Molecular basis of distinct oestrogen responses in ... · P

26:1Endocrine-Related Cancer

E Baxter et al. Mechanisms of oestrogen response

31–46

-17-0563

RESEARCH

Molecular basis of distinct oestrogen responses in endometrial and breast cancer

Eva Baxter1, Karolina Windloch1, Greg Kelly1, Jason S Lee1, Frank Gannon1* and Donal J Brennan2,3,4*1Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia2UCD School of Medicine, Catherine McAuley Research Centre, Mater Misericordiae University Hospital, Dublin, Ireland3Cancer Biology and Therapeutics Laboratory, UCD Conway Institute of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin, Ireland4Systems Biology Ireland, University College Dublin, Belfield, Dublin, Ireland

Correspondence should be addressed to E Baxter or D J Brennan: [email protected] or [email protected]

*(F Gannon and D J Brennan share last authorship)

Abstract

Up to 80% of endometrial and breast cancers express oestrogen receptor alpha (ERα). Unlike breast cancer, anti-oestrogen therapy has had limited success in endometrial cancer, raising the possibility that oestrogen has different effects in both cancers. We investigated the role of oestrogen in endometrial and breast cancers using data from The Cancer Genome Atlas (TCGA) in conjunction with cell line studies. Using phosphorylation of ERα (ERα-pSer118) as a marker of transcriptional activation of ERα in TCGA datasets, we found that genes associated with ERα-pSer118 were predominantly unique between tumour types and have distinct regulators. We present data on the alternative and novel roles played by SMAD3, CREB-pSer133 and particularly XBP1 in oestrogen signalling in endometrial and breast cancer.

Introduction

Over 1000 oestrogen-responsive genes have been identified in vitro, predominantly using the luminal breast cancer cell line MCF7, and these are involved in a variety of processes such as the cell cycle, proliferation and transcription (Frasor et al. 2003, Laganière et al. 2005, Carroll et al. 2006). Although anti-oestrogens are a key component of systemic breast cancer treatment, they are not routinely used in endometrial cancer due to lack of proven efficacy (Carlson et al. 2014). Tamoxifen is a selective oestrogen receptor modulator, acting as an oestrogen receptor alpha (ERα) antagonist in the breast and as an agonist in the endometrium, increasing the risk of developing endometrial cancer (Cohen 2004). Tissue-specific effects of tamoxifen are suggested to be due to

differential ERα coregulator recruitment (Shang & Brown 2002).

Approximately 80% of endometrial cancers express ERα. These tumours, traditionally designated type I (Bokhman 1983), are low-grade, oestrogen-dependent endometrioid carcinomas with a good prognosis. About 70% of breast cancers express ERα and these are typically of the luminal subtype (Perou et al. 2000). The prognosis of luminal breast tumours varies widely but molecular classification has aided in predicting clinical outcome (Sørlie et al. 2001, van’t Veer et al. 2002, Paik et al. 2004).

Classical oestrogen signalling involves oestrogen binding to ERα, which binds DNA at oestrogen response elements (EREs) and recruits multiple coregulators to

Endocrine-Related Cancer (2019) 26, 31–46

1

Key Words

f breast cancer

f CREB

f ERα

f endometrial cancer

f oestrogen

f SMAD3

f XBP1

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32E Baxter et al. Mechanisms of oestrogen response


modulate gene expression (Shang et al. 2000, Métivier et al. 2003). Oestrogen-mediated phosphorylation of ERα at serine residue 118 (ERα-pSer118) enhances transcriptional activation of ERα (Ali et al. 1993) by directing recruitment of ERα and coregulators to target promoters (Duplessis et al. 2011). Mutation of this serine residue disrupts both ligand-dependent and ligand-independent activity of ERα (Ali et al. 1993, Duplessis et al. 2011). Oestrogen may also regulate transcription via non-classical mechanisms as ERα can bind other transcription factors already bound to target genes to modulate their activity. Examples include AP-1 (Dahlman-Wright et al. 2012), NF-kB (Stein & Yang 1995) and Sp-1 (Porter et al. 1997). Oestrogen can also induce the mobilisation of intracellular calcium (Improta-Brears et al. 1999) and activate the MAPK and PI3K pathways independent of ERα (Migliaccio et al. 1996, Simoncini et al. 2000).

