EZH2 and histone deacetylase inhibitors induce apoptosis in triple negative breast cancer cells by differentially increasing H3 Lys27 acetylation in the BIM gene promoter and enhancers

Abstract

Enhancer of zeste homolog 2 (EZH2), a subunit of polycomb repressive complex 2, is a histone methyl-transferase and is considered to work cooperatively with histone deacetylases (HDACs) in the same protein complex to mediate gene transcription repression by increasing histone H3 Lys²⁷ trimethylation (H3K27me3), in particular in the nucleosome (s). EZH2 is overexpressed in numerous types of cancer, including triple negative breast cancer (TNBC), a subtype of breast cancer, which there are no effective treatment options for. Thus, inhibition of EZH2 may be harnessed for targeted therapy of this disease. The present study demonstrated that co-treatment with an EZH2 inhibitor and a HDAC inhibitor additively induced apoptosis in two TNBC cell lines, namely MDA-MB-231 and MDA-MB-436. The increased rate of cell death was associated with an elevation of B cell lymphoma-2 like 11 (BIM) expression level, a pro-apoptotic protein at the protein and mRNA expression levels in these two cell lines. The expression of forkhead box O1 (FOXO1), a known upstream transcriptional activator of BIM, was upregulated in both cell lines by the HDAC inhibitor, and the effect was more pronounced in MDA-MB-436 cells with higher phosphorylation levels of protein kinase B, a negative regulator of FOXO1, compared with MDA-MB-231 cells. Conversely, FOXO1 expression was inhibited following treatment with the EZH2 inhibitor, suggesting that EZH2 and HDAC inhibitors induced BIM expression via a FOXO1-independent mechanism. The present study further revealed that the EZH2 inhibitor, but not the HDAC inhibitor, induced high levels of H3K27 acetylation (H3K27ac) in the BIM promoter. By contrast, compared with the effect of the EZH2 inhibitor, HDAC inhibitor treatment resulted in an increase in H3K27ac at two BIM enhancers. Collectively, the results of the present study indicated that EZH2 and HDACs act differentially on H3K27ac levels in the nucleosome at the promoter and enhancer regions of the BIM gene. Through the upregulation of BIM, co-treatment with EZH2 and HDAC inhibitors had a pronounced therapeutic effect on TNBC cells, suggesting that co-targeting EZH2 and HDAC proteins represents a viable therapeutic option for the treatment of TNBC.

Keywords: B cell lymphoma-2 like 11; chemotherapy; enhancer of zeste 2 polycomb repressive complex 2 subunit; forkhead box O1; histone H3 Lys27 acetylation; histone H3 Lys27 trimethylation; histone deacetylases; triple negative breast cancer.

Figure 1.

EZH2 and HDAC inhibitors induce morphological changes in MDA-MB231 and MDA-MB-436 cells. (A) MDA-MB-231 and (B) MDA-MB-436 cells were treated with GSK126 (15 µM), LBH589 (2.5 nM) or both for 24 h. Cells growing under normal conditions (DMSO) were spindle-like and attached well to the culture dish. Conversely, following treatment with these inhibitors, certain cells were detached and became round, which is indicative of apoptotic cell death. Scale bars, 100 µm. EZH2, enhancer of zeste 2 polycomb repressive complex 2 subunit; HDAC, histone deacetylase; DMSO, dimethyl sulfoxide; GSK, GSK126; LBH, LBH589.

Figure 2.

EZH2 and HDAC inhibitors induce apoptotic death in triple negative breast cancer cells. (A) MDA-MB-231 cells were treated with vehicle (DMSO), GSK126, LBH589 or both GSK126 and LBH589 for 24 h. Cells were harvested and stained with PI, followed by fluorescence-activated cell sorting analysis. Apoptotic cells (PI intensity <2N) were determined by CellQuest software (version 5.1; BD Biosciences, Franklin Lakes, NJ, USA). (B) The percentage of apoptotic cells in the indicated groups was determined from two replicates. Data are represented as the mean ± standard deviation (n=2). *P<0.05, **P<0.01, ***P<0.001 compared with the control (DMSO) group. EZH2, enhancer of zeste 2 polycomb repressive complex 2 subunit; HDAC, histone deacetylase; DMSO, dimethyl sulfoxide; PI, propidium iodide; N, copy number of unreplicated genome; GSK, GSK126; LBH, LBH589.

