Biochimica et Biophysica Acta 1839 (2014) 297–305

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FOXM1 and androgen receptor co-regulate CDC6 gene transcription and DNA replication in prostate cancer cells Youhong Liu a, Zhicheng Gong b, Lunquan Sun a, Xiong Li a,⁎ a b

Center for Molecular Medicine, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan Province 410008, People's Republic of China Department of Pharmacy, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan Province 410008, People's Republic of China

a r t i c l e

i n f o

Article history: Received 20 September 2013 Received in revised form 19 February 2014 Accepted 20 February 2014 Available online 27 February 2014 Keywords: Transcription factor Transcription regulation DNA replication FOXM1 AR

a b s t r a c t CDC6 is a key component of the DNA replication initiation machinery, and its transcription is regulated by E2F or androgen receptor (AR) alone or in combination in prostate cancer (PCa) cells. Through both overexpression and knockdown approaches, we found that in addition to its effects on the E2F pathway, the cell proliferation specific transcription factor FOXM1 stimulated CDC6 transcription in cooperation with AR. We have identified a forkhead box motif in the CDC6 proximal promoter that is occupied by FOXM1 and is sufficient to drive FOXM1-regulated transcription. Indirectly, FOXM1 elevated AR protein levels and AR dependent transcription. Furthermore, FOXM1 and AR proteins physically interact. Using synchronized cultures, we observed that CDC6 expression is elevated near S phase of the cell cycle, at a time coinciding with elevated FOXM1 and AR expression and CDC6 promoter occupancy by both AR and FOXM1 proteins. Androgen increased the binding of AR protein to CDC6 promoter, and AR and FOXM1 knockdown decreased AR binding. These results provided new evidence for the regulatory mechanism of aberrant CDC6 oncogene transcription by FOXM1 and AR, two highly expressed transcription factors in PCa cells. Functionally, the cooperation of FOXM1 and AR accelerated DNA synthesis and cell proliferation by affecting CDC6 gene expression. Furthermore, siomycin A, a proteasome inhibitor known to inhibit FOXM1 expression and activity, inhibited PCa cell proliferation and its effect was additive to that of bicalutamide, an antiandrogen commonly used to treat PCa patients. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Prostate cancer (PCa) is the most commonly diagnosed malignancy and the second leading cause of cancer death in men in the United States [1]. An estimated 238,590 new cases and 29,720 deaths were projected for 2013 [2]. The growth of PCa cells depends on androgen activating androgen receptor (AR). AR is a member of the nuclear hormone receptor super-family that regulates the expression of target genes in a ligandinducible manner. It plays an important role in prostate gland development and also contributes to PCa progression [3]. AR mediates androgen-induced signaling by binding to the specific cis-regulatory regions of DNA sequences, known as androgen responsive elements (AREs) in both enhancer and promoter regions of AR-target genes, followed by the recruitment of basic transcription machinery as well as co-regulators to modulate transcription. Forkhead box M1 (FOXM1) is a cell proliferation-specific transcription factor that promotes G1/S and G2/M cell cycle transition [4]. FOXM1 and CDC25A proteins interact to regulate cell cycle progression [5]. FOXM1 expression is cell cycle specific, and FOXM1 protein is ⁎ Corresponding author. E-mail address: [email protected] (X. Li).

http://dx.doi.org/10.1016/j.bbagrm.2014.02.016 1874-9399/© 2014 Elsevier B.V. All rights reserved.

degraded during the mitotic exit [6]. Elevated expression of FOXM1 has been detected in tumor biopsies from PCa patients, especially hormone-refractory and metastatic prostate tumors [7]. Ectopic overexpression of FOXM1 accelerates the cell proliferation of PCa cells in TRAMP and LADY transgenic mice, and promotes the initiation and development of PCa [8]. Recent evidence suggests that FOXM1 may be involved in DNA replication, and that silencing FOXM1 blocked the cell cycle transition from G1 to S phase [9]. Siomycin A, an antibiotic thiazole compound, was identified as an inhibitor of FOXM1, down-regulating its transcriptional activity as well as its protein and mRNA abundance [10]. Siomycin A has shown significant anti-tumor properties in PCa cells by inducing apoptosis and inhibiting anchorage-independent growth [11]. Cell division cycle 6 (CDC6) is an essential regulator of DNA replication. It helps form a pre-replication complex at the origins of DNA replication in early G1 phase and initiates DNA replication during S phase. CDC6 is also involved in checkpoint mechanisms that coordinate S phase and mitotic entry. By tightly coupling DNA replication and the cell cycle S-M checkpoint, CDC6 ensures that the entire genome is replicated only once in each cell division [12]. The deregulation of CDC6 results in aberrant DNA replication, DNA damage and genomic instability [13]. CDC6 gene transcription is regulated by E2F transcription factors [14]. In PCa cells, AR has been identified as a transcription factor

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regulating the gene transcription of CDC6 by binding to the promoter region of CDC6 gene [15]. Additionally, androgen regulates the gene transcription of CDC6 through interactions between AR and E2F1 and E2F3 transcription factors [16]. We identified a potential FOX cis-regulatory binding motif in the CDC6 promoter, 5′-TGTTTGTT-3′ [17]. It is not determined if the motif is occupied by FOXM1 and how FOXM1 and AR, when highly co-expressed in the same PCa cells, co-regulate CDC6 gene expression. In the present report, we found that in addition to its effects on the E2F pathway, FOXM1 regulates CDC6 gene transcription through both direct effects and cooperation with AR. FOXM1 up-regulated CDC6 gene transcription by elevating AR levels in addition to direct binding to promoter and physically interacted with AR proteins. Androgen increased AR protein binding to the CDC6 promoter, and silencing FOXM1 decreased AR promoter binding. These results provided new evidence for the regulatory mechanism of aberrant CDC6 oncogene transcription by FOXM1 and AR in PCa cells. Additionally, we tested if knockdown of FOXM1 and AR inhibited DNA synthesis and cell proliferation by regulating CDC6. Finally, we evaluated the combined effect of a FOXM1 inhibitor, siomycin A, and the antiandrogen bicalutamide on cell proliferation.

