Potential epigenetic biomarkers of circulating tumour DNA to improve detection of endocrine therapy resistance in breast cancer

Abstract

Background and aims: Endocrine therapy resistance is a major clinical problem and leading cause of metastatic breast cancer (MBC) death. Epigenetic changes via aberrant DNA methylation play an important role in therapy resistance. This thesis aimed to investigate aberrant methylation in oestrogen responsive elements (EREs) as a biomarker of hormone therapy resistance using MCF7 BC cell lines resistant to tamoxifen (TAMR-MCF7) and fulvestrant (FULVR-MCF7), with a view to utilising these signatures for early detection of hormone therapy resistance through circulating-tumour DNA (ctDNA).

Methods: The four methylation conversion kits were compared for DNA recovery of to select the most efficient method for ctDNA methylation analysis. Aberrant DNA methylation was analysed in cell lines by shallow-depth whole genome bisulfite analysis (WGBS), and correlated with RNA-Seq data. Lastly, sets of primers were designed and validated the analysis of aberrant ctDNA methylation to apply to longitudinal plasma from patients with MBC.

Results: The Premium bisulfite kit from Diagenode was the optimal kit for methylation conversion. EREs were hypermethylated and oestrogen target genes significantly downregulated in hormone therapy resistant cell lines. The hypermethylation phenotype existed more in ERE enhancers than promoters. EREs were not the dominant responsive elements in the aberrant DNA methylation analysis. FOX and AP-1 responsive elements were the top hits for hypermethylation in both resistant cell lines, while TEAD and MYC responsive elements were hypomethylated in FULVR and TAMR, respectively. The designed methylated-specific assays for OSMR promoter, and the enhancer of CBX4, TFF1, TRAF7 TERT, and RASA3 validated the enriched methylation level of these regions at resistant cell line.

Conclusions: Results generated in this thesis has identified potential candidate regions that can be applied to longitudinal ctDNA samples from MBC patients to determine whether aberrant ctDNA methylation in EREs can be analysed as a marker of endocrine resistance.