To date, few studies have examined the molecular basis of ERα or oestrogen signalling in endometrial cancer and compared these findings to their more extensively studied roles in breast cancer. Previous reports used the endometrial cancer cell line Ishikawa (Johnson et al. 2007, Tamm-Rosenstein et al. 2013), or ECC-1 which has since been entered in the Register of Misidentified Cell Lines by the International Cell Line Authentication Committee (Korch et al. 2012). Chromatin immunoprecipitation sequencing (ChIP-seq) analysis of human tumours demonstrated that ERα binding sites were highly conserved between tamoxifen-associated endometrial tumours and breast tumours (Droog et al. 2016), but differed from endometrial tumours from non-tamoxifen users (Droog et al. 2017). We sought to investigate the molecular basis for differential oestrogen responses in endometrial and breast cancer, focusing on non-tamoxifen-associated endometrial tumours. Using publicly available data from TCGA, we used ERα-pSer118 as a marker of transcriptional activation of ERα and identified genes associated with ERα-pSer118 in endometrial and breast tumours. We also analysed ERα coregulator expression and recruitment to target genes in cell lines. Our results suggest that the cancer type-specific effects of oestrogen are due to localised interplay between ERα and other factors.

Materials and methods

TCGA data

Publicly available level 3 RNA-sequencing raw counts and reverse phase protein array (RPPA) data were downloaded from TCGA data portal. Endometrial tumours were

selected based on endometrioid histology, grades 1 or 2 and copy number alteration (CNA) clusters 1–3 (n = 138). CNA cluster 4 was excluded as these tumours are more similar to non-endometrioid or serous tumours (Cancer Genome Atlas Research Network et al. 2013). Tumours from women with a prior or other malignancy or tamoxifen treatment were excluded. ERα-positive breast tumours annotated as being luminal A and/or B according to both PAM50 and RPPA clusters were selected (n = 128) (Cancer Genome Atlas Research Network 2012). Using RPPA data, the ratio of ERα-pSer118 to ERα was calculated (Supplementary Table 1, see section on supplementary data given at the end of this article). Differential gene expression was performed in R (version 3.1.1) with P < 0.05 after multiple testing correction. Genes that were not expressed (counts = 0) in at least half the tumours were filtered out. edgeR (Robinson et al. 2010), DESeq2 (Love et al. 2014) and limma with sample weighting (Smyth 2004, Liu et al. 2015) were used with default settings.

Transcription factor analysis

ERα ChIP-seq data for six tamoxifen-naïve endometrioid endometrial tumours (GSE94031 Droog et al. 2017), eight ER+PR+HER2− breast tumours (GSE32222 Ross-Innes et al. 2012), Ishikawa and MCF7 cells (GSE23893 Joseph et al. 2010) were downloaded. Genes were designated as having an ERα-binding event if an ERα ChIP-seq peak was located within the gene body or 20 kb of the transcription start site (Fullwood et al. 2009) in the relevant cancer type. DNA sequences from −2500 bp to +500 bp were extracted using the Regulatory Sequence Analysis Tools with ENSEMBL database version 89 (Thomas-Chollier et al. 2011). Transcription factor-binding site enrichment was performed using F-Match 1.0 in the Gene Regulation suite (GeneXplain GmbH, Wolfenbüttel, Germany).

Functional analysis of genes

Predicted upstream regulators were modelled using Ingenuity Pathway Analysis (Qiagen), only direct relationships were considered. An activation z-score >2 (activated) or <−2 (inhibited) is considered significant (Ingenuity Systems white paper). Gene Set Enrichment Analysis was conducted in the GenePattern suite (Reich et al. 2006).

