Carcinogenesis vol.31 no.3 pp.489–495, 2010 doi:10.1093/carcin/bgp305 Advance Access publication September 7, 2009
Dietary omega-3 polyunsaturated fatty acids suppress expression of EZH2 in breast cancer cells Manjari Dimri, Prashant V.Bommi, Anagh A.Sahasrabuddhe, Janardan D.Khandekar and Goberdhan P.Dimri Department of Medicine, NorthShore University HealthSystem Research Institute, 1001 University Place, Evanston, IL 60201, USA To whom correspondence should be addressed. Department of Medicine, Room 304, 1001 University Place, NorthShore University HealthSystem Research Institute, Evanston, IL 60201, USA. Tel: þ1 224 364 7521; Fax: þ1 224 364 7402; Email:
[email protected]
The polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), is overexpressed in several human malignancies including breast cancer. Aberrant expression of EZH2 has been associated with metastasis and poor prognosis in cancer patients. Despite the clear role of EZH2 in oncogenesis and therapy failure, not much is known about chemotherapeutics and chemopreventive agents that can suppress its expression and activity. Here, we show that dietary omega-3 (v-3) polyunsaturated fatty acids (PUFAs) can regulate the expression of EZH2 in breast cancer cells. The treatment of breast cancer cells with v-3 PUFAs, but not v-6 PUFAs, led to downregulation of EZH2. Studies using proteosome inhibitor MG132 suggested that v-3 PUFAs induce degradation of the PcG protein EZH2 through posttranslational mechanisms. Furthermore, downregulation of EZH2 by v-3 PUFAs was accompanied by a decrease in histone 3 lysine 27 trimethylation (H3K27me3) activity of EZH2 and upregulation of E-cadherin and insulin-like growth factor binding protein 3, which are known targets of EZH2. Treatment with v-3 PUFAs also led to decrease in invasion of breast cancer cells, an oncogenic phenotype that is known to be associated with EZH2. Thus, our studies suggest that the PcG protein EZH2 is an important target of v-3 PUFAs and that downregulation of EZH2 may be involved in the mediation of anti-oncogenic and chemopreventive effects of v-3 PUFAs.
human malignancies. For example, an overexpression of EZH2 is found in patients with breast cancer, prostate cancer and other neoplasias (5–12). Importantly, it has been shown that EZH2 is a marker for aggressive breast cancer and that the expression of EZH2 increases in histologically normal breast epithelium of patients who are at a higher risk of developing breast cancer (5,11). The primary histone PTM activity associated with EZH2 is trimethylation of histone 3 lysine 27 (H3K27me3) (13). Thus, an overexpression of EZH2 in cancer cells lead to an increased H3K27me3 activity (14,15). Importantly, overexpression of EZH2 is known to be associated with metastasis, poor prognosis and therapy failure in breast and prostate cancer patients (7,12,15). Although few recent reports suggest that the expression of EZH2 is regulated by microRNA-101 in cancer cells (16,17), detailed transcriptional, posttranscriptional and posttranslational mechanisms regulating EZH2 expression are not clearly understood. At present, chemotherapeutics and chemopreventive agents that can be used to target EZH2 also remain largely unidentified. Chemopreventive agents such as dietary polyunsaturated fatty acids (PUFAs) are known to influence the development and progression of breast cancer and other cancers (18,19). It is generally agreed that omega-3 (x-3) and omega-6 (x-6) PUFAs have paradoxical effect on cancer risk; x-3 PUFAs apparently are associated with lower risk of breast cancer, whereas x-6 PUFAs are associated with the higher risk of breast cancer (18–21). Importantly, the lower ratio of x-6:x-3 PUFAs in diets is thought to provide a protective effect against breast cancer and other cancers (18–21). Several laboratories have studied the effect of PUFAs on growth and proliferation of breast cancer cells. In general, x-3 PUFAs have been shown to inhibit the proliferation of breast cancer cells in culture and in animal models of breast cancer, whereas x-6 PUFAs have been shown to enhance proliferation of breast cancer cells and increase tumorigenesis in vivo in animal models (22–26). Although PUFAs are thought to inhibit or enhance cancer cell proliferation by mediating the regulation of expression of genes that are involved in lipid and cellular metabolism, the molecular targets of PUFAs are not very well understood (27). In this paper, we show that one of the important molecular targets of x-3 PUFAs is the PcG protein EZH2, whose overexpression has been linked to several types of cancers including breast cancer.
