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Originally published In Press as doi:10.1074/jbc.M603906200 on June 28, 2006

J. Biol. Chem., Vol. 281, Issue 35, 25167-25176, September 1, 2006
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The Forkhead Box M1 Protein Regulates the Transcription of the Estrogen Receptor {alpha} in Breast Cancer Cells*Formula

Patricia A. Madureira{ddagger}, Rana Varshochi{ddagger}, Demetra Constantinidou{ddagger}, Richard E. Francis{ddagger}, R. Charles Coombes{ddagger}, Kwok-Ming Yao§, and Eric W.-F. Lam{ddagger}1

From the {ddagger}Cancer Research-United Kingdom Laboratories, Department of Oncology, MRC Cyclotron Building, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, United Kingdom, §Department of Biochemistry, The University of Hong Kong, 3/F Laboratory Block, The Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, China

Received for publication, April 24, 2006 , and in revised form, June 28, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we have identified the Forkhead transcription factor FoxM1 as a physiological regulator of estrogen receptor {alpha} (ER{alpha}) expression in breast carcinoma cells. Our survey of a panel of 16 different breast cell lines showed a good correlation (13/16) between FoxM1 expression and expression of ER{alpha} at both protein and mRNA levels. We have also demonstrated that ectopic expression of FoxM1 in two different estrogen receptor-positive breast cancer cell lines, MCF-7 and ZR-75–30, led to up-regulation of ER{alpha} expression at protein and transcript levels. Furthermore, treatment of MCF-7 cells with the MEK inhibitor U0126, which blocks ERK1/2-dependent activation of FoxM1, also repressed ER{alpha} expression. Consistent with this, silencing of FoxM1 expression in MCF-7 cells using small interfering RNA resulted in the almost complete abrogation of ER{alpha} expression. We also went on to show that FoxM1 can activate the transcriptional activity of human ER{alpha} promoter primarily through two closely located Forkhead response elements located at the proximal region of the ER{alpha} promoter. Chromatin immunoprecipitation and biotinylated oligonucleotide pulldown assays have allowed us to confirm these Forkhead response elements as important for FoxM1 binding. Further co-immunoprecipitation experiments showed that FoxO3a and FoxM1 interact in vivo. Together with the chromatin immunoprecipitation and biotinylated oligonucleotide pulldown data, the co-immunoprecipitation results also suggest the possibility that FoxM1 and FoxO3a cooperate to regulate ER{alpha} gene transcription.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The biological effects of estrogen are primarily mediated through two nuclear steroid receptors, estrogen receptors {alpha} and beta (ER{alpha} and ERbeta)2 (1–3). Estrogen receptors play a major role in regulating the growth, survival, and differentiation of normal and malignant breast epithelial cells. In normal mammary epithelium, ERs are rarely expressed in a large proportion of cells or at high levels (4, 5). Approximately 60–80% of all breast cancers overexpress ER{alpha}, and ~70% of those respond to endocrine treatment, with further increased risk of breast cancer development in benign mammary tissue. These findings suggest that ER{alpha} plays a major role in breast cancer initiation and progression (4). ERbeta appears to have an opposing function to ER{alpha} in tumor growth (6), and low levels of ER{alpha} predict resistance to Tamoxifen therapy in breast cancer (7).

ER{alpha} functions as a classical transcription factor as well as a signal transducer. Estrogen binding activates ER{alpha} through phosphorylation, dissociates it from chaperonin proteins, and alters its conformation. Several kinases in the mitogenic signaling pathways can also phosphorylate and consequently activate ER{alpha} through a ligand-independent manner. Hormone-bound ERs dimerize and bind to estrogen response elements present in the promoter regions of target genes, including c-fos, c-jun, c-myc, TGF-{alpha}, retinoic acid receptor {alpha}1, and progesterone receptor A, etc., to activate gene transcription (8–12). ER{alpha} has also been shown to modulate gene transcription through alternative regulatory DNA sequences, such as AP-1, NF-{kappa}B, and Sp-1 binding sites (13). Many of these ER-regulated genes, including IGFR1, cyclin D1, c-myc, and the anti-apoptotic gene bcl-2, are important for cell proliferation and survival (14–17). Recent studies also suggest that a pool of ERs are located in the plasma membrane and cytoplasm. Direct binding of ER{alpha} to a diversity of membrane/cytoplasmic signaling molecules have been observed, and they include the p85 regulatory subunit of class I phosphoinositide 3-kinase, Src tyrosine kinase, and the insulin-like growth factor 1 (13). Activation of these pathways by estrogen through the ER initiates cell survival and proliferation signals via phosphorylation and activation of Akt and MAPK. Additionally, these signaling molecules are able to phosphorylate the ER and its co-regulators to augment nuclear ER signaling (13). In summary, the genomic and non-genomic actions of ER{alpha} play a crucial role in breast epithelial cell proliferation and survival.

Endocrine therapies aimed at blocking the action of estrogens have been the most effective and widely used methods for treating ER{alpha}-positive breast cancers. These therapeutic approaches involve blocking estrogen binding to ER using antiestrogens such as tamoxifen, inhibiting estrogen synthesis using aromatase inhibitors such as exemestane, and reducing ER protein levels using "pure" anti-estrogens such as fluvestrant (ICI 182,780 or Faslodex) (13). Nevertheless, a proportion of ER{alpha}-positive tumors do not respond to hormone treatment at all (de novo resistance), and the majority of those that initially respond eventually become resistant (acquired resistance). Most resistant tumors remain ER{alpha}-positive, indicating a continued role for ER{alpha} in breast cancer cell survival and proliferation (18, 19).