Cell culture

Ishikawa (Sigma-Aldrich), JHUEM14 (RIKEN, Tsukuba, Japan) and MCF7 (ATCC) cells were grown in phenol

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red-free DMEM (Sigma-Aldrich) supplemented with 10% charcoal-stripped foetal bovine serum (Sigma-Aldrich) and 1% penicillin/streptomycin (Life Technologies) for 72 h, prior to stimulation with 10 nM β-oestradiol (Sigma-Aldrich), 2 ng/mL or 20 ng/mL recombinant human TGFβ (R&D Systems) or vehicle for 3 h. shXBP1 (5′-GGCATCTCAAACCTGCTTTCA-3′) or shNS (non-silencing control used in Casciello et al. 2017) was transfected into Ishikawa using GeneIn (MTI-GlobalStem, MD, USA), or MCF7 using SuperFect (Qiagen) for 48 h prior to stimulation with oestrogen. Cell lines were STR profiled and mycoplasma tested.

Western blotting

Protein was extracted in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH8, 0.5 mM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol) and boiled at 100°C for 5 min in Laemmli buffer (2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.002% bromophenol blue, 0.125 M Tris–HCl). Proteins were separated by 10% SDS-PAGE and transferred onto a PVDF membrane (Millipore). After blocking in 5% skimmed milk, membranes were incubated with primary antibody overnight, followed by horseradish peroxidase-conjugated anti-rabbit secondary antibody (Cell Signaling Technology, 7074). Bands were visualised using enhanced chemiluminescent substrate (GE Healthcare) on the ImageQuant LAS 500 (GE Healthcare). Band quantification was performed using ImageJ (National Institutes of Health, USA).

RNA extraction and quantitative real time PCR (qRT-PCR)

RNA was extracted using TRIzol (Life Technologies) or the RNeasy mini kit (Qiagen) and reverse transcribed using SuperScript III (Life Technologies). qRT-PCR was conducted using SYBR green (Life Technologies) on the ABI ViiA7 (Applied Biosystems) for 40 cycles (95°C for 15 s, 60°C for 60 s) in triplicate. Transcript levels were normalised to HPRT and GAPDH. Primers were selected from PrimerBank (Wang et al. 2012) and synthesised by Sigma-Aldrich (Supplementary Table 3A).

RNA sequencing

RNA was quality-controlled (LabChip GX, PerkinElmer) and libraries were constructed using the TruSeq RNA kit (Illumina) and sequenced on the NextSeq500 with 1 × 75 bp reads (The University of Queensland, Brisbane,

Australia). Sequences were aligned to the human genome hg38 using STAR (version 2.4.1c) on default settings except the following parameters: --outFilterMultimapNmax 10 --outSAMunmapped within --outSAMtype BAM SortedByCoordinate --limitBAMsortRAM 34359738368. BAM files from the same sample were merged, sorted and indexed using samtools (version 0.1.19-96b5f2294a). A BED file containing genomic coordinates for RefSeq genes was downloaded from the UCSC genome browser (28th March 2016) and tags falling in those exonic regions were counted and tallied using bedtools (version 2.17.0; parameters -split -S -D). The highest expressing transcript of duplicate transcripts was retained. Raw counts were normalised using trimmed mean of M-values (Robinson & Oshlack 2010) in R (version 3.1.1). Data is deposited at Gene Expression Omnibus (GSE107693).

Chromatin immunoprecipitation

Cells were fixed with 1% formaldehyde for 10 min before the addition of glycine to 100 mM. After washing twice with ice cold PBS, cells were harvested in collection buffer (100 mM Tris pH 9.4 and 10 mM dithiothreitol) and incubated at 30°C for 15 min. Cells were washed consecutively with PBS, Buffer I (10 mM EDTA pH 8, 0.5 m EGTA pH 8, 10 mM HEPES pH 6.5 and 0.25% Triton X-100) and Buffer II (1 mM EDTA pH 8, 0.5 mM EGTA pH 8, 10 mM HEPES pH 6.5 and 200 mM NaCl), and then lysed in lysis buffer (10 mM EDTA pH 8, 50 mM Tris pH 8.1, 1% SDS and protease inhibitor cocktail (cOmplete mini EDTA-free, Roche)) before being sonicated six times for 10 s each at 70% power (Branson Ultrasonics, CT, USA). After diluting with dilution buffer (2 mM EDTA pH 8, 20 mM Tris pH 8.1, 150 mM NaCl, 1% Triton X-100 and protease inhibitor cocktail), 2 μg antibody and protein A/G magnetic beads (Millipore) were incubated at 4°C overnight. 10% of the sample was retained as input. After washing six times with wash buffer (1 mM EDTA pH 8, 50 mM HEPES pH 7.6, 0.5 mM LiCl, 1% NP-40 and 0.7% sodium deoxycholate), chromatin was eluted twice in elution buffer (100 mM NaHCO3 and 1% SDS). Cross-links were reversed after the addition of NaCl to 200 mM and incubation at 65°C overnight. Chromatin was purified using the Gel Extraction Kit (Qiagen) and subjected to qPCR. Primers were designed to be within 500 bp of the transcription start site (Supplementary Table 3B). For ChIP re-ChIP assays, the first ChIP was conducted for ERα. Chromatin was eluted using 10 mM dithiothreitol at 37°C for 30 min before being diluted 20-fold in dilution buffer and proceeding with the second ChIP.