Introduction Polycomb group (PcG) proteins are evolutionarily conserved from Drosophila to human and are important regulators of chromatin remodeling and gene silencing (1,2). These proteins also regulate cell cycle progression and proliferation and differentiation of cells (1,2). By assembling together, PcG proteins form polycomb repressive complexes (PRCs), which possess histone posttranslational modifications (PTMs) activities (2). PRC1 ubiquitinates histone 2A at lysine 119 residue (H2A-K119Ub modification), whereas PRC2 trimethylates histone 3 at lysine 27 residue (H3K27me3 modification) (2). These histone modifications triggered by PRCs lead to compaction of chromatin and silencing of important tumor suppressors, developmental regulators and differentiation-specific genes (3,4). An aberrant expression of PcG proteins, in particular BMI1 and enhancer of zeste homologue 2 (EZH2), is associated with several
Abbreviations: AA, arachidonic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; EZH2, enhancer of zeste homologue 2; H3K27me3, histone 3 lysine 27 trimethylation; H3K9me3, trimethylation of K9 residue of H3; HMTases, histone methyl transferases; IGFBP3, insulin-like growth factor binding protein 3; IP, immunoprecipitation; LA, linoleic acid; mAb, monoclonal antibody; RT–PCR, reverse transcription–polymerase chain reaction; PcG, polycomb group; PRC, polycomb repressive complex; PTM, posttranslational modification; PUFA, polyunsaturated fatty acid.
Materials and methods Cells, cell culture methods and fatty acid treatment of cells MCF10A, MCF7, T47D, MDA-MB-231 and other breast cancer cells were obtained from American Type Culture Collection (Manassas, VA) and cultured as described previously (28). Two x-3 PUFAs [eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA)] and two x-6 PUFAS [linoleic acid (LA) and arachidonic acid (AA)] were obtained from Cayman Chemicals (Ann Arbor, MI). These fatty acids were dissolved in ethanol (EtOH). For fatty acid treatment, cells were grown to a confluence of 70–80%, starved for 24 h in 0.5% serum containing medium and then treated with x-3 and x-6 PUFAs for 3–8 h. After the treatment, cells were processed for reverse transcription–polymerase chain reaction (RT–PCR) and western blot analyses and invasion and immunofluorescence assays as required. RT–PCR analysis RT–PCR analysis was performed as described previously (29). Briefly, total RNA was isolated from mock or treated cell lines, and complementary DNA synthesis was carried out using a kit (USB Corporation, Cleveland, OH). The complementary DNAs were polymerase chain reaction amplified using primer specific for EZH2 and b-actin in a Rotor-Gene 6000 series polymerase chain reaction system (QIAGEN, Valencia, CA). The primer sequences were as follows: EZH2-F, 5#-TTCTCAAGATGAAGCTGACAGAAGAGGG-3#; EZH2-R, 5#-TGAAGCTAAGGCAG CTGTTTCAGAGG-3#; b-actin-F, 5#-AGGCGGACTA TGACTTAGTTGCGTTACACC-3# and b-actin-R, 5#-TGGCAAGGGACTTCCTGTAACAACGC-3#.