A recent study suggested that ER{alpha} transcription is regulated by FoxO3a (Forkhead box class O, 3a), a member of the Forkhead family of transcription factors (20). FoxO3a activity is negatively regulated by the phosphoinositide 3-kinase signaling pathway. Activation of phosphoinositide 3-kinase by growth factors leads to phosphorylation and inactivation of FoxO3a by Akt (also termed PKB (protein kinase B)) (21). When activated, FoxO3a up-regulates p27Kip1, p130, and bim and down-regulates cyclin D1/2 and bcl-XL expression to mediate cell cycle arrest and apoptosis (22–25). Because ER{alpha} expression is associated with breast cancer initiation and progression and FoxO3a activation with cell cycle arrest and/or apoptosis, it is implausible that FoxO3a would be the principal positive regulator of ER{alpha} transcription. Because all members of the Forkhead transcription factors share a similar DNA binding domain, we reasoned that other family member(s) could be the main physiological regulator(s) of ER{alpha} transcription.

Forkhead box (Fox) M1 is a transcription factor ubiquitously expressed in proliferating cells and a key regulator of both G1/S and G2/M phases of the cell cycle (26–30). FoxM1 is localized mainly in the cytoplasm in late G1 and S phases; nuclear translocation occurs during entry into the G2/M phase and is associated with FoxM1 phosphorylation by ERK1/2 (31). In this study, we have examined the possibility that FoxM1 regulates ER{alpha} expression and explored the mechanism involved.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Transfections, and Cell Lines—The human breast carcinoma cell lines MCF-7, ZR-75–1, ZR-75–30, 734 B, MDA-MB-175, CAMA1, MDA-MB-231, MDA-MB-453, MDA-MB-469, HMT 3552, HBL-100, SKBR-3, SKBR-7, BT-474, BT-549, T47-D, and CAL-51 were obtained from the American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 2 mM glutamine, and 100 units/ml penicillin/streptomycin in a humidified incubator in an atmosphere of 10% CO2 at 37 °C. MCF-7 Tet-on FoxM1 cells were selected and maintained in the presence of 500 µg/ml of zeocin (Invitrogen).

Plasmids—The FoxO3a expression vector pLPC-FoxO3a(wt) and pLPC-FoxO3a(A3) and the FoxM1 expression vector pcDNA3-FoxM1 have previously been described (22, 32). The ER{alpha} promoter constructs pGL3-proA and pGL3-proB were kindly provided by Prof. Shin-Ichi Hayashi (Saitama Cancer Center Research Institute, Saitama, Japan) (33). The psiRNA-FoxO3a expression vector was generated by cloning small synthetic oligonucleotides encoding two complementary sequences of 19 nucleotides, TCACTGCATAGTCGATTCA, into the Invivogen psiRNA plasmid (Autogen Bioclear, Wiltshire, UK). The pTER-FoxM1 siRNA expression vector was generated by cloning the two complementary hairpin sequences of CACGCAAGTAGTGGCCATC into the BglII and HindIII sites of the pTER vector provided by Dr. Marc van de Wetering (Department of Immunology, University Medical Center, Utrecht, The Netherlands) (34). The respective control RNA interference vectors were generated from nucleotide sequences with the middle 2 bases mutated (i.e. FoxM1-TCACTGCCCAGTCGATTCA and FoxO3a-TCACTGCACCGTCGATTCA). The pcDNA4-FoxM1 vector used for the establishment of MCF-7 Tet-on FoxM1 MCF-7 cells was generated by PCR amplification of the human FoxM1 cDNA (GenBankTM accession number U83113 [GenBank] ) using a high-fidelity polymerase (Clontech) and subcloning into the HindIII/XbaI restriction sites of pcDNA4TM4T/O (Invitrogen) mammalian expression plasmid.

Western Blotting and Antibodies—Western blotting was performed on whole cell extracts prepared by lysing cells with a 2x packed cell volume of Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 10 mM NaF, 1 mM sodium orthovanadate, and protease inhibitors ("Complete" protease inhibitor mixture)), as instructed by the manufacturer (Roche Applied Science). Protein concentration was determined by Bio-Rad Dc protein assay. 25 µg of protein was size-fractionated using SDS-PAGE and electrotransferred onto Protran nitrocellulose membranes (Schliecher & Schuell). Antibodies recognizing FoxO3a phosphorylated at Thr-32 and total FoxO3a (06–951) were purchased from Upstate (Dundee, UK). Mouse monoclonal antibody against FoxO3a (F-1304) was purchased from Sigma. Antibodies against p27Kip1 (C-19), Bim (H-191), Cdc25b, Cdc25c (C-20), cyclin B1 (H-433), Plk (F-8), actin (I-19), FoxM1 (C-20), FoxM1 (H-300), and ER{alpha} (F-10) were purchased from Santa Cruz Biotechnology. Antibodies against phospho-p44/42 MAPK (Thr-202/Tyr-204), total MAPK, phospho-Akt (Ser-473), and total Akt were purchased from Cell Signaling Technologies (Hitchin, UK). Primary antibodies were detected using horseradish peroxidase-linked anti-mouse, anti-goat, or anti-rabbit conjugates (DAKO, Ely, UK), as appropriate, and visualized using the ECL detection system (Amersham Biosciences).

