DNA methylation of oestrogen-regulated enhancers defines endocrine sensitivity in breast cancer

Andrew Stone, Elena Zotenko, Warwick J Locke, Darren Korbie, Ewan K A Millar, Ruth Pidsley, Clare Stirzaker, Peter Graham, Matt Trau, Elizabeth A Musgrove, Robert I Nicholson, Julia M W Gee, Susan J Clark, Andrew Stone, Elena Zotenko, Warwick J Locke, Darren Korbie, Ewan K A Millar, Ruth Pidsley, Clare Stirzaker, Peter Graham, Matt Trau, Elizabeth A Musgrove, Robert I Nicholson, Julia M W Gee, Susan J Clark

Abstract

Expression of oestrogen receptor (ESR1) determines whether a breast cancer patient receives endocrine therapy, but does not guarantee patient response. The molecular factors that define endocrine response in ESR1-positive breast cancer patients remain poorly understood. Here we characterize the DNA methylome of endocrine sensitivity and demonstrate the potential impact of differential DNA methylation on endocrine response in breast cancer. We show that DNA hypermethylation occurs predominantly at oestrogen-responsive enhancers and is associated with reduced ESR1 binding and decreased gene expression of key regulators of ESR1 activity, thus providing a novel mechanism by which endocrine response is abated in ESR1-positive breast cancers. Conversely, we delineate that ESR1-responsive enhancer hypomethylation is critical in transition from normal mammary epithelial cells to endocrine-responsive ESR1-positive cancer. Cumulatively, these novel insights highlight the potential of ESR1-responsive enhancer methylation to both predict ESR1-positive disease and stratify ESR1-positive breast cancer patients as responders to endocrine therapy.

