Oncogene (2010) 29, 2292–2301 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc SHORT COMMUNICATION NFAT3 inhibits breast cancer cell motility by targeting the Lipocalin 2

M Fouge` re1, B Gaudineau1, J Barbier2, F Guaddachi1, J-P Feugeas1, D Auboeuf2 and S Jauliac1

1CNRS UMR7212, INSERM U944, Universite´ Paris Diderot, Institut d’He´matologie, Hoˆpital Saint-Louis, Paris Cedex 10, France and 2INSERM U685/AVENIR, Centre Hayem, Institut d’He´matologie, Hoˆpital Saint-Louis, Paris Cedex 10, France

NFAT1 and NFAT5 act as pro-invasive and pro- are also present in non-immune cells (Ho et al., 1998; migratory transcription factors in breast carcinoma, Molkentin et al., 1998; Ranger et al., 2000; Jauliac et al., contributing to the formation of metastases. We report 2002; Hill-Eubanks et al., 2003; Benedito et al., 2005) that NFAT3 is specifically expressed in estrogen and regulate a variety of signaling pathways involved in a positive (ERA þ ) breast cancer cells. We show that cell growth and development (Xanthoudakis et al., 1996; NFAT3 inhibits by itself the invasion capacity of ERA þ Baksh et al., 2002; Chuvpilo et al., 2002). Owing to their breast cancer cells and needs to cooperate with ERA to function in critical signaling pathways controlling cell inhibit their migration. Conversely, NFAT3 downregula- fate, one could expect that disturbing signaling of tion results in actin reorganization associated with NFAT factors may impact on carcinogenesis. Indeed, increased migration and invasion capabilities. NFAT3 molecular pathways involving specific members of the signaling reduces migration through inhibition of Lipoca- NFAT family were recently highlighted in the migratory lin 2 (LCN2) . Collectively, our study and invasive capacities of breast cancer cells (Jauliac unravels an earlier unknown NFAT3/LCN2 axis that et al., 2002). Moreover, there is growing evidence for a critically controls motility in breast cancer. function of the NFAT factors in carcinogenesis (Man- Oncogene (2010) 29, 2292–2301; doi:10.1038/onc.2009.499; cini and Toker, 2009). Genomic amplification of NFAT1 published online 25 January 2010 has been observed in pancreatic cancer (Holzmann et al., 2004), and ectopic activation of NFAT2 induces Keywords: NFAT3; LCN2; a; moti- over-expression of c- (Buchholz et al., 2006). Ectopic lity; breast expression of constitutively activated NFAT2 in pre- adipocytes induces their transformation and tumors in nude mice (Neal and Clipstone, 2003). Furthermore, NFAT5 expression level is a prognostic marker in breast Introduction cancer (Ayers et al., 2004) or in non-small cell lung cancer patients (Zhong et al., 2004). Breast cancer is a leading cause of morbidity in women The ability of tumor cells to form metastases is one of worldwide. The main reason of death for these patients the critical determinants of malignancy. Lipocalin 2 is not the primary tumor, but distant metastases, which (LCN2) is a secreted protein of the Lipocalin family are directly linked to the migratory and invasive (Flower, 1996). LCN2 can be induced under diverse phenotype of the cancer cells (Friedl and Wolf, 2003). inflammatory conditions (Nielsen et al., 1996) and has a Invasion is a complex process that requires both (i) the function in cell survival (Devireddy et al., 2001). High capacity of the cells to destroy and reorganize the extra- LCN2 expression levels are associated with malignancy cellular matrix and (ii) their capacity to migrate, the in different cancers including ovarian (Lim et al., 2007), latter being necessary, but not sufficient, for invasion. pancreatic (Furutani et al., 1998), lung and colon The family of NFAT transcription factors comprises cancers (Friedl et al., 1999). Recently, LCN2 has been five (NFAT1 to 5). NFAT1, NFAT2, NFAT3 and implicated in tumorigenesis and formation of metastases NFAT4 were first identified as transcription in murine breast cancer models (Shi et al., 2008; Leng factors and bind the human IL-2 promoter after et al., 2009; Yang et al., 2009). A potential function for activation (Shaw et al., 1988), whereas NFAT5 is LCN2 in the migratory capacity of colon cells (Playford induced by osmotic stress (Lopez-Rodriguez et al., et al., 2006) and tissue invasion by leukemia cells (Leng 1999). It is now well documented that NFAT proteins et al., 2008) has also been documented. The LCN2 gene is specifically downregulated in estrogen receptor a positive (ERA þ ) breast cancer patients (Stoesz et al., Correspondence: Dr S Jauliac, CNRS UMR7212, INSERM U944, 1998) and has been reported as a predictor of breast Universite´Paris Diderot, Institut d’He´matologie, Hoˆpital Saint-Louis, cancer progression (Bauer et al., 2008; Provatopoulou 1 avenue Claude Vellefaux, Paris Cedex 10 75475, France. E-mail: [email protected] et al., 2009). It has been clearly shown that expression of Received 20 April 2009; revised 9 November 2009; accepted 11 ERA is associated with more differentiated, less invasive December 2009; published online 25 January 2010 tumors. Moreover, numerous clinical studies suggest NFAT3/LCN2 axis inhibits breast carcinoma motility M Fouge`re et al 2293 that estrogens and their receptors protect the patients et al., 1988; Platet et al., 2000). Moreover, when ERA þ against the apparition of metastases (Li et al., 2001), and breast carcinoma cell lines are re-implanted in mice, breast carcinomas cell lines that express the ERA are tumors arise only in the presence of estrogen and they less invasive than cells lacking its expression (Thompson are poorly metastatic compared with the cells that do

