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

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.

ARTICLE

Received 18 Mar 2015 | Accepted 6 Jun 2015 | Published 14 Jul 2015

Andrew Stone1,2,*, Elena Zotenko1,2,*, Warwick J. Locke1,2, Darren Korbie3, Ewan K.A. Millar4,5,6,7, Ruth Pidsley1,2, Clare Stirzaker1,2, Peter Graham7,8, Matt Trau3, Elizabeth A. Musgrove2,4,9, Robert I. Nicholson10,Julia M.W. Gee10 & Susan J. Clark1,2

Expression of oestrogen receptor (ESR1) determines whether a breast cancer patient receives endocrine therapy, but does not guarantee patient response. The molecular factors that dene 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.

1 Epigenetics Research Program, Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia.

2 Faculty of Medicine, St Vincents Clinical School, UNSW, NSW 2052 & St Vincents Hospital, Sydney, New South Wales 2010, Australia. 3 Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Queensland 4072, Australia. 4 Translational Breast Cancer Research, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia. 5 Department of Anatomical Pathology, South Eastern Area Laboratory Service, St George Hospital, Kogarah, Sydney, New South Wales 2217, Australia. 6 School of Medicine and Health Sciences, University of Western Sydney, Campbelltown, Sydney, New South Wales 2560, Australia. 7 Faculty of Medicine, UNSW, Kensington, New South Wales 2052, Australia. 8 Department of Radiation Oncology, Cancer Care Centre, St George Hospital, Kogarah, Sydney, New South Wales 2217, Australia. 9 Wolfson Wohl Cancer Research Centre, University of Glasgow, Glasgow G61 1BD, UK. 10 Breast Cancer Molecular Pharmacology Group, School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Wales CF10 3NB, UK. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to S.J.C. (email: mailto:[email protected]

Web End [email protected] ).

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DOI: 10.1038/ncomms8758 OPEN

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

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8758

The steroid hormone oestrogen activates the oestrogen receptor (ESR1) to mediate a variety of functions that are central to the normal development and maintenance of

multiple tissues1. The unique transcriptional response to oestrogen in each tissue-specic cell subtype is, in part, regulated by the epigenome2. Differential DNA methylation and chromatin remodelling serve to dictate accessibility to functional, oestrogen-responsive regions of the genome, and thus dene endocrine response3,4. Inappropriate activation of the ESR1 signalling network in mammary epithelial cells initiates neoplastic transformation and drives ESR1-positive breast cancer1. Patients with this disease commonly receive adjuvant endocrine therapy, which serves to inhibit ESR1 signalling1,5. Although endocrine therapy reduces the risk of disease recurrence, a third of patients acquire drug resistance and experience disease relapse6. Thus, endocrine sensitivity of both normal breast cells and breast cancer cells is dynamic, raising the hypothesis that global epigenetic reprogramming of oestrogen-responsive regions of the genome can modulate endocrine sensitivity and contributes to the onset of ESR1-positive breast cancer and the acquisition of endocrine resistance.

While recent studies have provided excellent proof of principle that the DNA methylation prole of mammary epithelial cells is altered in early carcinogenesis7, and further modied in cell models of endocrine-resistant breast cancer8,9, they do not address how these changes could directly affect endocrine sensitivity. Here we identify DNA methylation as a key determinant of endocrine response in breast cancer. We show that differential DNA hypermethylation occurs predominantly at oestrogen-responsive enhancer, not promoter regions, and is associated with reduced ESR1 binding and decreased gene expression of key regulators of ESR1 activity. In addition, we demonstrate that the methylation status of these regulatory regions is associated with endocrine resistance in human disease, thus providing a novel mechanism by which endocrine response is abated in ESR1-positive breast cancers.

