Sequential DNA methylation changes are associated with DNMT3B overexpression in colorectal neoplastic progression
- Ashraf E K Ibrahim1,2,
- Mark J Arends1,
- Ana-Luisa Silva1,
- Andrew H Wyllie1,
- Liliana Greger1,
- Yoko Ito2,
- Sarah L Vowler2,
- Tim H-M Huang3,
- Simon Tavaré2,
- Adele Murrell2,
- James D Brenton4
- 1Department of Pathology, Division of Molecular Histopathology, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK
- 2Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge, UK
- 3Human Cancer Genetics Program, Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, Columbus, Ohio, USA
- 4Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Hills Road, Cambridge, UK
- Correspondence to Ashraf E K Ibrahim, Department of Pathology, Division of Molecular Histopathology, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK;
Contributors AEKI conceived of the study and carried out the experiments, participated in bioinformatics and statistical analysis and drafted the manuscript. MJA participated in the study design and drafting the manuscript. ALS participated in carrying out the experiments. AHW participated in the study design and drafting the manuscript. LG participated in the statistical analysis. YI participated in carrying out the experiments. SLV participated in the statistical analysis.TH participated in the study design and material support. ST performed the bioinformatics analysis and participated in drafting the manuscript. AM participated in the study design and drafting the manuscript. JDB supervised the study, participated in the study design and drafting the manuscript. All authors read and approved the final manuscript.
- Revised 5 October 2010
- Accepted 6 October 2010
- Published Online First 10 November 2010
Background and aims Although aberrant methylation of key genes in the progression of colorectal neoplasia has been reported, no model-based analysis of the incremental changes through the intermediate adenoma stage has been described. In addition, the biological drivers for these methylation changes have yet to be defined. Linear mixed-effects modelling was used in this study to understand the onset and patterns of the methylation changes of SFRP2, IGF2 DMR0, H19, LINE-1 and a CpG island methylator phenotype (CIMP) marker panel, and they were correlated with DNA methyltransferase 3B (DNMT3B) levels of expression in a sample set representative of colorectal neoplastic progression.
Methods Methylation of the above CpG islands was measured using quantitative pyrosequencing assays in 261 tissue samples. This included a prospective collection of 44 colectomy specimens with concurrent normal mucosa, adenoma and invasive cancer tissues. Tissue microarrays from a subset of 64 cases were used for immunohistochemical analysis of DNMT3B expression.
Results It is shown that the onset and pattern of methylation changes during colorectal neoplastic progression are locus dependent. The CIMP marker RUNX3 was the earliest CpG island showing significant change, followed by the CIMP markers NEUROG1 and CACNA1G at the hyperplastic polyp stage. SFRP2 and IGF2 DMR0 showed significant methylation changes at the adenomatous polyp stage, followed by the CIMP markers CDKN2A and hMLH1 at the adenocarcinoma stage. DNMT3B levels of immunohistochemical expression increased significantly (p<0.001) from normal to hyperplastic and from adenomatous polyps to carcinoma samples. DNMT3B expression correlated positively with SFRP2 methylation (r=0.42, p<0.001, 95% CI 0.25 to 0.56), but correlated negatively with IGF2 DMR0 methylation (r=0.26, p=0.01, 95% CI −0.45 to −0.05). A subset of the CIMP panel (NEUROG1, CACNA1G and CDKN2A) positively correlated with DNMT3B levels of expression (p<0.05).
Conclusion Hierarchical epigenetic alterations occur at transition points during colorectal neoplastic progression. These cumulative changes are closely correlated with a gain of DNMT3B expression, suggesting a causal relationship.
Funding AI was funded by a Cancer Research UK Bobby Moore fellowship.
Competing interests None.
Ethics approval This study was conducted with the approval of the Cambridgeshire 2 Research Ethics Committee.
Provenance and peer review Not commissioned; externally peer reviewed.