ERA+ ERA-

ERA+ERA- ERA+ DNA ladder BT-474 MCF-7 MDA-MB-435 MDA-MB-231 ZR-75-1 600 bp T-47D NFAT1

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Figure 1 For caption please see page 2294.

Oncogene NFAT3/LCN2 axis inhibits breast carcinoma motility M Fouge`re et al 2294 not express ERA (Price et al., 1990). Indeed, some (Figure 1B, upper left panel) or of NFAT1 and NFAT2 studies have reported that NFAT3 is specifically present (Supplementary Figure S2). Such treatment did not lead in breast cancer patients who express ERA (Gruvberger to cell apoptosis, as assayed by annexin V labeling et al., 2001; Zhang et al., 2005). (unpublished data). The absence of NFAT3 resulted in In this report, we unravel a new regulatory circuit, dramatic actin redistribution and condensation at the wherein NFAT3 is an anti-motility factor that acts by cell periphery, with formation of membrane ruffles and blocking LCN2 expression in ERA þ breast cancer. lamella structures (Figure 1B, lower panels b and c) typical of migrating cells. Indeed, both the invasive and migratory capacities of the NFAT3-depleted cells were increased relative to cells transfected with control Results siRNA (Figure 1B, upper right panel). As a control, ectopic expression of the constitutive active N-terminal NFAT genes are differentially expressed in ERA þ deletion mutant of NFAT3, DNFAT3 (Molkentin et al., and ERA breast cancer cells À 1998), not targeted by the siRNA for NFAT3, was We used six distinct breast cancer cell lines to evaluate sufficient to prevent increased invasion elicited by the distribution of NFAT genes relative to ERA depletion of endogenous NFAT3 (Supplementary expression (ERA : MCF-7, T-47D, ZR-75-1, BT-474; þ Figure S3). We, therefore, tested whether higher NFAT3 ERAÀ: MDA-MB-231, MDA-MB-453) and a melano- level in ERA þ cells could impact invasion and ma cell line MDA-MB-435 described earlier as a breast migration. Indeed, when NFAT3 and DNFAT3 were cancer cell line (Ellison et al., 2002). Using primers that transiently transfected in ERA þ breast cancer cells, specifically amplified the different NFAT splice iso- the levels of ectopic NFAT3 was clearly upregulated forms, we found that RNA of every gene—but compared with the endogenous NFAT3 level (Supple- NFAT3—was similarly detected independently of the mentary Figure S4) and this resulted in decreased cells’ ERA status (Figure 1A, left panel). Remarkably, invasion and migration (Figure 1C). NFAT3 expression at the RNA as well as at the These data indicate that, in contrast to NFAT1 and protein level was restricted to ERA cells (Figure 1A, þ NFAT5 (Jauliac et al., 2002), NFAT3 is an anti-invasive right panel), whereas neither NFAT1 nor NFAT2 and anti-migratory factor for ERA þ cells. proteins were detected in ERA þ cells (Supplementary Figures S1 and S2); NFAT1 protein was present only in ERAÀ cells, NFAT2 protein was not detected in the NFAT3 cooperates with ERA to block migration ERAÀ and ERA þ cells, and NFAT5 and NFAT4 We then evaluated whether NFAT3 interacted with proteins were found in both types of cells (Supple- ERA to elicit its anti-invasive and anti-migratory effects. mentary Figure S1). We first attempted to downregulate ERA expression with siRNA in ERA þ cells. Unfortunately, the cells NFAT3 regulates actin organization and inhibits invasion died in the absence of ERA (data not shown). To and migration circumvent this difficulty, we used ERAÀ cells in which To evaluate whether NFAT3 protein had an impact on ERA could be re-introduced or not. In this model, cell morphology, ERA þ cancer cells were treated with NFAT3 clearly inhibited invasion in the absence of two different siRNA, which reduced NFAT3 expression ERA (Figure 2a, left panel), but it had no effect on by 90% without affecting that of NFAT5 or of NFAT4 ERAÀ cells’ migration (Figure 2a, right panel). How-