ResultsMethylation of enhancer loci in endocrine-resistant cells. To interrogate DNA methylation remodelling as a critical component of acquired endocrine resistance, we performed methylation proling in duplicate using the Innium Human-Methylation 450 beadchip, on ESR1-positive hormone sensitive MCF7 cells, and three different well-characterized endocrine-resistant MCF7-derived cell lines; tamoxifen-resistant (TAMR)10, fulvestrant-resistant (FASR)11 and oestrogen deprivation-resistant (MCF7X)12 cells. Density plots showing the correlation between the DNA methylation prole of parent MCF7 cells and individual endocrine-resistant cell lines indicate that the MCF7X and TAMR cells, which are both ESR1 positive10,12, predominantly gained DNA methylation as indicated by the increased density of points above the trend line. In contrast, FASR cells, which are ESR1 negative11, exhibited both hyper and hypomethylation events relative to parent MCF7 cells as indicated by a symmetrical density distribution (Fig. 1ac). We rst sought to identify the common differential DNA methylation events present in each of the three uniquely derived endocrine-resistant cell models by carrying out paired analyses (that is, each endocrine-resistant cell line versus MCF7 parent control) and overlapping the data (Fig. 1d). We found that across the individual resistant cell lines, 14,749 CpG probes were commonly hypermethylated (false discovery rate, FDRo0.01), whereas only 192 probes exhibited shared hypomethylation (FDRo0.01; Fig. 1d).

To comprehensively characterize the functional genomic location of differential methylation observed in the endocrine-resistant cell

models, we used ChromHMM segmentation of the MCF7 genome (previously described in Taberlay et al.13; Fig. 1e). Strikingly, signicant enrichment of commonly hypermethylated probes was exclusively observed in enhancer regions of the genome (n 3,932 probes, Poo0.0001; hypergeometric test; Fig. 1e). We

next sought to determine whether the enhancer regions identied as being more heavily methylated in all endocrine resistance models were regulated by the ESR1 in the parental MCF7 cells. Using reprocessed, publically available MCF7 ESR1 (ref. 14), GATA3 (ref. 15) and FOXA1 ChIP-Seq data16 (two transcription factors closely associated with ESR1 activity), we found that enhancer-specic CpG-hypermethylated probes were enriched in ESR1-binding sites by approximately sixfold, FOXA1-binding sites by vefold and GATA3-binding sites by eightfold (Poo0.0001;

hypergeometric test; Fig. 2a). The greatest number of hyper-methylated enhancer probes were found to overlap ESR1-binding sites (n 801), which represents B20% of all hypermethylated

probes in enhancer regions. Signicantly, 47% (379 out of 801) of the hypermethylated enhancer probes that were located within an ESR1-binding site were also located within a FOXA1 and/or GATA3-binding site (Fig. 2b), which is particularly noteworthy since these transcription factors cooperatively modulate ESR1-transcriptional networks by forming a functional enhanceosome17.

Enhancer DNA hypermethylation and diminished ESR1 binding. Having dened a subset of ESR1-binding sites that overlap enhancer regions which contain hypermethylated loci in multiple models of endocrine resistance (see Methods section; n 856 sites,

Supplementary Data 1), we sought to determine whether DNA methylation affected the intensity of ESR1 binding at these sites. Using MCF7 and TAMR ESR1 ChIP data14, we compared the change in ESR1 binding signal intensity at ESR1-enhancer sites that contained (a) hypermethylated probe(s) to that of all other ESR1-enhancer sites (Fig. 2c). At methylated ESR1-enhancer sites, there was a 2.29-log-fold reduction in ESR1 binding in TAMR compared with MCF7 cells. In contrast, at all other ESR1-enhancer-binding sites, there was a 0.52-log-fold reduction in ESR1 binding in TAMR compared with MCF7 cells. Thus, increased methylation at ESR1-enhancer sites is associated with reduction in ESR1 binding (Poo0.0001; t-test; Fig. 2c). Four illustrative examples show the loss of ESR1 binding in the TAMR cells at enhancer regions that are more heavily methylated in the endocrine-resistant versus the parent MCF7 (Fig. 2d). The examples include enhancer regions located within the gene body of death-associated protein 6 (DAXX), golgi to ER trafc protein 4 homologue (GET4; a member of the BAG6-UBL4A-GET4 DNA damage response/cell death complex18), ESR1 itself and nuclear receptor co-repressor 2 (NCOR2; Fig. 2d).