Immunoprecipitation Assay—Cells were washed in phosphate-buffered saline and lysed with 500 µl of Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Tris, pH 7.4, 10 mM sodium molibdate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, and Complete Mini protease inhibitors (Roche Applied Science)) for 15 min on ice. The cell lysates were precleared for 1 h with protein G-Sepharose, incubated with specific antibodies for 1 h, and then with 50% slurry of protein G-Sepharose for 1 h. The beads were washed five times with 500 µl of lysis buffer and analyzed by Western blotting.

Transfection and Luciferase Reporter Promoter Assay—MCF-7 cells were cultured in 96-well plates until 60% confluent. The cells were transfected using FuGENE 6 reagent (Roche Applied Science) according to the manufacturer's instructions for 24 h, washed twice in phosphate-buffered saline, and then harvested for firefly/Renilla luciferase assays using the Dual-Luciferase reporter assay system (Promega, Southampton, UK).

Mutagenesis—Mutagenesis of putative Forkhead binding sites of the ER{alpha} promoter was carried out using the QuikChange® site-directed mutagenesis kit (Stratagene, Cambridge, UK). The sequences for primers used for creating point mutations and deletions are essentially those described for the DNA pulldown assays plus franking regions to give oligonucleotides of 42 bases.

Real-time Quantitative PCR (RT-qPCR)—Total RNA was isolated using the RNeasy kit (Qiagen, Crawley, UK). 1 µg of total RNA was reverse-transcribed using the Superscript first strand synthesis system for RT-qPCR (Invitrogen), and the resulting first strand cDNA was used as the template in the real-time quantitative PCR analysis. All measurements were performed in triplicate. The mRNAs analyzed were FoxO3a, FoxM1, ER{alpha}, and GAPDH, which served as an internal control and was used to normalize for variances in input cDNA. The following gene-specific primer pairs were designed using the ABI Primer Express software: FoxO3a-sense, 5'-TCTACGAGTGGATGGTGCGTT-3' and FoxO3a-antisense, 5'-CGACTATGCAGTGACAGGTTGTG-3'; FoxM1-sense, 5'-TGCAGCTAGGGATGTGAATCTTC-3' and FoxM1-antisense, 5'-GGAGCCCAGTCCATCAGAACT-3'; ER{alpha}-sense, 5'-CAGATGGTCAGTGCCTTGTTGG-3' and ER{alpha}-antisense, 5'-CCAAGAGCAAGTTAGGAGCAAACAG-3'; GAPDH-sense, 5'-TCCCATCACCATCTTCCA-3' and GAPDH-antisense, 5'-CATCACGCCACAGTTTCC-3'. The specificity of each primer was determined using the NCBI BLAST software module. Detection of FoxO3a, FoxM1, and ER{alpha} transcription was performed with SYBR Green (Applied Biosystems, Brackley, UK) and an ABI PRISM 7700 sequence detection system (Applied Biosystems, UK) using the relative standard curve method.

Gene Silencing with Small Interfering RNAs—MCF-7 cells cultured in 100-mm plates were transfected with 15 µg of pTER-FoxM1siRNA or psi-FoxO3a plasmid using FuGENE 6 reagent (Roche Applied Science). Cells were collected 72 h after transfection either for Western blot analysis or RT-qPCR.

Immunoprecipitation Assay—Cells were washed in phosphate-buffered saline and lysed with 500 µl of Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Tris, pH 7.4, 10 mM sodium molibdate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, and Complete Mini protease inhibitors (Roche Applied Science) for 15 min on ice. The cell lysates were precleared for 1 h with protein G-Sepharose and incubated with specific antibodies for 1 h and then with 50% slurry of protein G-Sepharose for 1 h. The beads were washed five times with 500 µl of lysis buffer and analyzed by Western blotting.

Chromatin Immunoprecipitation (ChIP) Assay—ChIP assay was performed as described previously (25) using MCF-7 cells grown to 70% confluence. DNA fragments were purified using the QIAquick Spin Kit (Qiagen, Crawley, UK). For PCR, one-twenty-fifth of the extracted DNA was used and amplified in 25 PCR cycles using specific primers. The following primers were used: B1-sense(–3215/–3193), 5'-AGGCACCACTGTCACCAACAAA-3' and B1-antisense(–2887/–2865), 5'-AAGCCTCCATTGGGTGTCATGT-3'; B2-sense(–2755/–2731), 5'-CCACTGGGAAATGAGAGACCTCGT-3' and B2-antisense(–2438/–2414), 5'-GGGCCAGTAAGGCATTTGATCCAC-3'; A1-sense(–1018/–994), 5'-TCCTAGCCCAAGTGAACCGAGAAG-3' and A1-antisense(–637/–613), 5'-AGAGGAAGAAACTGAGGTCCTGGC-3'; A2-sense(–437/–417), 5'-GTAGTCCTCCCCAGGGTCAT-3' and A2-antisense(–244/–224), 5'-CCTTTAGCAGATCCTCGTGC-3'; and A3-sense(–210/–190), 5'-GCCGTGAAACTCAGCCTCTA-3' and A3-antisense(–46/–26), 5'-TACTGGTCTCCCGAGCTCAT-3'.

