Wedelolactone disrupts the interaction of EZH2-EED complex and inhibits PRC2-dependent cancer.

Polycomb repressive complex 2 (PRC2), which is responsible for the trimethylation of H3K27 (H3K27me3), plays a part in tumorigenesis, development and/or maintenance of adult tissue specificity. The pivotal role of PRC2 in cancer makes it a therapeutic target for epigenetic cancer therapy. However, natural compounds targeting the enhancer of zeste homolog 2 (EZH2) - embryonic ectoderm development (EED) interaction to disable PRC2 complex are scarcely reported. Here, we reported the screening and identification of natural compounds which could disrupt the EZH2-EED interaction. One of these compounds, wedelolactone, binds to EED with a high affinity (KD = 2.82 μM), blocks the EZH2-EED interaction in vitro, induces the degradation of PRC2 core components and modulates the expression of detected PRC2 downstream targets and cancer-related genes. Furthermore, some PRC2-dependent cancer cells undergone growth arrest upon treatment with wedelolactone. Thus, wedelolactone and its derivatives which target the EZH2-EED interaction could be candidates for the treatment of PRC2-dependent cancer.


IntroductIon
Cancer is a major public health problem in the world. In the United States, estimated new cancer cases and cancer deaths in 2014 are 1,665,540 and 585,720, respectively [1]. Recurrent somatic mutations in numerous epigenetic regulators in various cancers draw much attention and highlight the fact that we have now entered an era of epigenetic cancer therapies [2,3]. The epigenomic landscape features different machinery in transcriptionally active versus silent regions [4]. Polycomb group (PcG) proteins, conserved chromatin proteins, are widely deployed in higher eukaryotes to implement gene silencing [5].
In this study, we used the Biacore 3000 and competitive co-immunoprecipitation (co-IP) assay to screen for small-molecule inhibitors which could disturb the binding of EZH2 to EED from the natural products library. Two compounds, epigallocatechingallate (EGCG) and wedelolactone, were identified and further studied. Interestingly, EGCG has been reported by Subhasree Roy Choudhury's group with a function to negatively regulate PRC2 [22]. In addition to disrupt PRC2, we found that wedelolactone also induce the degradation of PRC2 core components and modulate the expression of PRC2 targets and cancer-related genes. Moreover, we observed that wedelolactone could inhibit the proliferation and migration, induce cell cycle arrest and apoptosis of PRC2 dependent cancer cells. Our results provide evidences that EZH2-EED interaction is a target for the treatment of PRC2-dependent cancer and wedelolactone is a candidate for modifications in the future. results screen for natural compounds disrupting the eed-eZH2 interaction EED was reported to bind the N-terminal sequence of EZH2 (residues 39-68) [20], so natural compounds which could bind to EED might disrupts the EZH2-EED interaction. Then we used the SPR platform Biacore 3000 to screen for natural compounds that bind to EED. Fresh recombinant EED was covalently immobilized on a CM5 sensor chip as ligand before detection. Natural compounds were diluted in PBS buffer and injected as analyte. The response unit (RU) of each compound was collected and was showed in Figure 1A.
Then, we performed competitive coimmunoprecipitation (co-IP) experiments to identify EED-EZH2 disruptors among natural compounds with RU higher than 50. In these disruptors, we found that 1E7 (EGCG) and 2D7 (wedelolactone) with the concentration of 5 µM could disrupt the interaction between EZH2 and EED significantly ( Figure 1B). In order to exclude the potential influence of other proteins in the process, we translated Myc-EZH2 and Myc-His-EED in vitro using the reticulocyte lysate system and performed competitive co-IP assays to investigate the effects of 2D7 on the interaction between EZH2 and EED. The results showed that 2D7 blocked the binding of EZH2 to EED efficiently ( Figure 1C), suggesting a direct inhibition of 2D7 on the association of these two proteins.
As dismantling the PRC2 complex could result in the decrease of protein stability and further depletion of PcG members [21], we examined whether wedelolactone treatment altered the levels of EZH2 and EED. As shown in Figure 1D, wedelolactone treatment reduced the protein levels of these two core PRC2 components in human hepatocellular carcinoma cell lines HepG2, human monocytic leukemia cells THP1 and human myeloid leukemia cell lines K562.