Enhancer DNA hypermethylation and related gene expression. Since the vast majority of ESR1-enhancer-binding sites identied as hypermethylated in the endocrine-resistant cell lines compared with the parent MCF7 cells were intragenic (that is, 617 out of 856, 72% with at least partial overlap; Supplementary Data 1), we next sought to determine if the DNA methylation of these regions correlated with the expression of the genes in which they were located (or closest TSS if intergenic) in human breast cancer. Using RNA-seq and HM450 methylation data derived from TCGA breast cohort19 (n 459 patients), we determined that out

of the 856 ESR1-enhancer-binding sites of interest, hyper-methylation of 328 sites (that is, 38% of ESR1-enhancer sites) correlated with the reduced expression of the genes with which they were most closely associated (Spearmans correlation coefcient; Po0.001; Supplementary Data 2). The 328 ESR1-enhancer-binding sites represented 291 unique genes (including

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1.0

1.0

r2 = 0.895 r2 = 0.91 r2 = 0.848

3,932

1.0

0.8

0.8

0.8

0.6

MCF7X

TAMR

FASR

0.6

0.6

0.4

0.4

0.4

0.2

0.2

0.2

0.0

0.0

0.0

0.0 0.2 0.4 0.6

0.8 1.0

0.0 0.2 0.4 0.6 0.8 1.0

0.0 0.2 0.4 0.6 0.8 1.0

MCF7

MCF7

MCF7

Common endocrine resistant specific methylation events

FDR < 0.01 TAMR MCF7X FASR Hypermethylated 43,724 42,198 44,092

Hypomethylated 3,945 4,505 24,496

4

* Hyper

Hypo

Hypermethylation

TAMR MCF7X

FASR

14,950 8,143 9,249

5,882

14,749

10,056

13,405

Fold enrichment (observed/expected)

3

Hypomethylation

TAMR MCF7X

FASR

2

2,884

213 2,841

718

10

766

656

192

1,259

22,389

1

3,638

65

5

12

960

7

4,747

93

0

Promoter

Enhancer

Transcribed

CTCF

Repressed

Heterochromatin

Figure 1 | Genome-wide DNA methylation proling of endocrine-resistant MCF7 cell models. (ac) A colorimetric density plot showing correlation between the HM450 methylation prole 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 prole 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; Pearsons coefcient), 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 (FDRo0.01). (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 annotation13. 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 signicant enrichments (P valueoo0.0001;

hypergeometric test) are marked with an asterisk. The numbers of commonly hyper/hypomethylated probes located within each specic region are presented in the respective column.

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 specically 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 valueoo0.0001; hypergeometric test). The numbers of commonly hyper/hypomethylated probes located within each specic region are presented in the columns. (b) A Venn diagram showing the overlap of enhancer-specic 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 (*Poo0.0001; 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 followsblue, enhancer; yellow, transcribed; green, promoter; light blue, CTCF; and burgundy, transcribed. The HM450 b 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-specic hypermethylation are highlighted by the blue boxes.

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those presented in Fig. 2d; Supplementary Data 3). Gene set enrichment analysis revealed that these genes were over-represented in gene sets upregulated by ESR1 activation, downregulated in the acquisition of endocrine resistance and gene sets lowly expressed in basal versus luminal disease, thus suggesting that such genes were critical drivers of oestrogen-driven tumours (Supplementary Fig. 1a). Interestingly, using unsupervised clustering analysis, this gene set (n 291) straties

ESR1-positive and ESR1-negative breast cancer patients (Supplementary Fig. 1b). Cumulatively, this indicates that the

methylation events occurring throughout the acquisition of endocrine resistance are serving to facilitate an oestrogen-independent phenotype reective of a breast cancer subtype that is refractory to endocrine therapy.

ESR1-enhancer methylation denes breast cancer subtype. We next sought to determine whether ESR1-enhancer hypermethylation was indicative of breast cancer subtype. We assessed the median methylation of all hypermethylated ESR1-enhancer

10

Loss of ESR1 binding

5

*

Fold enrichment (observed/expected)

8

*

393

ESR1 FOXA1

GATA3

logFC (TAMR vs MCF7)

