Enhancer of Zeste Homologue 2 (EZH2) Down-regulates RUNX3 by Increasing Histone H3 Methylation

Abstract

Overexpression of enhancer of zeste homologue 2 (EZH2) occurs in various malignancies and is associated with a poor prognosis, especially because of increased cancer cell proliferation. In this study we found an inverse correlation between EZH2 and RUNX3 gene expression in five cancer cell lines, i.e. gastric, breast, prostate, colon, and pancreatic cancer cell lines. Chromatin immunoprecipitation assay showed an association between EZH2 bound to the RUNX3 gene promoter, and trimethylated histone H3 at lysine 27, and HDAC1 (histone deacetylase 1) bound to the RUNX3 gene promoter in cancer cells. RNA interference-mediated knockdown of EZH2 resulted in a decrease in H3K27 trimethylation and unbound HDAC1 and an increase in expression of the RUNX3 gene. Restoration of RUNX3 expression was not associated with any change in DNA methylation status in the RUNX3 promoter region. RUNX3 was repressed by histone deacetylation and hypermethylation of a CpG island in the promoter region and restored by trichostatin A or/and 5-aza-2-deoxycytidine. Immunofluorescence staining confirmed restoration of expression of the RUNX3 protein after knockdown of EZH2 and its restoration resulted in decreased cell proliferation. In vivo, an inverse relationship between expression of the EZH2 and RUNX3 proteins was observed at the individual cell level in gastric cancer patients in the absence of DNA methylation in the RUNX3 promoter region. The results showed that RUNX3 is a target for repression by EZH2 and indicated an underlying mechanism of the functional role of EZH2 overexpression on cancer cell proliferation.Three members of the Runt-related (RUNX) family of genes, RUNX1, RUNX2, and RUNX3, play pivotal roles in normal development and neoplasia. All RUNX family members share the central Runt domain, which is well conserved and recognizes a specific DNA sequence, but each has relatively divergent N-and C-terminal regions (1). RUNX3 is involved in neurogenesis (2, 3) and thymopoiesis (4, 5) and functions as a tumor suppressor gene in gastric cancer (6, 7). Failure to express RUNX3 because of a combination of hemizygous deletion and DNA hypermethylation of the RUNX3 promoter region has been found in about 60% of primary gastric cancer specimens (7). RUNX3-R122C is a mutation located in the conserved Runt domain that was discovered in a case of gastric cancer and it abolishes the tumor suppressive activity of RUNX3 (7). Subsequent studies have revealed that RUNX3 inactivation is not limited to gastric cancer, and frequent inactivation of RUNX3 due to DNA hypermethylation has been reported in various other cancers, including lung cancer (8), hepatocellular carcinoma (9), breast cancer (10), colon cancer (11), pancreatic cancer (12), bile duct cancer (12), prostate cancer (13), and laryngeal cancer (10). Thus, RUNX3 is primarily inactivated by epigenetic silencing, rather than by mutations or deletions, suggesting that RUNX3 can be reactivated and serve as a good gene for drug targeting.Enhancer of zeste homologue 2 (EZH2) 2 is one of the ...

Claudin-4 overexpression is associated with epigenetic derepression in gastric carcinoma

“…This also suggests that CLDN4 is not a direct target of EZH2 in contrast to RUNX3 or E-cadherin the expressions of which have been reported to be directly regulated by EZH2. 41,42 As shown in Figure 5e, DZNep/TSA caused a significant increase in active H3K4me3, H3Ac and H4Ac along with producing a significant reduction in H4K20me3 and H3K9me3 levels in SNU638 cells (Po0.05). Unexpectedly, H3K27me3 was not decreased by DZNep/TSA, suggesting that loss of the EZH2-mediated H3K27me3 mark in SNU638 cells may not be responsible for CLDN4 derepression by DZNep/TSA.…”

RUNX3 is multifunctional in carcinogenesis of multiple solid tumors

“…More specifically, reintroduction of miR-101 or knockdown of EZH2 expression was sufficient to reduce H3K27 trimethylation and increase RUNX3 mRNA levels. In the same vein, Fujii et al (2008) found that EZH2 binds to the RUNX3 promoter, resulting in upregulation of H3K27 methylation and concomitant downregulation of RUNX3 expression (Fujii et al, 2008). Recently, Lee et al (2009) extended this finding by showing that hypoxia-induced upregulation of G9a histone methyltransferase and HDAC1 expression also cause epigenetic RUNX3 silencing through H3K9 methylation and decreased H3 acetylation at RUNX3 promoter.…”

“…Although it is unclear what causes aberrant methylation of RUNX3, RUNX3 was recently reported to be silenced in gastric, breast, prostate, colon, and pancreatic cancer cell lines by another epigenetic mechanism-enhancer of Zeste Homologue 2 (EZH2)-mediated histone methylation (Fujii et al, 2008). The EZH2 oncogene is a component of Polycomb-repressor complex 2, which is required for stem cell pluripotency and self-renewal.…”

The expression of RUNX3 in colorectal cancer is associated with disease stage and patient outcome

“…Mislocalisation of RUNX3 protein to the cytoplasm is another mechanism by which RUNX3 can be inactivated in gastric and breast cancers Lau et al, 2006). Overexpression of the enhancer of zeste homologue 2 (EZH2) protein was recently shown to downregulate RUNX3 expression by increasing histone H3 methylation, thus providing yet another mechanism for inactivation of RUNX3 (Fujii et al, 2008). As might be expected, if RUNX3 were behaving as a tumour suppressor, the decreased expression of this protein in gastric (Wei et al, 2005), lung (Araki et al, 2005) and oesophageal (Sakakura et al, 2007) cancers has been associated with worse patient outcome.…”