(Santa Cruz, CA). The synergistic androgen methyltrienolone (R1881) was purchased from PerkinElmer (Waltham, MA). Bicalutamide and siomycin A were purchased from Sigma Aldrich. 2.4. Transient transfection LNCaP cells underwent DNA transfection using a Fugene 6 HD Transfection Kit (Promega, Madison, MI). siRNA transfection was performed using DharmaFECT Transfection Reagent (Thermo Fisher Scientific, Wilmington, DE). The experimental procedure followed the protocols provided by the manufacturer. 2.5. RT-PCR The total RNA was extracted using the RNeasy Mini QIAcube Kit in the QIAcube Robotic workstation (Qiagen, Valencia, CA). One microgram of total RNA was used for reverse transcription to cDNA using the High Capacity RNA to cDNA Master Mix (Applied Biosystems, Carlsbad, CA). The RT-PCR was run in the Applied Biosystems 7500 Fast and 7500 Real-Time PCR system. The primers specific for FOXM1, AR, CDC6, E2F1, E2F2, E2F3 genes and GAPDH were designed by Primer 3 software (version 0.4.0).

2. Materials and methods 2.6. Dual luciferase reporter gene assays 2.1. Cell culture LNCaP and HEK 293T cells were purchased from American Type Culture Collection, Manassas, VA. LNCaP cells were maintained in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/ streptomycin. HEK 293T cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin.

The cells were transfected with the plasmids as described in the individual experiments for 48 h. The plasmid pRL-SV40 (SV40 enhancer driven Renilla gene) was used as the transfection control. The cells were treated with R1881 in charcoal-stripped FBS (CSS) for 16 h. The cells were lysed with passive lysis buffer by the dual luciferase assay kit (Promega). The luciferase activities were tested in a luminometer. The results were expressed as the ratio of firefly to Renilla luciferase values.

2.2. Plasmids and siRNA 2.7. ChIP-qPCR FOXM1, AR and CDC6 cDNAs in the pCMV-XL5 vector were purchased from Origene (Rockville, MD). pGL3-CDC6-Luc, containing a CDC6 gene promoter from − 1700 bp to + 7 bp of the transcription start sites, was a gift from Dr. Joseph Nevins at Duke University. A primer was first designed containing the transcription start sites of CDC6 (around 20 bp) and enzymes XhoI and HindIII. The annealing of the primer and its complemental primer was performed and the fragment was inserted into a pGL3-basic vector to form pGL3-CDC6-TSS. The CDC6 promoter fragments containing the FOX binding motif or both FOX and ARE binding motifs were generated by PCR using pGL3CDC6-Luc as the template, and the fragments were digested by Nhe I and XhoI and inserted into pGL3-CDC6-TSS to form pGL3-CDC6-FOX or pGL3-CDC6-FOX-AR. The FOX binding motif in pGL3-CDC6-FOX was mutated using the site-directed mutagenesis kit from Finnzyme (Vantaa, Finland). FOXM1 cDNA was subcloned in the p3×FLAG-CMV-14 vectors (Sigma Aldrich, St. Louis, MI) at the HindIII and XbaI sites. The AR cDNA plasmid was purchased from Origene and subcloned inframe in the pCMV3Tag9 expression vector (Agilent, Santa Clara, CA) at the BamHI and XhoI restriction sites. pGL3-AR-Luc, containing an AR gene promoter/enhancer from −5400 bp to +800 bp of the transcription start sites, was a gift from Dr. Donald J. Tindall at Mayo Clinic. ONTarget plus SMARTpool small interfering RNA (siRNA) for the targets of AR, FOXM1, and non-targeting siRNA control was purchased from Dharmacon Inc. (Lafayette, CO) via Thermo Fisher Scientific. 2.3. Antibodies and chemicals Specific antibodies against CDC6 and AR were purchased from Cell Signaling (Danvers, MA). Antibodies against FOXM1, FOXA1, Flag, Myc, E2F1, E2F2, E2F3, and horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG were purchased from Santa Cruz Biotechnology

LNCaP cells were transfected with or without siRNA of AR (si-AR), siRNA of FOXM1 (si-FOXM1) or non-targeting control (si-CTR) in CSS for 48 h, followed by the treatment with or without R1881 for 16 h. The cells were harvested and used for ChIP assays performed using Chromatrap Pro-A Spin Columns from the Porvair Filtration Group Ltd. (Ashland, VA). For the immunoprecipitation of formaldehyde crosslinked chromatin-protein complexes, the antibodies against FOXM1, FOXA1 and AR were used or IgG was used as the negative control. The same amount of chromatin without antibody incubation was used as the input control. The reaction mixtures were eluted in Chromatrap Pro-A spin columns following the manufacturer's protocol. All ChIP experiments were repeated at least three times. 2.8. Co-immunoprecipitation HEK-293T cells (1 × 106) were co-transfected with 4 μg of p3×FLAGCMV-14-FOXM1 (or control p3×FLAG-CMV-14) and 4 μg of pCMV3Tag9-AR (or control pCMV-3Tag9) using Lipofectamine 2000. After 48 h, cells were lysed in M-PER containing protease and phosphatase inhibitors. Soluble fractions were incubated with anti-FLAG M2 antibody resin (Sigma) overnight at 4 °C with end-over-end mixing. The resin was washed three times in BupH TBS, pH 7.2 (Thermo Fisher) containing 0.1% Tween 20 and eluted with 3×FLAG peptide (Sigma). The eluted protein complex was then analyzed by Western blotting. To detect the endogenous protein interactions of FOXM1 and AR, LNCaP cells were treated with R1881 in CSS for 16 h. Nuclear extracts of LNCaP cells were pre-cleared with protein G sepharose (Sigma Aldrich) and normal rabbit IgG overnight at 4 °C with end-over-end mixing. The cell lysates were incubated with anti-FOXM1 or anti-AR antibody (Santa Cruz) overnight at 4 °C and then with protein G sepharose

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for 1 h at 4 °C. The resin was washed three times, and the eluted protein complex was analyzed using anti-AR or anti-FOXM1 antibody (Santa Cruz) by Western blotting.