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Antibodies

CREB (Cell Signaling Technology, 9197), CREB phospho-Ser133 (Cell Signaling Technology, 9198), ERα (Santa Cruz Biotechnology, sc-543), ERα phospho-Ser118 (Santa Cruz Biotechnology, sc-12915-R), FOXA1 (Santa Cruz Biotechnology, sc-22841), GAPDH (Cell Signaling Technology, 5174), GATA3 (Santa Cruz Biotechnology, sc-268), IgG control (Santa Cruz Biotechnology, sc-2027), SMAD3 (Abcam, ab28379), SMAD3 phospho-Ser423/425 (Abcam, ab52903) and XBP1 (Abcam, ab198999).

Mouse xenograft model

Six-to-seven-week-old female C.B.17-SCID mice (Animal Resources Centre, Perth, Australia) either had a 0.72mg/pellet 90-day release 17β-oestradiol pellet (Innovative Research of America, FL, USA) implanted or underwent a sham procedure. Approximately 5 days later, 5,000,000 cells in a 1:1 mix of PBS and phenol red-free Matrigel (Corning, NY, USA) were injected subcutaneously into the flank. Tumour size was measured using digital callipers. All animal procedures were conducted in accordance with Australian National Health and Medical Research regulations on the use and care of experimental animals and were approved by the QIMR Berghofer Animal Ethics Committee (ethics approval A1406-610M, P2052).

Statistical analyses

Univariate and multivariate survival analyses were conducted in SPSS version 24. Cox multivariate analysis included genes of choice dichotomised based on median expression, age and grade. Heatmaps were drawn in R (version 3.1.1) using the gplots package. All other statistical tests were conducted and graphs were drawn in GraphPad Prism version 7.02. Survival curves were analysed using the log-rank (Mantel–Cox) test. The distribution of categorical variables between groups was tested by Fisher’s exact or chi-square tests. With five mice in each group and a two-sided alpha of 0.05, minimum 80% power was achieved for each comparison.

Results

ERα-pSer118 is a prognostic factor in endometrial but not breast tumours

Using phosphorylation of ERα at serine residue 118 (ERα-pSer118) as a marker of ERα-mediated transcription, TCGA data indicated that in endometrial tumours, high ERα-pSer118 levels were associated with significantly worse progression-free survival (P = 0.004; Fig. 1A) and overall survival (P = 0.028; Fig. 1B). After correcting for age, which was inversely correlated with ERα-pSer118 levels (P = 0.007; Table 1A), and grade, a known prognostic factor for endometrial cancer (Kosary 1994), high ERα-pSer118 levels remained an independent predictor of reduced

Figure 1ERα-pSer118 is a prognostic factor in endometrial tumours. In TCGA endometrial tumours, high levels of ERα-pSer118 (≥Median) are associated with significantly worse (A) progression-free survival and (B) overall survival vs low levels of ERα-pSer118 (<Median). In TCGA breast tumours, ERα-pSer118 levels are not associated with (C) disease-free or (D) overall survival.



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progression-free survival (Table 2). Similar to endometrial tumours, high levels of ERα-pSer118 also correlated with younger age in breast tumours (P < 0.001; Table 1B). However, ERα-pSer118 levels were not associated with survival in breast tumours (Fig. 1C and D).