121

*

114

0

6

801

159

*

99

28

4

422

5

107

2

10 Hypermethylated

ESR1 enhancer sites

All other ESR1

enhancer sites

0

ESR1 FOXA1 GATA3

422

DAXX NCOR2

GET4

ESR1

chr6

p25.2

chr7

p22.2

chr6

p25.2

chr12

p13.32

p24.3

p23

p22.3

p22.2

p21.33

p21.2

p21.3

p21.2

p15.3

p15.1

p24.3

p23

p22.3

p22.2

p21.33

p21.2

p21.1

p13.2

p12.3

p12.1

33,287 kb

33,288 kb

33,289 kb

921,000 bp 922,000 bp

152,124 kb 152,126 kb

124,845 kb

124,850 kb

RefSeq genes

MCF7 ChromHMM

MCF7 450K

MCF7X 450K

TAMR 450K

FASR 450K

MCF7 ESR1 ChIP

TAMR ESR1 ChIP

GET4

Promoter Enhancer [0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 4.00]

[0 4.00]

[0 4.00]

[0 4.00]

DAXX

NCOR2

Enhancer

Transcribed [0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.50]

[0 1.50]

[0 1.50]

[0 1.50]

ESR1

Heterochromatintranscribed Enhancer CTCF Enhancer

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 2.50]

[0 2.50]

[0 2.50]

Transcribed

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 1.00]

[0 8.00]

[0 8.00]

[0 8.00]

[0 8.00]

[0 2.50]

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Methylation

0.2 0.8

Normal Luminal A Lum B ESR1neg

**

**

0.8

** *

Median methylation ()

0.6

0.4

0.2

*P < 0.05

DAXX enhancer region DAXX promoter region

**P << 0.0001

Normal

LumA LumB ESR1neg

DAXX

DAXX

6,292 bp

1.0

LumA

Normal

LumB

0.8

ESR1neg

Methylation ()

0.6

0.4

0.2

0.0

cg01893963

cg03477252

cg04399147

cg07905975

cg09365002

cg09597022

cg16315106

cg17251196

cg21521230

cg22904406

cg23911291

cg24498636

cg26500914

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; *Po0.05, **Poo0.0001; MannWhitney 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 prole of 801

ESR1-enhancer-specic 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 scaleblue for low levels and red for high levels of methylation. (c) Box plots showing distribution of methylation b 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).

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probes (n 801) in TCGA normal (n 97), luminal A (n 301),

luminal B (n 52) and ESR1-negative (n 105) patient HM450

data (Fig. 3a). In normal breast tissue (which is reported to be B7% ESR1 positive20), the median methylation of the ESR1-enhancer sites was highest, while median DNA methylation was signicantly reduced in luminal A disease (Poo0.0001; Mann

Whitney U-test), which is indicative of its endocrine-responsive state. Interestingly, median ESR1-enhancer methylation was greater in luminal B patients compared with luminal A patients (P 0.017; MannWhitney U-test), who are almost twice as likely

to acquire endocrine resistance21. In ESR1-negative disease, median methylation was higher than in luminal disease (versus luminal A, Poo0.0001; versus luminal B, Poo0.0001;

MannWhitney U-test; Fig. 3a). A heatmap highlights the hypomethylated status of the ESR1-enhancer sites in luminal A disease relative to normal breast tissue and the other breast cancer subtypes (Fig. 3b). This trend is clearly illustrated at the DAXX enhancer region in which each CpG within the ESR1-binding site was hypomethylated in luminal A disease compared with normal tissue and luminal B and ESR1-negative cancer (Fig. 3c). Critically, no such variability was apparent at the DAXX promoter region (1,000 bp upstream and 100 bp downstream of the transcription start site; Fig. 3c), suggesting a signicant regulatory effect of increased methylation at the enhancer locus.

ESR1-enhancer hypermethylation predicts endocrine failure. Given that ESR1-enhancer hypermethylation is prevalent in acquired endocrine resistance in vitro (Figs 1e and 2a-d) and in molecular subclassications of breast cancer that are intrinsically less responsive to endocrine therapy (Fig. 3ac), we next sought to determine the methylation status of a panel of these loci in ESR1-positive (luminal A) breast cancer samples from patients with different outcomes. Primary samples were sourced from patients who received endocrine therapy for 5 years and either experienced relapse-free survival (RFS; 414 years) or those who had relapsed (o6 years), dened as no relapse-free survival (n/RFS). Matched local relapse samples were also compared with the primary n/RFS patient samples. All patients received the same endocrine therapy (tamoxifen; anonymized patient data is given in Supplementary Data 4). Using a multiplex bisulphite-PCR resequencing methodology specically devised for formalin-xed, parafn-embedded (FFPE)-derived DNA22, the methylation of multiple CpG sites across a panel of nine oestrogen-responsive

enhancer regions was interrogated (technical duplicate correlates for all amplicons investigated are shown in Supplementary Fig. 2). These enhancer regions included those located within DAXX, MSI2, NCOR2, RXRA and C8orf46 (Fig. 4ae) and enhancer regions located within GATA3, ITPK1, ESR1 and GET4 (Supplementary Fig. 3ad). The assay was repeated with DNA extracted from biological duplicates of the endocrine-resistant cell lines and the parent MCF7 cells to ensure its viability