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partly by regulating AR gene transcription in addition to its cooperation with AR. 3.2. FOXM1 protein directly binds to the FOX motif in the CDC6 promoter

2.9. DNA replication assay LNCaP cells were transfected with FOXM1 siRNA and AR siRNA alone or in combination, together with pCMV-XL5 or pCMV-XL5-CDC6 plasmid DNA. Non-targeting control siRNA was used as the control. The cells were starved of serum for 48 h and synchronized to G0/G1 stage. The cells re-entered the cell cycle after 10% CSS with or without R1881 add-back. The cells were harvested 16 h after serum add-back and DNA synthesis was evaluated with ClickiT® EdU Flow Cytometry Assay Kits (Invitrogen, Carlsbad, CA). 2.10. Cell proliferation assay LNCaP cells were transfected with FOXM1 siRNA and AR siRNA alone or in combination, together with pCMV-XL5 or pCMV-XL5-CDC6 plasmid DNA for 48 h. Non-target control siRNA was used as the control. Then the cells were treated with or without R1881 for 16 h. The cells were stained with trypan blue and cell number was counted 48 h after androgen addition. The experiments were performed in quadruplicate. LNCaP cells were cultured in 10% CSS, and treated with bicalutamide and/or siomycin A at the dose window shown in Fig. 6. R1881 was used together with or without drug treatment. Cell proliferation was assessed by MTS assay (Promega, Beijing, China) 72 h after drug treatments. 2.11. Statistical analysis The Microsoft Excel Program was used to calculate SD and statistically significant differences between samples using ANOVA or Student's t test. 3. Results 3.1. CDC6 gene transcription is positively regulated by FOXM1 in cooperation with AR and FOXM1 regulates AR gene expression An AR binding element (ARE) motif was recently identified in the CDC6 promoter region, and modulates CDC6 gene transcription [15]. FOXM1, like AR, is involved in DNA replication and G1/S cell cycle transition [9,18,19]. To clarify if FOXM1, similar to AR, regulates CDC6 gene expression, we tested CDC6 mRNA and protein levels when FOXM1 or AR was knocked down by siRNA alone or in combination in ARpositive and androgen sensitive LNCaP PCa cells. Knockdown of FOXM1 or AR significantly decreased CDC6 mRNA levels by around 43% and 38%, and double knockdown of FOXM1 and AR further decreased CDC6 mRNA levels by around 76%. It is interesting that the knockdown of FOXM1 also decreased AR mRNA levels by around 40% (Fig. 1A). Consistent with the mRNA level, knockdown of AR and/or FOXM1 decreased CDC6 protein levels and the knockdown of FOXM1 decreased AR protein levels as well (Fig. 1B). R1881, a synthetic androgen and an AR agonist, significantly increased CDC6 mRNA levels. The knockdown of FOXM1 or AR reversed R1881-increased CDC6 mRNA levels, and knockdown of FOXM1 and AR further reversed R1881increased CDC6 mRNA levels (Fig. 1C). To validate the impact of FOXM1 on AR gene expression, we assessed AR mRNA and protein expression levels when FOXM1 was elevated by the transient transfection of plasmids expressing FOXM1. FOXM1 significantly elevated AR mRNA and protein levels (Fig. 1D and E). The results were confirmed when pGL3-AR-Luc containing AR promoter fragment was co-transfected with FOXM1-expressing plasmids. FOXM1 significantly increased AR promoter activity (Fig. 1F). These results suggested that FOXM1 regulates CDC6 gene expression

