(2010) 29, 2983–2995 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www..com/onc ORIGINAL ARTICLE FOXM1 is a transcriptional target of ERa and has a critical role in breast cancer endocrine sensitivity and resistance

J Millour1, D Constantinidou1, AV Stavropoulou1, MSC Wilson1, SS Myatt1, JM-M Kwok1, K Sivanandan1, RC Coombes1, RH Medema2, J Hartman3, AE Lykkesfeldt4 and EW-F Lam1

1Department of Surgery and Cancer, Imperial College London, London, UK; 2Department of Medical Oncology, University Medical Center Utrecht, Utrecht, The Netherlands; 3Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden and 4Department of Breast Cancer Research, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark

In this study, we investigated the regulation of FOXM1 Introduction expression by estrogen a (ERa) and its role in hormonal therapy and endocrine resistance. FOXM1 Breast cancer is the second most prevalent cause of and mRNA expression was regulated by ER- cancer death in the western hemisphere and displays a ligands, including estrogen, tamoxifen (OHT) and fulves- complex aeitology. The forkhead box (FOX) family trant (ICI182780; ICI) in breast carcinoma cell lines. member FOXM1 was previously reported to be elevated Depletion of ERa by RNA interference (RNAi) in MCF-7 in breast cancer as well as in carcinomas of other origins cells downregulated FOXM1 expression. Reporter (Pilarsky et al., 2004). FOXM1 is expressed in prolifer- assays showed that ERa activates FOXM1 ating adult tissues and in response to injury or repair through an estrogen-response element (ERE) located (Korver et al., 1997a, b; Ye et al., 1999; Leung et al., within the proximal promoter region. The direct binding 2001; Wang et al., 2002). FOXM1 has a critical role in of ERa to the FOXM1 promoter was confirmed in vitro by progression; expression of FOXM1 increases mobility shift and DNA pull-down assays and in vivo at G1 to S phase transition, and reaches maximal levels by chromatin immunoprecipitation (ChIP) analysis. Our in G2 to M phase, promoting M phase entry (Leung data also revealed that upon OHT treatment ERa recruits et al., 2001; Laoukili et al., 2005). FOXM1 controls the histone deacetylases to the ERE site of the FOXM1 expression of cell cycle regulatory , such as promoter, which is associated with a decrease in histone cyclin B1 and cyclin D1 (Leung et al., 2001; Wang et al., acetylation and transcription activity. Importantly, silen- 2001; Laoukili et al., 2005), in addition to essential cing of FOXM1 by RNAi abolished estrogen-induced for faithful segregation and , such MCF-7 cell proliferation and overcame acquired tamox- as Nek-2, KIF20A, Cdc25B, Aurora B kinase, Survivin, ifen resistance. Conversely, ectopic expression of FOXM1 centromere protein A (CENPA) and CENPB (Wang abrogated the cell cycle arrest mediated by the anti- et al., 2005). Thus, knockdown of FOXM1 with siRNA estrogen OHT. OHT repressed FOXM1 expression in results in delay in G2 and mitotic spindle defects, and endocrine sensitive but not resistant breast carcinoma cell induces mitotic catastrophe. lines. Furthermore, qRT–PCR analysis of breast cancer Previous work has shown that FOXM1 regulates ERa patient samples revealed that there was a strong and expression in breast cancer cell lines (Madureira et al., significant positive correlation between ERa and FOXM1 2006). Consistently, expression of the FOXM1 target mRNA expression. Collectively, these results show cyclin D1 is associated with ERa positivity in breast FOXM1 to be a key mediator of the mitogenic functions cancer (Butt et al., 2005). The strong correlation of ERa and estrogen in breast cancer cells, and also between the expression patterns of ERa and FOXM1 suggest that the deregulation of FOXM1 may contribute in breast cancer cells also suggested the possibility that to anti-estrogen insensitivity. FOXM1 could be an ERa-regulated gene (Madureira Oncogene (2010) 29, 2983–2995; doi:10.1038/onc.2010.47; et al., 2006). Consistent with this hypothesis, a cDNA published online 8 March 2010 microarray also identified FOXM1 as one of 344 estrogen-responsive genes in breast cancer cells (Cica- Keywords: breast cancer; forkhead; ; tiello et al., 2004). Estrogen is generally considered to be FOXM1; endocrine resistance; transcription mitogenic (Brunner et al., 1989), and exerts its action in the mammary epithelial cells primarily via ERa (Ali and Coombes, 2002). The expression of ERa is a good prognostic factor in breast cancer, as about two-thirds Correspondence: Professor EW-F Lam, CR-UK labs and Department of ERa-positive patients respond to treatment with anti- of Surgery and Cancer, Imperial College London, Hammersmith estrogens such as OHT, ICI or aromatase inhibitors Campus, Du Cane Road, London W12 0NN, UK. E-mail: [email protected] (Osborne and McGuire, 1979; Gapinski and Donegan, Received 6 July 2009; revised 22 January 2010; accepted 29 January 1980; Elkak and Mokbel, 2001). Treatment of breast 2010; published online 8 March 2010 cancer cells with anti-estrogens results in cell cycle arrest ERa regulates FOXM1 expression in breast cancer J Millour et al 2984 