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Antibodies and western blot analysis Various antibodies were obtained from commercial sources. The antibodies used in the study were monoclonal antibody (mAb) anti-EZH2 and mAb anti-E-cadherin from BD Biosciences (San Jose, CA), anti-b-actin from Sigma-Aldrich (St. Louis, MO), anti-IGFBP3 and anti-ubiquitin from Santa Cruz Biotechnology (Santa Cruz, CA), anti-H3K27me3 from Upstate Biotechnology (Lake Placid, NY), anti-H3K9me3 from Abcam (Cambridge, MA) and anti-H3K4me3 from Active Motif (Carlsbad, CA). For immunoprecipitation, anti-EZH2 mAb was obtained from Cell Signaling Technology (Danvers, MA). Western blot analyses of total cell extracts using antibodies that are capable of detecting various proteins were performed as described (29). For the western blot analysis of IGFBP3, equal number of cells were plated and treated in an equal volume of cell culture medium, as described previously. The medium was collected and spun down, and the proteins present in supernatant were precipitated using trichloroacetic acid. After incubation for 10 min and centrifugation at 13 000 r.p.m. for 5 min, the supernatant was removed and the pellet was washed twice with cold acetone and dried. The dried pellet was resuspended in 2 sample buffer containing b-mercaptoethanol and boiled for 5 min before loading on the gel. The half-life of EZH2 was determined using cyclohexamide treatment of cells as described (29). Briefly, MDA-MB-231 cells were mock- or DHApretreated for 8 h. After pretreatment, 100 lg/ml cyclohexamide was added at various time points (0–90 min) to stop synthesis of new proteins, and cell lysates were prepared and analyzed for the expression of EZH2 and b-actin. The normalized % residual EZH2 was plotted against different time points to determine the rate of proteolysis and the half-life of EZH2 with and without DHA.
data were analyzed using the Student’s t-test, and P , 0.05 was considered significant.
Results PUFAs differentially regulate EZH2 in breast cancer cells To study the possible effect of PUFAs on the expression of PcG proteins in breast cancer cells, we treated MDA-MB-231, T47D and MCF7 cells with x-3 and x-6 PUFAs. We chose to study the effect of two well-known x-3 PUFAs (DHA and EPA) and two x-6 PUFAs (LA and AA) for our study. After treating the breast cancer cells for 8 h with different PUFAs, the expression of EZH2 and a control gene was determined by western blot analysis. Our results indicated that the treatment of breast cancer cells with x-3 PUFAs (DHA and EPA) led to decrease in expression of EZH2. In contrast, x-6 PUFAs (LA and AA) had no effect on the expression of EZH2 (Figure 1A–C). As SUZ12 and EED, two other constituents of PRC2 are often coregulated with EZH2 (10,30,31), we also examined their expression in PUFAs-treated cells. Our results indicated that similar to EZH2, the expression of SUZ12 and EED are also downregulated by x-3 PUFAs but not by x-6 PUFAs (supplementary Figure S1 is available at Carcinogenesis Online).
Histone extraction For the extraction of total histones, Abcam histone extraction protocol (www.abcam.com) was adapted. Briefly, equal number of cells were plated and treated as described above. The cell pellet was lysed in cold lysis buffer [0.5% Triton X-100, 50 mM Tris (pH 7.5), 150 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid, 5 mM sodium butyrate, 1 mM phenylmethylsulfonyl fluoride, 1 lg/ml each of leupeptin and pepstatin, 1 mM sodium orthovanadate and 10 mM sodium fluoride] for 30 min and centrifuged at 8000 r.p.m. for 10 min at 4°C to spin down the nuclei. The supernatant was removed and the pellet washed in lysis buffer. The pellet was resuspended in 0.2 N HCl overnight at 4°C for acid extraction. The samples were centrifuged to pellet the debris and supernatant containing the histones was transferred to a clean tube. The protein was quantified and 2.5 lg of the protein was loaded on 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel for western blot analysis. Immunoprecipitation, immunofluorescence and invasion assays Immunoprecipitation (IP) and immunofluorescence