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

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.

Somatic Mutations, Allele Loss, and DNA Methylation of the Cub and Sushi Multiple Domains 1 (CSMD1) Gene Reveals Association with Early Age of Diagnosis in Colorectal Cancer Patients

“…Our data also show that CSMD1 DNA methylation is significantly positively correlated with both methylated ALX4 and the known PRC2 target RUNX3. Observing RUNX3 methylation in our data coincides with others who have reported that 21% of colorectal cancer have RUNX3 hypermethylation [48]. We conclude that CSMD1 silencing by PRC2-mediated histone modifications may have impacted the somatic mutation landscape through acquired DNA methylation [49], [50].…”

EZH2 Depletion Blocks the Proliferation of Colon Cancer Cells

“…These results are consistent with a growth-promoting role for EZH2 in colon cancer and are in contrast to a recent report indicating that the growth of colon cancer cells is not affected by siRNA-mediated EZH2 depletion [22].…”

Loss of RUNX3 expression is an independent adverse prognostic factor in diffuse large B-cell lymphoma

“…[28,29] EZH2 expression has also been linked by some investigators to RUNX3 expression in various cancer types. [3][4][5][6] However, our results suggest that EZH2 expression appears to be decoupled from RUNX3 downregulation. A majority of the DLBCL tumors in our study showed EZH2 expression, consistent with the literature.…”

“…[2] In particular, EZH2 has been shown to mediate epigenetic silencing of RUNX3 in malignancies including gastric, breast, prostate, colon, pancreatic, [3] neuroblastoma, [4] and cholangiocarcinoma. [5] However, other studies failed to find an association between RUNX3 downregulation and EZH2 expression in colorectal carcinoma [6] suggesting alternative mechanisms may exist for RUNX3 loss. A few studies have explicitly investigated this possibility, and present evidence for RUNX3 downregulation due to loss of function point mutation, [7] copy number loss, [8] and aberrant cytoplasmic localization.…”