at the G1/S phase and in some cases, cell death (WT-Trident), or its truncation mutants HindIII (Lykkesfeldt et al., 1986). However, approximately half (1.4 kb), and ApaI (300 bp) (Figure 2a). We observed a of the patients who initially respond to hormonal twofold increase of the WT-Trident FOXM1-luc activity therapy develop resistance and relapse, following long- after E2 treatment, and this induction by E2 was term treatment (Ali and Coombes, 2002; Goss et al., repressed in the presence of OHT (Figure 2a). The 2008; Yamashita, 2008). Our previous observation that highest increase (2.7-fold) by E2 was observed for the ERa-positive breast cancer cell lines express higher ApaI-luc activity (Figure 2a). The HindIII-luc activity levels of FOXM1 protein (Madureira et al., 2006) led us was upregulated to a lesser extent (1.5-fold) by E2 to hypothesize that FOXM1 expression is also regulated (Figure 2a). The response of the FOXM1 promoter to by ERa. In an effort to elucidate the mechanisms of ERa was further verified by co-transfection of COS-1 anti-estrogen action and resistance to endocrine therapy, cells with titrated amounts of pHEG0 and the WT- we investigated the regulation of FOXM1 by ERa and Trident-luc or ApaI-luc reporter plasmids (Figure 2a). its role in endocrine sensitivity and resistance in breast Both the WT-Trident and ApaI promoters were induced cancer cells. in a dose-dependent manner by increasing amounts of ERa in the presence of E2 (Figure 2a). It is notable that the control pS2 promoter showed maximum E2- stimulation with very low levels of ERa expression, Results supporting the notion that pS2 may be one of the most E2-sensitive genes (Masiakowski et al., 1982). Collec- ERa ligands modulate the expression of FOXM1 protein tively, these results indicate that the ApaI truncation is and mRNA levels the minimal proximal FOXM1 promoter responsive to To investigate whether the FOXM1 protein and mRNA ERa, consistent with a previous study identifying a levels are regulated by ERa, the ERa-positive breast similar region of the FOXM1 promoter that responds to cancer cell lines ZR-75-1 and MCF-7 and ERa-negative serum stimulation (Korver et al., 1997a). line MDA-MB-231 were treated with the ERa ligands E2, OHT and ICI for the times indicated and harvested for western blotting and qRT–PCR analysis. Treatment The ERE-like element at À45 bp of the FOXM1 promoter of estrogen-starved ZR-75-1 and MCF-7 cells with E2 confers responsiveness to ERa ligands enhanced the expression of FOXM1 protein and mRNA Analysis using the Transcription Element Search System (Figure 1). Within 8 h of E2 treatment, FOXM1 protein (TESS,http://www.cbil. upenn.edu/cgi-bin/tess/tess) and mRNA levels peaked and remained high for at least (Schug, 2008) revealed an ERE-like element (Bourdeau 48 h. By contrast, E2 treatment did not cause significant et al., 2004) located at À45 bp from the transcription changes in either FOXM1 protein or mRNA levels in start site. We next focused our studies on the character- the ERa-negative control MDA–MB-231 cells. Treat- ization of the ERE-like element at À45 bp in the ApaI ment of both the ZR-75-1 and MCF-7 cells with the promoter fragment and introduced point mutations partial antagonist OHT resulted in a reduction in using site-directed mutagenesis by base-substitution in FOXM1 protein levels that was noticeable within 4 h, both arms of the ERE-like palindrome of the ApaI reaching minimal levels within 24 h post-treatment. Yet truncation (Bourdeau et al., 2004) (Figure 2b). For again, treatment with OHT had little effect on the comparison, a single point mutation that does not expression of FOXM1 protein and mRNA in MDA- grossly disrupt the ERE-consensus was also introduced MB-231 cells. Similar to OHT, incubation with the pure in one arm of the ERE-like element in mERE1 antagonist ICI, which causes the degradation of ERa, (Figure 2b). The activity of the wild-type as well as the resulted in a reduction in the FOXM1 expression, with mutated ApaI-luc constructs mERE1, mERE3 and both the protein and mRNA levels completely vanished mERE4 was examined by co-transfection assays in within 24 h post-treatment. We also studied the ability COS-1 cells with and without pHEG0, in the presence or of OHT or ICI to antagonize E2-regulated FOXM1 absence of E2 (Figure 2b). The results showed that the upregulation, and the result showed that the effects of mERE1-luc construct showed similar responsiveness to OHT or ICI following E2 stimulation are similar to ERa and E2 induction as the wild-type ApaI-luc. those under normal growth conditions (Supplementary In contrast, both the mERE3-luc and mERE4-luc Figure S1). In summary, the FOXM1 protein and mutants lost the majority of their responsiveness to E2 mRNA levels were upregulated by ERa agonists and (Figure 2b). Together these co-transfection results downregulated by ERa antagonists in ERa-positive, but indicate that the ERE-like element located at À45 bp not ERa-negative breast cancer cell lines, suggesting confers the responsiveness to ERa, further confirming that FOXM1 is regulated by ERa. that FOXM1 is a target gene of ERa.