sPr detection of eGcG and wedelolactone binding to eed
Drug candidate is usually expected to bind its target with a high affinity [23]. Here SPR platform Biacore 3000 was used to monitor the direct interaction between wedelolactone/EGCG and EED. Fresh recombinant EED proteins were covalently immobilized on a dextran sensor chip as ligand before detection. Wedelolactone/ EGCG was serially diluted in a vehicle of 1% DMSO in PBS buffer and injected as analyte to flow liquid phase. The sensorgrams had shown direct binding between wedelolactone ( Figure 2A)/EGCG ( Figure 2B) and EED molecule in a dose-dependent manner. Evaluated by BIA evaluation software, the equilibrium dissociation constant (K D ) value of wedelolactone (2D7)/EGCG (1E7) to EED is 2.82 μM and 15.1 μM, respectively ( Table 2).

Wedelolactone modulates Prc2 targets and tumor-related genes expression in Prc2dependent cancer cells
PRC2 has been reported to participate in silencing a myriad of target genes which are important in tumorigenesis and cancer progression. Several PRC2 target genes harbor tumor suppression function. For example, the DOC-2/DAB2 interactive protein(DAB2IP), a growth inhibitor, is involved in the tumor necrosis factor-mediated JNK signaling pathway leading to cell apoptosis [24]; Beta-2 adrenergic receptor(ADRB2) activation in xenograft mouse models inhibits prostate cancer tumor growth in vivo [25]; Loss of CDKN2A contributes to the loss of control over cell cycle, the bypass of critical senescent signals and is associated with progression to malignant disease [26] and GADD45A (growth arrest and DNA-damage-inducible, alpha) involves in cell cycle and apoptosis [27].
Dysregulation of EZH2 alters the expression of many cancer related genes [28,29]. For instance, targeting EZH2 could deplete HOXA9 and Meis1 levels in THP1 cells and disrupt the biological synergy between the two genes in inducing myeloid leukemia [30]. Moreover, sensitivity of cancer cells to the EZH2 inhibitors is partly dependent on PTEN and p53 [31,32].
To explore the regulation of wedelolactone treatment on the expression of these PRC2 target genes and cancer related genes, HepG2, THP1 and K562 cells were treated with 50 μM wedelolactone for 24 h. Total RNA were extracted and the mRNA levels of the above genes were analyzed by quantitative real-time PCR. As shown in Figure 3A, wedelolactone treatment significantly induces the expression of GADD45A, DAB2IP, ADRB2, CDKN2A and p53 while represses Meis1 expression in HepG2 cells. As shown in Figure 3B, wedelolactone significantly repressed HOXA9 and Meis1 expression while enhances the expression of GADD45A and p53 in THP1 cells. At the meantime, the expression of GADD45A, PTEN and p53 were activated after treatment with wedelolactone in  HepG2 cells (A), THP1 cells (b) and K562 cells (c) were treated with 50 μM wedelolactone for 24 h. Total RNA was isolated and qRT-PCR was performed with specific primers for the indicated target genes. Quantification results were shown as folds of control and expressed as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. leukemia cell lines K562 ( Figure 3C). Together, our results indicated that PRC2 targets and tumor-related genes which were involved in apoptosis and cell cycle arrest were modulated by wedelolactone.
We first examined the effects of wedelolactone on cell proliferation. As shown in Figure 4A, 50 μM of wedelolactone treatment repressed the proliferation of HepG2, THP1 and K562 cells. Since many drugs have been shown to inhibit cancer cells through induction of apoptosis, we then detected the apoptotic ratio in cells with or without wedelolactone treatment by Annexin V-FITC/PI double staining assay. As shown in Figure  4B, the presence of wedelolactone significantly increased apoptosis in HepG2, THP1 and K562 cells.
It is well-known that most anticancer agents, such as vinblastine and paclitaxel, have been documented to arrest cell cycle [35]. So we examined the effect of wedelolatctone on cell cycle distribution of HepG2, THP1 and K562 cells. Compared with control, there was an accumulation of cell population in S and G2/M phase after wedelolactone exposure in HepG2 cells and the proportion of K562 cells in S phase were increased after wedelolactone exposure ( Figure 4C). However, it exhibited no significant effect on THP1 cells.
Recently, several studies about EZH2 regulating cell invasion in various types of cancer showed that one of the major EZH2 PRC2-dependent function is promoting cell invasion [36][37][38]. Additionally, the migration of K562 and THP1 cells are scarcely reported. So we interrogated the effect of wedelolactone on the migration of HepG2 cells.
To determine whether wedelolactone inhibits cell migration in HepG2 cells, we performed Transwell migration assays. As shown in Figure 4D, the presence of wedelolactone significantly suppressed cell migration in HepG2 cells. Together, our data suggested that wedelolactone inhibited proliferation, induced apoptosis and cell cycle arrest and suppressed cell migration of PRC2-depedent cancer cells.

