure 5 .

 5 Figure 5. ERα and EGFR silencing abrogates the up-regulation of E-cadherin by OHT and EGF.

Figure 6 .

 6 Figure 6. OHT and EGF induce the recruitment of ERα on the Sp1 site within E-cadherin

Figure 7 .

 7 Figure 7. OHT and EGF induce cell-cell adhesion in three-dimensional cultures.

Table 1 .

 1 Effect of OHT and EGF on cell aggregation in MCF7 breast cancer cells. MCF7 cells were cultured as three-dimensional spheroids in medium without serum and treated as reported. The extent of aggregation was scored by measuring the spheroid diameters with an ocular micrometer. The values represent a sum of spheroids in 10 optical fields under x10 magnification. The results are mean mean ± S.D. from at least three experiments. Representative three-dimensional cultures are shown in Fig. 7.

Acknowledgments

This work was supported by MURST, Ex 60%, AIRC grants 2007 , 2008 , MFAG 2008. .


Theodoros N Sergentanis
email: tsergentanis@sni.gr.

Konstantinos P Economopoulos

GSTT1 and GSTP1 polymorphisms and breast cancer risk: a metaanalysis

Keywords: GSTT1, GSTP1, glutathione S-transferase, polymorphism, breast cancer

Cytosolic glutathione S-transferase (GST) comprises multiple isoenzymes; studies have principally examined mu-1 (GSTM1: null/present), theta-1 (GSTT1: null/present) and pi-1 (GSTP1 Ile105Val) gene polymorphisms concerning breast cancer risk. Regarding GSTT1 and GSTP1 polymorphisms, studies remain controversial and no recent meta-analysis has appeared. This meta-analysis aims to examine whether GSTT1 and GSTP1 polymorphisms are associated with breast cancer risk. Separate analyses were performed on Chinese and non-Chinese populations, in an attempt to investigate race-specific effects. Eligible articles were identified by a search of MEDLINE bibliographical database for the period up to August 2009. Regarding GSTT1 null/present genotype, 41 case-control studies were eligible (16,589 breast cancer cases and 19,995 controls); 30 case-control studies were eligible for GSTP1 Ile105Val (16,908 cases and 20,016 controls). Pooled odds ratios (OR) were appropriately derived from fixed-effects or random-effects models. At the overall analysis, the null GSTT1 genotype was associated with elevated breast cancer risk (pooled OR=1.114, 95%CI: 1.035-1.199, random effects). However, the association seemed confined to non-Chinese populations (33 studies, pooled OR=1.128, 95%CI: 1.042-1.221, random effects), given that the association was not significant in the subset of Chinese studies (eight studies, pooled OR=1.061, 95%CI: 0.875-1.286, random effects). Regarding GSTP1 Ile105Val, no statistically significant associations were detected in non-Chinese populations (25 studies). On the other hand, the GG genotype was associated with increased breast cancer risk in Chinese populations (five studies, pooled OR=1.297, 95%CI: 1.023-1.645, fixed effects); accordingly, the recessive model yielded statistically significant results (pooled OR=1.273, 95%CI: 1.006-1.610, fixed effects). In conclusion, polymorphisms of both 3 GSTT1 and GSTP1 genes seem associated with elevated breast cancer risk in a racespecific manner. Given the small number of Chinese studies, the finding on GSTP1 Ile105Val merits further investigation.

Introduction

Cytosolic glutathione S-transferase (GST) comprises multiple isoenzymes; studies have principally examined mu-1 (GSTM1), theta-1 (GSTT1) and pi-1 (GSTP1) genes concerning breast cancer risk. GSTs catalyze reactions between glutathione and lipophilic compounds with electrophilic centers, leading to neutralization of toxic compounds [START_REF] Hengstler | Polymorphisms of Nacetyltransferases, glutathione S-transferases, microsomal epoxide hydrolase and sulfotransferases: influence on cancer susceptibility[END_REF]. GSTM1, GSTT1 and GSTP1 genes have been found polymorphic in the population, i.e. "present" or "null" genotypes may characterize an individual regarding GSTM1 and GSTT1, whereas a single-nucleotide polymorphism (A-to-G transition) has been identified in the exon 5 of the GSTP1 gene (Ile105Val) [START_REF] Kano | Structure and expression of a human class pi glutathione S-transferase messenger RNA[END_REF].