The FOXM1 promoter responds to ERa and ligands ERa binds directly to the ERE-like element To investigate if the regulation of FOXM1 by ERa is at of the FOXM1 promoter in vitro the promoter level, ERa-negative COS-1 cells were We next tested the in vitro binding of ERa to the ERE- transiently transfected with the ERa expression con- like site by electrophoretic mobility shift assay with struct pHEGO, with a luciferase reporter gene under nuclear lysate from MCF-7 cells. From the electro- the control of the proximal 2.4 kb FOXM1 promoter phoretic mobility shift assay, it was clear that ERa binds

Oncogene ERa regulates FOXM1 expression in breast cancer J Millour et al 2985 ZR-75-1: E2 OHT ICI 0 4 8 24 48 0 4 8 24 48 04 8 24 48 (h)

FOXM1

ERα

Tubulin

1.0 0.8 0.6 0.4 0.2

FOXM1/L19 RNA 0

MCF-7: 0 4 8 24 48 0 4 8 24 48 04 8 24 48 (h)

FOXM1

ERα

Tubulin

0.25 0.20 0.15 0.10 0.05 FOXM1/L19 RNA 0

MDA-MB-231: 0 4 8 24 48 0 4 8 24 48 04 8 24 48 (h)

FOXM1

ERα

Tubulin

1.4 1.2 1.0 0.8 0.6 0.4 0.2 FOXM1/L19 RNA 0.0 Figure 1 Expression of FOXM1 and ERa in response to E2, OHT and ICI treatments in breast cancer cell lines. ZR-75-1, MCF-7 and MDA-MB-231 cells were cultured in 5% double-charcoal striped FCS and phenol red-free medium for 24 h before stimulated with E2. Breast cancer cells cultured in 10% FCS and phenol red medium were also treated with OHT or ICI. At times indicated, cells were collected and analysed for FOXM1, ERa and tubulin expression by western blotting. FOXM1 mRNA levels of these cells were also analysed by qRT–PCR, and normalized with L19 RNA expression. Total RNA (2 mg) isolated using the RNeasy Mini kit (Qiagen, Crawley, UK) was reverse transcribed using the Superscript III reverse transcriptase and random primers (Invitrogen, Paisley, UK), and the resulting first strand cDNA was used as template in the real-time PCR. All experiments were performed in triplicate. The following gene-specific primer pairs were designed using the ABI Primer Express software: FOXM1-sense: 50-TGCAGCTAGGGAT GTGAATCTTC-30 and FOXM1-antisense: 50-GGAGCCCAGTCCATCAGAACT-30;ERa-sense: 50-CAGATGGTCAGTGCCTTG TTGG-30 and ERa-antisense: 50-CCAAGAGCAAGTTAGGAGCAAACAG-30; L19-sense 50-GCGGAAGGGTACAGCCAAT-30 and L19-antisense 50-GCAGCCGGCGCAAA-30. Specificity of each primer was determined using NCBI BLAST module. Real time PCR was performed with ABI PRISM 7700 Sequence Detection System using SYBR Green Mastermix (Applied Biosystems, Brackley, UK). The qRT–PCR results shown are representative of three independent experiments. to the wild-type ERE-like site of ApaI, but not the that the complex contains ERa (Figure 3a). This ERa mutated mERE3 site (Figure 3a). This complex of ERa complex could be competed off by excess amounts of a and WT ERE biotin-oligonucleotide was further super- classical consensus ERE oligonucleotide, but not by a shifted with a specific anti-ERa antibody, indicating mERE3 ApaI site oligonucleotide. Finally, unlabelled

Oncogene ERa regulates FOXM1 expression in breast cancer J Millour et al 2986 FOXM1 WT ERE oligonucleotide was successful in in Figure 3b, ERa had much higher affinity for the wild- competing off the ERa binding on the consensus ERE type ERE than the mutant mERE3. ERa-binding to the oligonucleotide. To show that ERa binds to the ERE- FOXM1 ERE could be competed away by molar excess like site of ApaI we used the biotin-labelled oligonucleo- of wild-type ERE, but not the mutant mERE. We next tides in pull-down assays. ERa binding was analysed by extended our pull-down assays to MCF-7 and ZR-75-1 immunoblotting with an anti-ERa antibody. As shown cells in the absence or presence of OHT, ICI and E2

a Full-length luc Hind III luc

ApaI luc

20 18 control 16 E2 14 12 E2+OHT 10 8 6 4 2

Relative Luciferase Units 0 ERα: -++ -+-+-+-

pGL3-basicpGL3-FOXM1 pGL3-FOXM1 pGL3-FOXM1 pGL3-ERE (Trident) (Hind III) (ApaI) -pS2

50 45 control 40 E2 35 30 25 20 15 10 5 Relative LuciferaseUnits 0 ERα (ng): 0200.1 1 10 0200.1 1 10 0200.1 1 10

pGL3-FOXM1 pGL3-FOXM1 pGL3-ERE (Trident) (ApaI) -pS2

b

FOXM1(ApaI) ) 4 20 18 16 control 14 E2 12 10 8 6 4 2 0 ERα : - + - + - + - + - + - +

Relative LuciferaseUnits (x10 pGL3- pGL3- pGL3- pGL3- pGL3- pGL3- basic WT mERE1 mERE3 mERE4 ERE -PS2 -FOXM1(ApaI)

Oncogene ERa regulates FOXM1 expression in breast cancer J Millour et al 2987 treatments (Figure 3c). Western blot analysis was first FOXM1 and ERa mRNA levels in 69 frozen archival performed to establish the expression patterns of ERa in clinical samples, of which 21 were from benign disease cytoplasmic and nuclear fractions of MCF-7 and ZR-75-1 patients, 39 from patients with breast tumours and nine cells, also with or without OHT, ICI or E2 treatment from healthy individuals (Supplementary Figure S3). (Supplementary Figure S2). The results confirmed our Preliminary studies showed that despite FOXM1 previous data that both OHT and ICI inhibit ERa mRNA expression being associated with tumour status, activity, whereas ICI, but not OHT, represses ERa grade, size and menopausal status, the differences were expression. In the pull-downs, ERa binding on the not significant, possibly reflecting the relatively small biotin-WT ERE was effectively competed by 10 Â molar sample size (Supplementary Figure S4). Analysis of excess of unlabelled WT ERE, and not mERE3, unadjusted data revealed no significant correlation oligonucleotides. We also probed for the recruitment of between the expression levels of FOXM1 and ERa HDAC to the ERE site upon OHT, ICI or E2 treatment mRNA (Figure 4a). This is not entirely unexpected as in MCF-7 cells, and the results revealed that HDAC2 was high levels of FOXM1 mRNA expression (upper recruited to the ERE site upon OHT but not ICI or E2 quartile) have previously been shown to be associated treatment (Figure 3c). Taken together these results with breast cancer progression, poor prognosis and showed that ERa binds specifically to the ERE-like endocrine resistance in a number of microarray studies element of the FOXM1 promoter in vitro and that HDAC (Martin et al., 2008). We next re-evaluated the data is recruited to the ERE site upon OHT treatment. qafter excluding patient samples with high levels of FOXM1 expression. By leaving out patient samples with FOXM1 mRNA levels above the 75th percentile ERa and HDAC bind specifically on the ERE-like value (FOXM1/L19 mRNA ¼ 40.700) (Figure 4b), we element of ApaI in vivo found a significant correlation between FOXM1 and We further tested the in vivo binding of ERa on the ERa mRNA expression (Figure 4c). In cases where FOXM1 promoter in the MCF-7 and ZR-75-1 cell lines there were high levels of FOXM1 mRNA expression, we by chromatin immunoprecipitation assays (ChIP), also found that FOXM1 expression is independent of following treatment of the cells with either OHT or ERa mRNA levels, suggesting FOXM1 is no longer ICI (Figure 3d). The DNA immunoprecipitated by an regulated by ERa (Figure 4d). These results validate our anti-ERa antibody was amplified using the FOXM1 tissue culture findings in breast patient samples, ERE primers to show in vivo binding of ERa on the confirming the crucial relationship between ERa and ERE-like site of FOXM1 promoter at À45 bp, in FOXM1 expression. In breast cancer samples with high agreement with the in vitro results. Occupancy of FOXM1 expression, the relationship between ERa and FOXM1 promoter by ERa was enhanced in ZR-75-1, FOXM1 mRNA expression is uncoupled, suggesting but not MCF-7 cells following treatment with OHT, and ERa no longer controls FOXM1 expression. This also a reduction in ERa binding was observed in MCF-7 and supports the conjecture that uncoupling of the regu- ZR-75-1 cells following treatment with ICI. The ChIP lation of FOXM1 by ERa may have a role in endo- assays also showed that upon OHT treatment there was crine resistance. an increase in HDAC1 and HDAC2 recruitment and a corresponding decrease in acetylated histones H3 and H4 associated with the FOXM1 promoter, indicating ERa silencing downregulates FOXM1 expression whereas that OHT treatment caused the recruitment of HDAC, FOXM1 knock-down inhibits estrogen-induced cell which confers transcriptional repression to the ERE proliferation region of the FOXM1 promoter (Figure 3e). To provide further evidence for the regulation of FOXM1 by ERa, we investigated the effect of ERa Significant positive correlation between FOXM1 and ERa silencing on FOXM1 expression in proliferating MCF-7 mRNA levels in breast cancer patient samples cells. As shown in Figure 5a, the ERa siRNA effectively To determine the clinical relevance of the relationship knocked down the expression of ERa, which in turn also between ERa and FOXM1 expression, we analysed the resulted in a downregulation of FOXM1 at the protein