Figure 1 NFAT3 RNA and protein expression is specifically restricted to ERA þ breast carcinoma cells and modulates actin organization and cell motility. (A, left panel) Total RNA was extracted by the Trizol reagent from ERAÀ (MDA-MB-231) and ERA þ (T-47D, ZR-75-1, MCF-7) breast cancer cells and the ERAÀ MDA-B-435 melanona cell line. cDNA synthesis was prepared using the superscript reverse transcriptase. For each NFAT isotype, PCR analysis was performed on the same cDNA sample with gene-specific primers. Primers were designed to identify all splice variants of the NFAT gene and arrows indicate the positions of the different splice products (see Supplementary Information). (A, right panel) ERAÀ (MDA-MB-231, MDA-MB-453), MDA-MB-435 and ERA þ (T-47D, ZR-75-1) cells were lysed in RIPA buffer (50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF, 1 mM NaF, 1 mM Na3VO4) supplemented with proteases inhibitors. The lysates were resolved by SDS–PAGE and immunoblotted with anti-NFAT3 and anti-actin; (*) indicates a non-specific band. (b, left panel) Western blot for NFAT3, NFAT4, NFAT5 and actin of total lysates of T-47D cells 48 h post-transfection with siRNA directed against endogenous NFAT3 (15 nM). (B, right panel) T-47D cells transiently transfected with control siRNA or siRNA directed against endogenous NFAT3 (siRNA NFAT3) with Dharmafect-1 for 48 h as indicated by the manufacturer; 48 h post-transfection, cells were tested for their ability to invade and migrate. Invasion and migration assays were performed essentially as described (Jauliac et al., 2002) using insert with 8-mm membrane chambers coated with Matrigel for invasion, but not for migration. (B, lower panel) T47-D cells were grown on coverslips in 12-well plates and fixed with 4% paraformaldehyde in phosphate-buffered saline and permeabilized with 0.1% Triton X-100 48 h post-transfection with siRNA directed against endogenous NFAT3 (siRNA1 or siRNA2). The actin cytoskeleton was stained with rhodamine phalloidin. Images were acquired by confocal microscopy on a Zeiss LSM 510 META confocal laser microscope (Zeiss) with a Plan Apochromat 63X N.A.1.4 objective using the LSM 510 software (Zeiss). Arrows point to membrane ruffles and lamella structures (B, C). Bar scale, 10 mm. (C) T-47D cells transiently transfected with NFAT3 or DNFAT3 vectors, both tagged with the T7 epitope, or a control vector (vector), were tested for the ability to invade and migrate. Immunoblotting with an anti-T7 (NFAT3 or DNFAT3) antibody is shown. In all cases, the ratio of invasion and migration is presented as fold invasion or migration relative to the empty control vector or control siRNA. All results are representative of three independent experiments (bars, s.e.; *Po0.05; **Po0.01).