Attention has been drawn at a meta-analytical level upon those polymorphisms; a recent meta-analysis has demonstrated that GSTM1 null genotype is associated with elevated breast cancer risk [START_REF] Yu | A functional polymorphism in the promoter region of GSTM1 implies a complex role for GSTM1 in breast cancer[END_REF]. Interestingly enough, the latest meta-analyses on the association between GSTT1, GSTP1 genotypes and breast cancer risk appeared in 2004 [START_REF] Vogl | Glutathione Stransferases M1, T1, and P1 and breast cancer: a pooled analysis[END_REF][START_REF] Egan | Genetic polymorphisms in GSTM1, GSTP1, and GSTT1 and the risk for breast cancer: results from the Shanghai Breast Cancer Study and metaanalysis[END_REF]; the above meta-analyses had been based upon a small number of studies available at that time period (at best 15 case-control studies) and had pointed to null associations. Since then, more than 20 additional case-control studies have been published regarding both GSTT1 and GSTP1 [7, 8, 11-15, 17, 20-32] polymorphisms and breast cancer risk, with contradictory findings. Under the light of the substantial improvement in the power of the meta-analysis, the need for an up-todate meta-analysis has become evident. This meta-analysis aims to examine whether GSTT1 and GSTP1 polymorphisms status is associated with breast cancer risk. Separate analyses were performed on Chinese and non-Chinese populations, in an attempt to investigate racespecific effects.

Methods

Trial Identification

Eligible articles were identified by a search of MEDLINE bibliographical database for the period up to August 2009 (last search: August 11, 2009) using combinations of the following keywords: "glutathione-S tranferase", "GSTT1", "GSTP1", "polymorphism", "genotype", "variant", "breast cancer". In addition, we checked all the references of relevant reviews and eligible articles that our search retrieved.

Language restrictions were not used and two investigators (KPE and TNS), working independently, searched the literature and extracted data from each eligible casecontrol study.

Eligible Studies and Data Abstraction

All case-control studies with any sample size examining the association between breast cancer and GSTT1 null genotype or GSTP1 Ile105Val were considered eligible for this meta-analysis. For each one of the eligible case-control studies the following data were collected: journal name, year of publication, inclusion and exclusion criteria, demographic characteristics of the population being studied, menopausal status, frequencies of genotypes in cases and controls for each of the above genotypes/polymorphisms.

Statistics

Based on the genotype frequencies in cases and controls, crude odds ratios (OR) as well as their standard errors (SE) were calculated. Regarding GSTT1, the ORs pertained to null genotype carriers vs. present (positive) genotype carriers.

Concerning GSTP1, four different ORs were calculated: i) AG vs AA (heterozygous carriers), ii) GG vs AA (homozygous carriers), iii) G allele carriers (AG and GG grouped together) vs. AA (dominant model) and iv) GG genotype vs. A allele carriers (AG and AA grouped together) (recessive model). Separate analyses were performed in Chinese and non-Chinese populations.

The fixed-effects model (Mantel-Haenszel method), as well as the random effects (DerSimonian Laird) model, were used to calculate the pooled OR. Between-study heterogeneity and between-study inconsistency were assessed by using Cochran Q statistic and by estimating I 2 respectively [START_REF] Higgins | Measuring inconsistency in meta-analyses[END_REF]. In case no significant heterogeneity was detected, the fixed effects model was chosen. Evidence of publication bias was determined using Egger's formal statistical test [START_REF] Egger | Bias in metaanalysis detected by a simple, graphical test[END_REF] and by visual inspection of the funnel plot. For the interpretation of Egger's test, statistical significance was defined as p<0.1. Meta-analysis was performed using the STATA "metan" command.

In addition, meta-regression was performed to assess whether Odds Ratio (OR) was associated with publication year. The exponentiated coefficient is provided, since the dependent variable in the meta-regression model is log(OR). Meta-regression was performed with the "metareg" STATA command. Analyses were conducted using STATA 10.0 (STATA Corp. College Station, TX, USA).