Figure 2 ERa induces the transcriptional activity of the human FOXM1 gene through an ERE consensus proximal to the transcription start site. (a) Effect of treatment with E2 and expression of ER on FOXM1 promoter activity. Schematic representation of the full-length, HindIII and ApaI FOXM1-luciferase reporter constructs. In upper panel, COS-1 cells cultured in 5% double- charcoal striped FCS and phenol red-free medium were transiently transfected with 20 ng of either the empty pGL3-basic, pGL3- FOXM1(Trident), pGL3-FOXM1(ApaI), or the control pGL3-ERE-pS2 promoter/reporter and 0 or 10 ng of ERa expression vector (pHEGO) in the absence or presence of E2 and with OHT treatment in the presence of E2 induction (E2 þ OHT). Cells were harvested 24 h after transfection and assayed for luciferase activity. All relative luciferase activity values are corrected for cotransfected Renilla activity. All data shown represent the averages of data from three independent experiments, and the error bars show the s.d.. In lower panel, COS-1 cells were transfected with pGL3-FOXM1(Trident), pGL3-FOXM1(ApaI) or pGL3-ERE promoter/reporter constructs, together with increasing amounts (0, 0.1, 1, 10 and 20 ng) of ERa expression vector (pHEGO), and processed as described above. (b) Schematic representation of the ApaI FOXM1-luciferase reporter construct, showing the consensus, the wild-type and the mutant ERE (mERE) sequences. COS-1 cells were transfected with pGL3-basic, pGL3-FOXM1(ApaI) wild-type (WT) or mutant ERE, or the control pGL3-ERE-PS2 promoter/reporter and with or without E2 treatment and 20 ng of ERa expression vector. The transfected cells were processed and assayed as described above.

Oncogene ERa regulates FOXM1 expression in breast cancer J Millour et al 2988 level. Conversely, FOXM1 knockdown also resulted in a substantial decrease in FOXM1 mRNA level decrease in ERa expression, consistent with our (Figure 5a). FOXM1 mRNA expression was not previous finding (Madureira et al., 2006). qRT–PCR completely abolished by ERa silencing; this is likely to analysis of the knocked-down cells also revealed a be due to the fact that FOXM1 transcription is regulated

a Biotin- WT ERE α +anti-ER +cons ERE Biotin-mutERE 3 +mutERE 3

b mutERE 3 Consensus ERE

Biotin- Biotin- mutERE 3 WT ERE: + + + + + + +

ERα

c Competition

WT ERE: - + -+- - - + -+- -+- -+- - mutERE3: --+ --+ --+ --+ --+ --+

ZR-75-1 IB:ERα

MCF-7 IB:ERα

MCF-7 IB:HDAC2

control OHT control ICI control E2 (High Exp)

d input IgGαERα input IgG αERα FOXM1 ERE (-184 to+4 bp)

MCF-7 control (-1157 to -1257 bp)

FOXM1 ERE (-184 to+4 bp) ZR-75-1 control (-1157 to -1257 bp) OHT: --+ -++ ICI: --+ -++

e MCF-7 Input IgG αHDAC1 αHDAC2

FOXM1 ERE (-184 to+4 bp) OHT: -+-+-+-+

Input IgG αAcH3 αAcH4

FOXM1 ERE (-184 to+4 bp)

OHT: -+-+-+-+

Oncogene ERa regulates FOXM1 expression in breast cancer J Millour et al 2989 by other proliferative signals as well (McGovern et al., that FOXM1 knockdown could be combined with OHT 2009). The complete downregulation of FOXM1 at the to cause further decreases in the rate of cell prolifera- protein level suggests the possibility of alternative tion. Together these results clearly show that the mechanisms of regulation of FOXM1 expression by knockdown of FOXM1 sensitizes the resistant cells to ERa (Major et al., 2004; Ma et al., 2005; Luscher- OHT and diminishes the responsiveness to E2. Firzlaff et al., 2006; Wierstra and Alves, 2006b). We next investigated the role of FOXM1 in mediating the proliferative function of ERa by studying the effects of Dysregulated FOXM1 is associated with breast cancer FOXM1 knockdown on estrogen-dependent growth in endocrine resistance MCF-7 cells. MCF-7 cells were estrogen-starved for 48 h As FOXM1 is essential for ERa’s proliferative function, then induced to proliferate with E2 in the presence or uncoupling FOXM1 from ERa regulation could result absence of FOXM1 siRNA. Western blot analysis in resistance to endocrine therapy in breast cancer cells. showed that FOXM1 was successfully silenced by the To test this hypothesis, we studied the expression siRNA, and that in the FOXM1 knocked-down cells of FOXM1 in hormone sensitive as well as resistant there was also a significant decrease in ERa expression MCF-7 cells, after treatment with OHT (Figure 6a). (Figure 5b). This is in agreement with our previous Consistent with our earlier results, OHT treatment of finding that FOXM1 regulates ERa expression (Madur- the sensitive MCF-7 cells caused a drastic reduction in eira et al., 2006). The results of the cell proliferation FOXM1 protein level over 72 h. In contrast, despite a assay showed that FOXM1 silencing completely abol- small transient decline in FOXM1 level at 8 h, no ished the mitogenic effects of E2 in the ERa-positive significant decreases in FOXM1 protein level were MCF-7 cells, indicating that FOXM1 is essential for observed in MCF-7 TAMR4 and MCF-7 TAMR7 (two the estrogen-dependent growth of MCF-7 cells, and independently generated) OHT-resistant clones over the mediates the proliferative function of ERa (Figure 5b). time course. Consistent with the western blot results, To investigate the potential role of FOXM1 in qRT–PCR analysis showed that OHT repressed hormonal resistance, we silenced FOXM1 expression FOXM1 mRNA expression in the parental MCF-7 using siRNA in the MCF-7 TAMR4 cells, an OHT- cells, but not in the MCF-7 TAMR4 and MCF-7 TAMR7 resistant MCF-7 clone (Lykkesfeldt et al., 1986, 1994), lines. These results suggested a role for FOXM1 in and studied its effects on cell proliferation (Figure 5c). mediating the endocrine resistance in breast cancer cells. Interestingly, the proliferation assays showed that the To examine this further, we next investigated if a MCF-7 TAMR4 was responsive to E2 re-stimulation, constitutively active FOXM1 (DN-FOXM1) could and knockdown of FOXM1 decreased the ability of E2 confer resistant to OHT in MCF-7 cells (Figure 6b). to induce the proliferation of the MCF-7 TAMR4 cells. The parental MCF-7 cells and one of the resistant lines, Notably, these cells with FOXM1 knockdown cultured MCF-7 TAMR4, were also included as controls. Western in the absence or presence of E2 had the same blot analysis revealed that the FOXM1 level declined in proliferation rate as steroid-depleted cells, suggesting the parental MCF-7 cells on treatment with OHT, but E2 targets FOXM1 to induce proliferation in this not in the resistant cells or MCF-7 cells expressing the system. In addition, our data also showed that OHT deregulated FOXM1. Consistently, the expression of could antagonize the proliferative function of E2, and FOXM1 targets, including Polo-like kinase-1 (PLK-1)