studies and invasion assays were carried out as described (28). Briefly, for IP, MDA-MB-231 cells were mock- or DHA-treated in presence or absence of MG132 for 8 h. Cell lysates were prepared in cold lysis buffer containing 0.5% Triton X-100, 50 mM Tris (pH 7.5), 150 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1 lg/ml each of leupeptin and pepstatin, 1 mmol/l sodium orthovanadate and 10 mM sodium fluoride. IP was performed using a mAb against EZH2 (Cell Signaling Technology) and immunoglobulin G (negative control). Of each sample used for IP, 10% of total extract was also analyzed for EZH2 input. The immunoblot analysis was carried out using antiubiquitin and anti-EZH2 antibodies. For immunofluorescence studies, cells were plated in chamber slides and treated with PUFAs or mock treated with EtOH as described above. Cells were fixed in 4% formaldehyde, permeabilized with 0.5% Triton X-100 for 5 min and coimmunostained with anti-EZH2 (BD Biosciences) and H3K27me3 (Upstate Biotechnology) followed by staining with Alexa Fluor 488-conjugated and Alexa Fluor 594-conjugated secondary antibodies, respectively (Molecular Probes, Eugene, OR). The slides were mounted with Vectashield mounting medium containing 4#,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). After staining, cells were photographed (40) using a Nikon Eclipse 80i confocal microscope and colocalization was studied by merging different colors [Alexa Fluor 488 (EZH2), Alexa Fluor 594 (H3K27me3) and 4#,6-diamidino-2-phenylindole]. Invasion assays were carried out using the 24-well BD Biocoat Matrigel Invasion Chambers (BD Biosciences) according to the manufacturer’s recommendations. Briefly, 2.5 104 cells in 0.2 ml medium were added to the top chambers of Matrigel-coated or control wells with medium containing 5% fetal bovine serum in the bottom chamber. After 8 h at 37°C, the cells on the topside were removed by scraping, and the invaded cells were fixed in methanol at 20°C and visualized using the Diff-Quik stain (Dade Behring, Deerfield, IL). Live images were taken with a 10 magnification for counting the cells that invaded through the Matrigel. The experiments were done in triplicates and
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Fig. 1. x-3 PUFAs but not x-6 PUFAs downregulate EZH2 in MDA-MB231 (A), MCF7 (B) and T47D (C) breast cancer cells. These cell lines were treated with EPA, DHA, LA or AA and analyzed for the expression of EZH2 by western blot analysis as described in Materials and Methods. The relative expression (Rel. Exp.) of EZH2 in PUFA- and mock-treated cells was determined by densitometric analysis of EZH2 signal in each lane and normalizing it to b-actin signal of the respective lane. The densitometric analysis was performed using Image J software (National Institutes of Health, Bethesda, MD).
Omega-3 PUFAs regulate EZH2 expression
Omega-3 PUFAs posttranslationally regulate the expression of EZH2 To further determine the possible mechanism for the downregulation of EZH2 by x-3 PUFAs and its effect on EZH2 activity and oncogenic properties of breast cancer cells, we chose to perform detailed studies in MDA-MB-231 cells. First, we performed RT–PCR analysis of PUFAs-treated and mock-treated MDA-MB-231 cells. RT–PCR analysis indicated that DHA and EPA treatment did not significantly alter messenger RNA levels of EZH2 in MDA-MB-231 cells (Figure 2A) and MCF7 and T47D cell lines (data not shown), suggesting that these x-3 PUFAs may posttranslationally but not transcriptionally regulate the expression of EZH2. To confirm our assumption, we treated MDA-MB-231 cells with DHA or EPA and a 26S proteosome inhibitor MG132 to block proteosome-mediated degradation of EZH2. We found that blocking proteosome-mediated degradation of EZH2 by MG132 could restore its previous levels in treated cells (Figure 2B). Since the proteosome-mediated protein degradation pathways involve ubiquitination of the target protein prior to degradation, we determined whether DHA treatment of cells lead to an increased ubiquitination of EZH2. We immunoprecipitated EZH2 from DHAtreated and mock-treated cells and determined the extent of EZH2 ubiquitination by immunoblot analysis using mAb against ubiquitin. The results indicated that DHA treatment of cells resulted in ubiquitination of EZH2 (Figure 2C). We also found that ubiquitination of EZH2 was further enhanced upon treatment with MG132, which
