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. 2008 Apr;99(4):738-46.
doi: 10.1111/j.1349-7006.2008.00743.x.

Enhancer of zeste homolog 2 downregulates E-cadherin by mediating histone H3 methylation in gastric cancer cells

Satoshi Fujii  1 , Atsushi Ochiai
Affiliations

Enhancer of zeste homolog 2 downregulates E-cadherin by mediating histone H3 methylation in gastric cancer cells

Satoshi Fujii et al. Cancer Sci. 2008 Apr.

Abstract

Overexpression of enhancer of zeste homolog 2 (EZH2), an epigenetic repressor, occurs in various malignancies and is associated with poor prognosis; however, the functional role of EZH2 overexpression in cancer versus non-cancerous tissue remains unclear. In this study, we found an inverse correlation between EZH2 and E-cadherin gene expression in gastric cancer cells. Knockdown of EZH2 by short interfering RNA in gastric cancer cells resulted in a restoration of the E-cadherin gene. We showed that the EZH2 complex existed with histone H3 and Lys27, which were methylated on E-cadherin promoter regions in gastric cancer cells. The restoration of E-cadherin was not involved in the change of the DNA methylation status in the E-cadherin promoter region. Immunofluorescence staining confirmed the expression of E-cadherin protein present in the cell membrane was restored after knockdown of EZH2, resulting in changing the cancer phenotype, such as its invasive capacity. In vivo, the relationship of inverse expression between EZH2 protein and E-cadherin protein was observed at the individual cellular level in gastric cancer tissue. This study provides into the mechanisms underlying the functional role of EZH2 overexpression in gastric cancer cells and a new modality of regulation of E-cadherin expression in silencing mechanisms of tumor suppressor genes. Our present study paves the way for exploring the blockade of EZH2 overexpression as a novel approach to treating cancer.

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Figures

Figure 1
Figure 1
Profiling of enhancer of zeste homolog 2 (EZH2) mRNA expression in gastric cancer cell lines, gastric cancer tissue, and non‐cancerous mucosa. (a) EZH2 mRNA from cancer cells including gastric, prostate, and breast cancer cell lines was determined by quantitative real‐time reverse transcription–polymerase chain reaction (RT‐PCR). After normalizing with glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), the ratio of each cell line (mean ± SD) carried out in duplicate from two different experiments, was calculated based on the standard curve. (b) Western blot analysis showed expression of EZH2 protein in 12 gastric cancer cell lines. (c) The level of E‐cadherin mRNA expression in 12 gastric cancer cell lines was confirmed by quantitative real‐time RT‐PCR. (d) EZH2 mRNA from 22 pairs of gastric cancer and non‐cancerous gastric mucosa was determined by quantitative real‐time RT‐PCR. The values from each tissue sample were calculated as above. The significant difference between gastric cancer tissue and non‐cancerous mucosa was determined by Mann–Whitney U‐test.
Figure 2
Figure 2
Restoration of E‐cadherin mRNA in MKN1 gastric cancer cell line after knockdown of enhancer of zeste homolog 2 (EZH2) accompanied by chromatin remodeling without DNA methylation in the promoter region of the E‐cadherin gene. (a) Restoration of E‐cadherin mRNA analyzed by reverse transcription–polymerase chain reaction (RT‐PCR) in MKN1 cells was visualized with 6% polyacrylamide gel electrophoresis. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; siRNA, short interfering RNA. (b) The restored expression level of E‐cadherin mRNA was quantified by real‐time RT‐PCR. (c) Chromatin immunoprecipitation (ChIP) assay was carried out using DNA–protein complex isolated from MKN1 cells transfected with EZH2 siRNA for 96 h and immunoprecipitated (IP) with various antibodies. The PCR product of each IP DNA and input DNA was visualized with 6% polyacrylamide gel electrophoresis. The number under each gel is the ratio of IP DNA versus input DNA quantified. A, antibody; B, no antibody; C, input. Individual ChIP assays were repeated at least twice to confirm the reproducibility of the PCR‐based experiment. The results of ChIP assays are shown as a diagram for each antibody and statistical analyses (student's t‐test) were carried out (H3K27me3; H3‐Lys‐27 trimethylation, H3K9me2; H3‐Lys‐9 dimethylation). (d) Methylation‐specific polymerase chain reaction analyses from the DNA of MKN1 cells transfected with EZH2 siRNA (e) or negative control siRNA (c), using primer sets that specifically amplify either unmethylated (U) or methylated (M). Control templates from human genomic placenta DNA, treated with SssI methylase (S) or untreated (P), are shown. Ma, 100 bp DNA ladder marker. (e) Combined bisulfite restriction analysis (COBRA) from the DNA of MKN1 and MKN28 cells transfected with EZH2 siRNA (E) or negative control siRNA (C), using primer sets for the E‐cadherin gene. MKN28 was a positive control for E‐cadherin expression. PCR products were digested with specific restriction enzymes including SnaBI or TaqI. (f) Bisulfite sequencing of the E‐cadherin CpG island. Top, map of the CpG island. Twenty‐two individual CpG dinucleotides are indicated as vertical lines. Primer, location of bisulfite sequencing primers. Numbers indicate positions relative to transcription start. TS, transcriptional start. Bottom, each circle indicates a CpG site in the primary sequence, and each line of circles represents analysis of a single cloned allele. Black circles, methylated CpG sites; white circles, unmethylated CpG sites.
Figure 3
Figure 3
Knockdown of enhancer of zeste homolog 2 (EZH2) restores E‐cadherin expression and induces the membranous translocation of β‐catenin from the nucleus. (a) The re‐expression of E‐cadherin was also detected by Western blot analysis, without changing β‐catenin expression after EZH2 knockdown. (b) Human gastric cancer MKN1 cells transfected with EZH2 short interfering RNA (siRNA) show the restoration and membranous distribution of E‐cadherin (E‐cadherin, red; EZH2, green). (c) MKN1 cells transfected with EZH2 siRNA showed the translocation of β‐catenin from the nucleus to the membrane (β‐catenin, red; EZH2, green).
Figure 4
Figure 4
Effect of E‐cadherin restored by the knockdown of enhancer of zeste homolog 2 (EZH2) on the invasive capacity of human gastric cancer MKN1 cells. (a) The invading cells were quantitated by dissolving stained cells with colorimetric reading of the optical density (OD) at 560 nm. (b) Forty‐eight hours after transfection with EZH2 short interfering RNA (siRNA) or control siRNA, cells were re‐seeded in new dishes at a concentration of 1.0 × 105 cells, then enumerated. Bars, SD; points, mean of three independent experiments.
Figure 5
Figure 5
Correlation between enhancer of zeste homolog 2 (EZH2) expression, expression of E‐cadherin, and the localization of β‐catenin in gastric cancer tissues. The expression of these proteins was evaluated by immunohistochemistry using 114 human gastric adenocarcinomas. (a) EZH2 is normally expressed in the neck region, the proliferative zone for gastric mucosa. HE, hematoylin–eosin. (b) High expression of EZH2 correlated with loss of E‐cadherin expression and nuclear and cytoplasmic localization of β‐catenin. (c) No expression of EZH2 correlated with membranous localization of E‐cadherin or β‐catenin. Lymphoblasts served as appropriate positive controls for EZH2. (d) The relationship between EZH2 expression and negative or membranous localization of E‐cadherin expression in 114 gastric cancer tissues (χ2‐test, P < 0.0001). Bar, 100 µm (a–c).
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