Oncogene (2010) 29, 2983–2995 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.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 receptor a (ERa) and its role in hormonal therapy and endocrine resistance. FOXM1 Breast cancer is the second most prevalent cause of protein 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 gene (Pilarsky et al., 2004). FOXM1 is expressed in prolifer- assays showed that ERa activates FOXM1 transcription 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 cell cycle 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 proteins, 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 genes essential cing of FOXM1 by RNAi abolished estrogen-induced for faithful chromosome segregation and mitosis, 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; estrogen receptor; 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.