blocks degradation of ubiquitinated proteins. Thus, our data showed that DHA induced EZH2 degradation via proteosome-mediated pathways in breast cancer cells. An increased turnover of EZH2 upon treatment with DHA was further confirmed by analysis of half-life of EZH2 protein. The half-life of EZH2 in mock- and DHA-treated MDA-MB-231 cells was determined by inhibiting the synthesis of the new protein using cyclohexamide at different time points and determining the remaining EZH2 at each time point by western blot analysis. The results indicated that half-life of EZH2 in mock-treated cells is 60 min as against 30 min in DHA-treated cells (Figure 2D). Thus, the downregulation of EZH2 by DHA treatment probably occurred because of its increased turnover in breast cancer cells. Downregulation of EZH2 by x-3 PUFAs results in decreased H3K27me3 To determine the functional significance of downregulation of EZH2 by DHA and EPA, we examined H3K27me3 activity of PRC2, which depends on the expression of EZH2. It is known that EZH2 is often overexpressed in cancer cells including breast cancer cells. We confirmed that overexpression of EZH2 in MCF7 and MDA-MB231 correlates with increased levels of H3K27me3 (supplementary Figure S2 is available at Carcinogenesis Online). Next, we examined whether downregulation of EZH2 by x-3 PUFAs resulted in decreased H3K27me3 levels as a direct measurement of PRC2 activity.
Fig. 2. x-3 PUFAs posttranslationally regulate the expression of EZH2. (A) RT–PCR analysis of mock-, EPA-, DHA-, LA- and AA-treated MDA-MB-231 cells was performed using primers specific for EZH2 and b-actin as described in Materials and Methods. The relative expression of EZH2 transcript was determined by densitometry as described in Figure 1. (B) MDA-MB-231 cells were either pretreated with MG132 for 60 min or mock-treated with dimethyl sulfoxide (solvent), followed by treatment with EtOH, EPA, DHA, LA and AA for 8 h in the presence or absence of MG132. After treatment, western blot analysis for EZH2 and b-actin was performed and the relative expression of EZH2 was determined by densitometry as described in Figure 1. (C) MDA-MB-231 cells were mock treated or DHA treated in the presence or absence of MG132 for 8 h, and IP was performed using a mAb against EZH2 and immunoglobulin G (negative control). Immunoblot (IB) analysis was performed using anti-ubiquitin and anti-EZH2 antibodies. Of the total extract used for IP, 10% of each sample was also analyzed for EZH2 input. (D) Half-life of EZH2 in mock- and DHA-treated (for 8 h) MDA-MB-231 cells was determined by blocking synthesis of new proteins using 100 lg/ml cyclohexamide (CHX) at different time points (0–90 min) and rate of EZH2 proteolysis was determined by western blot analysis of remaining EZH2 protein at each time point and normalizing it to b-actin using densitometry as described in Figure 1 and Materials and Methods.
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First, we isolated total histones by acid extraction. Total histones were fractionated by polyacrylamide gel electrophoresis and probed for H3K27me3 using a specific rabbit polyclonal antibody against it. The results of the western blot analysis showed that the treatment of the cells with x-3 PUFAs, but not x-6 PUFAs, led to decreased levels of H3K27me3 (Figure 3A). Although EZH2 is primarily re-
Fig. 3. x-3 PUFAs, but not x-6 PUFAs, downregulate H3K27me3 and H3K9me3 in MDA-MB-231 cells. (A) Total histones were extracted from mock- or EPA-, DHA-, LA- and AA-treated cells (8 h) using acid extraction as described in Materials and Methods. Ponceau staining of the membrane was done to visualize total histones, and western blot analysis was performed to detect total H3 and H3K27me3 levels. (B) Total histones were analyzed for the expression of H3K9me3 and total H3 by western blot analysis.