Results

Figure 1 graphically illustrates the trial flow chart. Out of the 161 abstracts retrieved through the search criteria, 94 were irrelevant, nine articles [START_REF] Zheng | Glutathione S-transferase M1 and T1 genetic polymorphisms, alcohol consumption and breast cancer risk[END_REF][START_REF] Lee | N-acetyltransferase (NAT1, NAT2) and glutathione S-transferase (GSTM1, GSTT1) polymorphisms in breast cancer[END_REF][START_REF] Park | Alcohol consumption, glutathione Stransferase M1 and T1 genetic polymorphisms and breast cancer risk[END_REF][START_REF] Park | Reproductive factors, glutathione Stransferase M1 and T1 genetic polymorphism and breast cancer risk[END_REF][START_REF] Van Der Hel | GSTM1 null genotype, red meat consumption and breast cancer risk (The Netherlands)[END_REF][START_REF] Gago-Dominguez | Marine n-3 fatty acid intake, glutathione S-transferase polymorphisms and breast cancer risk in post-menopausal Chinese women in Singapore[END_REF][START_REF] Yang | Genetic polymorphisms in glutathione-S-transferase genes (GSTM1, GSTT1, GSTP1) and survival after chemotherapy for invasive breast carcinoma[END_REF][START_REF] Samson | Role of GSTM1 (Null/Present), GSTP1 (Ile105Val) and P53 (Arg72Pro) genetic polymorphisms and the risk of breast cancer: a case control study from South India[END_REF][START_REF] Justenhoven | Breast cancer: a candidate gene approach across the estrogen metabolic pathway[END_REF] were excluded because they were conducted on overlapping populations with other eligible studies [START_REF] Park | Combined effect of GSTM1, GSTT1, and COMT genotypes in individual breast cancer risk[END_REF][START_REF] Ceschi | The effect of cyclin D1 (CCND1) G870A-polymorphism on breast cancer risk is modified by oxidative stress among Chinese women in Singapore[END_REF][START_REF] Rajkumar | TGFbeta1 (Leu10Pro), p53 (Arg72Pro) can predict for increased risk for breast cancer in south Indian women and TGFbeta1 Pro (Leu10Pro) allele predicts response to neo-adjuvant chemo-radiotherapy[END_REF][START_REF] The | Genetic polymorphisms in phase I and phase II enzymes and breast cancer risk associated with menopausal hormone therapy in postmenopausal women[END_REF][START_REF] Lee | Cruciferous vegetables, the GSTP1 Ile105Val genetic polymorphism, and breast cancer risk[END_REF][START_REF] Zheng | Cigarette smoking, glutathione-stransferase M1 and t1 genetic polymorphisms, and breast cancer risk (United States)[END_REF][START_REF] Van Der Hel | NAT2 slow acetylation and GSTM1 null genotypes may increase postmenopausal breast cancer risk in long-term smoking women[END_REF] (these excluded articles represent smaller studies performed on subsets of larger eligible studies), five studies [START_REF] Nedelcheva Kristensen | Single tube multiplex polymerase chain reaction genotype analysis of GSTM1, GSTT1 and GSTP1: relation of genotypes to TP53 tumor status and clinicopathological variables in breast cancer patients[END_REF][START_REF] Sweeney | Association between survival after treatment for breast cancer and glutathione Stransferase P1 Ile105Val polymorphism[END_REF][START_REF] Ambrosone | Polymorphisms in glutathione S-transferases (GSTM1 and GSTT1) and survival after treatment for breast cancer[END_REF][START_REF] Van Der Hel | Cumulative genetic defects in carcinogen metabolism may increase breast cancer risk (The Netherlands)[END_REF][START_REF] Van Emburgh | Polymorphisms in drug metabolism genes, smoking, and p53 mutations in breast cancer[END_REF] were excluded given that they have not included controls in their study design, two articles [START_REF] Vogl | Glutathione Stransferases M1, T1, and P1 and breast cancer: a pooled analysis[END_REF][START_REF] Dunning | A systematic review of genetic polymorphisms and breast cancer risk[END_REF] were reviews/meta-analyses, and three studies [START_REF] Sgambato | Glutathione S-transferase (GST) polymorphisms as risk factors for cancer in