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.