Figures

Figure 1. Genome-wide DNA methylation profiling of…
Figure 1. Genome-wide DNA methylation profiling of endocrine-resistant MCF7 cell models.
(a–c) A colorimetric density plot showing correlation between the HM450 methylation profile of the endocrine-resistant MCF7X (a), TAMR (b) and FASR (c) cells and the parent (endocrine-sensitive) MCF7 cells. The plots show that while the methylation profile of the endocrine-resistant cell lines is strongly correlated with the parent MCF7 cells (MCF7X, r2=0.895; TAMR, r2=0.91; FASR, r2=0.848; Pearson's coefficient), both the MCF7X and TAMR cells predominantly gain DNA methylation, whereas the FASR cells exhibit both hyper- and hypomethylation events relative to parent MCF7 cells. (d) A Venn diagram showing the overlap of HM450 methylation probes that are more heavily methylated in multiple endocrine-resistant cells compared with the parent MCF7 cells (FDR<0.01). (e) A bar plot showing the association of differentially methylated HM450 probes that were common to all endocrine-resistant cell lines (compared with the parent MCF7 cells) across functional/regulatory regions of the genome as determined by MCF7 ChromHMM annotation. The height of the bars represents the level of enrichment measured as a ratio between the frequency of hypermethylated (dark blue) or hypomethylated (light blue) probes overlapping a functional element over the expected frequency if such overlaps were to occur at random in the genome. Statistically significant enrichments (P value<<0.0001; hypergeometric test) are marked with an asterisk. The numbers of commonly hyper/hypomethylated probes located within each specific region are presented in the respective column.
Figure 2. ESR1 regulation of enhancer sites…
Figure 2. ESR1 regulation of enhancer sites commonly hypermethylated in endocrine-resistant cell models.
(a) A bar plot showing the association of HM450 probes that were more heavily methylated in endocrine-resistant cell models (compared with MCF7 cells) and also specifically located in enhancer regions, across ESR1-, FOXA1- and GATA3-binding sites in MCF7 cells. The height of the bars represents the enrichment measured as a ratio between the frequency of hypermethylated probes in enhancers overlapping a transcription factor binding site over the expected frequency if such overlaps were to occur at random across the genome (*P value<<0.0001; hypergeometric test). The numbers of commonly hyper/hypomethylated probes located within each specific region are presented in the columns. (b) A Venn diagram showing the overlap of enhancer-specific HM450 methylation probes that are more heavily methylated in multiple endocrine-resistant cell models (compared with MCF7 cells) across ESR1-, FOXA1- and GATA3-binding sites. (c) A box plot showing the log-fold change (logFC) in ESR1 binding signal at ESR1-enhancer sites that contain at least one commonly hypermethylated probe (yellow box) and all other ESR1-enhancer sites that overlap a HM450 probe (grey box) in TAMR cells compared with the parent MCF7 cells. The mean logFC in ESR1 binding at hypermethylated ER-enhancer sites is −2.29 and the mean logFC of all other ESR1-enhancer sites is −0.52 (*P<<0.0001; t-test). (The whiskers of the box plot extend to the most extreme data point, which is no more than 1.5 × interquartile range from the box). (d) IGV screen shots to illustrate the loss of ESR1 binding in TAMR cells compared with the parent MCF7 cells in enhancer regions that overlap methylation probes that are more heavily methylated in the endocrine-resistant cell models. The MCF7 ChromHMM regions are colour coded as follows—blue, enhancer; yellow, transcribed; green, promoter; light blue, CTCF; and burgundy, transcribed. The HM450 β values are shown for the MCF7 (green), MCF7X (burgundy), TAMR (orange) and FASR cells (red) and are representative of biological duplicates. ESR1 ChIP data (blue) is presented in duplicate for both MCF7 and TAMR cells. The ESR1 enhancers that overlap the regions of endocrine-resistant-specific hypermethylation are highlighted by the blue boxes.
Figure 3. Association between ESR1-enhancer methylation and…
Figure 3. Association between ESR1-enhancer methylation and breast cancer subtype.
(a) A box plot showing the median methylation of all HM450 probes that overlap an enhancer region, an ESR1-binding site and demonstrate hypermethylation in endocrine-resistant versus parental MCF7 cells (n=801 probes), in normal breast tissue (green; n=97), luminal A (light blue; n=301), luminal B (dark blue; n=52) and ESR1-negative (red; n=105) breast cancer (data obtained from TCGA breast cancer cohort; *P<0.05, **P<<0.0001; Mann–Whitney U-test). (The whiskers of the box plot extend to the most extreme data point, which is no more than 1.5 × interquartile range from the box). (b) A heatmap showing the methylation profile of 801 ESR1-enhancer-specific HM450 probes that are more heavily methylated in endocrine-resistant versus parent MCF7 cells in normal breast tissue (green; n=97), luminal A (light blue; n=301), luminal B (dark blue; n=52) and ESR1-negative (red; n=105) breast cancer. Columns are patient samples and rows are HM450 probes. The level of methylation is represented by a colour scale—blue for low levels and red for high levels of methylation. (c) Box plots showing distribution of methylation β values in normal n=97 (green), luminal A (light blue; n=301), luminal B (dark blue; n=52) and ESR1-negative (red; n=105) breast cancer samples across HM450 probes overlapping the ESR1-binding site located within the DAXX enhancer (Chr6: 33288112-33288670; left panel) and the DAXX promoter region (1,000 bp upstream and 100 bp downstream of the transcription start site; Chr6: 33290693-33291793; right panel). (The whiskers of the box plots extend to the most extreme data point, which is no more than 1.5 × interquartile range from the box).
Figure 4. ESR1-enhancer DNA hypermethylation in acquired…
Figure 4. ESR1-enhancer DNA hypermethylation in acquired endocrine resistance in human breast cancer.
(a–e) (Left panel) A scatter plot showing the methylation of individual CpG sites across the ESR1-enhancer region of interest ((a)-DAXX—Chr6: 33288296-33288372; (b)-MSI2—Chr17: 55371693-55371786; (c)-NCOR2—Chr12: 124844786-124844883; (d)-RXRA—Chr9: 137252867-137252967; (e)-C8orf46—Chr8: 67425069-67425134) in three primary luminal A breast cancers from patients that received adjuvant endocrine therapy and exhibited RFS (green), three primary luminal A breast cancers from patients that relapsed following adjuvant endocrine therapy, defined as no n/RFS (blue) and their matched local relapse (red). Each dot represents the % methylation at an individual CpG site for a single patient and the lines represent the average methylation for the region in primary RFS (green), primary n/RFS (blue) and matched recurrent tumours (red). (Right panel) Box plots showing the distribution of methylation values across the ESR1-enhancer region depicted in the left panel for RFS (green), prognosis/RFS (blue) and matched recurrent tumours (red); P values correspond to t-test comparison between RFS versus n/RFS, and n/RFS versus relapse tumours. (The whiskers of the box plots extend to the most extreme data point, which is no more than 1.5 × interquartile range from the box).

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Source: PubMed

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