Oncogene NFAT3/LCN2 axis inhibits breast carcinoma motility M Fouge`re et al 2295 INVASION MIGRATION 1.4 1.2 - ERA ERA-DBD* ERA- A/B 1.4 1.0 1.2 ERA- 0.8 1.0 0.8 Ratio 0.6 *

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Oncogene NFAT3/LCN2 axis inhibits breast carcinoma motility M Fouge`re et al 2296 ever, when ERA was expressed together with NFAT3, mentary Figure S5), and treatment of the cells with this resulted in the inhibition of migration (Figure 2a, siRNA-targeted NFAT3 completely blocked endogen- right panel, ERA). Neither a DNA-binding defective ous NFAT3 expression in both compartments mutant of ERA (ERA-DBD*) nor a mutated form of (Figure 3a, middle panel). To confirm this transcrip- ERA lacking the A/B domain (ERA-DA/B), required tional link, we repeated the same experiment as in for the hormone-independent action of ERA (Dutertre Figure 3a, left panel, in the presence of a transcriptional and Smith, 2003; Fowler et al., 2004), were able to inhibitor (actinomycin D), and now increase of LCN2 cooperate with NFAT3 (Figure 2a, right panel), mRNA by NFAT3 depletion was completely abolished suggesting that NFAT3 inhibition of migration in the (Figure 3a, right panel). As we found a transcriptional presence of ERA is hormone independent and relies on link between LCN2 mRNA expression and NFAT3, we active gene transcription. Indeed, when migration and look at the LCN2 promoter and identify six potential invasion of ERA þ cells were assessed in the presence of NFAT-binding sites (Supplementary Figure S6). We estradiol (E2) (Figure 2b) or of an anti-estrogen (ICI) realized ChIP experiments to evaluate whether NFAT3 (Figure 2c) in media without red phenol toward could interact directly with the LCN2 promoter and did NIH3T3 medium produced in stripped serum in media not find any direct binding on the LCN2 promoter, without red phenol, NFAT3 still blocked migration and suggesting that these NFAT-binding sites are not invasion. All together, these results indicate that the NFAT3-binding sites in vivo (data not shown). We, cooperation of ERA with NFAT3 to inhibit the therefore, made the hypothesis that NFAT3 might migration is hormone independent. regulate indirectly, by an unknown factor, the expres- These results highlight two distinct pathways to sion of the LCN2 mRNA. We cloned the LCN2 gene regulate breast cancer cell motility: (i) migration is promoter and fused it to the Luciferase gene. We found regulated by NFAT3 in cooperation with ERA, but not that NFAT3 downregulation increased LCN2 promoter by ERA alone and (ii) invasion is regulated by NFAT3 activity by threefold relative to cells transfected by alone indicating that genes targeted by ERA might not control siRNA (Figure 3b, left panel). Conversely, be key genes to regulate invasion. expression of DNFAT3 in ERA þ and ERAÀ cells (Figure 3b, middle and right panel) was sufficient to repress LCN2 promoter activity independently of ERA NFAT3 inhibits the expression of LCN2 expression (Figure 3b, right panel). One other possibility We realize gene chip experiments with NFAT3 siRNA for ERA to cooperate with NFAT3 would be that in ERA þ T47D breast cancer cells to find potential NFAT3 regulates ERA transcriptional activity and, NFAT3 target genes. We found several genes that are therefore, participates in the regulation of some ERA’s regulated by NFAT3 downregulation by more than target genes to modulate LCN2 protein stability. We twofold (95% confidence level). Of these genes, LCN2,a excluded this hypothesis, as NFAT3 was not able to gene associated with breast cancer metastasis, was modulate ERA activity measured on an ERE element upregulated two- to fourfold in the absence of NFAT3 fused to the Luciferase gene in our model (Supplemen- (data not shown). LCN2 has been implicated in the tary Figure S7). Therefore, we favor the hypothesis that formation of metastases in murine breast cancer models ERA targets independently of NFAT3-specific ERA’s (Shi et al., 2008; Leng et al., 2009; Yang et al., 2009) and target genes, yet to be identified, to cooperate with the it has been identified as one of the genes downregulated downregulation of LCN2 expression to inhibit migra- in ERA þ breast cancers (Stoesz et al., 1998). To tion. To confirm the expression of LCN2 at the protein validate the results from our gene chip analysis, we level, we performed immunoblotting analysis on cell performed quantitative real-time PCR (Q-PCR) on total lysates of ERA þ and ERAÀ cells. Western blotting RNA from the ERA þ T-47D breast cancer cells. As confirmed that LCN2 protein was expressed at higher shown in Figure 3a, left panel, in the absence of NFAT3 levels in ERAÀ breast cancer cells and was below the (siRNA NFAT3), cells display a threefold increased antibody sensitivity in ERA þ (Figure 3c, left panel). To expression of LCN2 mRNA compared with control cells further characterize the LCN2 protein expression in (control siRNA). These results suggest that NFAT3 breast cancer cells, we performed, on the conditioned might regulate LCN2 mRNA transcription. When we mediums of ERA þ and ERAÀ cells, an LCN2 ELISA look at the cellular distribution of endogenous NFAT3, assay more sensitive than the western blot. Indeed, the we found that it localized both in the cytosol and results indicate that ERAÀ breast cancer cells express nucleus, as for ectopic NFAT3 and DNFAT3 (Supple- fourfold more of the level of LCN2 protein found in

