Enrichment of CpG island shore region hypermethylation in epigenetic breast field cancerization

ABSTRACT

While changes in DNA methylation are known to occur early in breast carcinogenesis and the landscape of breast tumour DNA methylation is profoundly altered compared with normal tissue, there have been limited efforts to identify DNA methylation field cancerization effects in histologically normal breast tissue adjacent to tumour. Matched tumour, histologically normal tissue of the ipsilateral breast (ipsilateral-normal), and histologically normal tissue of the contralateral breast (contralateral-normal) were obtained from nine women undergoing bilateral mastectomy. Laser capture microdissection was used to select epithelial cells from normal tissue, and neoplastic cells from tumour for genome-scale measures of DNA methylation with the Illumina HumanMethylationEPIC array. We identified substantially more CpG loci that were differentially methylated between contralateral-normal and tumour (63,271 CpG loci q < 0.01), than between ipsilateral-normal and tumour (38,346 CpG loci q < 0.01). We identified differential methylation in ipsilateral-normal relative to contralateral-normal tissue (9,562 CpG loci p < 0.01). In this comparison, hypomethylated loci were significantly enriched for breast cancer-relevant transcription factor binding sites including those for ESR1, FoxA1, and GATA3 and hypermethylated loci were significantly enriched for CpG island shore regions. In addition, progression of shore hypermethylation was observed in tumours compared to matched ipsilateral normal tissue, and these alterations tracked to several well-established tumour suppressor genes. Our results indicate an epigenetic field effect in surrounding histologically normal tissue. This work offers an opportunity to focus investigations of early DNA methylation alterations in breast carcinogenesis and potentially develop epigenetic biomarkers of disease risk.

Abbreviations

DCIS: ductal carcinoma in situ; GO: gene ontology; OR: odds ratio; CI: confidence interval; TFBS: transcription factor binding site; LOLA: Locus Overlap Analysis

KEYWORDS:

• Field cancerization
• DNA methylation
• epigenetics
• breast cancer
• normal breast
• contralateral breast

Acknowledgments

The authors thank all the women who donated tissue to this study.

Authors’ Contributions

MEM conceived and designed the analytical approach, performed statistical analyses, interpreted the results, and wrote and revised the manuscript. AJT conceived and designed the analytical approach, performed statistical analyses, interpreted the results, and wrote and revised the manuscript. LAS designed the analytical approach, interpreted the results, and revised the manuscript. OMW designed the analytical approach, interpreted the results, and revised the manuscript. CM assisted with microdissection, isolated DNA, interpreted the results, and revised the manuscript. KJG sectioned tissue, performed microdissections, interpreted results, and revised the manuscript. SSS conceived and designed the approach, interpreted the results, and revised the manuscript. GMC reviewed normal and tumor tissue, marked areas for microdissection, interpreted the results, and revised the manuscript. RMJ reviewed normal and tumor tissue, interpreted the results, and revised the manuscript. CNO conceived and designed the approach, interpreted the results, and revised the manuscript. BCC conceived and designed the approach, oversaw project development, interpreted the results, and revised the manuscript. KFA conceived and designed the approach, oversaw project development, interpreted the results, and revised the manuscript. All authors read and approved the final manuscript.

Availability of Data and Materials

The datasets generated and analyzed during the current study are available in the Gene Expression Omnibus under the accession number GSE133985 (https://www.ncbi.nlm.nih.gov/geo/).

Disclosure Statement

The authors declare that they have no competing interests.

Ethics Approval and Consent to Participate

Written informed consent was obtained from all subjects. Approval for this research was obtained from the Baystate Health Institutional Review Board (protocol number 568088).

Supplementary Material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by funds of the Burroughs-Wellcome/Dartmouth Big Data in the Life Sciences Training Program to MEM. Office of the U.S. Director of the National Institutes of Health under award number T32LM012204 to AJT. COBRE Center for Molecular Epidemiology at Dartmouth P20GM104416, and R01CA216265 to BCC. R01CA230478-01A1 and ROH-BCR151672 to KFA.