DAXX

Chr 6

P = 1e06

P = 1.85e08

DAXX

Pos.

(Mb)

33.28828 33.28832 33.28836

33.2883 33.28834 33.28838

100

0

DNA methylation % DNA methylation % DNA methylation % DNA methylation % DNA methylation %

80

100

80

DNA methylation %

60

40

20

0

Primary (RFS) Primary (n/RFS) Relapse

60

40

20

Relapse

Primary (RFS)

Primary (n/RFS)

MSI2

0

Chr 17

100

0

MSI2

P = 0.008 P = 0.02

55.37168

55.3717

55.37172

55.37174

55.37176

55.37178

55.3718

Pos.

(Mb)

80

100

60

80

40

DNA methylation %

DNA methylation %

DNA methylation %

60

20

40

20

Primary (RFS)

Primary (n/RFS)

Relapse

Primary (RFS) Primary (n/RFS) Relapse

NCOR2

0

Chr 12

100

0

NCOR2

P = 0.002 P = 0.02

Pos.

(Mb)

124.84478 124.84482 124.84486

124.8448 124.84484 124.84488

80

100

60

80

40

60

20

40

20

Primary (RFS) Primary (n/RFS) Relapse

Primary (RFS)

Primary (n/RFS)

RXRA

0

Chr 9

100

0

RXRA

P = 0.003

P = 0.008

Figure 4 | ESR1-enhancer DNA hypermethylation in acquired endocrine resistance in human breast cancer. (ae) (Left panel) A scatter plot showing the methylation of individual CpG sites across the ESR1-enhancer region of interest ((a)-DAXXChr6: 33288296-33288372; (b)-MSI2 Chr17: 55371693-55371786; (c)-NCOR2Chr12: 124844786-124844883; (d)-RXRAChr9: 137252867-137252967; (e)-C8orf46Chr8: 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, dened 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 than1.5 interquartile range from the box).

Pos.

(Mb)

137.25286 137.2529 137.25294

137.25288 137.25292 137.25296

137.25298

80

100

60

80

40

60

20

40

20

Primary (RFS) Primary (n/RFS) Relapse

Primary (RFS)

Primary (n/RFS)

C8orf46

Chr 8

C8orf46

P = 0.03 P = 0.01

100

0

Pos.

(Mb)

67.42506 67.4251

67.42508 67.42512

67.42514

80

100

80

DNA methylation %

60

40

20

0

Primary (RFS) Primary (n/RFS) Relapse

60

40

20

Primary (RFS)

Primary (n/RFS)

Relapse

Relapse

Relapse

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(Supplementary Fig. 4ai; technical duplicate correlates for all amplicons investigated are shown in Supplementary Fig. 5). The average methylation levels detected at all enhancer loci were signicantly higher in the recurrent tumours compared with the matched primary (n/RFS) tumours (DAXX, Po0.0001; ESR1,

Po0.0005; RXRA, Po0.005; GET4, NCOR2, GATA3, MSI2, Po0.01; C8orf46, ITPK1, Po0.05; t-test), conrming that DNA methylation at ESR1-responsive enhancers is acquired in resistant disease (Fig. 4 and Supplementary Fig. 3). The difference in DNA methylation between RFS and n/RFS primary tumours was less considerable, although a statistically signicant difference was observed for DAXX, Po0.0001; RXRA, Po0.01; C8orf46,

P 0.01; NCOR2 and MSI2 (Po0.05; t-test) enhancer regions

(Fig. 4).