Figure 2 NFAT3 alone inhibits invasion and cooperates with ERA to block migration. (a) MDA-MB-231 cells were transiently transfected with NFAT3, DNFAT3, ERA (67kD), ERA-DA/B (46kD), ERA-DBD*(67kD) or control (vector) vectors alone or in combination; 24 h after transfection, cells were harvested, resuspended in serum-free media containing 0.1% BSA, added in triplicates to the inserts and allowed to invade or migrate toward NIH3T3-conditioned media. Immunoblottings with anti-T7 (NFAT3 or DNFAT3) and anti-ERA antibodies are shown. (b, c) T-47D cells were transiently transfected with NFAT3, DNFAT3 or control vectors (vector) and cultured with stripped serum in media without red phenol for 24 h; 24 h later, cells were harvested, resuspended in serum-free media without red phenol containing 0.1% BSA with E2 at 10 nM or ICI at 100 nM or respective vehicule (Ethanol, DMSO), added in triplicates to the inserts, and allowed to invade or migrate toward NIH3T3-conditionned media produced in media without red phenol. Immunoblottings with the anti-T7 (NFAT3 or DNFAT3) antibody are shown. All results are representative of three independent experiments (bars, s.e.; *Po0.05; **Po0.01).

Oncogene NFAT3/LCN2 axis inhibits breast carcinoma motility M Fouge`re et al 2297 ERA þ cells (Figure 3c, middle panel). To ensure the included in the assay (Figure 3c, middle panel). These LCN2 ELISA specificity, conditioned mediums of the results confirmed that LCN2 protein was expressed in cells treated with siRNA directed against LCN2 were both ERA þ and ERAÀ cells. Moreover, as for the

t=0 t=6h LCN2 mRNA * Actinomycin D -- + 3.0 * ERA+ 2.0 2.0 control siRNA * siRNA NFAT3 * 1.5 1.0 kD Arbitrary Units CCNN

0.0 130 control siRNA 1.0 kD siRNA NFAT3 110 Arbitrary Units 130 WB: α-NFAT3 0.5 WB: α-NFAT3 45 0.0 WB: α-actin control siRNA control siRNA control siRNA siRNA NFAT3 siRNA NFAT3 siRNA NFAT3

LCN2 promoter LCN2 LCN2 4.0 promoter promoter 1.2 1.2 3.5 * * 1 1 3.0 0.8 2.5 0.8 * * 0.6 ERA- 2.0 0.6 ERA+ * 1.5 0.4 0.4 1.0 0.2

0.2 Relative Luciferase units Relative Luciferase units 0.5 0.0 Relative Luciferase units vector ΔNFAT3 ΔNFAT3 0.0 0.0 ERα control siRNA vector ΔNFAT3 kD kD siRNA NFAT3 kD 70 130 70 WB: α-T7 α 70 WB: α-NFAT3 WB: -T7 WB: α-ERA 45 45 45 α WB: α-actin WB: -actin WB: α-actin

LCN2 protein LCN2 protein 2.5 7 * * * * 6 2

5 1.5 kD ERA+ ERA- 4 ERA+ 25 ng/ml 1 * ng/ml 3 WB: α-LCN2 2 0.5 45 WB: α-actin 1 0 control siRNA 0 siRNA NFAT3 kD control siRNA control siRNA 130 siRNA LCN2 siRNA LCN2 WB: α-NFAT3 ERA+ ERA- 45 WB: α-actin Figure 3 For caption please see page 2298.