In the present report we identified a FOX binding motif 5′-TGTTTG TT-3′ according to the literature [17], as well as a binding motif of androgen-response element (ARE, 5′-AGAACATAATATTCT-3′) at the CDC6 gene (NM_001254.3) promoter by using the TESS Transcription Elements Search System (http://www.cbil.upenn.edu/cgi-bin/tess/ tess) (Fig. A1). To test the hypothesis that FOXM1 transcription factor regulates CDC6 gene transcription by directly binding to the FOX motif TGTTTGTT at the CDC6 gene promoter, we assayed the protein–DNA binding of FOXM1 in LNCaP cells using chromatin immunoprecipitation (ChIP)-qPCR. FOXM1-bound chromatins were captured by ChIP assay using FOXM1 antibody, and quantitative PCR was used to test the binding of FOXM1 protein to CDC6 gene promoter. The primers were designed spanning the TGTTTGTT binding motif (primer set A) or lacking the binding motif (primer set B). To test if the FOX binding motif at the CDC6 promoter was occupied by other members in the FOX protein family, we used FOXA1 antibody as the control. FOXM1 and FOXA1 express at a relatively high levels in LNCaP cells (Fig. A2). Both FOXM1 and FOXA1 proteins bound to the DNA fragment containing the FOX binding motif to various degrees (8.14-fold for FOXM1 and 3.62-fold for FOXA1), but did not bind to the fragment without the binding motif (Fig. 2A). The results confirmed that the FOX binding motif TGTTTGTT at the CDC6 promoter is commonly occupied by both FOXM1 and FOXA1 proteins to different degrees. 3.3. The FOX binding motifs are sufficient to drive FOXM1-activated gene transcription We tested the roles of the FOX binding motif TGTTTGTT in CDC6 gene transcriptional activation using promoter deletion and site-directed mutagenesis assays. The promoter fragments containing the FOX binding motif were generated on the basis of pGL3-CDC6 containing full length CDC6 cDNA (pGL3-CDC6-FOX). As shown in Fig. 2B, FOXM1 significantly increased the luciferase activity of pGL3-CDC6-FOX (7.2-fold), even more than pGL3-CDC6, which contains the full-length of CDC6 promoter (6.1-fold), while FOXA1 increased the luciferase activity of promoter fragments to a lesser degree (2.9-fold). Next, we mutated the FOX binding sequence from TGTTTGTT to TGCCCGTT in pGL3CDC6-FOX. We compared the luciferase activities responding to the ectopic FOXM1 or FOXA1 protein between the CDC6 promoter fragments containing wild-type or mutated FOX binding motifs. The mutation of the FOX binding motif significantly blocked FOXM1-increased luciferase activity (from 61.2 down to 14.2), as well as FOXA1-increased luciferase activity (from 26.4 down to 9.2). The data validated that the binding of FOXM1 to the FOX motif is sufficient to drive FOXM1-activated gene transcription of CDC6. 3.4. FOXM1 and AR coordinate the regulation of CDC6 promoter activity It has been reported that CDC6 gene expression is modulated in an androgen-dependent fashion, with transcriptional activation mediated by the ARE motif in the CDC6 promoter [15]. Additionally, we identified that the FOX binding motif was occupied by FOXM1 and promoted the gene transcription of CDC6. To test whether FOXM1 and AR transcription factors coordinate the regulation of CDC6 gene transcription, we constructed a CDC6 promoter fragment containing FOX and ARE motifs, but not E2F motif in the pGL3-CDC6 vector (pGL3-CDC6-FOX-AR, Fig. 2C). FOXM1 and/or AR cDNA encoding plasmids were cotransfected with pGL3-CDC6-FOX-AR into LNCaP cells. Forty-eight hours post-transfection, the cells were treated with or without R1881 for an additional 16 h. The effects of FOXM1 and/or AR on the promoter activity of pGL3-CDC6-FOX-AR were evaluated by luciferase assay.

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Fig. 1. CDC6 gene expression is positively regulated by FOXM1 in cooperation with AR. A. LNCaP cells were transfected with siRNA of FOXM1 (si-FOXM1) or AR (si-AR) alone or in combination. Non-target siRNA was used as control (si-CTR). Forty eight hours post-transfection, mRNA levels were analyzed by RT-PCR using specific primers against FOXM1, AR and CDC6, with GAPDH used as a control. The results were expressed as mean ± S.E. of triplicate reactions. B. Protein was extracted by RIPA cell lysis buffer, and 30 mg of protein was probed by Western blot with antibodies against FOXM1, AR and CDC6. The antibody against β-actin was used as a loading control. C. LNCaP cells were transfected with si-FOXM1 or si-AR alone or in combination. Non-target siRNA was used as a control (si-CTR). Cells were cultured in media with 10% CSS for 48 h, followed by treatment with 10 nM R1881 for an additional 16 h. The mRNA levels were analyzed by RT-PCR using specific primers against CDC6 and GAPDH as a control. The results were expressed as mean ± S.E. of triplicate reactions. D.–E. LNCaP cells were transfected with pCMV-XL5-FOXM1 for 48 h. pCMV-XL5 was used as the vector control. D. The cells were cultured in media with 10% CSS, and the mRNA level of CDC6 gene was evaluated by RT-PCR. GAPDH was used as the control. E. The CDC6 protein level was evaluated by Western blot. F. FOXM1 regulates AR promoter activity. LNCaP cells were transfected with pGL3-AR-Luc together with pCMV-XL5-FOXM1. pCMV-XL5 was used as the control, and pRL-SV40 expressing Renilla luciferase was used as an internal control. The cells were cultured in 10% CSS for 64 h. The cells were assayed for dual luciferase activity. Data were normalized to Renilla luciferase activities and presented as the mean ± SD of triplicate reactions.

FOXM1 and AR each significantly increased, and FOXM1 plus AR further increased, the promoter activity. Androgen (R1881) further increased the FOXM1 and/or AR-increased promoter activity (Fig. 2C). On the other hand, in the presence or absence of androgen, FOXM1 siRNA and AR siRNA each significantly decreased, and their combination further decreased the promoter activity (Fig. 2D). These results confirmed that FOXM1 and AR co-coordinated the regulation of CDC6 gene promoter activity. 3.5. FOXM1 transcription factor regulates CDC6 gene expression via the E2F pathway It has been reported that CDC6 gene transcription is regulated by E2F/retinoblastoma (Rb) protein [14]. In PCa cells, AR regulates CDC6 gene transcription by direct binding to the promoter [15] or through interactions between AR and E2F1 and E2F3 [16]. In the present study, we tested the influence of FOXM1 on the gene expression of E2F family members. As shown in Fig. A3, the knockdown of FOXM1 by siomycin A, a proteasome inhibitor known to inhibit FOXM1 expression and activity, significantly down-regulated the mRNA and protein levels of E2F1, E2F2 and E2F3, which are known activators of CDC6 gene transcription. The regulation of E2F1, E2F2 and E2F3 by FOXM1 was confirmed by siRNA knockdown. Therefore, in addition to the above mechanisms where FOXM1 directly binds to CDC6 promoter and indirectly regulates AR gene expression and cooperates with AR, FOXM1 also regulated CDC6 gene transcription through the E2F pathway. 3.6. FOXM1 protein physically interacts with AR protein Both FOXM1 and AR are highly expressed in LNCaP cells, and coordinated the regulation of CDC6 gene transcription by direct binding to individual motifs in the CDC6 promoter, which suggested a possible physical interaction between FOXM1 and AR proteins. We assessed

the protein interaction by using co-immunoprecipitation. FOXM1 was tagged with 3×FLAG by cloning the gene into the p3×Flag-CMV14 vector, and AR was tagged with 3×MYC by cloning the gene into the pCMV3TAG9 vector. HEK 293T cells were co-transfected with plasmids as shown in Fig. 3A. FOXM1-3×FLAG protein was immunoprecipitated from the soluble fraction with an anti-FLAG resin. AR-3×MYC protein was co-immunoprecipitated, indicating that it is complexed through protein–protein interactions with FOXM1-3×FLAG (Fig. 3A). The endogenous interaction of FOXM1 and AR proteins was also detected in LNCaP cells. The nuclear protein was extracted when LNCaP cells were stimulated with R1881 for 16 h. As shown in Fig. 3B, androgen activated AR and translocated the protein to the nuclei, thereby increasing the accumulation of AR protein inside the nuclei. FOXM1 protein was detected in the protein complex captured by the AR antibody, but undetected in the IgG-captured protein complex. In the meantime, AR protein was also detected in the protein complex captured by the FOXM1 antibody, but undetected in the IgG-captured protein complex. The results indicated that endogenous FOXM1 actively interacted with AR protein within the nuclei when AR was activated by androgen. 3.7. The levels of FOXM1 and AR proteins coincide with CDC6 mRNA and protein during the cell cycle LNCaP cells were rendered quiescent by serum withdrawal for 48 h, and then stimulated by medium containing 10% CSS. The cells were harvested for cell cycle analysis at a series of time points after serum addition. As shown in Fig. A4, most cells were at G0/G1 phase at 4 h, at S phase at 20 h and at G2/M phase at 28 h. We assessed the levels of FOXM1, AR and CDC6 proteins at G0/G1 phase, S phase and G2/M phase. The levels of FOXM1 and AR proteins increased at S phase compared to the G0/1 and G2/M phases, and correlate with CDC6 protein levels during the cell cycle (Fig. 4A). The mRNA level of CDC6 gene also increased at S phase (Fig. 4B). The correlation of protein levels of

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Fig. 2. FOXM1 protein binds to the CDC6 promoter, and FOXM1 and AR coordinate the regulation of CDC6 promoter activity. A. The FOXM1-bound chromatins of LNCaP cells were captured by ChIP assay using anti-FOXM1 antibody. Anti-FOXA1 and IgG were used as the controls. Quantitative PCR was performed using the primer sets shown, and the PCR products were detected by electrophoresis. Primer set A was designed to span the FOX binding motif, and primer set B lacking FOX binding motif was used as a control. B. The FOX binding motif confers CDC6 gene transcriptional regulation of FOXM1 and FOXA1. The CDC6 promoter fragments containing the full length (pGL3-CDC6) and FOX binding motif (pGL3-CDC6-FOX) were generated. LNCaP cells were transfected with pGL3-CDC6 or pGL3-CDC6-FOX, together with pCMV-XL5-FOXM1 or pCMV-XL5-FOXA1. pCMV-XL5 vector was used as the control. pRL-SV40 expressing Renilla luciferase was used as an internal control. The cells were assayed for dual luciferase activity 48 h after DNA transfection. Data were normalized to Renilla luciferase activities and presented as the mean ± SD of triplicate reactions. C. The CDC6 promoter fragments containing FOX and ARE binding motifs were generated on the basis of pGL3-CDC6 (pGL3-CDC6-FOX-AR). LNCaP cells were transfected with pGL3-CDC6-FOX-AR, together with pCMV-XL5-FOXM1 and/or pCMV-XL5-AR. pCMV-XL5 vector was used as the control. The cells were cultured in media containing 10% CSS. Forty-eight hours post-transfection, the cells were treated with or without synthetic androgen R1881 (10 nM) for an additional 16 h. pRL-SV40 expressing Renilla luciferase was used as an internal control. The cells were assayed for dual luciferase activity. Data were normalized to Renilla luciferase activities and presented as the mean ± SD of triplicate reactions. D. LNCaP cells were transfected with pGL3-CDC6-FOX-AR, together with si-FOXM1 and/or si-AR, with the non-target siRNA used as the control (si-CTR). The cells were cultured in media with 10% CSS. Forty-eight hours post-transfection, the cells were treated with or without 10 nM R1881 for an additional 16 h and assayed for luciferase activity. The results were expressed as the mean ± S.E. of triplicate reactions.

FOXM1 and AR and CDC6 mRNA levels suggested that the degree of FOXM1 and AR protein binding to the CDC6 promoter may change at each cell cycle phase. We tested the binding activity of FOXM1 and AR proteins to the CDC6 promoter at each cell cycle phase. Consistent with the protein levels of FOXM1 and AR, the binding degree of FOXM1 and AR proteins to the CDC6 promoter increased at S phase compared to the G0/G1 and G2/M phases (Fig. 4C and D). The increase of FOXM1 and AR protein levels and their binding to the CDC6 promoter possibly conferred increased mRNA levels of CDC6 at the S phase.

AR binding to the ARE binding motifs at the CDC6 promoter, androgen increased AR binding activity around 3-fold. In reverse, AR occupation of the ARE site decreased when AR protein level was reduced by siRNA, even in the presence of androgen (Fig. 4F). It is interesting that decreasing the FOXM1 protein level resulted in a decline of AR protein binding to the CDC6 promoter. Neither androgen nor AR siRNA influences the binding of FOXM1 transcription factor to CDC6 promoter (Fig. 4G). 3.9. The knockdown of FOXM1 and/or AR functionally inhibited DNA replication and cell proliferation partly through CDC6

3.8. The levels of FOXM1 affected the DNA binding activity of AR FOXM1 and AR proteins interact. It is unknown how FOXM1 and AR proteins interact to affect their individual DNA binding to the CDC6 promoter, which directly decides the CDC6 gene transcription level. We tested the binding activity of AR and FOXM1 proteins to the CDC6 gene promoter when the nuclear protein levels of AR were elevated by androgen stimulation, or the levels of AR or FOXM1 were decreased by siRNA knockdown. The protein levels of FOXM1 and AR within the nuclei were first assessed by immunoblot. Androgen increased the nuclear protein levels of AR. Consistent with the data in Fig. 1, the knockdown of FOXM1 by siRNA also decreased the protein levels of AR (Fig. 4E). The FOXM1 or AR-bound chromatin was captured by ChIP assay using antibodies against FOXM1 or AR in LNCaP cells, with IgG used as the negative control. The binding activity of FOXM1 or AR proteins to the CDC6 promoter was evaluated by quantitative real-time PCR. For

FOXM1 and AR regulated CDC6 gene transcription alone and in combination. CDC6 is an essential factor for DNA replication in eukaryotic cells. We tested if down-regulation of FOXM1 and AR functionally influenced CDC6-regulated DNA replication and cell proliferation. We first assessed DNA replication when FOXM1 and/or AR were down-regulated by siRNA knockdown. LNCaP cells were starved of serum for 48 h after siRNA transfection, and synchronized to G0/G1 stage. Around 30–40% of the control cell population was at S phase 16–20 h after 10% CSS add-back (Fig. A1). The cells were stained with EdU, and the positive cells were analyzed by flow cytometry. Knockdown of either FOXM1 or AR alone significantly decreased DNA synthesis (12.5% or 9.2%), and double knockdown of FOXM1 and AR further decreased DNA synthesis (23.8%). Furthermore, we tested if CDC6 cDNA reversed the siRNA-decreased DNA synthesis. Co-transfection of siRNAs of FOXM1 and/or AR and CDC6 cDNA rescued DNA synthesis (Fig. 5A). The results indicated that FOXM1 and AR regulated DNA

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Fig. 3. FOXM1 protein physically interacts with AR protein. A. The interaction between FOXM1 and AR proteins was determined by co-immunoprecipitation. HEK 293T cells were cotransfected with p3×Flag-CMV14-FOXM1 (FOXM1-3×Flag) or the empty vector control p3×Flag-CMV14, along with pCMV3Tag9-AR (AR-3×MYC) or the empty vector control (pCMV3Tag9). Lysates were immunoprecipitated with anti-Flag agarose resin, separated by PAGE, and electroblotted to PVDF. Western blot analysis showed that AR-3×MYC coimmunoprecipitated with FOXM1-3×Flag, indicating that these two expressed proteins interact. B. The endogenous interaction of FOXM1 and AR proteins was detected by coimmunoprecipitation. LNCaP cells were cultured in media with 10% CSS for 48 h, followed by treatment with or without R1881 (10 nM) for an additional 16 h. The nuclear protein was extracted, and the FOXM1 protein complex was immunoprecipitated by FOXM1 antibody. Then AR protein was identified by Western blot using anti-AR antibody. The AR protein complex was immunoprecipitated by AR antibody, and then FOXM1 protein was identified by Western blot using anti-FOXM1 antibody.

synthesis through influencing CDC6. Androgen (R1881, 1 nM) slightly enhanced DNA synthesis, and the effect was weakened when FOXM1 or AR was down-regulated, indicating that FOXM1 or androgenactivated AR participated in DNA synthesis by regulating CDC6. We next tested the influence of FOXM1 and AR knockdown on LNCaP cell proliferation. Cell proliferation was significantly suppressed by FOXM1 or AR knockdown (around 40% or 46%), and the double knockdown of FOXM1 and AR further suppressed cell proliferation

(to around 75%, P b 0.01, Fig. 5B). It has been reported that androgenactivated AR enhanced cell proliferation and G1/S cell cycle progression by upregulating CDC6 gene transcription [15]. Since FOXM1 is another cell proliferation-specific transcription factor regulating the cell cycle regulatory genes [9], we hypothesized that similar to AR, the knockdown of FOXM1 would decrease cell proliferation partly by affecting CDC6 gene expression. As shown in Fig. 5B, in the presence or absence of androgen the co-transfection of CDC6 cDNA and siRNA of

Fig. 4. FOXM1 and AR protein levels and binding of protein–DNA coincide with CDC6 mRNA and protein levels during cell cycle phases. LNCaP cells were rendered quiescent by serum withdrawal for 48 h, and then stimulated by medium containing 10% CSS to re-enter the cell cycle. Cells were harvested for cell cycle analysis at a series of time points after the serum addition. A. The levels of FOXM1, AR and CDC6 proteins at G0/G1 phase, S phase and G2/M phase were assessed by immunoblot. B. The mRNA levels of CDC6 at G0/G1 phase, S phase and G2/M phase were assessed by RT-PCR. C. and D. The binding of FOXM1 or AR proteins to the CDC6 promoter was tested by ChIP-PCR assay. FOXM1 or AR-bound chromatins of LNCaP cells at each cell cycle phase were prepared along with anti-FOXM1 or AR antibodies. The ChIP assays were performed in Chromatrap Pro-A Spin Columns by following the manufacturer's manual. DNA was analyzed via quantitative real-time PCR. The primers spanning the FOXM1 or AR binding sequence were designed, and primers lacking the FOXM1 and ARE binding sites were used as controls. All ChIP experiments were repeated at least three times. E.–G. The levels of FOXM1 affect the DNA binding activity of AR to CDC6 promoter. LNCaP cells were transfected with AR-siRNA or FOXM1-siRNA. The cells were cultured in media with 10% CSS for 48 h followed by treatment with or without 10 nM R1881 for an additional 16 h. The non-targeted siRNA was used as control (si-CTR). E. The nuclear proteins were extracted and the protein levels of FOXM1 and AR within the nuclei were assessed by immunoblot. F.–G. Chromatin was extracted by ChIP assay using FOXM1 or AR antibodies. IgG was used as the control. The binding activity of FOXM1 or AR proteins was tested by quantitative real-time PCR. The FOXM1 or AR binding activity was evaluated by comparing the CT value of the samples vs. the input. F. AR binding to ARE; G. FOXM1 binding to FOX consensus motif.

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Fig. 5. Knockdown of FOXM1 and/or AR significantly suppress DNA replication and cell proliferation partly through CDC6. LNCaP cells were transfected with si-FOXM1 or si-AR alone or in combination for 48 h. Non-target siRNA was used as the control (si-CTR). pCMV-XL5 or pCMV-XL5-CDC6 plasmids were co-transfected with siRNAs to test the influence of CDC6 on the siRNA-suppressed DNA replication and cell proliferation. A. The same number of LNCaP cells (1 × 106/well) was cultured in serum-free media for 48 h after transfection, and re-entered the cell cycle after 10% CSS with or without R1881 (1 nM) add-back. The cells were harvested 16 h after serum add-back and DNA synthesis was evaluated. Cells were cultured with EdU (5-ethynyl-2′-deoxyuridine), and the positive cells at S phase were analyzed by flow cytometry. B. The same number of cells (1 × 106/well) was cultured in media containing 10% CSS for 48 h, followed by treatment with or without synthetic androgen R1881 (1 nM) for an additional 16 h. The cell number was counted and cell viability was evaluated 48 h after androgen addition. The cell viability ratio of treatments to the control represents the cell proliferation rate.

FOXM1 and/or AR reversed siRNA-induced suppression of cell proliferation. Androgen (R1881, 1 nM) accelerated cell proliferation, and the effect was weakened when FOXM1 or AR was down-regulated. The results indicate that in addition to AR, FOXM1 regulates cell proliferation by affecting CDC6 gene expression. 3.10. Siomycin A significantly suppressed LNCaP cell proliferation when combined with bicalutamide Bicalutamide is an anti-androgenic drug competitively inhibiting the binding of dihydrotestosterone (DHT) to androgen receptor (AR),

thus interfering with androgen action. Anti-androgenic compounds have been commonly used to treat PCa patients, but many patients develop to androgen-refractory status and become resistant to antiandrogenic compounds [19]. We tested if bicalutamide suppresses cell proliferation better when combined with siomycin A, a macrocyclic thiazole antibiotic and FOXM1 inhibitor reported to have an anti-tumor effect on PCa [10]. Doses of siomycin A and bicalutamide as shown in Fig. 6 were used to treat LNCaP cells, and cell proliferation was assessed by MTS assay 72 h after drug treatments. Siomycin A or bicalutamide alone suppressed cell proliferation in a dose–response fashion. From the single treatment results, we identified the best doses of siomycin

Fig. 6. Siomycin A inhibits prostate cancer cell proliferation, and is additive to the anti-androgen bicalutamide. LNCaP cells (1 × 104/well in 96-well plate) were treated with bicalutamide or siomycin A at dose windows as shown in the figure, in media containing 10% CSS. The cells were co-treated with or without R1881 (1 nM) and cell viability was assessed by MTS assay 72 h after drug treatment. From the single treatment results, the best doses of siomycin A (0.125 μM) and bicalutamide (25 μM) were identified to test the effects of combining the two compounds. A. The combination of bicalutamide (25 μM) with a series of doses of Siomycin A. B. The combination of siomycin A (0.125 μM) with a series of doses of bicalutamide. Log10(M) was used (X-axis, M represents the drug concentration). C. Model describing the regulatory mechanism of aberrant CDC6 oncogene transcription by FOXM1 and AR in PCa cells.