sponsible for H3K27me3 activity, it can also partially regulate trimethylation of K9 residue of H3 (H3K9me3) (32). Hence, we also determined H3K9me3 in control and PUFAs-treated MDA-MB-231 cells. We found that x-3 PUFAs also downregulated H3K9me3, although the extent of downregulation was modest compared with downregulation of H3K27me3, whereas x-6 PUFAs had no effect on it (Figure 3B). Next, we performed coimmunostaining of mock- and DHA-treated breast cancer cells using antibodies specific to EZH2 and H3K27me3. The results of coimmunostaining experiment suggested that downregulation of EZH2 by x-3 PUFAs results in a corresponding decrease in nuclear H3K27me3 on a single-cell basis in MDA-MB-231 cells (Figure 4) and MCF7 and T47D cells (supplementary Figure S3A and B is available at Carcinogenesis Online). As dietary manipulation can potentially regulate other histone PTMs (33), we also determined H3K4me3 levels by western blot analysis. Our data indicated that H3K4me3 levels were also slightly downregulated by x-3 PUFAs but not x-6 PUFAs in MDA-MB-231 cells (supplementary Figure S4 is available at Carcinogenesis Online). Downregulation of EZH2 by x-3 PUFAs is accompanied by induction of E-cadherin and IGFBP3 Recently, it was reported that one of the important transcriptional targets of EZH2 is E-cadherin, a potential tumor suppressor (34,35). Specifically, it was shown that an overexpression of EZH2 represses the expression of E-cadherin and that EZH2 knockdown induces an expression of E-cadherin in breast cancer cells. To determine whether downregulation of EZH2 by x-3 PUFAs results in a upregulation of E-cadherin in breast cancer cells, we performed western blot analysis of mock- and DHA-treated MDA-MB-231 cells. Western blot analysis confirmed our inference that the downregulation of EZH2 by DHA results in upregulation of E-cadherin in MDA-MB-231 cells, which express virtually undetectable E-cadherin under normal growth conditions (Figure 5A). Another important direct target of EZH2 in cancer cells is IGFBP3, which has been shown to be repressed by
Fig. 4. x-3 PUFAs, but not x-6 PUFAs, downregulate H3K27me3, which correlates with EZH2 downregulation by x-3 PUFAs in MDA-MB-231 cells. Cells were treated with 40 lM PUFAs or mock treated with EtOH in chamber slides, fixed and coimmunostained using antibodies specific for EZH2 (BD Biosciences) and H3K27me3 (Upstate Biotechnology) and stained with nuclear stain 4#,6-diamidino-2-phenylindole (DAPI). After staining, cells were photographed (40) using a Nikon Eclipse 80i confocal microscope and colocalization was studied by merging different colors [Alexa Fluor 488 (EZH2), Alexa Fluor 594 (H3K27me3) and DAPI].
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cells with x-3 PUFAs may result in downregulation of EZH2 and decrease in the invasion potential of breast cancer cells. To confirm that EZH2 downregulation by x-3 PUFAs indeed results in decreased cancer cell invasion, we treated MDA-MB-231 cells with DHA and determined the invasion potential of DHA- and mock-treated cells. We found that DHA treatment of breast cancer cells led to a significantly decreased cancer cell invasion (Figure 5C). Discussion
Fig. 5. x-3 PUFAs upregulate E-cadherin and IGFBP3 and inhibits invasion in MDA-MB-231 cells. (A) Upregulation of E-cadherin by DHA in MDAMB-231 cells was determined by the western blot analysis using an antibody specific to E-cadherin (BD Biosciences). (B) Equal number of MDA-MB231 cells were plated and treated with EtOH or PUFAs for 8 h in equal volume of cell culture medium. IGFBP3 expression in the trichloroacetic acid-precipitated supernatant of treated cells was determined by western blot analysis using an anti-IGFBP3 antibody (Santa Cruz Biotechnology) as described in Materials and Methods. Ponceau staining of the blot was done to confirm the loading of supernatant proteins. The major band in the Ponceau stained blot probably represents immunoglobulin G from the serum constituent of the medium. (C) Effect of x-3 and x-6 PUFAs on invasion in MDA-MB-231 cells was determined by Matrigel Invasion assays using 24well BD Biocoat Matrigel Invasion Chambers (BD Biosciences) as described in Materials and Methods. Columns indicates mean of triplicate samples; bars represents SD; P , 0.002, P , 0.003.