Figure 3.

Treatment of triple negative breast cancer cells with EZH2 and HDAC2 inhibitors induces PARP cleavage and BIM protein expression. (A) MDA-MB-231 and (B) MDA-MB-436 cells were treated with vehicle (DMSO), GSK126, LBH589 or both GSK126 and LBH589 as indicated for 24 h. Cells were then harvested for western blot analysis using the indicated antibodies. ERK2 expression was used as a protein loading control. Similar results were obtained in two independent experiments. EZH2, enhancer of zeste 2 polycomb repressive complex 2 subunit; HDAC, histone deacetylase; PARP, poly (ADP-ribose) polymerase; BIM, B cell lymphoma-2 like 11; DMSO, dimethyl sulfoxide; ERK2, extracellular signal-related kinase 2; IB, immunoblot.

Figure 4.

EZH2 and HDAC inhibitors induce BIM mRNA expression in triple negative breast cancer cells. (A) MDA-MB-231 and (B) MDA-MB-436 cells were treated with vehicle (DMSO), GSK126, LBH589 or both GSK126 and LBH589 as indicated for 24 h. Cells were then harvested, and BIM mRNA expression was analyzed by reverse transcription-quantitative polymerase chain reaction, using GAPDH as an internal control. All data are presented as the mean ± standard deviation (error bar) from three replicates. **P<0.01, ***P<0.001 compared with the control (DMSO) group. EZH2, enhancer of zeste 2 polycomb repressive complex 2 subunit; HDAC, histone deacetylase; BIM, B cell lymphoma-2 like 11; DMSO, dimethyl sulfoxide; GSK, GSK126; LBH, LBH589.

Figure 5.

Effect of EZH2 and HDAC2 inhibitors on FOXO1 protein expression in triple negative breast cancer cells. (A) MDA-MB-231 and (B) MDA-MB-436 cells were treated with vehicle (DMSO), GSK126, LBH589 or both GSK126 and LBH589 as indicated for 24 h. Cells were then harvested for western blot analysis using the indicated antibodies. ERK2 expression level was used as the protein loading control. Non-specific western blotting bands are indicated by *, a phenomenon observed in HeLa cells (http://www.cellsignal.com/products/primary-antibodies/akt-antibody/9272?N=4294956287&Ntt=akt&fromPage=plp). Similar results were obtained in two independent experiments. EZH2, enhancer of zeste 2 polycomb repressive complex 2 subunit; HDAC, histone deacetylase; DMSO, dimethyl sulfoxide; ERK2, extracellular signal-related kinase 2; AKT, protein kinase B; p-, phosphorylated; FOXO1, forkhead box O1; PTEN, phosphatase and tensin homolog.

Figure 6.

EZH2 and HDAC inhibitors increase H3K27ac expression levels in the BIM gene promoter and enhancers in triple negative breast cancer cells. (A) University of California at Santa Cruz genome browser screen shots display ChIP-seq signals of H3K27ac, the enhancer histone mark H3K4me1 and the promoter histone mark H3K4me3 obtained from the public data generated from a different cancer cell type (prostate cancer LNCaP cells). Reverse transcription-quantitative polymerase chain reaction analysis of ChIP DNA using (B) primers for the promoter, (C) putative enhancer-1 and (D) enhancer-2. DNA was immunoprecipitated by control IgG or anti-H3K27ac antibody from MDA-MB-231 and MDA-MB-436 cells treated with vehicle (DMSO), GSK126, LBH 589 or both GSK126 and LBH589. Cells were harvested for ChIP assay at 24 h after treatment. *P<0.05, **P<0.005 compared with the control (DMSO) group. H3K27me1, histone H3 Lys²⁷ methylation; H3K27me3, histone H3 Lys²⁷ trimethylation; H3K27ac, histone H3 Lys²⁷ acetylation; EZH2, enhancer of zeste 2 polycomb repressive complex 2 subunit; HDAC, histone deacetylase; DMSO, dimethyl sulfoxide; BIM/BCL211, B cell lymphoma-2 like 11; GSK, GSK126; LBH, LBH 589; G+L, GSK126 and LBH 589; ChIP, chromatin immunoprecipitation; IgG, immunoglobulin G.