Figure 3 ERa binds directly to the ERE on the FOXM1 promoter. (a) Biotinylated wild-type ERE or the mutant ERE3 oligonucleotides were incubated with MCF-7 cell lysates in the presence or absence of 10 Â molar excess of non-biotinylated ERE3 or consensus ERE oligonucleotides or an anti-ERa antibody. In brief, gel shift assays (20 ml total volume) contained 3 ı` g nuclear extracts and 50 ng/ml poly (dI-dC) in 1 Â binding buffer and 20 pmol unlabelled oligonucleotides. For the supershifts, 1 ml of the anti-ERa (Santa Cruz; H222) antibody were included in the reaction mix. The mixtures were pre-incubated at room temperature for 15 min, followed by addition of 100 fmol of biotin-labelled double-stranded estrogen response element (ERE-conc. 50-GCCGATTGGCGA CGTTCGGTCACGCTGACCTTAACGCTCCGCCGGCG-30 50-CGCCGGCGGAGCGTTAAGGTCACGCTGACCGAACGT CGCCAATCGGC-30), (ERE-wt 50-GCCGATTGGCGACGTTCCGTCACGTGACCTTAACGCTCCGCCGGCG-30,50-CGCCGG CGGAGCGTTAAGGTCACGTGACGGAACGTCGCCAATCGGC-30), or (mERE3 50-GCCGATTGGCGACGTTCCGTAACG TTACGTTAACGCTCCGCCGGC-30,50-CGCCGGCGGAGCGTTAACGTAACGTTACGGAACGTCGCCAATCGGC-30), and incubation at room temperature for 20 min, followed by a 30-min incubation on ice. Protein-DNA complexes were separated on 5% 0.5 Â TBE polyacrylamide gels. The electrophoretic transfer of the binding reactions to the nylon membrane, and the detection of the biotin labelled DNA by chemiluminescence, were performed according to the PIERCE kit’s protocols. (b) Nuclear extracts from MCF- 7 cells were incubated with biotinylated oligonucleotides representing region of the FOXM1 promoter containing the ERE or the mutated ERE3 site in the absence or presence of molar excess of non-biotinylated ERE3 or consensus ERE oligonucleotides. Proteins binding to the biotinylated oligonucleotides were pulled-down using streptavidine agarose beads and analysed by western blot using specific antibodies as indicated. (c) The nuclear extracts (Supplementary Figure S1) from MCF-7 and ZR-75-1 cells with or without OHT or ICI for 24 h were also examined by pull-down assays using biotinylated wild-type or mutant (mERE3) oligonucleotides as described above. (d) Chromatin immunoprecipitation (ChIP) analysis of the human FOXM1 promoter. MCF-7 and ZR-75-1 cells untreated or treated with ICI or OHT for 24 h were used for ChIP assays using IgG, anti-ERa antibodies as indicated. After crosslink reversal, the co-immunoprecipitated DNA was amplified by PCR using primers amplifying the FOXM1 ERE containing region (À184/ þ 4) and a control region (À1157/À1257), and resolved in 2% agarose gel. (e) MCF-7 untreated or treated with OHT for 24 h were used for ChIP assays using IgG antibodies against acetylated H3 and H4, HDAC1 and HDAC2 as described above. Representative data from three independent experiments are shown.