Genes associated with ERα-pSer118 are predominantly unique between endometrial and breast tumours

To understand the possible biological basis for high ERα-pSer118 levels being a poor prognostic factor only in endometrial and not breast tumours, we investigated the gene expression profiles associated with ERα-pSer118 in each tumour type. Genes that were consistently differentially expressed between the highest and lowest quartiles of ERα-pSer118 levels using three statistical

methods (edgeR, DESeq2 and limma with sample quality weighting) were designated as ERα-pSer118-associated genes. Overall, 293 genes were associated with ERα-pSer118 in endometrial tumours (Supplementary Fig. 1A and Supplementary Table 4A), the majority of which (77%) were upregulated in endometrial tumours with high ERα-pSer118 levels.

In breast tumours, 602 genes were associated with ERα-pSer118 (Supplementary Fig. 1B and Supplementary Table 4B). 58% of these genes were upregulated in breast tumours with high ERα-pSer118 levels, which was a significantly reduced proportion compared to endometrial tumours (58 vs 77%, P < 0.001). Only 31 genes were associated with ERα-pSer118 in both endometrial and breast tumours (Supplementary Fig. 1C). Most ERα-pSer118-associated genes were thus different between the two tumour types.

Table 1 Characteristics of TCGA (A) endometrial and (B) breast tumours with low (<median) and high (≥median) levels of ERα-pSer118.

<Median (n = 69) ≥Median (n = 69)P-Valuen % n %

A Age (years) Mean 64 59 0.007

Range 38–89 31–88 Race White 56 81 59 86 0.228

Non-White 13 19 7 10Unknown 0 0 3 4

Stage 1 56 81 54 78 0.828>1 12 17 14 20Unknown 1 1 1 1

MSI status High 18 26 28 41 0.195Low 4 6 3 4Stable 47 68 38 55

POLE Yes 3 4 4 6 0.696No 55 80 41 59Not assigned 11 16 24 35

CNA cluster 1 24 35 18 26 0.1322 33 48 29 423 12 17 22 32

mRNA cluster Mitotic 3 4 2 3 0.344Hormonal 30 43 33 48Immunoreactive 30 43 19 28Not assigned 6 9 15 22

<Median (n = 64) ≥Median (n = 64) P-Valuen % n %

B Age (years) Mean 66 57 <0.001

Range 34–89 29–88 Race White 41 64 49 77 0.590

Non-White 8 13 7 11Unknown 15 23 8 13

Stage 1 9 14 11 17 0.808>1 54 84 53 83Unknown 1 2 0 0



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Gene set enrichment analysis (GSEA) indicated that all the hallmark gene sets enriched in the ERα-pSer118-associated genes in endometrial tumours were also enriched in the ERα-pSer118-associated genes in breast tumours (FDR < 0.05; Table 3), indicating similarities in some molecular processes between the two tumour types. Interestingly, GSEA only identified a significant enrichment of oestrogen response in breast tumours, suggesting that the ERα-pSer118-associated genes in endometrial tumours are not recognised in current databases to be associated with oestrogen response.

The majority of ERα-pSer118-associated genes in either tumour type had at least one ERα binding event as assessed using publicly available ChIP-seq data from tumours (Fig. 2A). However, significantly more genes in breast tumours had ERα binding events (85% vs 70%, P < 0.0001), suggesting that ERα may bind indirectly to target genes more frequently in endometrial tumours.

ERα-associated networks are distinct between tumour types

We sought to further examine the regulation of ERα-pSer118-associated genes in endometrial and breast tumours by analysing: (1) their upstream regulators, (2) ERα co-regulator expression and (3) transcription factor-binding site enrichment.