Oncogene NFAT3/LCN2 axis inhibits breast carcinoma motility M Fouge`re et al 2298 LCN2 mRNA, NFAT3 downregulation by siRNA was 2008) (Supplementary Figure S9). Indeed, in the sufficient to increase LCN2 protein level in ERA þ cells presence of LCN2, NFAT3 was still able to inhibit (Figure 3c, right panel). invasion for ERA þ and ERAÀ cells (Figures 4c and d, We next examined a panel of patients’ gene expression left panels), but its effect on migration was then data that compared ERA þ and ERAÀ breast cancers completely reversed for both cell types (Figures 4c and (Bertheau et al., 2007) and confirmed that NFAT3 was d, right panels). LCN2 did not reverse the inhibition of mainly highly expressed among ERA þ patients, whereas migration elicited by a dominant negative form of Rac1 LCN2wasratherupregulatedinERAÀ patients and (Supplementary Figure S10), suggesting that down- downregulated in ERA þ patients (Figure 4a). regulation of LCN2 is specifically required for NFAT3 All together, these results show that the LCN2 gene is to block migration. indirectly repressed by NFAT3 in ERA þ breast cancer Altogether, these results show that LCN2 down- cells and is more expressed both in breast cancer cell lines regulation occurs downstream of NFAT3 to achieve the and breast cancer patients lacking NFAT3 and ERA. inhibition of migration.

LCN2/NFAT3 axis regulates migration and invasion As LCN2 expression can be inhibited by NFAT3, we Discussion then examined whether LCN2 contributed to NFAT3- dependent regulation of the invasive and migratory Metastasis formation is a complex process that requires capacities of breast cancer cells. Reducing endogenous cancer cell migration and invasion, the former being a LCN2 was sufficient to decrease invasion in both later event necessary, but not sufficient, for invasion to ERA þ and ERAÀ cells (Figure 4b, ERA þ and occur. Here, we show that NFAT3 is specifically ERAÀ) without any effect on apoptosis (Supplementary expressed in ERA þ cells and inhibits both these cells’ Figure S8). A recent study indicates that reducing migration and invasion capacities. Downstream endogenous LCN2 by siRNA induces an increase of NFAT3, we further identify a key target gene, LCN2, ERA expression (Yang et al., 2009); we did not see such transcriptionaly repressed by NFAT3. Recently, LCN2 effects on ERA expression in ERA þ T-47D cells has been implicated in the pro-invasive and pro- treated with the siRNA of LCN2 (Supplementary migratory capacities of breast carcinoma (Shi et al., Figure S8). In contrast to invasion, lack of endogenous 2008; Yang et al., 2009). In our hands, blocking LCN2 LCN2 was not sufficient to reduce the migratory expression was indeed sufficient to recapitulate the capacity of ERAÀ cells (Figure 4b, ERAÀ, right panel), effects of NFAT3 on both these capacities. Conversely, although it was sufficient in this respect as regards recombinant LCN2 was sufficient to rescue the inhibi- ERA þ cells (Figure 4b, ERA þ , left panel). Thus, tion of migration elicited by NFAT3. These data decreasing endogenous LCN2 expression parallels, in unravel that the migratory capacity of ERA þ cells is both ERA þ and ERAÀ cells, the effect of NFAT3 on mediated by a critical NFAT3/LCN2 axis. Adding back migration and invasion. recombinant LCN2 on ERA þ cells was not able to To assess whether LCN2 downregulation was re- recapitulate the effect of downregulation of NFAT3 on quired for NFAT3 to reduce the migration and invasion actin reorganization (data not shown), suggesting that capacities of breast cancer cells, we examined whether LCN2 might target other migration-regulating mechan- adding recombinant human LCN2 to the medium isms. Indeed, one study has shown that LCN2 is able to would be sufficient to reverse such NFAT3 effects. We activate the ERK pathway (Gwira et al., 2005) and verify that effectively LCN2 entered the cells and was ERK seems to be an important actor of breast cancer active by testing known targets of LCN2 (Zhang et al., cell migration (Krueger et al., 2001; Irie et al., 2005).