DiscussionOur results support a model whereby ESR1-responsive enhancer DNA methylation is a fundamental unifying characteristic that denes endocrine sensitivity in breast cancer. Interestingly, previous studies interrogating DNA methylation changes in endocrine-resistant cell models have predominantly reported ESR1-regulated promoter methylation8,9,2326. Our study is the rst to combine in depth MCF7 ChromHMM annotation and genome-wide methylation data from multiple resistance models to more comprehensively characterize global differential methylation across diverse genomic regions. We show for the rst time that the methylation status of enhancers is associated with the inhibition of ESR1 binding in vitro and with the reduced expression of critical regulators and effectors of ESR1 activity in human disease. The identication of ESR1-responsive enhanceosome hypermethylation is both novel and considerably pertinent in the context of endocrine resistance, since genome-wide positional analyses dening the set of cis-regulatory elements that recruit ESR1 in breast cancer cells have revealed its predominant recruitment to enhancers as opposed to promoter regions3,2730. Enhancers are more common than promoters in the mammalian genome31 and can regulate gene transcription from tens to thousands of kilobases away by promoting communication with target promoters through chromatin looping32,33. In our study, the majority of ESR1-regulated enhancer regions identied as hypermethylated in the resistant cells were located within gene bodies. Strikingly, hypermethylation of these enhancer regions was frequently correlated with reduced expression of the host gene, which is in line with recent studies that have shown that over half of all enhancer regions are located within a gene body and that the activation of these enhancers can indeed affect the transcription of the host gene34,35. Examples of genes whose expression inversely correlated with ESR1-enhancer DNA methylation include DAXX and GET4, which have been previously associated with roles in apoptosis18,36. It is conceivable that the loss of expression of genes associated with pro-apoptotic functions facilitates the progression of endocrine resistance by reducing the efcacy of apoptotic signalling pathways activated by endocrine therapies37.

Importantly, the ESR1-responsive enhancer hypermethylation events identied in the endocrine-resistant cell lines were also differentially methylated in endocrine-sensitive and endocrine-resistant breast cancer patient samples. Therefore, it is feasible that ESR1-responsive enhancer methylation status is reective of endocrine dependence and could potentially be used to stratify patients as responders to endocrine therapy. For example, NCOR2, a gene whose expression has previously been associated with metastasis-free survival in 620 lymph node-negative patients with ESR1-positive breast cancer38, was shown to negatively

correlate with ESR1-enhancer methylation. In the present study, NCOR2 enhancer methylation was signicantly higher in the poor (non-relapse-free) prognosis patients, compared with the good (relapse-free) prognosis primary luminal A breast cancer patients. Critically, however, in matched recurrent tumours, enhancer DNA methylation was further increased, supporting the hypothesis that the endocrine-resistant methylation prole is acquired, rather than pre-existing, limiting its potential prognostic value. Intriguingly, it could be a combination of both acquired and intrinsic methylation differences that give rise to endocrine-resistant disease. One possible explanation is that sparse, or seeding methylation at ESR1-responsisive enhancer sites in primary tumours could reect a propensity to gain extensive methylation that spreads as resistance develops, which then becomes rmly established in recurrent disease (as discussed in ref. 39). Further characterization of ESR1-responsive enhancer methylation in endocrine-resistant disease will hereafter be an important area of future investigation, as will be the assessment of its potential predictive and prognostic application in breast cancer.

Methods

Cell culture and HumanMethylation450K array. MCF7 breast cancer cells and the corresponding endocrine-resistant sub-cell lines were kindly given to our laboratory by Dr Julia Gee (Cardiff University, UK). In brief, MCF7 cells were maintained in RPMI-1640-based medium containing 5% (v/v) fetal calf serum (FCS). TAMR MCF7 cells were generated by the long-term culture of MCF7 cells in phenol red-free RPMI medium containing 5% charcoal stripped FCS and 4-OH-tamoxifen (1 10 7 M; TAM). FASR MCF7 cells were generated by the long-term

culture of MCF7 cells in phenol red-free RPMI medium containing 5% charcoal stripped FCS and fulvestrant (1 10 7 M; FAS). Long-term oestrogen-deprived

MCF7 (MCF7X) cells were generated by the long-term culture of MCF7 cellsin phenol red-free RPMI medium containing 5% charcoal stripped FCS. Endocrine-resistant sub-lines were established and characterized following 6 months endocrine challenge/oestrogen deprivation exposure1012. All cell lines were authenticated by short-tandem repeat proling (Cell Bank, Australia) and cultured for o6 months after authentication. Genomic DNA was extracted using the Qiagen DNeasy Blood and Tissue kit according to the manufacturers instructions. HumanMethylation450K arrays were carried out by the Australian Genome Research Facility (AGRF; Melbourne, Australia).