dIscussIon
Wedelolactone is an essential active compound of Eclipta prostrate. It has been reported to possess various biological functions, including the inhibition of IKK kinase, K+-ATPase activity, hepatitis virus C RNA-polymerase, phospholipase A2 , 5-lipoxygenase and DNA topoisomerase IIα [39]. Also, it exhibits anticancer function in some cancers, such as prostate cancer, breast cancer and so on [39]. The growth inhibition effects of wedelolactone on tumor cells were believed to be accomplished through its inhibition of IKK, the androgen receptor [40], or topoisomerase II. But its function on the EED-EZH2 interaction and the PRC2 activity is unknown.
In the present study, we present a new method to identify inhibitors targeting PRC2. Firstly, Biacore 3000 was used to screen for natural compouds which could bind to EED. Then the competitive co-IP experiment was performed to further identify PRC2 disruptors. By this way, we identified that EGCG and wedelolactone could bind to EED and target the EZH2-EED interaction.
Tumor suppressors refer to a large group of molecules that can prevent cancer through controlling cell www.impactjournals.com/oncotarget Wedelolactone decreased the number of migration cells compared with control cells (original magnification ×200). Quantification results were shown as folds of control and expressed as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. division, promoting apoptosis, helping DNA damage repair and suppressing metastasis [41]. So reactivating tumor suppressors which are silenced by PRC2 will contribute to the inhibition of carcinoma cells proliferation. Indeed, wedelolactone could activate PRC2 downstream tumor suppression genes such as DAB2IP, ADRB2, CDKN2A and GADD45A (Figure 3A), thus it serves as a mechanism for its inhibition on PRC2-dependent cancer cells. In fact, not all the target genes can be influenced by wedelolactone (data not shown).
In conclusion, we identified that wedelolactone could bind to EED and target PRC2, thereby modulate its targets and cancer-related genes. As a consequence, wedelolactone exhibits anti-cancer effects by inducing proliferation and migration inhibition, apoptosis and cell cycle arrest of PRC2-dependent cancer cells. So it could serve as a candidate for the treatment of PRC2dependent cancer. Also, our work verified the possibility to the development of anti-cancer agents by disrupting the association of core PRC2 components EZH2 and EED.

Abs and reagents
The primary antibodies used in this study were as follows: anti-Myc (sc-40), anti-EED (sc-28701) and anti-β-actin (sc-47778) were purchased from Santa Cruz Biotechnology. Anti-EZH2 (#3147S) was from Cell Signaling Technology, anti-His (#TA-02) was from ZSGB-BIO and anti-trimethyl histone H3 (Lys27) antibody (ABE44-S) was from Millipore. Wedelolactone was purchased from National Institutes for Food and Drug Control (NIFDC, China). Dimethylsulfoxide (DMSO) was obtained from Sigma-aldrich (USA). Protein G beads and GST beads were purchased from Santa Cruz Biotechnology and GE Healthcare, respectively.

binding detection based on sPr platform
The interaction between compound and protein was detected by surface plasmon resonance platform Biacore 3000 (GE Healthcare). Fresh EED protein was diluted to 100 μg/ml in 10 mM acetate buffer (pH 5.0), and then immobilized as ligand in the NHS/EDC pre-activated CM5 sensor chip, following blocking by ethanolamine.

competitive co-immunoprecipitation assay
Cell lysates from 293T cells transfected with Myc-EZH2 and Myc-His-EED were incubated with anti-His antibody, protein G beads and natural compound with corresponding concentration or DMSO overnight at 4ºC. The beads were then washed three times and boiled to be used for WB.