a highly homogeneous population from southern Italy[END_REF][START_REF] Mitrunen | Combined COMT and GST genotypes and hormone replacement therapy associated breast cancer risk[END_REF][START_REF] Sweeney | Association between a glutathione S-transferase A1 promoter polymorphism and survival after breast cancer treatment[END_REF] were excluded due to other reasons (two of them [START_REF] Sgambato | Glutathione S-transferase (GST) polymorphisms as risk factors for cancer in a highly homogeneous population from southern Italy[END_REF][START_REF] Mitrunen | Combined COMT and GST genotypes and hormone replacement therapy associated breast cancer risk[END_REF] was excluded due to reporting reasons, i.e. no reporting of the relevant genotype frequencies, whereas the other [START_REF] Sweeney | Association between a glutathione S-transferase A1 promoter polymorphism and survival after breast cancer treatment[END_REF] was excluded for examining the association between GSTA1 polymorphism and survival after breast cancer treatment). As a result, 48 case-control articles [5-32, 44, 45, 55-72] were included in this meta-analysis; 41 case-control studies [5-28, 44, 45, 55-59, 61-65, 67-71] were included in the meta-analysis on GSTT1 genotype (16,589 breast cancer cases and 19,995 controls) and 30 case-control studies [7, 8, 11-15, 17, 20-32, 60, 62, 64-68, 70, 72] on GSTP1 polymorphism (16,908 cases and 20,016 controls). Evidently, the sum of studies surpasses the number of eligible articles, as more than one study were presented per article. In addition, 33 case-control studies were included in the metaanalysis on GSTT1 genotype on non-Chinese subjects (14,139 cases and 16,465 controls) [7, 9, 12-15, 17, 19-28, 44, 45, 55-58, 61-65, 67-71] and eight case-control studies on Chinese subjects (2,450 cases and 3,530 controls) [START_REF] Egan | Genetic polymorphisms in GSTM1, GSTP1, and GSTT1 and the risk for breast cancer: results from the Shanghai Breast Cancer Study and metaanalysis[END_REF][START_REF] Park | Combined effect of GSTM1, GSTT1, and COMT genotypes in individual breast cancer risk[END_REF][START_REF] Ceschi | The effect of cyclin D1 (CCND1) G870A-polymorphism on breast cancer risk is modified by oxidative stress among Chinese women in Singapore[END_REF][START_REF] Cheng | Breast cancer risk associated with genotype polymorphism of the catechol estrogen-metabolizing genes: a multigenic study on cancer susceptibility[END_REF][START_REF] Chang | Glutathione S-transferase polymorphisms associated with risk of breast cancer in southern Taiwan[END_REF][START_REF] Chang | A case-control study on serum organochlorines residues, genetic polymorphisms of glutathione S-transferase T1 and the risks of breast cancer[END_REF][START_REF] Li | Using MSR model to analyze the impact of gene-gene interaction with related to the genetic polymorphism of metabolism enzymes on the risk of breast cancer[END_REF][START_REF] Wu | Association of DNA-protein crosslinks and breast cancer[END_REF]. Accordingly, 25 case-control studies [7, 12-15, 17, 20-28, 32, 60, 62, 64-69, 70, 72] were included in the meta-analysis on GSTP1 genotype on non-Chinese subjects (12,652 cases and 14,843 controls) and five case-control studies on Chinese subjects (4,256 cases and 5,173 controls) [START_REF] Ceschi | The effect of cyclin D1 (CCND1) G870A-polymorphism on breast cancer risk is modified by oxidative stress among Chinese women in Singapore[END_REF][START_REF] Chang | Glutathione S-transferase polymorphisms associated with risk of breast cancer in southern Taiwan[END_REF][START_REF] Kim | Genetic polymorphism of glutathione S-transferase P1 and breast cancer risk[END_REF][START_REF] Lee | Cruciferous vegetables, the GSTP1 Ile105Val genetic polymorphism, and breast cancer risk[END_REF][START_REF] Sakoda | Glutathione S-transferase M1 and P1 polymorphisms and risk of breast cancer and fibrocystic breast conditions in Chinese women[END_REF].