Biotinylated Oligonucleotide Pulldown Assay—Nuclear extracts were prepared from cultured cells using the high-salt buffer (20 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and Complete Mini protease inhibitors (Roche Applied Science) as described previously. After diluting with 2 volumes of low-salt lysis buffer without NaCl, 50 µg of the cell extracts were incubated at 30 °C for 10 min with either 0.1 nmol of the 5'-biotinylated double-stranded wild-type or mutant oligonucleotides (Invitrogen) previously coupled to streptavidin-agarose beads (Sigma) in the presence or absence of an appropriate amount of competitor non-biotinylated oligonucleotides for 1 h at 4 °C and then for 10 min at 30 °C. The beads were washed six times with phosphate-buffered saline and subjected to SDS-PAGE and Western blot analysis.

The sequences of oligonucleotides used for pulldown assays were as follows: S1S2-sense(–3174/–3138), 5'-CAATATTTATTTATATCCAGTATTTATTTTCAATAC-3' and S1S2-mutsense, 5'-CAATATCTGTCTGTATCCAGTGTCTGTCTTCAATAC-3'; S4-sense(–2631/–2595), 5'-ATTTTTCACATGTTTACAGAAAGCAGTCAACTGAGC-3' and S4-mutsense, 5'-GTCTTCCACACTTCTGCAGAAAGCAGTCAACTGAGC-3'; S5-sense, 5'-GTACTGTGGTCCAACATAAACACACAAGTCAGGCTGAG-3' and S5-mutsense, 5'-GTACTGTGGTCCAACCTTCTGCACAAGTCAGGCTGAG-3'; S6S7-sense(–385/–313), 5'-GAGGAGGGGGAATCAAACAGAAAGAGAGACAAACAGAGATATATC-3' and S6S7-mutsense, 5'-GAGGAGGGGGGGGCGGGCAGAGAGAGGGGCGGGCAGAGATATATC-3'; S8-sense(–130/–84), 5'-ATCGAGTTGTGCCTGGAGTGATGTTTAAGCCAATGTCAGGGCAAGG-3' and S8-mutsense, 5'-ATCGAGTTGTGCCTGGAGTGGGGGCCGGGCCGGGGGCAGGGCAAGG-3'.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
FoxM1, but Not FoxO3a, Expression Correlates with ER{alpha} Expression at the Transcriptional Level in Breast Cancer Cell Lines—To explore the possibility that FoxM1 regulates ER{alpha} expression, we first analyzed the correlation between the levels of expression of FoxM1 or FoxO3a and ER{alpha} in a panel of 16 different breast cell lines by Western blotting. As shown in Fig. 1A, 13 of the 16 breast carcinoma cell lines showed good correlations between the levels of FoxM1 and ER{alpha} expression. In contrast, only 6 of the 16 cell lines demonstrated positive correlation between the levels of FoxO3a and ER{alpha} expression. We next examined the mRNA levels of ER{alpha} in these cells by RT-qPCR (Fig. 1B). The result showed that, although the expression level of FoxM1 protein correlated with that of ER{alpha} mRNA in 13 of the 16 cell lines, only 6 of the 16 cell lines analyzed showed significant correlations between FoxO3a and ER{alpha} mRNA. Because the activity of FoxO3a is negatively regulated by Akt phosphorylation, the phosphorylation status of Akt was also studied using specific antibodies against phosphorylated and total Akt. The result again failed to account for the lack of correlation between FoxO3a and ER{alpha} expression. These data suggest that FoxM1, rather than FoxO3a, is the physiological regulator of ER{alpha} transcription in breast cancer cells.


Figure 1
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  		FIGURE 1.
FoxM1, but not FoxO3a, expression correlates with ER{alpha} expression at the transcriptional level in breast carcinoma cell lines. A, 16 different cancer cell lines, as indicated, were analyzed by Western blot using specific antibodies against ER{alpha}, FoxM1, FoxO3a, P-Akt, Akt, and actin, and their correlations were examined. B, ER{alpha} mRNA of these breast carcinoma cells was analyzed by RT-qPCR using the ABI PRISM 7700 sequence detection system and correlated with FoxM1 and FoxO3a expression. The expression levels of ER{alpha} protein and mRNA were correlated with the expression levels of FoxO3a and FoxM1 proteins in individual cell lines (1–16). + indicates positive correlation, and – denotes no correlation for each cell line.

 
Ectopic Expression of FoxM1, but Not FoxO3a, Results in Up-regulation of ER{alpha} Expression—To test the hypothesis that FoxM1, but not FoxO3a, regulates ER{alpha} expression, we transiently transfected the ER{alpha}-positive breast cancer cell line ZR-75–30 with different amounts of expression plasmids encoding for FoxM1 and a constitutively active FoxO3a(A3) and analyzed the expression levels of ER{alpha} and known FoxM1 and FoxO3a targets at 48 h after transfection by Western blotting. These results showed that ectopic expression of FoxM1 increased the expression levels of the FoxM1 targets Plk and Cdc25c (Fig. 2A). We also observed a moderate but significant increase in ER{alpha} expression in the ZR-75–30 cells overexpressing FoxM1 but not those overexpressing FoxO3a(A3). To further investigate whether FoxM1 was regulating the transcription of ER{alpha}, we analyzed these cells by RT-qPCR. These results showed an ~2-fold increase in ER{alpha} transcription when FoxM1 was overexpressed, which was not observed when FoxO3a(A3) was overexpressed (Fig. 2A).