Using Ingenuity Pathway Analysis, SMAD3, part of the TGFβ signalling pathway, was predicted to be an activated upstream regulator in both tumour types (P < 0.01, Fig. 2B). This suggested that ERα signalling may stimulate TGFβ signalling. ERα was predicted to be a significant upstream regulator of ERα-pSer118-associated genes in breast but not endometrial tumours, which as indicated earlier from our analyses using GSEA, suggests that the ERα-pSer118-associated genes in endometrial tumours are not recognised in current databases to be ERα targets. Alternatively and potentially consistent with our earlier finding that ERα-binding events occur less frequently in ERα-pSer118-associated genes in endometrial tumours (Fig. 2A), ERα may indirectly bind target genes in endometrial tumours, possibly via alternative coregulators. We therefore sought to investigate whether known ERα coregulators (Supplementary Table 2) were similarly expressed between endometrial and breast tumours. Using unsupervised clustering, ERα coregulator expression stratified tumours by tissue of origin (Fig. 2C). Most ERα coregulators were consistently differentially expressed between endometrial and breast tumours (75/86; Supplementary Fig. 1D), with the classic ERα co-activators FOXA1 and GATA3 being the most differentially expressed (Supplementary Fig. 1E).

Finally, we compared transcription factor-binding site enrichment in the ERα-pSer118-associated genes in endometrial vs breast tumours. ERα-pSer118-associated genes in endometrial tumours were significantly enriched in binding sites for multiple members of the CREB/ATF family of transcription factors (Fig. 2D), whilst genes in breast tumours were significantly enriched in binding sites for several known oestrogen-responsive genes (Fig. 2D). This suggests that CREB/ATF family members may play an important role in ERα signalling specifically in endometrial tumours.

Table 2 ERα-pSer118 is an independent prognostic factor in endometrial tumours.

Univariate MultivariateHR 95% CI P-Value HR 95% CI P-Value

ERα-pSer118 (<median vs ≥median)

6.69 1.49–30.00 0.013 7.03 1.51–32.76 0.013

Age (continuous) 1.00 0.96–1.05 0.891 1.03 0.98–1.08 0.325Grade (grade 1 vs grade 2) 3.63 1.01–13.03 0.048 3.12 0.87–11.24 0.082

Table 3 Hallmark gene sets significantly enriched in ERα-pSer118 associated genes in endometrial and breast tumours (FDR < 0.05).

Hallmark gene set Endometrial Breast

Allograft rejection No YesAngiogenesis Yes YesApical junction Yes YesApical surface No YesApoptosis No YesCoagulation No YesComplement No YesEpithelial-mesenchymal transition Yes YesHedgehog signalling No YesHypoxia No YesIL2-STAT5 signalling No YesIL6-JAK-STAT3 signalling No YesInflammatory response No YesKRAS signalling Yes YesMyogenesis Yes YesOestrogen response early No YesOestrogen response late No Yesp53 pathway No YesTNFA signalling via NFkB No YesUV response Yes Yes



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Cell lines confirm differences in oestrogen response networks between endometrial and breast cancer

ERα-positive endometrial cancer cell lines Ishikawa and JHUEM14 (both endometrioid histology) and the luminal breast cancer cell line MCF7 were used to validate our findings from TCGA tumour data. In a mouse xenograft model, oestradiol significantly increased tumour growth of all three cell lines (P < 0.01, Supplementary Fig. 2A).

ERα phosphorylation at serine-118 was increased by oestrogen in all three cell lines (Supplementary Fig. 2B).

As our earlier analysis of TCGA tumour data had implicated involvement of SMAD3 in both tumour types, we sought to investigate whether oestrogen activated TGFβ signalling. Oestrogen did not increase SMAD3 phosphorylation in any of the cell lines (Supplementary Fig. 2B). Conversely, TGFβ did not affect phosphorylation of ERα (Supplementary Fig. 2B). Cyclin D1 (CCND1),

Figure 2Genes associated with ERα-pSer118 in endometrial and breast tumours have distinct regulators. (A) ERα-binding events are significantly enriched in the 602 ERα-pSer118-associated genes in breast tumours vs the 293 ERα-pSer118-associated genes in endometrial tumours (P < 0.0001, Fisher’s exact test). Data are shown for at least one ERα-binding event present in at least one tumour of the relevant type. (B) Predicted activated (activation z-score >2) and inhibited (activation z-score <−2) upstream regulators of the genes associated with ERα-pSer118 in each tumour type identified by Ingenuity Pathway Analysis (P < 0.05). (C) Unsupervised clustering of 86 known ERα coregulators in endometrial and breast tumours demonstrates that ERα coregulator expression differentiates between tumour types. FOXA1 and GATA3 are marked with arrows. (D) Transcription factor-binding site enrichment in the 293 ERα-pSer118-associated genes in endometrial tumours vs the 602 ERα-pSer118-associated genes in breast tumours indicates that binding sites for multiple members of the CREB/ATF family of transcription factors (marked with an asterisk) are significantly enriched in endometrial tumours (P < 0.01). The top five factors significantly enriched in each tumour type are shown.