HM450 analysis. Two biological replicates per conditionMCF7, TAMR, MCF7X or FASRwere proled on Illuminas HumanMethylation450K array. Raw HM450 data was preprocessed and background normalized with the Biconductor min package40 using preprocess Illumina(..., bg.correct TRUE,

normalize controls, reference 1); resulting M values were used for statistical

analyses and b values for heatmap visualizations and clustering. Differential methylation analysis of the preprocessed data was performed using the Bioconductor limma package.

Genomic segmentation and annotation. The ChromHMM segmentation of the MCF7 genome was obtained from Taberlay et al.13. Enhancer (Enhancer and Enhancer CTCF) and Promoter categories (Promoter, Promoter CTCF and

Poised Promoter) were collapsed into a single Enhancer and Promoter state respectively for the purposes of our analysis. RefSeq transcript annotations were obtained from UCSC genome browser41,42.

ChIP-seq data acquisition and analysis. ESR1 ChiP-seq data for ESR1 in MCF7 and TAMR14 was utilized in this study. Reads were mapped to genome build HG19 (GRCh37) with bowtie and mismatched (43 mismatched bases), multiple mapping and duplicate reads were excluded from downstream analysis. ESR1 enrichment peaks were identied with the HOMER software suite43 using the ndPeaks utility (-style factor -fragLength 200 -size 300 -F 0 -L 0 -C 0 -poisson 1e-06) on each experiment separately. We merged the resulting peaks to produce a ground set of 120,735 regions for subsequent analysis. Active ESR1 regions were identied in MCF7 by comparing the distribution of reads overlapping the ground set of ESR1 regions in the three MCF7 ESR1 experiments (GSM798423, GSM798424 and GSM798425) and MCF7 input experiment (GSM798440) with edegR44. This yielded 54,265 active ESR1 regions in MCF7 (FDRo0.05). A similar strategy was applied to TAMR data to yield 49,511 ESR1 regions in TAMR cells. Regions of differential ESR1 binding were identied by comparing the distribution of sequence reads in MCF7 and TAMR across the ground set of ESR1 regions using edgeR and potential variation in copy number was accounted for using DiffBind14. This analysis resulted in 24,711 regions with statistical signicant gain (FDR 5%)

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8758

and 32,343 regions with statistical signicant loss (FDR 5%) of ESR1 binding in TAMR cells as compared to MCF7 cells. ESR1 peaks overlapping HM450 probes were assigned to the nearest RefSeq transcript (o20 kb distance) for the purposes of gene expression analysis. Raw MCF7 GATA3 and FOXA1 ChIP-Seq data was obtained from Theodorou et al.15 and Hurtado et al.16, respectively. Data were processed in the same manner as outlined for ESR1 ChIP-seq above.

Gene set enrichment analysis. GSEA was performed against the Molecular Signatures Database v4.0 (MSigDB)45 C2 Collection. Enrichment was assessed by hypergeometric testing as implemented in the R stats package.

TCGA data acquisition. DNA methylation analysis utilized clinical data available through the TCGA Breast Invasive Carcinoma cohort19. Raw HM450 methylation data (level 1) were obtained from the TCGA data portal (normal samples 97,

ESR1-positive tumours 353 and ESR1-negative tumours 105). ESR1-positive

tumours were further divided into luminal A (lumA 301) and luminal B

(lumB 52) populations using progesterone receptor (PR) expression, such that

lumA were ESR1 /PR and lumB were ESR1 /PR . Processed RNA-Seq

expression data (level 3) were obtained from TCGA data portal (588 ESR1 positive tumours with 73 matched normals and 174 ESR1 negative samples with 19 matched normals).