Induction of FoxM1 Expression in an MCF-7 Tet-on System Results in an Increase in the Levels of ER{alpha} Expression/Transcription—To further confirm that FoxM1 regulates ER{alpha} expression, we generated an MCF-7 cell line (MCF-7 Tet-on FoxM1) stably transfected with the pTet-On (Clontech) and pcDNA4-FoxM1 plasmids in which the FoxM1 expression was inducible by the addition of doxycycline. As shown in Fig. 2B, treatment of this cell line with 2 µg/ml doxycycline increased the expression of FoxM1 with time, and this was accompanied by an increase in the expression of ER{alpha} and known FoxM1 targets such as Cdc25c and Plk. There was a modest down-regulation of FoxM1 expression at 24 h, and this was probably because of the negative cell proliferative effect of doxycycline. Nevertheless, this was accompanied by a similar decline in ER{alpha}, Cdc25c, and Plk expression, further confirming our hypothesis that FoxM1 regulates ER{alpha} expression. Notably, the level of FoxO3a expression remained constant up to 48 h and declined moderately at 72 h after doxycycline treatment, suggesting that the induction of ER{alpha} by FoxM1 is not mediated through modulating FoxO3a expression. It is notable that the level of ER{alpha} induction was lower in the ZR-75–1 cells compared with the MCF7 cells. The reason is unclear, but it is likely to be due to the fact that not all of the ZR-75–1 cells would be expressing FoxM1 at high levels during transient transfection, whereas the MCF7 (Tet-on FoxM1) is a clonal cell line in which all of the cells would express FOXM1 at high levels following induction. Moreover, the low level of ER{alpha} expression in the ZR-75–30 cells might also be the result of post-transcriptional regulation. To further address whether the regulation of ER{alpha} by FoxM1 was at the transcriptional level, we analyzed the ER{alpha} RNA level by RT-qPCR. The result showed an increase in FoxM1 transcription of 3.5- and 7.0-fold at 48 and 72 h after treatment with doxycycline, correlating with 2.5- and 4.7-fold increases in ER{alpha} transcript level. Similarly, the FoxO3a mRNA level remained constant throughout the time course. These results indicate that FoxM1 regulates ER{alpha} expression at the transcription level in the MCF-7 breast cancer cell line.


Figure 2
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  		FIGURE 2.
Expression of FoxM1, but not FoxO3a, results in up-regulation of ER{alpha} expression. A, ZR-75–30 cells were transfected with 0, 1, or 2 µg of either pcDNA3-FoxM1 or pLPC-FoxO3a(A3), and 48 h after transfection, the cells were analyzed by Western blot using specific antibodies as indicated (top panel). Gene transcripts of these cells were analyzed by RT-qPCR using the ABI PRISM 7700 sequence detection system (lower panels). B, MCF-7 Tet-on FoxM1 cells in which FoxM1 expression is inducible by the addition of doxycycline were treated with 2 µg/ml doxycycline for the times indicated and analyzed by Western blot using specific antibodies (top panel). The mRNA of these cells was analyzed by RT-qPCR using the ABI PRISM 7700 sequence detection system (lower panels).

 
Inhibition of FoxM1 Activity Leads to Down-regulation of ER{alpha} Expression—We next studied whether inhibition of FoxM1 activity would lead to down-regulation of ER{alpha} expression. To this end, we treated MCF-7 cells with 10 µM U0126 and performed a time course experiment as indicated in Fig. 3A. Western blot analysis showed down-regulation of the known FoxM1 targets Cdc25c and Cyclin B1 as early as 1 h after treatment. This was accompanied by a mobility shift and decrease in FoxM1 levels. The faster migration species of FoxM1 observed after treatment with U0126 probably reflects the dephosphorylated and inactive form of this protein. We also observed a similar down-regulation of ER{alpha} expression but only at 3 h after treatment. This probably highlights the fact that the half-life of ER{alpha} protein is longer than those of Cdc25c and cyclin B1. The levels of expression of FoxO3a and the FoxO3a targets Bim and p27Kip1 were constant throughout the time course, indicating that there was no change in FoxO3a activity. These results provide further evidence that FoxM1 is a physiological regulator of ER{alpha} expression in breast cancer cells. To extend these observations, we next studied the effects of FoxM1 and FoxO3a silencing on the expression of ER{alpha} in MCF-7 cells. To this end, we transfected siRNA vectors targeting FoxM1 or FoxO3a into MCF-7 cells and analyzed the protein expression at 72 h post-transfection by Western blotting. The results showed that abolishing FoxM1 expression in MCF-7 cells led to the abrogation of expression of ER{alpha} as well as FoxM1 targets, such as Cyclin B1, Plk, Cdc25b, and Cdc25c (Fig. 3B). Silencing of FoxO3a in MCF-7 cells resulted in the down-regulation of the known FoxO3a targets Bim and p27Kip1. Surprisingly, we also detected a moderate down-regulation of ER{alpha} and FoxM1 expression in these cells when compared with mock and control siRNA-transfected MCF-7 cells. The down-regulation of ER{alpha} in cells transfected with FoxO3a siRNA could be mediated through down-regulation of FoxM1 expression. The results also indicated that there were no significant changes in the expression and activity of ERK1/2 and Akt, which are upstream regulators of FoxM1 and FoxO3a, respectively. These results together established that ER{alpha} is a physiological transcriptional target of FoxM1.