Figure 3 LCN2 gene expression is inhibited by NFAT3. (a, left panel) Quantification of LCN2 mRNA in ERA þ T-47D cells transiently transfected with control siRNA or siRNA directed against endogenous NFAT3. Quantitative real-time PCR (Q-PCR) was performed using the SYBR Green PCR Master Mix on a Roche LightCycler according to the manufacturer’s instruction. Immunoblottings with an anti-NFAT3 or an anti-actin antibody are shown. (a, middle panel) Immunobloting with an anti-NFAT3 of cytosolic (C) and nuclear extracts (N) of control siRNA or siRNA NFAT3 transfected T-47D cells with the NE-PER kit as directed by the manufacturer, different phosphorylation states of NFAT3 are present between the brackets in the control siRNA treated cells. (a, right panel) Quantification of LCN2 mRNA in ERA þ T-47D cells transiently transfected with control siRNA or siRNA directed against endogenous NFAT3; 36 h post-transfection (t ¼ 0), vehicule (À) or actinomycin D at 2 mg/ml ( þ ) were added to the cells for 6 h. After the 6 h of treatment, total RNA was isolated and Q-PCR was performed as described earlier. (b) Cells were cotransfected with the pCS4-(n)-b-gal plasmid and the LCN2 promoter plasmid and cotransfected with DNFAT3 or siRNA targeting endogenous NFAT3 (T-47D cells (ERA þ ); left and middle panel) or DNFAT3/ERA (MDA-MB-231 cells (ERAÀ); right panel) or control vector using Exgen 500; 48 h later, cells were analyzed for Luciferase and b-gal activities using the Luciferase assay and Galacton-plus system, and measured in a luminometer. Quantification of LCN2 promoter Luciferase activity normalized against that of b-galactosidase activity is shown. Immunoblottings with anti-T7 (DNFAT3), anti-ERA or anti-actin antibodies are shown. (c, left panel) Immunoblotting with anti-LCN2 or anti-actin antibodies in T-47D (ERA þ ) and MDA-MB-231 (ERAÀ) total cell lysates are shown; (*) indicate a non-specific band. (c, middle panel) An LCN2 ELISA was performed, as directed by the manufacturer, on 50 ml of conditioned mediums for ERA þ and ERAÀ cells either transiently transfected with a control siRNA or an siRNA-targeted endogenous LCN2 for 48 h. (c, right panel) An LCN2 ELISA was performed on 50 ml of conditioned mediums for ER þ cells either transiently transfected with a control siRNA or an siRNA-targeted endogenous NFAT3 for 48 h. All results are representative of three independent experiments (bars, s.e.; *Po0.05; **Po0.01).

Oncogene NFAT3/LCN2 axis inhibits breast carcinoma motility M Fouge`re et al 2299

257812310 6780 78 71 47 69 82 5118 17 74 4 19 72 33 61 30 76 68 24 21 60 42 4615 34 49 27 38 39 22 25 patients ERA LCN2 NFAT3

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212531_at LCN2 3934 204 98 -106 0.0098 213345_at NFAT3 4776 149 220 71 0.00003

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ERA- 1.4 - LCN2 1.4 - LCN2 1.2 1.2 kD Vector 1.0 1.0 NFAT3/ERA