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progesterone receptor (PGR), trefoil factor 1 (TFF1) and X-box-binding protein 1 (XBP1) were oestrogen responsive in MCF7 cells (Supplementary Fig. 2C). PGR was consistently oestrogen-responsive in both endometrial cell lines, whereas CCND1 and XBP1 were not and TFF1 was not expressed. TGFβ reduced the oestrogen-mediated induction of PGR and XBP1 in Ishikawa, and a similar trend was seen in JHUEM14, but not in MCF7 cells (Supplementary Fig. 2C). Serpin family E member 1 (SERPINE1) is a TGFβ-responsive gene (Supplementary Fig. 2C). Oestrogen reduced the TGFβ-mediated induction of SERPINE1 in JHUEM14 and MCF7 cells but not in Ishikawa cells. It therefore appeared that there is some crossover between oestrogen and TGFβ signalling in both endometrial and breast cancer cell lines.

To investigate the possibility of a more direct interaction between SMAD3 and oestrogen signalling, we conducted chromatin immunoprecipitation (ChIP).

This demonstrated that, like ERα (Fig. 3A), SMAD3 was recruited to target genes in all three cell lines, and this recruitment was promoted by oestrogen at select genes, particularly in Ishikawa cells (Fig. 3B). Furthermore, ChIP re-ChIP experiments indicated that ERα and SMAD3 were co-recruited to target genes in all three cell lines, and this was promoted by oestrogen to select genes in Ishikawa cells (Fig. 3C). Co-recruitment of ERα and SMAD3 may thus explain why SMAD3 is so intimately linked with oestrogen signalling in both cancer types.

To assess the role of SMAD3 in oestrogen signalling in vivo, we divided TCGA tumours into quartiles of SMAD3 expression. In both endometrial and breast tumours, altered SMAD3 expression was associated with the altered expression of select oestrogen-responsive genes (Fig. 4A), consistent with SMAD3 being involved in oestrogen signalling in both tumour types. Furthermore, high levels of SMAD3 were associated with significantly

Figure 3ERα and SMAD3 are recruited to target genes in both cancer types. ChIP assays demonstrate that both (A) ERα and (B) SMAD3 are recruited to the promoters of both oestrogen-responsive and TGFβ-responsive genes in both endometrial and breast cell lines, and this is promoted by oestrogen, predominantly in Ishikawa cells. (C) ChIP of ERα followed by re-ChIP with SMAD3 indicates that ERα and SMAD3 are co-recruited to target genes in all three cell lines, but this is only promoted by oestrogen in Ishikawa cells. HPRT is a negative control. Mean ± s.e.m. of two independent experiments is shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Welch’s t-test).



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worse progression-free survival in endometrial but not breast tumours (P = 0.013; Fig. 4B) and remained an independent predictor after Cox multivariate analysis (P = 0.003; Table 4A), suggesting a unique role for SMAD3 in endometrial cancer.

Similar to our findings in TCGA tumours, significantly more of the ERα-pSer118-associated genes identified from tumours had ERα-binding events in breast cells than in endometrial cells (48 vs 6%, P < 0.0001; Fig. 5A). Furthermore, ERα coregulator expression stratified cell lines by tissue of origin (Supplementary Fig. 3A). Seventeen ERα coregulators were consistently differentially expressed between endometrial and breast cancer cells (Fig. 5B and Supplementary Fig. 3B). Eleven of these 17 coregulators were also differentially expressed in TCGA tumours and these included FOXA1 and GATA3 (Supplementary Fig. 3C), suggesting that the differences in co-regulator expression explains the variable oestrogen response seen between the two cancer types. However, ChIP experiments showed that, despite their low expression in endometrial cancer, FOXA1 (Fig. 5C) and GATA3 (Fig. 5D) recruitment to target genes was similar in all three cell lines with oestrogen promoting recruitment to select genes.