Oncogene ERa regulates FOXM1 expression in breast cancer J Millour et al 2990 All values downregulated by 48 h in MCF-7 cells, and the decline 4 3 in cyclin A levels occurred in a comparatively slower R4 3 Spearman kinetics in TAM and DN-FOXM1 cells. Consistent r = 0.1976 2 with this, the CDK2 activity-specific pRB(Thr821) 2 p = 0.1089 antibody showed that the repression of cyclin A/E– 1 1 CDK2 activity coincided with the downregulation of FOXM/L19 RNA 0.70 FOXM1/L19 RNA cyclin A expression in these MCF-7 cells. Given that 0 0 cyclin A-CDK2 phosphorylation of FOXM1 can 012345 FOXM1 activate its transcriptional activity (Wierstra and Alves, ERα/L19 RNA 2006a; Park et al., 2008; Laoukili et al., 2008a, b), this FOXM1 mRNA values < 0.70 FOXM1 mRNA values > 0.70 finding suggests another potential mechanism by which 4 FOXM1 activity can be downregulated by OHT. As 0.6 Spearman cyclin A and -E functions downstream of cyclin D1, 3 r = 0.1863 p = 0.4741 0.4 (Fung and Poon, 2005; Myatt and Lam, 2007; Tashiro 2 Spearman et al., 2007), which is a transcription target of FOXM1, r = 0.4469 0.2 it is likely that the constitutively active DN-FOXM1 p = 0.0001 1 FOXM1/L19 RNA FOXM1/L19 RNA upregulates cyclin D1 to sustain cyclin A expression and 0.0 0 G1 to S cell cycle phase progression upon OHT 012345 012345 treatment in the DN-FOXM1 MCF-7 cells. Consistent ERα/L19 RNA ERα/L19 RNA with this, the western blot analysis showed that the Figure 4 Significant correlation between ERa and FOXM1 cyclin D1 level and its associated activity revealed by the expression in human breast samples. Expression of ERa mRNA CDK4/6 phospho-pRB(Ser807/811) antibody were and FOXM1 mRNA in non-cancerous breast biopsies and overexpressed in the DN-FOXM1- and maintained in malignant breast epithelial tissues. RNA was isolated from R4 epithelial cells purified from normal breast tissues and primary the TAM -MCF-7 cells. In addition, ectopic expression tumours and subjected to qRT–PCR with FOXM1, ERa and L19 of FOXM1 also increases the expression levels of cyclin primers. (a) Graph shows the FOXM1 and ERa mRNA levels of D1 in both MCF-7 (Supplementary Figure S5) and the tumour samples after normalization with L19 RNA levels. No MDA-MB-231 cells (Supplementary Figure S6). Given significant correlation is seen using Pearson’s correlation when considering all values (n ¼ 69; Spearman r ¼ 0.198; P ¼ 0.109). the well-documented role of cyclin D1 in endocrine Significance is defined as Po0.05. Line is linear regression, shown resistance (Lundgren et al., 2008; Wang et al., 2008; for illustrative purposes. (b) Box and whisker plot showing the Finn et al., 2009; Yamashita et al., 2009; Zwart et al., distribution of values for FOXM1. Box edges represent 25th and 2009) and G1/S transition (Fung and Poon, 2005; Myatt 75th percentiles; middle line is the median, whereas plus shows the and Lam, 2007; Tashiro et al., 2007), our data also mean. The whiskers represent the 10th and 90th percentiles, whereas outliers are shown as dots. The 75th percentile for support a role for FOXM1 in mediating breast cancer FOXM1 mRNA values is 0.7002. (c) The right hand graph shows endocrine sensitivity and resistance at least in part the FOXM1 and ERa mRNA levels of the tumour samples with through modulating cyclin D1 expression. We per- FOXM1/L19 mRNA levels below the upper quartile. Correlation formed cell cycle analysis of the MCF-7, MCF-7TAMR4 analysis was performed between FOXM1 and ERa mRNA. A significant and positive association was found between FOXM1 and MCF-7 DN-FOXM1 cells following treatment with and ERa mRNA levels in breast patient samples with FOXM1 OHT (Figure 6c). The results showed that OHT caused mRNA level below the upper quartile (n ¼ 52; Spearman r ¼ 0.447; a predominantly G1 cell cycle arrest in the parental P ¼ 0.0001). Po0.05 was considered statistically significant. MCF-7 cells, but had comparatively little effects on the (d) The lower left-hand graph shows the FOXM1 and ERa mRNA cell cycle distribution of the MCF-7TAMR4 and MCF-7 levels of the tumour samples with FOXM1/L19 mRNA levels in the upper quartile (0.7) of high levels of FOXM mRNA expression. DN-FOXM1 cells. We also generated MCF-7 cells No significant correlation is found using Pearson correlation stably transfected with the wild-type FOXM1 (MCF- (n ¼ 17; Spearman r ¼ 0.186; P ¼ 0.474). Significance is defined as 7-FOXM1) and studied the effects of OHT on the Pp0.05. transfected pool as well as individual clones of MCF-7- FOXM1 cells (Supplementary Figure S5). Western blot analysis showed that overexpression of the full-length FOXM1 prevented the downregulation of FOXM1 and CDC25B, mirrored the expression patterns of targets such as CDC25b and PLK in response to FOXM1 in the three MCF-7 cell lines. It is notable tamoxifen treatment. The cell cycle analysis showed that there were no significant changes in either the ERa that these cells underwent the cell cycle arrest at G1, or the endogenous FOXM1 in the OHT-resistant cell concommitant with FOXM1 downregulation, at a line and the MCF-7 harbouring the active FOXM1. slower kinetics compared with MCF-7 cells transfected This is likely to be due to the positive feedback with the empty vector. This further suggests that mechanism between ERa and FOXM1, described here downregulation of FOXM1 is important for the G1/S and previously (Madureira et al., 2006). In agreement arrest and that FOXM1 may also be regulated by with the western blot results, qRT–PCR analysis showed tamoxifen at post-transcriptional levels. Collectively, that OHT repressed FOXM1 mRNA expression in these results indicate that FOXM1 has a role in MCF-7 cells but not in the MCF-7-TAMR4 and MCF-7- mediating the anti-proliferative effects of OHT via DN-FOXM1 cells. The western blot results also showed ERa, and that its deregulated expression contributes that cyclin A and not its catalytic partner CDK2 was towards the development of endocrine resistance.

Oncogene ERa regulates FOXM1 expression in breast cancer J Millour et al 2991 siRNA