At the overall analysis, the null GSTT1 genotype was associated with elevated breast cancer risk (pooled OR=1.114, 95%CI: 1.035-1.199, random effects).

Interestingly enough, stratification by race pointed to discrepancy between non-Chinese and Chinese studies. The association seemed confined to non-Chinese populations (pooled OR= 1.128, 95%CI: 1.042-1.221, random effects, Figure 2a), given that the association was not significant in the subset of Chinese studies (pooled OR= 1.061, 95%CI: 0.875-1.286, random effects, Figure 2b). Regarding GSTP1 Ile105Val, the results of the meta-analysis are presented in detail in the Table 1. No statistically significant associations were detected in non-Chinese populations. On the other hand, the GG genotype was associated with increased breast cancer risk in Chinese populations; accordingly, the recessive model yielded statistically significant results (Figure 3a and3b).

Meta-regression with publication year did not point to any modifying effect of publication year in the case of GSTT1 polymorphism (exponentiated coefficient= 0.994, 95%CI: 0.970-1.019, p=0.630). Accordingly, no modifying effects of publication year were observed at any approach regarding GSTP1 status (for heterozygous carriers: exponentiated coefficient= 0.993, 95%CI: 0.953-1.035, p=0.738; for homozygous carriers: exponentiated coefficient= 0.993, 95%CI: 0.916-1.076, p=0.864; for the dominant model: exponentiated coefficient= 0.995, 95%CI: 0.954-1.038, p=0.809; for the recessive model: exponentiated coefficient= 0.999, 95%CI: 0.939-1.063, p=0.970).

Concerning GSTT1 polymorphism, publication bias was significant in the overall meta-analysis (p=0.048), mainly due to the publication bias in non-Chinese studies (p=0.045); no significant publication bias was detected in Chinese studies (p=0.742). On the contrary, no significant publication bias was detected in any metaanalysis performed on GSTP1 polymorphism status (p=0.607 for heterozygous carriers, p=0.557 for homozygous carriers, p=0.787 for the dominant model, p=0.393 for the recessive model).

Discussion

The principal message of this meta-analysis is that polymorphisms of both GSTT1 and GSTP1 genes seem associated with elevated breast cancer risk in a race-specific manner. Specifically, GSTT1 null phenotype seems able to confer additional breast cancer risk in non-Chinese populations, whereas GSTP1 Ile105Val G allele seems associated with increased breast cancer risk in Chinese subjects following a recessive model.

Concerning GSTT1 null phenotype, it is worth mentioning that the present metaanalysis makes one step beyond the existing meta-analysis by Egan et al [START_REF] Egan | Genetic polymorphisms in GSTM1, GSTP1, and GSTT1 and the risk for breast cancer: results from the Shanghai Breast Cancer Study and metaanalysis[END_REF], which had been published five years ago and had pointed to marginal associations, characterized by the authors as "null results". In the present meta-analysis, under the light of a nearly 3-fold increase in the number of eligible studies, a clear association between GSTT1 null genotype and increased breast cancer risk became evident and seemed indeed confined to non-Chinese populations. Race-specific effects of GSTT1 polymorphism status may not seem surprising at a meta-analytical level, as racespecific associations have been described in meta-analyses examining lung [START_REF] Raimondi | Meta-and pooled analysis of GSTT1 and lung cancer: a HuGE-GSEC review[END_REF] and gastric [START_REF] Saadat | Genetic polymorphisms of glutathione S-transferase T1 (GSTT1) and susceptibility to gastric cancer: a meta-analysis[END_REF] cancer.

Similarly, the results concerning GSTP1 Ile105Val exhibited a race-specific pattern, but opposite to that demonstrated in GSTT1. The association between the G allele seemed confined in Chinese studies; nevertheless, given that the sample size of the study by Lee at al [START_REF] Lee | Cruciferous vegetables, the GSTP1 Ile105Val genetic polymorphism, and breast cancer risk[END_REF] is considerably larger than that of the remaining studies, the latter emerges as particularly influential. As a result, the need for additional studies on Chinese populations seems warranted, so as to establish the present findings.

