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Title: Exploring the transcriptional and translational implications of DNA methylation in breast cancer
Author: Shenker, Natalie
ISNI:       0000 0004 5917 6510
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2015
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The significance of DNA methylation beyond the gene promoter is little understood. High levels of intragenic methylation (IGM) correlate with active gene expression in lower organisms. Pyrosequencing assays across oestrogen receptor alpha (ESR1) in ESR1-positive and negative breast cancer cell lines showed higher IGM levels in ESR1-positive cells, with reduced ESR1 expression after demethylation with decitabine. However, different ESR1 IGM patterns were determined in breast cancer biopsies, probably because of cell heterogeneity. DNA methylation can reflect cellular environmental exposures. A methylation signature of smoking was determined in a European Prospective Investigation into Cancer and Nutrition epigenome-wide association study (EWAS, 450K array), in case-control pairs of breast (n=92) and colorectal cancer (n=95). Differentially methylated loci (p=1x107) were validated independently by pyrosequencing in peripheral mononuclear blood cells from 102 never, 45 former and 33 current smokers (n=180). Validated loci included intragenic loci in AHRR, involved in dioxin metabolism, and an intergenic region, 2q37, that was associated with breast cancer risk (p=0.003). Four loci comprised an index that outperformed the gold standard test for former smoking, cotinine. DNA methylation signatures and exposure levels vary between cell types, but few studies have exclusively investigated the target for breast carcinogenesis, the breast epithelial cell. Therefore, a proof-of-principle study into cells from breast milk samples (n=150 milk bank donors) characterised >97% as epithelial (EMA-positive) and >80% as ESR1-positive by flow cytometry. Extracted DNA and RNA underwent methylation, genotyping and expression analyses. ESR1 IGM patterns matched ESR1-positive tumour cell lines. Furthermore, 2q37 was hypomethylated in 19 former smokers compared to 69 non-smokers (p=0.026). To conclude, differentially methylated non-promoter loci may be functionally important, and useful as biomarkers, but tissue selection for prospective epigenetic studies should be carefully considered. Future large-scale breast milk sample collections will inform population-level studies into environmental exposures, breast cancer risk, and mammary biology.
Supervisor: Flanagan, James ; Brown, Robert Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available