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. 2010 Sep;31(9):1567-75.
doi: 10.1093/carcin/bgq147. Epub 2010 Jul 14.

The role of EZH2 and DNA methylation in the silencing of the tumour suppressor RUNX3 in colorectal cancer

Liudmila L Kodach  1 , Rutger J Jacobs, Jarom Heijmans, Carel J M van Noesel, Alexandra M J Langers, Hein W Verspaget, Daniel W Hommes, G Johan A Offerhaus, Gijs R van den Brink, James C H Hardwick
Affiliations

The role of EZH2 and DNA methylation in the silencing of the tumour suppressor RUNX3 in colorectal cancer

Liudmila L Kodach et al. Carcinogenesis. 2010 Sep.

Abstract

In gastric cancer, a new epigenetic mechanism of tumour suppressor loss has been suggested where the histone methyltransferase enhancer of zeste homolog 2 (EZH2) is responsible for loss of expression of RUNX3. This is consistent with EZH2 upregulation in multiple cancer types being associated with poor prognosis. We investigated whether EZH2 influences the expression of RUNX3 in colorectal cancer (CRC) and whether this is independent of methylation. We determined protein and messenger RNA (mRNA) levels of EZH2 and RUNX3 and assessed RUNX3 methylation with methylation-specific polymerase chain reaction using 72 human CRCs and 8 CRC cell lines. We assessed the effect of efficient RNA interference-mediated knockdown of EZH2 on RUNX3 levels, cell viability and H3K27 trimethylation of the RUNX3 promoter using chromatin immunoprecipitation. Despite higher levels of EZH2 and lower levels of RUNX3 in CRC specimens in general, no inverse correlation between EZH2 and RUNX3 in paired samples was found arguing against a major role for histone methylation in silencing RUNX3 in CRC. Conversely, downregulation of RUNX3 mRNA in the same tumours was associated with RUNX3 DNA methylation (P < 0.05). In cell lines, knockdown of EZH2 removed the repressive chromatin marks from RUNX3 but did not result in RUNX3 re-expression. However, it prevented the re-silencing of RUNX3 after the removal of demethylating agents. In conclusion, DNA methylation is primarily responsible for the transcriptional silencing of RUNX3 in CRC, but EZH2 and histone methylation are necessary for its methylation-dependent re-silencing after the removal of demethylating agents. These results would predict that inhibitors of EZH2 and histone methylation would enhance the effects of demethylating agents in cancer therapy.

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Figures

Fig. 1.
Fig. 1.
Expression of EZH2 and RUNX3 in CRC cell lines and CRC patient specimens. (A) Immunoblots of colon cancer cell lines for EZH2 and RUNX3 with actin as a loading control. (B) Methylation-specific PCR (MSP) analysis of the CpG island methylation status of the RUNX3 promoter region in CRC cell lines. PCR products specific for unmethylated (U) and methylated (M) CpG sites were analysed in 2.5% agarose gels. (C) Positive nuclear expression of EZH2 and RUNX3 in HCT116 and SW480 CRC cell lines by immunofluorescence. (D) Quantitative RT–PCR analysis of EZH2 and RUNX3 expression in CRC cell lines. (E) Quantitative RT–PCR analysis of EZH2 (linear scale) and RUNX3 expression (log10 scale) in CRC patients. (F) MSP for RUNX3 promoter region MSP in a subgroup of CRC patients. T1–T5 shows positive methylated and unmethylated signals, whereas T6–T8 shows only an unmethylated band.
Fig. 2.
Fig. 2.
Expression of EZH2 and RUNX3 in normal human colon and in CRC specimens using immunohistochemistry. (A) Expression pattern of EZH2 in normal human colon. (B) Expression pattern of RUNX3 in normal human colon. (C) Positive nuclear EZH2 staining in CRC. (D) Positive RUNX3 expression in CRC. (E) Overexpression of EZH2 in cancer tissue. (F) Negative expression of RUNX3 in cancer tissue. (G) EZH2 shows negative staining in 9 (12.5%), positive staining in 9 (12.5%) and is overexpressed in 54 (75%) of CRC patients. RUNX3 is not expressed in 46 (64%) and exhibits positive nuclear or cytoplasmatic staining in 23 (36%) of CRC patients.
Fig. 3.
Fig. 3.
Co-localized expression patterns of EZH2 and RUNX3 in CRC specimens. (A) Kaplan–Meier survival analysis of CRC patients according to the expression of EZH2 and RUNX3. No significant association was found. (B) Overexpression of EZH2 coincides with (C) negative expression of RUNX3. (D) Overexpression of EZH2 co-localizes with (E) strong nuclear positivity of RUNX3. (F) Immunofluorescence images of CRC cells stained with EZH2 and RUNX antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole. RKO and DLD1 cells show negative expression of RUNX3. The RUNX3 expression does not change after efficient EZH2 knockdown in DLD1 cells.
Fig. 4.
Fig. 4.
EZH2 knockdown is not sufficient to restore RUNX3 expression in CRC cell lines. (A) Immunoblots of colon cancer cell lines for EZH2 and RUNX3 with actin as a loading control. For every cell line, the first lane is loaded with negative control siRNA-transfected cell lysate and the second lane with EZH2 siRNA-transfected cell lysate. Despite undetectable levels of EZH2 protein after siRNA-mediated knockdown, RUNX3 protein levels remain unchanged. (B) Chromatin immunoprecipitation analysis of the RUNX3 promoter in DLD1 cells treated with control or EZH2 siRNA. Two different pairs of primers for RUNX3 promoter region were used (chromatin immunoprecipitations 1 and 2). Levels of tri-methylated histone 3 K27 are reduced by siRNA-mediated knockdown of EZH2. (C) Left panel. Quantification of the efficiency of the siRNA-mediated knockdown of EZH2 on mRNA levels. The EZH2 mRNA expression in negative control siRNA-transfected cells is set at 100%. (C) Right panel. Quantitative RT–PCR shows no differences in RUNX3 mRNA expression after EZH2 knockdown. (D) Quantitative RT–PCR analysis of RUNX3 mRNA levels in cells treated with 5-aza-2’-deoxycytidine (5-aza-dC) and combined treatment of 5-aza-dC with trichostatin A (TSA) shows a considerable increase in RUNX3 mRNA level.
Fig. 5.
Fig. 5.
(A, B, C and D) Efficient EZH2 knockdown does not affect cell growth or viability as assessed by the MTT assay in different CRC cell lines. (E) DLD1 cells were demethylated by treatment with 5-aza-dC and trichostatin A (TSA) for 3 days (72 h) leading to re-expression of RUNX3 mRNA. The same cells were subsequently transfected either with control siRNA or with EZH2 siRNA and the level of RUNX3 expression was evaluated 72 h after transfection (6 days after the beginning of the experiment). Knockdown of EZH2 significantly inhibits the re-silencing of RUNX3 after the removal of demethylating agents.
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