EZH2 (31). To determine whether downregulation of EZH2 by x-3 PUFAs is also accompanied by increase in IGFBP3, we collected supernatant of DHA-treated breast cancer cells and analyzed the expression of IGFBP3 by western blot analysis. We found that DHA treatment led to increase in IGFBP3 levels in MDA-MB-231 cells (Figure 5B). These data suggest that downregulation of EZH2 by x-3 PUFAs upregulate E-cadherin and IGFBP3 in breast cancer cells. Downregulation of EZH2 by x-3 PUFAs is accompanied by a decrease in the invasive phenotype in breast cancer cells The treatment of cancer cells with x-3 PUFAs is known to induce cell death in a proportion of cells (36,37). We confirmed that the treatment of breast cancer cells with DHA and EPA (x-3 PUFAs), but not LA and AA (x-6 PUFAs), results in a quantifiable cell death (data not shown). Although EZH2 conceivably could play a role in cell survival by inhibiting cell death, the oncogenic phenotype that is clearly associated with EZH2 is tumor invasion and metastasis (11,12,17,34). Accordingly, we hypothesized that treatment of invasive breast cancer
The exact role of PUFAs in breast cancer development, progression and prevention is not very well understood. PUFAs can mediate cancer development and progression through multiple mechanisms. For example, it was recently shown that x-3 PUFAs, but not x-6 PUFAs, induced cell death in a Bad-dependent manner in a mouse model of prostate cancer (38). Among other important regulators of growth, survival and apoptosis, x-3 PUFAs have been shown to induce growth inhibition of MDA-MB-231 cells by decreased AKT phosphorylation and reduced DNA-binding activity of nuclear factor-kappaB (37). Similarly, treatment of BT-474 and SKBr-3 breast cancer cell lines with x-3 PUFA has been shown to suppress the expression of human epidermal growth factor receptor 2/neu oncoprotein via regulation of human epidermal growth factor receptor 2/neu transcription (39). A recent microarray and quantitative RT-PCR-based gene expression study of x-3- and x-6-treated breast cancer cells suggest that PUFAs are involved in the regulation of a broad spectrum of genes involved in diverse biological functions such as nutrition, cell division, proliferation, apoptosis and metastasis (40). x-3 PUFAS also act as angiogenesis inhibitors; in particular, EPA and DHA are known to inhibit production of many important angiogenic mediators such as vascular endothelial growth factor, platelet-derived growth factor, platelet-derived endothelial cell growth factor, cyclo-oxygenase 2 and prostaglandin-E2 (41). Very recently, DHA and EPA were also shown to downregulate cell surface expression of a chemokine receptor CXCR4 and also significantly reduce CXCR4-mediated cell migration in MDA-MB-231 cells (42). The regulation of a wide spectrum of unrelated genes by PUFAs suggests that these dietary fatty acids may modulate chromatin structure, which can result in concurrent changes in expression of genes that are not necessarily related. It is very well established that the structure of chromatin is modulated by epigenetic mechanisms accompanied by histone PTMs such as histone methylation, histone acetylation and histone ubiquitination and methylation of specific regions/loci in the genome. We hypothesized that PUFAs and other fatty acids may work epigenetically by modulating histone PTMs. A variety of nuclear proteins are known to possess histone PTM activities, most notably PcG proteins. In the present study, we have provided a direct evidence that DHA and EPA, the two well-known x-3 PUFAs downregulate EZH2, a core component of PRC2, which trimethylates histone 3. Our data also indicate that x-3 PUFAs can downregulate SUZ12 and EED, the two other constituent proteins of PRC2. The mechanism of downregulation of SUZ12 and EED by x-3 PUFAs is yet to be determined. The downregulation of EZH2 by the RNA interference