Figure 3
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  		FIGURE 3.
Inhibition of FoxM1 activity and expression leads to down-regulation of ER{alpha} expression. A, MCF-7 cells were treated with 10 µM MEK inhibitor U0126 for the times indicated and analyzed by Western blot using specific antibodies. B, MCF-7 cells were transiently transfected with pTER-FoxM1 siRNA (left panel) or psiRNA-FoxO3a (right panel) plasmids. Seventy-two hours after transfection, the cells were analyzed by Western blot using specific antibodies as indicated.

 
FoxM1 Expression Activates the ER{alpha} Promoter-dependent Transcription Activity—To test whether FoxM1 regulates ER{alpha} at the gene promoter level, we next investigated the ability of FoxM1 and FoxO3a to activate the ER{alpha} promoter. To this end, the ER{alpha} promoters were transfected into COS and MCF-7 cells with various amounts of expression vectors for FoxM1, wild-type FoxO3a, or a constitutively active FoxO3a. The co-transfection experiments demonstrated that, although FoxM1 does not transactivate ER{alpha} promoter B (distal) efficiently, it effectively induced promoter A (proximal) activity in both COS and MCF-7 cells. Conversely, FoxO3a specifically transactivates ER{alpha} promoter B, but not promoter A, consistent with the previous report (20). However, it was also notable that the basal activity of promoter B was low when compared with A, suggesting that promoter A is the predominant promoter in these cell lines. Sequence analysis of the ER{alpha} promoters A and B identified a number of consensus Forkhead-responsive elements (FHREs), including sites S1, S2, and S4 on promoter B, previously identified by Guo and Sonenshein (20) to be inducible by FoxO3a (Fig. 4A). To then map the transcriptional elements responsible for mediating the induction by FoxM1, a series of deletion reporter constructs of the ER{alpha} promoters A and B were co-transfected with FoxM1 expression vector into COS cells and analyzed for transcriptional activity 24 h after transfection. Deletion of the region of the ER{alpha} promoter B (–4542 to –3038) containing the previously defined FHREs S1 and S2 reduced significantly the activation by FoxM1 from 1.3- to 1.1-fold. The average induction of the longest wild-type ER{alpha} promoter A (–1168/+190) after FoxM1 transfection was 3.3-fold, and the comparably low levels of induction by FoxM1 probably reflects the fact that the FoxM1 expression vector is not expressing a constitutively active form of FoxM1. Deletion of the 5' distal region (–1168/–513) only marginally reduced the ability of FoxM1 to induce the promoter activity. The fold of activation by FoxM1 reduced significantly to background level when the region (–513/+190) of the promoter containing three putative FoxM1 binding sites (S6, S7, and S8) was deleted. Similarly, a decrease in the level of induction was also observed when any one of the FHREs of the full-length ER{alpha} promoter A was mutated, suggesting that these FHRE sites are responsible for FOXM1 transactivation. Mutation of all three putative FHREs rendered the ER{alpha} promoter insensitive to FoxM1 induction. However, our transfection result also indicated that S8 is a weak FHRE compared with S6 and S7. Together, these transfection results suggested that the proximal ER{alpha} promoter FHREs S6, S7, and S8 primarily mediate the response to FoxM1 of the promoter.

FoxM1 and FoxO3a Bind to the Same Responsive Elements on the ER{alpha} Promoter—To further characterize these putative FHREs identified by promoter analysis and to demonstrate physical interaction of FoxM1 and FoxO3a with these sites, we used biotinylated oligonucleotides coupled to streptavidin-agarose beads to "pulldown" proteins interacting with different FHREs in MCF-7 cells and then analyzed the bound proteins by Western blotting. The result showed that FoxM1 bound to the S1/S2, S4, S6/S7, and S8 FHREs containing oligonucleotides but not to similar mutant oligonucleotides with the FHREs mutated, consistent with the transfection results. Surprisingly, FoxO3a was also shown to be interacting with the same FHREs. Similar to the deletion analysis results, we failed to demonstrate binding of FoxM1 or FoxO3a to the S5 FHRE. These interactions depend on the FHREs, as both the FoxM1 and FoxO3a binding could be competed off using increasing amounts of oligonucleotides containing the FHRE but not similar oligonucleotides with the FHRE mutated (Fig. 5A). These results together suggest that both FoxM1 and FoxO3a are recruited directly to the FHREs.

FoxM1 and FoxO3a Associate with the ER{alpha} Promoter in Vivo—We next performed ChIP assays to examine the in vivo occupancy of the ER{alpha} promoter by FoxM1 and FoxO3a. As demonstrated in Fig. 5B, both the anti-FoxM1 and anti-FoxO3a antibodies, but not the control antibodies (IgGs), precipitated the ER{alpha} promoter regions containing the S1/S2, S4, and S6/S7 FHREs. Consistent with the oligo pulldown result, the ChIP assay also illustrated that the anti-FoxM1 and anti-FoxO3a antibodies failed to precipitate promoter fragments containing the S5 site. Interestingly, although the pulldown experiment showed that the S8 FHRE could bind FoxM1 and FoxO3a, the ChIP assay failed to show either FoxM1 or FoxO3a binding to the promoter region containing S8 in vivo (Fig. 6). In general, the ChIP result confirms the findings from the co-transfection and pulldown experiments.