Multiplex bisulte-PCR resequencing of clinical FFPE DNA. Bisulte DNA conversions were performed using a manual protocol. For each conversion,

B100 ng was bisulte converted at a time. Conversion took place at 80 C for45 min in the presence of 0.3 M NaOH, 3.75 mM quinone and 2.32 M sodium metabisulte, as per the method of Clark et al.46. The multiplex bisulte-PCR reaction was performed as follows22. In brief, Promega HotStart GoTaq with Flexi buffer (M5005) was used with the following components at the indicated concentrations: 5 green (1 ), CES 5 , (0.5 , N.B. refer to ref. 47 for CES

recipe), MgCl2 (4.5 mM), dNTPs (200 mM each), primers (forward and reverse at 100 mM), Hot Start Taq (0.025 U ml 1), DNA (2 ng ml 1 ). All primers used are listed in Supplementary Data 5. Cycling conditions were: 94 C, 5 min; 12 cycles of (95 C, 20 s; 60, 1 min); 12 cycles of (94 C, 20 s; 65 C, 1 min 30 s); 65 C, 3 min, 10 hold. Agencourt XP beads were using to clean-up and concentrate the multiplex reaction for subsequent barcoding (that is, addition of Illumina p5/p7 sequences and sample-specic DNA barcodes). The barcoding PCR used the following reagents at the indicated nal concentrations in a 100-ml reaction: 1 GoTaq

Green Flexi buffer; 0.25 CES; 4.5 mM MgCl2; 200 mM dNTPs; 0.05 U ml 1

HotStart Taq; 25 ml of pooled template after Agencourt XP bead clean-up; and 20 ml MiSeq (Fluidigm PN FLD-100-3771). Cycling conditions were: 94 C, 5 min;9 cycles of (97 C, 15 s; 60 C, 30 s; 72 C, 2 min); 72 C, 2 min; 6 C, 5 min. MiSeq sequencing was performed used the MiSeq Reagent Kit v2, 300 cycle; PN MS-102-2002. Bioinformatic analysis started with adaptor trimming using Trim galore (options: --length 100). Mapping used the Bismark methylation mapping programme48 running Bowtie2 (ref. 49) (options: --bowtie2 -N 1 -L 15 --bam -p 2 --score L,-0.6,-0.6 --non_directional; bismark_methylation_extractor -s -merge_non_CpG comprehensive --cytosine_report). To reduce computational overhead, mapping took place against only those genomic regions which were being investigated, plus an additional 100 bp1 kb of anking sequence.

Clinical sample acquisition and DNA extraction. FFPE breast cancer samples were obtained from the St George Hospital, Kogarah, Australia (Ethics approval reference from St George Hospital Human Research Ethics Committee is 96/84). The deidentied haematoxylineosin-stained sections were reviewed by a pathologist and representative tumour areas were marked and blocks were cored accordingly. Genomic DNA was extracted using the Qiagen AllPrep DNA/RNA FFPE kit according to the manufacturers instructions.

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Acknowledgements

We thank Dr Jason Carroll and members of his lab for providing raw ChIP-Seq data used in this study, and Dr Brigid OGorman for her careful review of the manuscript. We also acknowledge the input of the late Professor Robert L. Sutherland. This work is supported by the National Breast Cancer Foundation (NBCF) programme and project grants and National Health and Medical Research Council (NHMRC 1029579) project grant and

NHMRC Fellowship (S.J.C.). In addition, J.M.W.G. received support from Breast Cancer Campaign UK and E.A.M. received support from The Cancer Institute NSW (11/CDF/3-26) and Cancer Research UK (C596/A18076).

Author contributions

A.S., E.Z., S.J.C. were responsible for the concept and designA.S., E.Z., D.K., W.J.L. and S.J.C. developed the methods A.S., E.Z., W.J.L., D.K., E.A.M. and S.J.C. were responsible for acquisition of data; A.S., E.K.A.M. and P.G. provided clinical samples (preparation of DNA); E.Z., W.J.L., A.S., D.K., R.P., S.J.C. and C.S. analysed and interpreted the data (for example, statistical analysis, biostatistics and computational analysis); all authors, with the exception of P.G., were responsible for writing, gures and review of the manuscript; E.A.M, C.S. and S.J.C supervised the study..

Additional information

Accession codes: Cell line HumanMethylation450K array data is available online at GEO (GSE69118).

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How to cite this article: Stone, A. et al. DNA methylation of oestrogen-regulated enhancers denes endocrine sensitivity in breast cancer. Nat. Commun. 6:7758 doi: 10.1038/ncomms8758 (2015).

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