An issue that has been raised in previous meta-analyses examining GSTT1 and GSTP1 status vis-à-vis breast cancer risk is the putative interaction with menopausal status. In the present meta-analysis, such subanalyses have not been presented after carefully taking into account that solely ten [START_REF] Park | Combined effect of GSTM1, GSTT1, and COMT genotypes in individual breast cancer risk[END_REF][START_REF] Chacko | Role of xenobiotic metabolizing gene polymorphisms in breast cancer susceptibility and treatment outcome[END_REF][START_REF] Steck | Interactions among GSTM1, GSTT1 and GSTP1 polymorphisms, cruciferous vegetable intake and breast cancer risk[END_REF][START_REF] Saxena | Detection and relevance of germline genetic polymorphisms in glutathione S-transferases (GSTs) in breast cancer patients from northern Indian population[END_REF][START_REF] Zheng | Cigarette smoking, glutathione-stransferase M1 and t1 genetic polymorphisms, and breast cancer risk (United States)[END_REF][START_REF] Zheng | GSTM1 and GSTT1 polymorphisms and postmenopausal breast cancer risk[END_REF][START_REF] Mitrunen | Glutathione S-transferase M1, M3, P1, and T1 genetic polymorphisms and susceptibility to breast cancer[END_REF][START_REF] Millikan | Glutathione S-transferases M1, T1, and P1 and breast cancer[END_REF][START_REF] Garcia-Closas | Glutathione S-transferase mu and theta polymorphisms and breast cancer susceptibility[END_REF][START_REF] Helzlsouer | Association between glutathione Stransferase M1, P1, and T1 genetic polymorphisms and development of breast cancer[END_REF] out of 41 studies on GSTT1 and nine [15, 28-32, 65, 67, 70] out of 30 studies on GSTP1 status have presented subanalyses on pre-and postmenopausal women. Indeed, the power of the above subsets of studies was so limited that the main associations were blurred (data not shown); as a result, proceeding to subanalyses on pre-and postmenopausal subjects did not seem of particular value.

In conclusion, this meta-analysis points to a positive association between GSTT1 null genotype and breast cancer risk in non-Chinese subjects; the positive association between GSTP1 G allele and elevated breast cancer risk in Chinese subjects is worth investigating further.

Figure 1 :

 1 Figure 1: Study flow chart explaining the selection of the 48 eligible case-control

Figure 2 :

 2 Figure 2: Forest plot for the overall association between null GSTT1 genotype and

Figure 3 :

 3 Figure 3: Forest plot for the overall association between the GSTP1 GG (Val/Val)

Figure Click here to download high resolution imageFigure Click here to download high resolution imageFigure

  Figure Click here to download high resolution image

Table 1 .

 1 Pooled ORs by race for heterozygous, homozygous carriers, dominant and recessive model. All pooled ORs were derived from random-effects models except for cells marked with F (fixed-effects model). Bold letters denote statistically significant results.

	Race	Heterozygous §	Homozygous §	Dominant model	Recessive model §
		(AG vs. AA)	(GG vs. AA)	(GG and AG vs. AA)	(GG vs. AA and AG)
		OR (95%CI)	Test for	OR (95%CI)	Test for	OR (95%CI)	Test for	OR (95%CI)	Test for
			heterogeneity		heterogeneity		heterogeneity		heterogeneity
	Overall (n=30)	1.004	p<0.001	0.991	p<0.001	1.013	p<0.001	0.992	p<0.001
		(0.886-1.137)		(0.788-1.246)		(0.899-1.142)		(0.826-1.192)
	Non-Chinese	1.001	p<0.001	0.980	p<0.001	1.007	p<0.001	0.979	p<0.001
	(n=25)
		(0.858-1.169)		(0.754-1.273)		(0.869-1.167)		(0.795-1.204)
	Chinese (n=5)	1.057 F	p=0.370	1.297 F	p=0.374	1.083 F	p=0.611	1.273 F
		(0.965-1.159)		(1.023-1.645)		(0.994-1.180)

(1.006-1.610) p=0

  .323 § the calculations were based on 23 non-Chinese and four Chinese studies, as the studies by Chang et al.[START_REF] Chang | Glutathione S-transferase polymorphisms associated with risk of breast cancer in southern Taiwan[END_REF], Steck et al.[START_REF] Steck | Interactions among GSTM1, GSTT1 and GSTP1 polymorphisms, cruciferous vegetable intake and breast cancer risk[END_REF] and McCarty et al.[START_REF] Mccarty | PAH-DNA adducts, cigarette smoking, GST polymorphisms, and breast cancer risk[END_REF] did not provide the necessary data.