approach, the treatment of cells with 3-deazaneplanocin A, a pharmacological inhibitor of EZH2, and histone deacetylase inhibitors have also been shown to downregulate SUZ12 and EED (10,30,31). It is possible that disruption of PRC2 complex due to proportionally less EZH2 indirectly affects the expressions of SUZ12 and EED. As expected, the downregulation of EZH2 and other PRC2 constituent proteins by x-3 PUFAs results in a downregulation of H3K27me3. In addition to H3K27me3, we also observed slight downregulation of H3K9me3 and H3K4me3 by x-3 PUFAs in breast cancer cells. H3K9me3 activity primarily depends on Suv39h histone methyl transferases (HMTases) and is associated with gene silencing, however, EZH2 could be partially involved in the regulation of H3K9me3 (32). Hence, somewhat decreased levels of H3K9me3 may be related to the downregulation of EZH2. On the other hand, H3K4me3 is generally associated with active transcription and is regulated by ASH1-like protein and mixed lineage leukemia proteins,
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which are homologues of Drosophila trithorax group of proteins (43). Since H3K4me3 is catalyzed by several HMTases, it is probably that H3K4me3 regulation by PUFAs is complex, and additional HMTases may be regulated by x-3 PUFAs. A detailed analysis of regulation of various HMTases and histone PTMs by PUFAs is yet to be performed. Nonetheless, EZH2 and PRC2-associated H3K27me3 are known to be involved in the regulation of several cancer-relevant targets in mammalian cells such as E-cadherin and IGFBP3. In the present study, we found that the expression of E-cadherin and IGFBP3 is upregulated by x-3 PUFAs, suggesting that downregulation of EZH2 and PRC2-associated H3K27me3 activity by DHA and EPA resulted in upregulation of these important potential tumor suppressors. Furthermore, treatment of breast cancer cells with x-3 but not x-6 PUFAs decreased breast cancer cell invasion, a tumor phenotype that is regulated by EZH2 and its targets including E-cadherin. It is tempting to speculate that decrease in invasive phenotype of breast cancer cells by x-3 PUFAs results because of the downregulation of EZH2 and EZH2-associated PRC2 activity. In summary, our studies suggest that anti-oncogenic effect of x-3 PUFAs, at least in part is mediated by the PcG targets of PUFAs such as EZH2. With respect to the mechanism of downregulation of EZH2 by DHA and EPA, our studies suggest that these x-3 PUFAs posttranslationally regulate the expression of EZH2 in breast cancer cells. The posttranslational regulation of EZH2 by x-3 PUFAs probably involves EZH2 ubiquitination and subsequent degradation by proteosome-dependent pathways. A detailed understanding of the mechanism of EZH2 proteolysis should help further in the identification of chemotherapeutics and chemopreventive agents that could be used to target EZH2 and PRCs to restore the expression of PcGsilenced tumor suppressors in cancer cells. Nevertheless, to the best of our knowledge, this is the first study that links the regulation of expression of PcG proteins, in particular EZH2, to chemopreventive agents such as dietary fatty acids. In summary, our data suggest that certain chemopreventive agents such as x-3 PUFAs may function by modulating the expression of PcG proteins, histone PTMs and chromatin modification, which collectively contribute to anti-oncogenic and chemopreventive properties of x-3 PUFAs. Supplementary material Supplementary Figures S1–S3 can be found at http://carcin .oxfordjournals.org/ Funding National Cancer Institute (RO1 CA094150); National Institutes of Health to G.P.D. Acknowledgements M.D. gratefully acknowledges support from Breast and Ovarian Cancer Research Program of the NorthShore University HealthSystem, Evanston, IL. Conflict of Interest Statement: None declared.
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