α Mock FOXM1 siRNA ER NS siRNA 3.0 MCF-7 TamR4 2.5 siRNA: n.s.FOXM1 n.s. FOXM1

siRNA 2.0

α 24h48h 72h 24h 48h 72h 1.5 0h 0h Mock NS siRNA ER Mock NS siRNA FOXM1 siRNA 1.0 FOXM1 FOXM1 0.5 FOXM1/L19 RNA ERα 0 ERα 3.0 Tubulin Tubulin 2.5 2.0 1.5 250 1.0 n.s. siRNA+E2 200 ERa/L19 RNA 0.5 ** 0 FOXM1 siRNA+E2 150 * n.s. siRNA+E2+OHT +E2 200 100 FOXM1 siRNA+E2+OHT NS siRNA: -++ - 180 % SRB unit at 0h FOXM1 siRNA: ++-+ 160 50 0h 24h48h 72h 140 FOXM1 120 100 250 ERα 80 n.s. siRNA+E2 200 ** Tubulin 60 NS siRNA+E2 % SRB unit at 0h 40 NS siRNA FOXM1 siRNA+E2 150 20 FOXM1 siRNA+E2 FOXM1 siRNA n.s. siRNA 00 100 0h 24h 48h % SRB unit at 0h FOXM1 siRNA Time 50 0h 24h48h 72h Time Figure 5 Effects of ERa and FOXM1 silencing on the expression of FOXM1 and response to E2 induction in MCF-7 cells. (a) MCF- 7 cells were transiently transfected with ERa, FOXM1 or control smart pool siRNA, and 72 h after transfection cells were analysed by western blot using specific antibodies as indicated and by qRT–PCR. (b) MCF-7 cells were transiently transfected with smart pool siRNA against FOXM1, incubated with E2 and analysed by western blotting. SRB assays were also performed on these cells, indicating that the knockdown of FOXM1 decreases the cell proliferation rate and renders MCF-7 cells unresponsive to E2 stimulation. (c) OHT-resistant TAMR4 MCF-7 cells were transiently transfected with smart pool siRNA against FOXM1 or control siRNA pool (non-specific/ns siRNA) and analysed by western blotting (upper panel). These transfected cells were incubated with or without E2 (middle panel), and with or without OHT in the presence of E2 (lower panel). SRB assays were performed on these cells, indicating that the knockdown of FOXM1 sensitizes the resistant TAMR4 MCF-7 cells to OHT and diminishes their responsiveness to E2 stimulation. Statistical analysis was performed on the proliferation results at 72 h. **denotes very significant, Po0.01 and *significant, Po0.05. The results show mean þ s.e.m. of triplicate measurements.

Discussion comes tamoxifen resistance in ‘acquired resistant’ MCF- 7 cells. Conversely, ectopic expression of a constitutively Expression of ERa expression in breast cancer cell lines active FOXM1 can abrogate the cell cycle arrest is regulated by FOXM1 (Madureira et al., 2006). In this mediated by OHT. Collectively, the results presented study, we investigated the reciprocal regulation of clearly confirm FOXM1 as a key mediator of the FOXM1 expression by ERa. Using breast carcinoma mitogenic functions of ERa and estrogen in breast cell lines, we showed that FOXM1 protein and mRNA cancer cells. expression are regulated by ER-ligands, including E2, To address the physiological significance of our OHT and ICI. We also found that depletion of ERa by finding, we studied the FOXM1 mRNA expression RNA interference in MCF-7 cells leads to downregula- and its relationship to ERa mRNA level in the breast tion of FOXM1 expression. Using reporter gene assays, cancer biopsy samples. After the exclusion of the data we showed that ERa activates FOXM1 transcription with high levels of FOXM1 expression (upper 25th through an ERE located at À45 bp upstream of the percentile), a statistically significant correlation between transcriptional start site (Bourdeau et al., 2004). The ERa and FOXM1 mRNA expression was detected. This direct binding of ERa to the FOXM1 promoter was is in agreement with a recent breast cancer patient confirmed in vitro by mobility shift and DNA pull-down microarray data set analysis indicating that high levels assays, and in vivo by ChIP analysis. Importantly, of FOXM1 mRNA expression (upper 25th percentile) silencing of FOXM1 by RNA interference abolishes are associated with poor prognosis in breast cancer estrogen-mediated MCF-7 cell proliferation and over- (Martin et al., 2008). The discordance between ERa and

Oncogene ERa regulates FOXM1 expression in breast cancer J Millour et al 2992 MCF-7 MCF-7 TAMR4 MCF-7 TAMR7 OHT: 0 8 24 48 72 0 8 24 48 72 0 8 24 48 72

FOXM1

ERα

Tubulin 1.0 1.2 0.8 R4 Δ 0.6 0.1 MCF-7 TAM MCF-7 MCF-7 N 0.4 0.8 0.2 0 0.6 FOXM1/L19 RNA OHT: 0 8 24 48 72 0 8 24 48 72 0 8 24 48 72 (h) 0.4

TAMR4 MCF-7 ΔN-FOXM1 FOXM1/L19 RNA 0.2 0 24 48 72 0 24 48 72 0 24 48 72 (h) 0.0 024487200 24 48 72 24 48 72 (h) FOXM1 ΔN-FOXM1 0h 24h 48h 72h 0.2 0.2 1.2 0.6 %<2N ERα 48.4 71.5 73.8 72.3 %G0/G1 17.0 9.1 8.0 7.8 %S MCF-7 CDC25B 34.4 19.2 17.0 19.3 %G2/M

PLK-1 0.3 2.1 2.6 1.9 47.2 61.3 59.3 60.7 P-PKB TAMR4 8.0 14.4 13.5 14.0 44.5 22.2 24.6 23.4 PKB cyclin A 0.4 0.2 0.2 1.0 68.7 65.0 58.1 68.7 ΔN-FOXM1 12.5 14.5 18.2 16.9 CDK2 18.4 20.3 23.5 13.7 cyclin D1 CDK4 P-pRb(cdk2 act) P-pRb(cdk4 act) pRb Tubulin

Figure 6 Expression of FOXM1 and cell cycle regulation in wild-type, OHT-resistant and constitutively active DN-FOXM1 expressing MCF-7 cells in response to OHT treatment. (a) MCF-7 and the resistant MCF-7-TAMR4 and -TAMR7 lines cultured in 10% FCS and phenol red medium were treated with OHT in a time course of 72 h. Cell lysates were prepared at the times indicated, and the expression of FOXM1, ERa and tubulin was analysed by western blotting. (b) MCF-7, MCF-7 TAMR4 and MCF-7 N-FOXM1 cells were treated with OHT in a time course of 72 h. Cell lysates were prepared at the times indicated, and the expression of FOXM1, ERa, CDC25B, P-PKB, total PKB and Tubulin was analysed by western blotting. (c) FOXM1 mRNA levels of these cells were also analysed by qRT–PCR and normalized to L19 RNA expression. (d) Cells were fixed at 0, 24, 48 and 72 h after treatment, and cell cycle phase distribution was analysed by flow cytometry after propidium iodide staining. Percentage of cells in each phase of the cell cycle (sub-G1, G1, S and G2/M) is indicated. Representative data from three independent experiments are shown.