In vitro translation assays
Myc-EZH2 and Myc-His-EED were translated in vitro with TNT T7 coupled reticulocyte lysate system (Promega, #L4611) according to the manufacturer's instructions. The in vitro translated products were used to perform competitive co-immunoprecipitation assay.

Western blotting
Cells were lysed and prepared with 1 X SDS Reducing sample buffer (CST, #7722) according to the manufacturer's instructions. Appropriate volume of sample was loaded onto the SDS-polyacrylamide gels and transferred to a PVDF membrane. After blocking, the membrane was incubated with the primary antibody overnight at 4ºC followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Bands were detected using enhanced chemiluminescence (Applygen, China).

rnA extraction and quantitative reverse transcriptase polymerase chain reaction (qrt-Pcr)
Total RNA was isolated from the cells using TRNzol (TIANGEN, China). cDNA was synthesized using the RevertAid First Strand cDNA Synthesis kit (Thermo Scientific, #K1622). Quantitative real-time PCR (qRT-PCR) was conducted using SYBR premix Ex Taq II (Takara, China). Thermal cycling was performed using an ABI 7300 real-time PCR machine (Applied Biosystems) as follows: 95°C for 30 s followed by 40 cycles of amplification for 5 s at 95°C, 31 s at 60°C. The primer sequences used for PCR are listed in Table 1.

cell cycle analysis
The effect of wedelolactone on cell cycle distribution was determined by flow cytometric analysis. The cells were treated with 50 μM wedelolactone for 24 h. Appropriate controls were also set up. After treatment, 1×10 5 floating and adherent cells were collected, washed with PBS and fixed with 70% ethanol. Staining for DNA content was performed using Cell Cycle Detection Kit (KGA512, KeyGEN BioTECH, China). Populations in G0/G1, S and G2/M phases were measured by BD FACSCalibur Flow Cytometry System with CellQuest Pro software (BD Bioscience). Data were analyzed using the ModFit 3.0 Software.

Detection of apoptotic cells by flow cytometry
Cells were plated in six-well plates at a density of 1x10 5 cells/ml and incubated overnight. Wedelolactone or DMSO was then added into each well and incubated for 48 h. Cells were collected and washed with PBS, followed by resuspension in 300 μl binding buffer at a concentration of 5x10 5 cells/ml. Mixed with Annexin V-FITC and propidium iodide (PI) according to the manufacturer's instructions. The mixed solution was incubated in the dark at room temperature for 15 min. Cell apoptosis analysis was performed using the BD FACSAriaII Flow Cytometry System (BD, USA) within 1 h. Data were analyzed using the FACSDiva Version 6.1 Software.

cell migration assay
HepG2 cells were treated with 25 µm wedelolactone or DMSO for 12h then the cells were trypsinized and replated onto the upper chamber of a Transwell filter with 8 µm pores (Costar) at 2x10 5 cells/well in serumfree medium. Medium supplemented with 10% FBS was placed in the bottom well, and the cells were then incubated for 24 h at 37°C in a humidified 5% CO 2 atmosphere. After the incubation, the chambers were removed, and migration cells on the bottom side of the membrane were fixed with methanol for 15 min and stained with gentian violet for 10 min. Each experiment was performed in triplicate, and the number of cells in five random fields on the underside of the filter was counted and averaged. The results were expressed as the migrated cell number.

statistical analysis
The data are presented as mean ± stand deviation (S.D.). Parametrical data were compared using Student's t test. One-way ANOVA analysis was used to determine the difference between independent groups. The differences between the variants were considered to be statistically significant if P < 0.05.

conFlIcts oF Interest stAteMent
The authors declare no conflict of interest.