NFAT3 130 Δ Vector 0.8 kD 0.8 * α 70 * Ratio WB: -T7 Ratio 0.6 * * 0.6 WB: α-T7 70 0.4 0.4 45 WB: α-ERA 0.2 WB: α-actin 0.2 45 0.0 0.0 vector NFAT3 vector NFAT3 Δ Δ WB: α-actin vector NFAT3 vector NFAT3 ERA ERA Figure 4 LCN2 gene expression is associated with an ERAÀ and NFAT3-negative status in breast cancer patients and downregulation of LCN2 gene expression by NFAT3 is a required limiting effect to inhibit migration and is dispensable to blunt invasion. (a) Hierarchical clustering based on LCN2 and NFAT3 genes in 37 tumors analyzed with Affymetrix-grade RNA. Annotations: ERA (immunohistochemistry); p, positive; n, negative; 2–25 refers to the earlier reported patients’ references (Bertheau et al., 2007). (b) Endogenous LCN2 was transiently knocked down in T-47D (ERA þ ) or MDA-MB-231 (ERAÀ) cells by transfecting siRNA targeting LCN2 or control siRNA for 48 h, and tested for the ability to invade and migrate. (c) T-47D (ERA þ )or(d) MDA- MB-231 (ERAÀ) cells were transiently transfected with NFAT3, DNFAT3, ERA or control (vector) vectors for 24 h, and tested for the ability to migrate and invade in the presence of 15 mM recombinant human LCN2 (LCN2) or its vehicle (PBS). Immunoblottings with anti-T7 (NFAT3 or DNFAT3), anti-ERA or anti-actin antibodies are shown. All results are representative of three independent experiments. (bars, s.e.; *Po0.05; **P o0.01).

However, further studies will be required to investigate Mechanistically, we show that NFAT3 has a specific the potential function of the ERK pathway downstream anti-invasive capacity in breast cancer cells and needs to LCN2 to regulate migration in breast cancer cells. cooperate with ERA to achieve inhibition of migration.

Oncogene NFAT3/LCN2 axis inhibits breast carcinoma motility M Fouge`re et al 2300 However, we could not confirm (data not shown) the Conflict of interest reports indicating that NFAT3 can associate with ERA and did not see any effects of NFAT3 on ERA The authors declare no conflict of interest. transcriptional activity (Zhang et al., 2005; Qin et al., 2008). Interestingly, although both ERA þ cell migra- tion and invasion were inhibited by LCN2 down- Acknowledgements regulation, the later was not enough to inhibit migration in ERAÀ cells, suggesting that cooperation We thank H de The´, D Auboeuf and members of their of other critical genes was then required. The data from laboratories for insightful discussions. We thank E Turpin, P Bertheau for providing their microarray data. We thank J L our study suggest that these genes that cooperate with Poyet for providing the T7-pcDNA3 vector and G lazennec for the downregulation of LCN2 by NFAT3 are certainly the ERa expression vector. We thank T Hoey for providing the genes targeted by ERA. In contrast, for invasion, rescue human NFAT3 expression vector. We thank J-C Gluckman by recombinant LCN2 was not able to prevent for reading the paper. We thank N Setterblad at the Service inhibition of invasion elicited by NFAT3, suggesting Commun d’Imagerie Cellulaire et Mole´culaire of the Institut that NFAT3 might target other key genes in addition Universitaire d’He´matologie IFR105 for confocal microscopy. to LCN2 to modulate the invasion. We could make the The Service Commun d’Imagerie Cellulaire et Mole´culaire is hypothesis that these LCN2-independent mechanisms supported by grants from the Conseil Re´gional d’Ile-de-France mediated by NFAT3 to inhibit the invasion relay on the and the Ministe` re de la Recherche. M Fouge` re was supported regulation of specific invasive genes similar to the matrix by a doctoral grant from INSERM Re´gion Ile-de-France and an ARC fellowship. B Gaudineau was supported by a grant remodeling proteases (Supplementary Figure S11). from the Cance´ropole Ile-de-France and J Barbier by a grant Our findings further highlight the versatility of the from the Research Ministry. This work was supported by the NFAT transcription factors family in modulating the INSERM AVENIR Program and grants from Ligue Natio- behavior of cancer cells and imply that perturbing this nale contre le Cancer, the Comite´de Paris of Ligue Nationale novel ERA/NFAT3/LCN2 axis could be a strategy to contre le Cancer, ARC, and the Comite´Tumeurs de la limit the aggressiveness of ERA þ breast cancer cells. Fondation de France and the Cance´ropole Ile-de-France.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene