CARM1 Regulates Estrogen-Stimulated Breast Cancer Growth Through Up-Regulation of E2F1 Seth Frietze,1 Mathieu Lupien,2,3 Pamela A

Research Article

CARM1 Regulates Estrogen-Stimulated Breast Cancer Growth through Up-regulation of E2F1 Seth Frietze,1 Mathieu Lupien,2,3 Pamela A. Silver,1 and Myles Brown2,3

1Department of Systems Biology, Harvard Medical School; 2Division of Molecular and Cellular Oncology, Department of Medical Oncology, Dana-Farber Cancer Institute; and 3Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts

Abstract proteins in breast cancer cell proliferation (11). Recently, the Estrogen receptor A (ERA) mediates breast cancer prolifer- coactivator protein AIB1 (also called ACTR/SRC-3 and NCoA3), ation through transcriptional mechanisms involving the which belongs to the family of p160 steroid receptor coactivators, recruitment of specific coregulator complexes to the pro- was shown to play an important role in breast cancer proliferation (12). AIB1 interacts with ligand-activated ERa and mediates the moters of cell cycle genes. The coactivator-associated arginine methyltransferase CARM1is a positive regulator of ER A- assembly of coactivator complexes containing chromatin-modifying mediated transcriptional activation. Here, we show that enzymes, including the protein arginine methyltransferase PRMT4/ CARM1 (13, 14). E2 stimulation induces the formation of a complex CARM1is essential for estrogen-induced cell cycle progression a in the MCF-7 breast cancer cell line. CARM1is specifically including CARM1, ER , and AIB1 on the E2-responsive promoter required for the estrogen-induced expression of the critical of the TFF1 gene where CARM1 is capable of methylating cell cycle transcriptional regulator E2F1whereas estrogen specific arginines on histone H3 (15). Although CARM1 has a role stimulation of cyclin D1is CARM1independent. Upon estro- in E2-mediated TFF1 gene activation, it is not known if CARM1 gen stimulation, the E2F1promoter is subject to CARM1- contributes to E2-induced breast cancer proliferation. dependent dimethylation on histone H3 arginine 17 Here, we show that CARM1 is necessary for the E2-induced (H3R17me2) in a process that parallels the recruitment of proliferation of breast cancer cells. We show that CARM1 is ERA. Additionally, we find that the recruitment of CARM1and specifically required for the E2-mediated expression of E2F1 and subsequent histone arginine dimethylation are dependent on not CCND1.UponE2 signaling, the nucleosomes that are positioned near the transcription start site of the E2F1 gene the presence of the oncogenic coactivator AIB1. Thus, CARM1 is a critical factor in the pathway of estrogen-stimulated breast undergo cell cycle variations in histone arginine dimethylation on A arginine 17 of histone H3 (H3R17me2), which correlate with the cancer growth downstream of ER and AIB1and upstream of a the cell cycle regulatory transcription factor E2F1. These recruitment of ER and E2F1 and increased E2F1 expression. studies identify CARM1as a potential new target in the Collectively, our data suggest that CARM1 plays a critical role in treatment of estrogen-dependent breast cancer. [Cancer Res breast cancer cell proliferation through the positive regulation of 2008;68(1):301–6] E2F1 expression, and thus may be an important new target in the treatment of estrogen-dependent breast cancer. Introduction Materials and Methods Human breast cancer proliferation is a complex process involving pathways that are controlled by hormones, growth factors, and Reagents and antibodies. E2 and 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) were purchased from Sigma. The other signaling molecules (1). The steroid hormone 17h-estradiol following antibodies were used: AIB1 (clone 34) was purchased from BD (E2) regulates gene expression through the activation of the Biosciences; CARM1 (M-16) and CARM1 from Santa Cruz Biotechnology a h a h estrogen receptors and (ER and ER ), which function as and Upstate Biotechnology, respectively; H3R17me2 from Upstate Biotech- ligand-regulated transcription factors (2). In breast cancer cells, nology; and calnexin from Stressgen. E2-activated ERa modulates the expression of key cell cycle regu- RNA interference. MCF-7 cells were transfected with small interfe- latory genes, including cyclin D1 (CCND1) and the transcription ring RNA oligonucleotide duplexes (siRNA) at a final concentration of factor E2F1 (3–6). These factors play important roles in cell cycle 50 nmol/L using LipofectAMINE 2000 (Invitrogen). Forty-eight hours after progression, directing the phosphorylation and inactivation of the transfection, cells were stimulated and harvested for analysis. Cells were retinoblastoma protein (Rb) and mediating the expression of genes hormone deprived for at least 3 days at the moment of stimulation. To involved in DNA replication and S phase entry (7), respectively. silence CARM1 expression, we used the ON-TARGETplus SMARTpool (Dharmacon; siCARM1-1) and Silencer siRNA (Ambion; siCARM1-2), sense Hence, drugs that block ERa action such as tamoxifen have been 5¶-GGAUAGAAAUCCCAUUCAAdTdT-3¶. AIB1 was silenced as described in widely used in the prevention and treatment of breast cancer (8). ref. 16. In the past few years, research interests have been directed Chromatin immunoprecipitation assays. Cells were hormone- toward understanding the roles of ERa (9, 10) and coregulatory deprived by culture for 3 to 4 days in phenol red–free medium (Invitrogen) supplemented with 5% charcoal-dextran–treated fetal bovine serum. Cells were stimulated with hormone for 45 min and cross-linked using 1% for- Note: Supplementary data for this article are available at Cancer Research Online maldehyde. Cells were lysed and sonicated three times for 10 s each at 12% (http://cancerres.aacrjournals.org/). amplitude (Fisher Sonic dismembrator, model 500). Immunoprecipitation, Requests for reprints: Myles Brown, Division of Molecular and Cellular Oncology, reverse cross-linking, and DNA purification were done as described (9). Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. Phone: 617-632- Immunoprecipitated DNA was quantified by real-time PCR. 3948; Fax: 617-582-8501; E-mail: [email protected]. I2008 American Association for Cancer Research. Real-time PCR. Real-time PCR was done using SYBR Green PCR Master doi:10.1158/0008-5472.CAN-07-1983 Mix (Applied Biosystems). The presence of a single amplicon was systemically www.aacrjournals.org 301 Cancer Res 2008; 68: (1).January 1, 2008

number was quantified with MTT viability assay after 3 days of E2 stimulation and is expressed as percent change relative to control (siCONTROL-transfected cells treated with vehicle). Data analysis. Data analysis was done using the Prism software. Statistical significance was determined using Student’s t test comparison for unpaired data and was indicated as follows: *, P < 0.05; **, P < 0.01.

Figure 1. Effect of CARM1 silencing in MCF-7 cells on E2-induced cell cycle progression. A, siRNA-mediated silencing of CARM1 in MCF-7 cells. Two different CARM1-specific siRNAs or a control siRNA were transfected into hormone-depleted MCF-7 cells. Extracts were collected and Western blotting was done withanti-CARM1 antibodies. Anti-calnexin was used as a loading control. B, cell cycle analysis of CARM1-depleted MCF-7 cells. MCF-7 cells were transfected as described in A. Forty-eight hours later, cells were treated with 10 nmol/L E2 or ethanol vehicle for 24 h and harvested to analyze DNA content by propidium iodide staining and flow cytometry. *, P < 0.05, versus siCONTROL-transfected cells treated withE 2. C, cell viability assays were done as described in Materials and Methods using MCF-7 cells transfected with the indicated siRNAs. Cell proliferation is expressed as the percentage of the cells compared withthesiCONTROL-transfected wells treated withE 2. Columns, mean from one representative experiment done in triplicate; bars, SE. **, P < 0.01, versus similarly treated siCONTROL-transfected cells. verified by dissociation curve analysis. See Supplementary Table S1 for a list of chromatin immunoprecipitation primers used in this study. Real-time reverse transcription-PCR. RNA isolation and real-time reverse transcription-PCR (RT-PCR) were done as described in ref. 17. Expression was normalized to RPS28 and glyceraldehyde-3-phosphate dehydrogenase. See Supplementary Table S1 for a list of target primers used in this study.

Western blotting. Western blot assays were done as described in ref. 17. Figure 2. CARM1 silencing affects E2-induced expression of E2F1. A to C, Cell cycle distribution and growth assays. MCF-7 cell cycle CARM1-specific or control siRNAs were transfected into hormone-depleted MCF-7 cells. Forty-eight hours after transfection, cells were treated with vehicle distribution was analyzed 24 h after stimulation with 10 nmol/L E2 or or 10 nmol/L E2 for the indicated time period, and RNA was isolated to measure vehicle using propidium iodide staining and flow cytometry as described in the expression of TFF1 (A), E2F1 (B), and CCND1 (C) genes by real-time ref. 17. For growth analysis, MCF-7 cells transfected with the indicated RT-PCR. Columns, mean of three independent replicates; bars, SD. Shown are siRNAs were replated 48 h after transfection (seeding density, 3 Â 104 cells results obtained from siCARM1-1 transfections; similar results were obtained P per well) in phenol red–free DMEM containing 5% charcoal-stripped serum from siCARM1-2 transfections. *, < 0.05, versus siCONTROL-transfected cells. D, after 48 hof transfection withsiCARM1 or control siRNA, MCF-7 and 10 nmol/L E2. Cells were supplemented with fresh medium containing cells were stimulated with10 nmol/L E 2 for 0, 24, and 48 hand whole-cell hormones every 48 h throughout the course of the experiment. The total cell extracts from MCF-7 cells were analyzed by Western blot for E2F1.

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Figure 3. Effect of CARM1 silencing on the E2-mediated expression of E2F1-responsive genes. siRNA-mediated silencing of CARM1 results in a reduced level of E2F1-responsive mRNAs. As in Fig. 2, CARM1 was silenced by siRNA transfection and the relative levels of CCNE1, CCNE2, CDC25A, and CCNA1 were determined by real-time RT-PCR. *, P < 0.05, versus siCONTROL- transfected cells.

Results program of ERa-positive breast cancer cells (25). We therefore investigated the effect of CARM1 silencing on the expression of CARM1is required for E 2-induced proliferation in ERA- positive breast cancer cells. CARM1 is thought to play an E2F1. As a positive control, CARM1 silencing reduced expression of important role in hormone signaling through coactivation of the TFF1 gene (Fig. 2A). Estrogen stimulation induced a 5- and nuclear receptor–mediated transcription and histone modification 7-fold increase in E2F1 mRNA after 3 and 12 h, respectively, and (18); however, this has been shown for a small number of target this effect was very significantly reduced by CARM1 silencing genes. Whereas both ERa and AIB1 have been shown to be (Fig. 2B). Interestingly, whereas CARM1 silencing impaired the involved in regulating cellular growth properties in both normal transcription of E2F1, the estrogen-induced transcription of the and breast cancer cells (19, 20), the mechanisms involving CARM1 CCND1 gene was unaffected by CARM1 silencing (Fig. 2C). in this process and its critical targets are not clearly defined. To Accordingly, we also observed that E2F1 protein expression levels explore the role of CARM1 in ERa-positive breast cancer cells, we were strongly reduced by CARM1 silencing (Fig. 2D). E2F1 expression examined the consequences of its suppression on the growth was also diminished when CARM1 was silenced in another breast stimulatory effect of E2 using MCF-7 cells as a model. Expression of cancer cell model, the T47D cell line (Supplementary Fig. S2). CARM1 was efficiently and specifically silenced in MCF-7 cells at CARM1silencing blocks the E 2 induction of E2F1-responsive the mRNA (data not shown) and protein levels (Fig. 1A) with either genes. Estrogen regulation of E2F1 is important for the regulation of two different CARM1 siRNA oligonucleotide duplexes. Upon of several cell cycle–associated genes. Having shown that CARM1 silencing of CARM1, we observed that the E2-mediated stimulation is important for the estrogen regulation of E2F1, we examined of MCF-7 cell cycle progression was strongly reduced (Fig. 1B). whether CARM1 silencing resulted in reduced expression of the Furthermore, we observe a decrease in the number of MCF-7 cells E2F1-responsive genes including CCNE1, CCNE2, CDC25A, and following growth stimulation with E2 when CARM1 was silenced CCNA1. As predicted in E2-stimulated MCF-7 cells in which CARM1 compared with a control siRNA (Fig. 1C). We found a similar effect was silenced, we observed a significant reduction in the levels of of CARM1 silencing on E2-induced proliferation of T47D cells, CCNE1 and CCNE2 mRNA, as well as CDC25A and CCNA1 mRNA, another model of ERa-positive breast cancer (Supplementary compared with the control (Fig. 3). Hence, these data support an Fig. S1). These data point to a critical role for CARM1 in the important role for CARM1 in the regulation of E2F1 and E2F1- proliferative response to estrogen in ERa-positive breast cancer cells. responsive genes. CARM1controls breast cancer proliferation by regulating CARM1directly mediates the E 2-induced activation of the E2F1expression. CARM1 has been shown to regulate the E2F1promoter. To examine the mechanism by which CARM1 is transcription of a small number of genes in response to distinct involved in the E2-mediated control of E2F1 transcription, we stimuli (21–24). To address whether CARM1 regulates the cell cycle carried out chromatin immunoprecipitation assays. Previous by modulating transcription of specific genes that are involved in studies have shown that a region up to À220 bp from the this process, we examined the effect of CARM1 depletion on the transcription start site is sufficient for full promoter activity expression of specific cell cycle genes. Previous results show that (26, 27). This region contains two palindromic E2F-binding sites, the key cell cycle regulator E2F1 and, subsequently, its downstream several putative binding sites for SP1, a putative binding site for target genes are critical for hormone regulation of the proliferative nuclear factor nB, and two canonical CAAT boxes. The E2F1 www.aacrjournals.org 303 Cancer Res 2008; 68: (1).January 1, 2008

promoter has neither a TATA motif nor an initiator element. We CARM1recruitment and E 2-mediated histone modification find that E2 enhances the recruitment of both ERa and E2F1 to the at the E2F1promoter are dependent on AIB1. It is thought that E2F1 promoter (Fig. 4A). In addition, we observe constitutive CARM1 is targeted to ERa-regulated genes via an interaction with recruitment of SP1 to the E2F1 promoter independent of E2 SRC/p160 members including AIB1 (28). To test the requirement of stimulation (Fig. 4A). Because both AIB1 and CARM1 are thought AIB1 for CARM1 recruitment and histone arginine dimethylation at to function in ERa- and E2F1-mediated transcriptional activation, the E2F1 promoter, we silenced AIB1 in MCF-7 cells using AIB1- we measured the enrichment of AIB1, CARM1, and H3R17me2 at specific siRNAs and carried out CARM1 and H3R17me2 chromatin the E2F1 promoter before and after E2 stimulation (Fig. 4B). immunoprecipitation assays. Following efficient silencing of AIB1, Following E2 treatment, the coactivators AIB1 and CARM1, as well we detect a significantly reduced recruitment of CARM1 to the as the CARM1-catalyzed histone modification H3R17me2, are E2F1 promoter compared with cells transfected with control siRNA enriched at the E2F1 promoter (Fig. 4B). These results indicate that (Fig. 5A) both in the absence of hormone and, more significantly, E2-mediated expression of E2F1 involves the recruitment of AIB1 after E2 stimulation. Importantly, the reduction of AIB1 had no and CARM1 to the E2F1 promoter and that this region of measurable effect on the levels of CARM1 protein (Fig. 5A, right). chromatin is subject to estrogen-dependent H3R17 dimethylation Additionally, silencing AIB1 reduced the levels of H3R17me2 at (H3R17me2). Interestingly, CARM1 was also recruited to the the E2F1 promoter. The silencing of AIB1 affects histone methy- promoter of several E2F1 target genes (CCNE1, CCNE2, CDC25A, lation to a similar degree as when CARM1 is silenced (Fig. 5B). and CCNA1) following E2 stimulation (Supplementary Fig. S3). This Thus, AIB1 functions in the recruitment of CARM1 and the sub- suggests that in addition to the role of CARM1 in regulating E2F1 sequent H3R17 dimethylation during transcriptional activation of expression, it may play a second role in the regulation of E2F1 E2F1 and may in part explain the oncogenic functions of AIB1. target genes. These results provide a direct link of CARM1 to cell cycle regulation through the transcriptional activation of E2F1 and E2F1 target genes. Discussion Estrogens elicit proliferative responses in breast cancer cells through ERa-mediated transcriptional mechanisms (29). CARM1 has been implicated in the positive regulation of ERa-mediated gene activation in response to estrogen signaling (28). Thus, a presumed but not fully tested model for CARM1 action is that it functions in the control of cell proliferation through the activation of key cell cycle genes. In the current study, we describe a specific role for CARM1 in regulating the cell cycle by inducing the expression of E2F1 in MCF-7 cells following E2 treatment. We show that CARM1 is required for the E2-mediated activation of E2F1 and for the induction of E2F1 target genes, including CDC25A, CCNA1, CCNE1, and CCNE2. These data are in agreement with a recent report showing that CARM1 functions in E2F1- mediated transactivation through AIB1 to positively regulate the expression of CCNE1 on cell cycle entry (22). Transfection of siRNA oligonucleotide duplexes into MCF-7 cells reduced the mRNA and protein levels of CARM1 by >70%. Reduced levels of CARM1 were sufficient to affect the E2-mediated activation of the TFF1 gene, suggesting that CARM1 was functionally silenced. Significantly, CARM1 silencing does not affect the E2-stimulation of CCND1, showing that CARM1 does not function in the entire E2 response but rather has an important role in targeting a subset of ERa targets including E2F1. The observed gene-specific effect of CARM1 is consistent with the reported promoter-specific role for CARM1 in the activation of specific nuclear factor nB–dependent genes (23). At the E2-responsive TFF1 promoter, CARM1 recruitment was shown to follow ERa recruitment. Thus, CARM1 is thought to be recruited by ERa to this promoter (15, 30). However, it is not known whether the recruitment of CARM1 occurs at additional a E2-responsive sites including nonclassical ER target sites. Here, Figure 4. E2-mediated E2F1 expression corresponds witharginine methylation we show that CARM1 is recruited to the E2F1 promoter, a of nucleosomal histones located at the E2F1 promoter. A, chromatin immunoprecipitation analysis of the human E2F1 promoter. Chromatin nonclassical ERa site (6). Our chromatin immunoprecipitation immunoprecipitation was done withprimers specific for theproximal portion of data confirm the recruitment of ERa to the promoter of E2F1 and the E2F1 promoter withantibodies against Sp1, E2F1, and ER a. Formaldehyde show that E2 induces the recruitment of both AIB1 and CARM1. cross-linked chromatin samples prepared from vehicle- and E2-treated MCF-7 cells were used for chromatin immunoprecipitation analyses with the indicated CARM1 targeting to the E2F1 promoter coincides with enhanced antibodies and analyzed by real-time PCR for the presence of the E2F1 levels of H3R17 dimethylation. Importantly, H3R17me2 at this promoter fragment as diagramed. Columns, mean of three independent replicates; bars, SD. B, chromatin immunoprecipitation analysis of the E2F1 site is undetectable in CARM1-depleted cells, signifying that CARM1 promoter withantibodies against AIB1, CARM1, and H3R17me2. is the main or sole enzyme responsible for the dimethylation

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Figure 5. Depletion of AIB1 inhibits CARM1 recruitment and E2F1 promoter methylation. A, siRNA-mediated depletion of AIB1 affects CARM1 recruitment to the E2F1 promoter. Specific siRNAs targeting AIB1 or control siRNAs were transfected into MCF-7 cells 48 hbefore E2 stimulation. CARM1 chromatin immunoprecipitation (ChIP) analyses for the E2F1 promoter were done as in Fig. 4. Western blot analysis of AIB1 and CARM1 was done withAIB1- and CARM1-specific antibodies. Calnexin was used as a loading control. *, P < 0.05, versus siCONTROL- transfected cells treated withE 2. B, depletion of CARM1 or AIB1 results in decreased levels of H3R17me2 at the promoter of E2F1. siRNAs targeting a nonspecific control, CARM1, or AIB1 were transfected into MCF-7 cells and H3R17me2 chromatin immunoprecipitation was done. *, P < 0.05, versus siCONTROL-transfected cells treated withE 2.

of histone H3 at arginine 17. We also observe a major defect in effect of ERa, AIB1, and CARM1 on the E2F1 promoter. In this CARM1 recruitment to the E2F1 promoter in AIB1-knockdown model (Fig. 6), the ability of estrogen acting through ERa and AIB1 cells and that histone dimethylation at H3R17 is eliminated in AIB1- to activate CCND1 (35) and subsequently inactivate Rb is silenced cells on E2 stimulation. These data are in agreement independent of CARM1, although the ability of estrogen to with a model in which p160 coactivators such as AIB1 serve as scaffold factors to target CARM1 to specific regulated sites where CARM1 can methylate histones and participate in gene activation. Methylation of histones by PRMTs is increasingly being acknowledged as an important aspect for the dynamic regulation of gene expression. For example, CARM1/PRMT4 and PRMT1, two type I PRMTs, have been proposed to play a role in gene activation and were shown to catalyze monomethylation and asymmetrical NG,NG-arginine dimethylation on histone H3R2, H3R17, H3R26, and H4R3, respectively (31). In contrast, PRMT5, a type II PRMT, was shown to catalyze histone H4 monomethylation and symmetrical NG,N1G-arginine dimethylation, a modification that coincides with gene repression (32, 33). Whereas it is recognized that specific PRMT enzymes are important transcriptional coregulators, it is unclear how these enzymes and their associated histone mod- ifications function to coordinately regulate gene expression in vivo. In this report, we reveal that the arginine methyltransferase CARM1 functions in breast cancer proliferation through specific gene activation. Aberrant expression of CARM1 has been linked to human prostate and breast cancers (21, 22, 34). Consistent with a study À À showing that CARM1 / murine embryonic fibroblasts display an altered capacity to enter the cell cycle upon serum stimulation (22), we show that CARM1 depletion also adversely affects breast cancer cell proliferation when stimulated with estrogen. Our finding that CARM1 is required for E2F1 expression, but not CCND1 expression, following estrogen simulation suggests that the pathway of estrogen-stimulated growth involves the direct transcriptional Figure 6. Model of E2-mediated breast cancer cell proliferation. www.aacrjournals.org 305 Cancer Res 2008; 68: (1).January 1, 2008

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 2008 American Association for Cancer Research. Cancer Research stimulate the cell cycle is dependent on CARM1 activation of E2F1 Grant support: National Institutes of Diabetes, Digestive, and Kidney Diseases grant R56DK074967 (M. Brown); NIH (P.A. Silver); National Cancer Institute (Dana- and several of its downstream targets. Taken together, these results Farber/Harvard Cancer Center Breast Cancer Specialized Program of Research suggest that CARM1 may represent an important target for Excellence Grant), National Cancer Institute grant P01CA8011105 (M. Brown), Dana- therapeutic intervention in hormone-dependent breast cancer. Farber Cancer Institute Women’s Cancers Program, and National Science Foundation predoctoral fellowship (S. Frietze). 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 Acknowledgments with 18 U.S.C. Section 1734 solely to indicate this fact. We thank I. Swinburne and J. Eeckhoute for critical reading of the manuscript and Received 6/5/2007; revised 10/2/2007; accepted 11/1/2007. the members of the Brown and Silver laboratory for helpful discussions.

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