title: 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.
authors.affiliation: Department of Gastroenterology and Hepatology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Liudmila L
authors.initials: LL
authors.lastname: Kodach
authors.forename: Rutger J
authors.initials: RJ
authors.lastname: Jacobs
authors.forename: Jarom
authors.initials: J
authors.lastname: Heijmans
authors.forename: Carel J M
authors.initials: CJ
authors.lastname: van Noesel
authors.forename: Alexandra M J
authors.initials: AM
authors.lastname: Langers
authors.forename: Hein W
authors.initials: HW
authors.lastname: Verspaget
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Hommes
authors.forename: G Johan A
authors.initials: GJ
authors.lastname: Offerhaus
authors.forename: Gijs R
authors.initials: GR
authors.lastname: van den Brink
authors.forename: James C H
authors.initials: JC
authors.lastname: Hardwick
mesh_terms: D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D050677:Core Binding Factor Alpha 3 Subunit / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D000071221:Enhancer of Zeste Homolog 2 Protein; D004797:Enzyme-Linked Immunosorbent Assay; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D016147:Genes, Tumor Suppressor; D006801:Humans; D007124:Immunoenzyme Techniques; D063151:Polycomb Repressive Complex 2; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D046888:Tissue Array Analysis; D014157:Transcription Factors / Q000502:physiology*
chemical_list: D050677:Core Binding Factor Alpha 3 Subunit; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; C493729:Runx3 protein, human; D014157:Transcription Factors; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2