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A (0.125 μM) and bicalutamide (25 μM) to test the effects of combining the two compounds. Bicalutamide (25 μM) significantly suppressed cell proliferation when combined with siomycin A at the dose window from 0.0625 μM to 4 μM (P b 0.01, Fig. 6A). On the other hand, siomycin A (0.125 μM) significantly suppressed cell proliferation when combined with bicalutamide at the dose window from 6.25 μM to 400 μM (P b 0.01, Fig. 6B). Androgen (R1881, 1 nM) accelerated cell proliferation, and the effect was weakened when androgen was used together with bicalutamide and siomycin A. The results showed that siomycin A is additive to bicalutamide for inhibiting the proliferation of PCa cells. These cell functional data support that FOXM1 and AR collaborate to regulate CDC6 gene expression, thereby promoting DNA replication and cell proliferation. 4. Discussion CDC6 gene expression is regulated by E2F proteins by direct binding to the promoter [14,20]. FOXM1 is a cell proliferation specific transcription factor that regulates cell cycle progression. We reported that FOXM1 regulates CDC25A gene transcription via direct promoter binding and indirect activation of E2F-dependent pathways [5]. In the present report, the knockdown of FOXM1 by siomycin A or siRNA decreased the gene expression of E2F1, E2F2 and E2F3, activators of CDC6 gene transcription, to various degrees (Fig. A3). It has been reported that AR and E2F1 and E2F3 proteins form a complex to regulate CDC6 gene transcription [16]. Therefore, FOXM1 and AR regulate CDC6 gene transcription in common through the E2F pathway. In the present study we reported that CDC6 gene expression was regulated by FOXM1 and AR transcription factors alone or in combination. A FOX cis-consensus motif (TGTTTGTT) has been identified in the CDC6 promoter and is occupied by FOXM1 protein. The ectopic FOXM1 only activated the CDC6 promoter fragment containing FOX binding motif, and the mutation of the FOX binding motif prevented activation. These data showed that the FOX binding motifs at the CDC6 proximal promoter are sufficient to drive FOXM1-activated CDC6 gene expression. In PCa cells, CDC6 gene expression is under the regulatory control of AR in a cell cycle dependent manner, and the ARE binding motif at the CDC6 promoter is responsible for androgen-dependent CDC6 transcription [15]. In addition, AR, as a component of the pre-replication complex, drives the progression of LNCaP cells from G1 to S phase through protein interaction with CDC6 [21]. The growth of PCa cells depends on androgen activating AR. AR protein is degraded at mitosis during each cell cycle to re-initiate DNA replication in the next cell cycle, and is considered as one of the licensing factors contributing to the initiation of DNA replication [22]. Similar to AR, FOXM1 expression is cell cycle specific, and FOXM1 protein is degraded during the mitotic exit [13]. Recent evidence suggests that FOXM1 may be involved in DNA replication, and that silencing of FOXM1 blocked the cell cycle transition from G1 to S phase [18]. The similarity of function supports a possible functional and structural interaction between AR and FOXM1 proteins. In the present report, FOXM1 and AR were found to coordinate CDC6 gene transcription in addition to the E2F pathways. Furthermore, we are the first to report that FOXM1 regulates CDC6 gene transcription by regulating AR gene expression. FOXM1 and AR proteins interacted within the nuclei when AR was activated by androgen. FOXM1 and AR proteins coordinated the CDC6 gene transcription level, highly expressed at S phase during the cell cycle. Several models have been proposed to dissect the mechanism by which two different transcription factors work together to regulate gene transcription [23]. However, it is unclear how FOXM1 and AR transcription factors, two interactive proteins co-expressing at high levels in the same cells, collaboratively bind to the individual consensus motifs to modulate CDC6 gene transcription. The binding of FOXM1 and AR proteins to the CDC6 promoter is relatively high at S phase. The protein levels of AR or FOXM1 influence their mutual binding to the CDC6

promoter. Androgen increased AR binding. Decline of either AR or FOXM1 protein levels resulted in the decrease of AR binding to the CDC6 promoter while the decreased AR did not change FOXM1 binding to the CDC6 promoter. These results indicating that FOXM1 protein levels influenced the DNA binding of AR protein to the CDC6 promoter might be explained by two possible mechanisms. The knockdown of FOXM1 decreased AR protein levels because FOXM1 regulated AR gene transcription. Recent reports have suggested that FOXA1 is a pioneer factor of AR protein and plays an important role in the transcriptional regulation of androgen-activated genes [24,25]. The data could not exclude a novel role of FOXM1 as a pioneer factor for AR protein binding to the CDC6 promoter. Further studies are required to clarify the exact roles of FOXM1 as a pioneer factor in the epigenetic regulation of androgen-activated genes such as CDC6. Functionally, the collaboration of FOXM1 and AR transcription factors likely enhances DNA synthesis and accelerates cell proliferation by regulating CDC6 gene expression, since knockdown of FOXM1 and AR alone or in combination reduced DNA synthesis and cell proliferation in a manner that can be overcome through CDC6 overexpression. Consistent with this data, siomycin A, a proteasome inhibitor known to inhibit FOXM1 expression and activity, inhibits prostate cancer proliferation and this effect is additive to that of the antiandrogenic compound bicalutamide. 5. Conclusions This study provided new evidence for the regulatory mechanism of aberrant CDC6 oncogene transcription by FOXM1 and AR in PCa cells (Fig. 6C). ● FOXM1 regulates CDC6 gene transcription through multiple mechanisms. 5.1. FOXM1 is recruited to the FOX binding motifs at the CDC6 proximal promoter; 5.2. FOXM1 up-regulates AR gene expression, thereby regulating CDC6 gene expression; 5.3. FOXM1 regulates the expression of E2F family members. ● FOXM1 and AR coordinate for CDC6 gene transcription regulation. 5.4. FOXM1 and AR proteins interact. 5.5. FOXM1 and AR proteins and CDC6 mRNA are elevated near S phase coinciding with elevated CDC6 promoter occupancy by both FOXM1 and AR. 5.6. Androgen increased AR protein binding to the CDC6 promoter, and silencing FOXM1 decreased AR binding. FOXM1 knockdown decreased AR expression. FOXM1 may act as a pioneer factor of AR in CDC6 gene transcription. Functionally, knockdown of FOXM1 and AR inhibited DNA synthesis and cell proliferation by regulating CDC6. Siomycin A, a FOXM1 inhibitor, has additive effects with the antiandrogen bicalutamide for the inhibition of cell proliferation. These data provided experimental evidence that a combined therapy of bicalutamide and siomycin A for PCa patients might contribute therapeutic benefits. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbagrm.2014.02.016. Abbreviations FOX forkhead box AR androgen receptor CDC cell division cycle ChIP chromatin immunoprecipitation qPCR quantitative Polymerase Chain Reaction PCa prostate cancer DHT dihydrotestosterone CSS charcoal-stripped FBS Rb retinoblastoma

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ARE DBD LBD

androgen-response element DNA binding domain ligand binding domain

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