Figure 4
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  		FIGURE 4.
Characterization of the human ER{alpha} promoter and its activity in response to FoxM1 and FoxO3a expression. Schematic representation of the human ER{alpha} promoter showing the putative FHREs (top panel). A, comparison of the ability of FoxO3a and FoxM1 to transactivate the human ER{alpha} promoters A and B. MCF-7 and COS cells were transiently transfected with the 10-µg human ER{alpha} promoter A or B constructs together with increasing amounts (0, 2, 10, 20, and 50 µg) of pLPCFoxO3a(wt), A3, or pcDNA3FoxM1. Cells were harvested 24 h after transfection and assayed for luciferase activity. Values are corrected for co-transfected Renilla activity. All data shown represent the average of three independent experiments, and the error bars show the standard deviation. B, cycling COS cells were transfected with different human ER{alpha} promoter-luciferase reporter constructs as indicated together with 20 µg of pcDNA3FoxM1. The COS cells were harvested 24 h and assayed for luciferase activity. All values were corrected for co-transfected Renilla activity. The folds of induction by FoxM1 are shown on the right.

 
FoxO3a Interacts with FoxM1 in Vivo—With the chromatin immunoprecipitation and biotinylated oligonucleotide pulldown data indicating that FoxM1 and FoxO3a bind to the same elements on the ER{alpha} promoter, we next investigated whether these two proteins could interact in vivo. To this end, we performed immunoprecipitation assays using antibodies specific for FoxM1 or FoxO3a. The co-precipitated proteins were then identified by Western blotting. These results showed that the endogenous FoxM1 and FoxO3a proteins co-precipitated with one another in MCF-7 cells, thus confirming in vivo interaction between FoxM1 and FoxO3a.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Estrogens are powerful mitogens essential for the initiation and progression of human breast and other gynecological cancers. Because ER{alpha} mediates the effects of estrogens, endocrine agents, such as Tamoxifen, Raloxifene, and Fulvestrant (ICI 182,780), are frequently used to block their synthesis or their activities in breast cancer treatment regimes. The successes of endocrine therapies for breast cancer treatments are often hampered by the development of resistance to hormonal treatments. Although enhanced growth factor signaling (which induces both genomic and non-genomic activities of ER{alpha}) is the principal mechanism of acquired resistance, the major reason for de novo resistance to endocrine therapy is loss of ER{alpha} expression. Thus, unraveling the molecular mechanisms by which ER{alpha} transcription is regulated might provide vital information not only for the development of novel therapeutic strategies against endocrine-resistant ER{alpha}-positive tumors but also for understanding the loss of ER{alpha} in de novo resistant diseases.

In this study, we have identified FoxM1 protein as a physiological regulator of ER{alpha} expression in breast carcinoma cells. Our survey of a panel of 16 different breast cancer cell lines showed a good correlation (13/16) between FoxM1 expression and expression of ER{alpha} at both the protein and mRNA levels. We have also demonstrated that ectopic expression of FoxM1 in two different ER-positive breast cancer cell lines, MCF-7 and ZR-75–30, led to up-regulation of ER{alpha} expression at the protein and mRNA transcript levels. Moreover, treatment of MCF-7 cells with the MEK inhibitor U0126, which blocks ERK1/2-dependent activation of FoxM1, also repressed ER{alpha} expression. Furthermore, silencing of FoxM1 expression in MCF-7 cells using siRNA resulted in the almost complete abrogation of ER{alpha} expression. We have also shown that FoxM1 can activate the transcriptional activity of human ER{alpha} promoter primarily through three closely located FHREs located at the proximal region of the ER{alpha} promoter.


Figure 5
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  		FIGURE 5.
In vitro and in vivo binding of FoxM1 and FoxO3a to the putative Forkhead-responsive elements on the ER{alpha} promoter. Shown is a schematic representation of the human ER{alpha} promoter showing the putative FHREs as well as the regions corresponding to sequences used for DNA pulldown and chromatin immunoprecipitation PCR analyses (top panel). A, oligonucleotides containing different putative FHREs of the human ER{alpha} promoter were generated with 5' ends either free or conjugated to biotin molecules. DNA-binding nuclear extracts prepared from cycling MCF-7 cells were incubated with 1 µg of biotin-conjugated wild-type (wt) and mutant (mut) oligonucleotides; see the first two lanes and wt in all other lanes). In lanes 3–5 and lanes 6–8, increasing amounts (1, 5, and 10x molar excess) of competing non-conjugated oligos were incubated with the mix. Proteins binding to the biotinylated oligonucleotides were pulled down using streptavidine-agarose beads, and the washed lysates were analyzed using SDS-PAGE followed by immunoblotting using specific antibodies against FoxO3a and FoxM1. B, protein-DNA complexes from cycling MCF-7 cells were formaldehyde cross-linked in vivo. Chromatin fragments from these cells were subjected to immunoprecipitation with antibodies against IgG (nonspecific), FoxM1, or FoxO3a as indicated. After cross-link reversal, the co-immunoprecipitated DNA was amplified by PCR using the indicated primers and resolved in 2% agarose gels.