FOXM1 mRNA expression in patient samples with high significant changes in the ERa or the endogenous FOXM1 expression probably indicates that in these FOXM1 levels in the MCF-7 cells expressing the active subjects FOXM1 expression is deregulated with their FOXM1 highlights an important positive feedback control of FOXM1 transcription by ERa overridden by mechanism between ERa and FOXM1, described here other mitogenic signals or genetic changes. Our findings and previously (Madureira et al., 2006). This makes that OHT represses FOXM1 expression in endocrine FOXM1 a particularly critical ERa target gene in breast sensitive but not resistant breast carcinoma cell lines, cancer development and endocrine resistance, as the and that ectopic expression of FOXM1 can abrogate the feedback loop will amplify the mitogenic action of anti-proliferative effects of OHT, further confirm that estrogens. It is likely this positive feedback transcrip- deregulation of FOXM1 may contribute to anti-estro- tional mechanism is uncoupled in ERa-negative breast gen insensitivity. The observation that there were no cancer, as ERa overexpression does not induce FOXM1

Oncogene ERa regulates FOXM1 expression in breast cancer J Millour et al 2993 expression and vice versa in the ERa-negative endo- assays were performed as described (Essafi et al., 2009). crine-resistant MDA-MB-231 breast carcinoma cells Antibodies for acetylated H3 (06–599) and H4 (06–866) were (Supplementary Figure S6). Previous studies have from Upstate (Milton Keynes, UK). shown that changes in epigenetic regulation, such as DNA methylation and histone acetylation, are respon- Quantitative real-time PCR (qRT–PCR) sible in silencing of the ERa gene in ER-negative human Frozen samples from patients who had undergone surgery at breast cancer cells (Yan et al., 2003). We also speculate Charing Cross Hospital (London, UK) were used for RNA that recruitment of ERa to target genes in vivo might extraction. The procedures for purification of normal human require ‘pioneering factors, such as FOXA1, which are breast epithelial cells have previously been described (Diaz- deregulated in ERa-negative cells (Carroll et al., 2005; Arias et al., 1993; Kothari et al., 2003; Fellowes et al., 2004). Carroll and Brown, 2006). This positive feedback FOXM1 and L19 transcript levels were quantified using the transcriptional mechanism may also account for the standard curve method. L19, a non-regulated ribosomal significant association between ERa and FOXM1 housekeeping gene was used as an internal control to normal- ize input cDNA. mRNA observed in breast tissue samples. Consistently, FOXM1 is one of eight overexpressed genes identified in solid tumours by comparison of different microarray Gene Silencing with Small Interfering RNAs (siRNAs) data sets (Pilarsky et al., 2004). In conclusion, our For gene silencing, cells were transiently transfected with results indicate that FOXM1 is a downstream target 50 nM of the siRNA SMARTpool reagents purchased from of ERa. Our findings also provide potential insights Dharmacon (Lafayette, CO, USA) using OligofectAMINE into the mechanism of anti-estrogen action and endo- (Invitrogen) according to the manufacturer’s instructions. crine resistance, and show that ER regulation of FOXM1 may also contribute to anti-estrogen respon- Statistical analysis siveness. Statistical analyses were performed using GraphPad Prism v5.0.

Materials and methods Site-directed mutagenesis Mutants of the FOXM1 promoter generated for this study Cell culture were made using the QuickChange7 Site-Directed Mutagenesis The human breast carcinoma MCF-7, ZR-75-1 and MDA- Kit (Stratagene, La Jolla, CA, USA) following the manufac- MB-231 cell lines and COS-1 cells, were maintained in DMEM turer’s protocol. supplemented with 10% FCS, 2 mM glutamine, and 100 units/ ml penicillin/streptomycin at 37 1C. The OHT-resistant MCF- 7-TAMR4 and TAMR7 cells and their growth conditions have Gel shift and pull-down assays using biotin-labelled previously been described (Lykkesfeldt et al., 1994; Madsen oligonucleotides et al., 1997).The cells were exposed to ERa ligands 10–8M Gel shift assays were performed following a modified version estradiol (E2), 10–6 M 4-OHT (OHT) or 10–7 M ICI prepared of the manufacturer’s protocol from the LightShift Chemilu- in ethanol, or only ethanol (vehicle control) for the indicated minescent electrophoretic mobility shift assay Kit (Pierce) times before harvesting. For steroid starvation, cells were (Essafi et al., 2009). Nuclear and cytoplasmic extracts were cultured in phenol-free DMEM/F-12 containing 5% double prepared and pull-down assays performed as described charcoal-stripped FCS. previously (Essafi et al., 2005, 2009).

Plasmids and transfections Cell cycle and proliferation assays The human FOXM1 promoter constructs have previously been Cell cycle analysis was performed using propidium iodide described (Korver et al., 1997b). Cells were transfected with staining alone, as previously described (Collado et al., 2000). the human FOXM1 promoter and Renilla (pRL-TK; Prome- Cell proliferation was determined using the sulforhodamine B ga, Southampton, UK) as internal transfection control using colorimetric assay (Skehan et al., 1990). Fugene-6 (Qiagen, Crawley, UK) as described (Kwok et al., 2008). Conflict of interest Western blotting, ChIP analysis and antibodies Western blotting was performed on whole cell extracts by The authors declare no conflict of interest. lysing cells in buffer as described (McGovern et al., 2009). Antibodies cyclin B (H-433), cyclin D1 (DCS-6), PLK (F8), tubulin (H235), ERa (F-10 and HC20), pS2 (FL-84), FOXM1 (C-20), Cyclin A (C-19), pRB (C-15), HDAC-1 (N-19) CDK2 Acknowledgements (M2), CDK4 (C-22) and pRB (C-15) were from Santa Cruz Biotechnology (Wiltshire, UK). The anti-phospho-pRB Grant support was given by Breast Cancer Campaign (Ser807/811) and HDAC2 (2540) antibodies were purchased (J Millour and EW-F Lam), Cancer Research UK (SS Myatt, from New England Biolabs (Hitchin, UK), the anti-phospho- K-K Ho, RC Coombes, EW-F Lam), Biotechnology pRB (Thr821) antibody from Biosource (London, UK), and and Biological Sciences Research Council (MSC Wilson and Cdc25b (DCS-162) from Abcam (Cambridge, UK). ChIP EW-F Lam).

Oncogene ERa regulates FOXM1 expression in breast cancer J Millour et al 2994 References

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

Oncogene