 
FoxO3a has previously been reported to regulate ER{alpha} transcription (20). Although we have confirmed in vitro and in vivo binding of FoxO3a to the ER{alpha} promoter and shown that overexpression of a constitutively active FoxO3a is able to transactivate region B of the ER{alpha} promoter in co-transfection studies, the results of our functional studies do not totally support the idea that FoxO3a is the primary physiological regulator of ER{alpha} transcription. We observed a poor correlation of 6/16 between FoxO3a expression levels and ER{alpha} expression in a larger panel of breast carcinoma cells compared with the previous study. Moreover, although ectopic expression of FoxM1 resulted in a significant increase in the level of ER{alpha} transcription in the ZR-75–30 breast cancer cell line, overexpression of the constitutively active FoxO3a did not lead to an up-regulation of ER{alpha} transcription in the same cell system. Although we did observe a moderate down-regulation of ER{alpha} expression in MCF-7 cells transfected with siRNA against FoxO3a, the decline in ER{alpha} expression could be a result of the down-regulation of FoxM1 expression as the level of FoxM1 expression was also significantly lowered in the same cells. Furthermore, given that FoxO transcription factors mediate cell cycle arrest and apoptosis whereas expression of ER{alpha} is associated with breast cancer cell proliferation, it is unlikely that FoxO3a is the primary physiological activator of ER{alpha} expression in breast cancer cells. However, paradoxically, a number of previous studies have shown that ER{alpha} and the proliferation marker Ki67 nuclear antigen are not usually expressed in the same breast tumor cells, leading to the suggestion that ER{alpha} regulates breast epithelial cell proliferation indirectly (35). Despite these earlier studies, more recent data demonstrates that ER{alpha} is expressed in proliferating breast epithelial cells and co-expresses with proliferative markers, including Ki67, MYC, cyclin D1, and stromal cell-derived factor (SDF)-1 (36). The failure in these earlier studies to demonstrate the co-expression of ER{alpha} and Ki67 in breast epithelial cells has been proposed to be due to the fact that the low mitotic index in these early samples and the short half-life and narrow expression window of Ki67 during the cell cycle have made the detection of transient co-expression of Ki67 and ER{alpha} difficult (36).


Figure 6
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  		FIGURE 6.
Analysis of FoxM1 and FoxO3a complexes in MCF-7 cells. Cell extracts prepared from cycling MCF-7 cells were immunoprecipitated (IP) with antibodies against FoxM1, FoxO3a, or a control antibody. The precipitated complexes were examined for FoxM1 and FoxO3a expression by Western blotting.

 
In exploring the mechanism by which FoxM1 regulates ER{alpha} expression, we performed mutation and deletion analyses of the ER{alpha} promoter and identified two FHREs located at the proximal region of the ER{alpha} promoter A to be primarily responsible for the transactivation of the ER{alpha} promoter by FoxM1. Further, our chromatin immunoprecipitation and biotinylated oligonucleotide pulldown assays indicate that FoxM1 binds directly to the FHREs of the ER{alpha} promoter to induce ER{alpha} transcription. Interestingly, these experiments also show that these FoxM1-bound FHREs are also occupied by FoxO3a. The significance of this observation is unclear, but it does suggest that FoxO3a could have a role in ER{alpha} transcription. One explanation is that FoxO3a could form a complex with FoxM1 to enhance FoxM1-dependent transcription activity. Supporting this hypothesis is the co-immunoprecipitation results showing that FoxO3a and FoxM1 interact in vivo. Indeed, different Forkhead subfamily members have been shown to form transcriptional complexes to regulate gene transcription. It has been demonstrated previously that FoxG1 can bind to FoxO3a in a Smad-containing complex to negatively regulate the cyclin-dependent kinase p21Cip1 expression (37). This observation not only indicates that Forkhead transcription factors can interact but also that they can cooperate to control target gene transcription.

In summary, we have presented comprehensive experimental evidence demonstrating that FoxM1 is a physiological regulator of ER{alpha} expression in breast cancer cells. We have also shown that FoxM1 regulates ER{alpha} expression at the transcriptional and promoter levels primarily through binding directly to FHREs located at the proximal region of the ER{alpha} promoter. Although we could detect binding of FoxO3a to the ER{alpha} promoter, we failed to demonstrate that FoxO3a can activate expression of endogenous ER{alpha} expression. However, our chromatin immunoprecipitation, biotinylated oligonucleotide pulldown, and co-immunoprecipitation results all suggest the possibility that FoxM1 and FoxO3a cooperate to regulate ER{alpha} gene transcription. Thus, despite an evident role of FoxM1 in ER{alpha} gene regulation, the function and mechanism of FoxO3a in regulating ER{alpha} expression in breast epithelial cells are still unclear and warrant further investigation.


   FOOTNOTES
 
* This work was supported by grants from Cancer Research United Kingdom, the Association for International Cancer Research, Leukemia Research Fund, Medical Research Council, Engineering and Physical Sciences Research Council, Fundação para a Ciência e a Tecnologia (Portugal), and the Research Grants Council of the Hong Kong Special Administrative Region (China) (HKU 7650/05M). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental data. Back

1 To whom correspondence should be addressed. Tel.: 44-20-8383-5829; Fax: 44-20-8383-5830; E-mail: eric.lam{at}imperial.ac.uk.

2 The abbreviations used are: ER, estrogen receptor; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; Fox, Forkhead box; siRNA, small interfering RNA; RT-qPCR, real-time quantitative PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ChIP, chromatin immunoprecipitation. Back


   ACKNOWLEDGMENTS
 
We thank Alice M. S. Cheung for constructing the pTER-hFoxM1 plasmid and Marco Da Costa for the psiRNA-hFoxO3a plasmid.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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