To assess the roles of FOXA1 and GATA3 in oestrogen signalling in vivo, TCGA tumours were divided into quartiles of expression, similar to analyses conducted earlier. In both endometrial and breast tumours, altered expression of either FOXA1 (Supplementary Fig. 4A) or GATA3 (Supplementary Fig. 4B) was associated with the altered expression of select oestrogen-responsive genes, suggesting that both FOXA1 and GATA3 are involved in oestrogen signalling in both tumour types. Neither cofactor was associated with survival (Supplementary Fig. 4C and D).

XBP1 has a distinct role in endometrial cancer

Our analysis of TCGA tumours had indicated that CREB/ATF family members may play an important role in oestrogen signalling specifically in endometrial cancer (Fig. 2D). CREB has previously been postulated to be involved in oestrogen signalling (Lazennec et al. 2001). Another family member, XBP1, was predicted by TCGA to be an important part of a regulatory signalling hub in hormonal endometrial tumours (Cancer Genome Atlas Research Network et al. 2013). A more recent TCGA study indicated higher enrichment of XBP1 in endometrioid

Figure 4SMAD3 is involved in oestrogen signalling in endometrial and breast tumours. (A) The expression of select oestrogen-responsive genes is significantly altered in both TCGA endometrial (left) and breast (right) tumours when comparing high SMAD3 (quartile 4) to low SMAD3 (quartile 1) expression. Mean ± s.e.m.; *P < 0.05, **P < 0.01, ****P < 0.0001 (Mann–Whitney U test). (B) High SMAD3 expression (>Median) is associated with significantly worse progression-free survival in endometrial tumours (left) compared to low SMAD3 expression (<Median). There is no difference in breast tumours (right).

Table 4 (A) SMAD3 and (B) XBP1 are independent prognostic factors in endometrial tumours.

Univariate MultivariateHR 95% CI P-Value HR 95% CI P-Value

A SMAD3 (<median vs >median) 4.43 1.21–15.98 0.023 7.75 1.96–30.54 0.003 Age (continuous) 1.00 0.96–1.05 0.891 1.03 0.98–1.09 0.211 Grade (grade 1 vs grade 2) 3.63 1.01–13.03 0.048 6.05 1.61–22.69 0.008B XBP1 (<median vs >median) 0.26 0.07–0.92 0.037 0.19 0.05–0.71 0.013 Age (continuous) 1.00 0.96–1.05 0.891 1.02 0.98–1.07 0.379 Grade (grade 1 vs grade 2) 3.63 1.01–13.03 0.048 4.74 1.30–17.33 0.019



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endometrial tumours compared to breast luminal tumours (Berger et al. 2018). We therefore hypothesised that CREB and/or XBP1 may be involved in the differential oestrogen responses of endometrial and breast cancers.

ChIP assays revealed that CREB was recruited to target genes in all three cell lines (Supplementary Fig. 5A). However, recruitment of the phosphorylated form of CREB, CREB-pSer133, which is required for transcriptional activity (Mayr & Montminy 2001), was increased by oestrogen in all three cell lines (Fig. 6A). ChIP re-ChIP indicated that CREB-pSer133 and ERα were co-recruited to target genes in all three cell lines, and this was promoted by oestrogen at select genes (Fig. 6B). XBP1 was also recruited to target genes in all three cell lines, but recruitment was increased at select genes with oestrogen only in endometrial and not MCF7 cells (Fig. 6C). ChIP re-ChIP indicated that XBP1 was co-recruited with ERα to target genes in all three cell lines and again, this was only increased with oestrogen in endometrial cells (Fig. 6D). These data point to tissue-specific differences in oestrogen-response specifically for XBP1 recruitment to chromatin.

To assess the roles of CREB and XBP1 in oestrogen signalling in vivo, TCGA tumours were divided into quartiles of expression, similar to previous analyses. Altered expression of either CREB (Fig. 7A) or XBP1

(Fig. 7B) was associated with altered expression of select oestrogen-responsive genes only in endometrial and not breast tumours. CREB was not associated with survival in either tumour type (Fig. 7C), whereas high levels of XBP1 were associated with significantly improved progression-free survival in endometrial (P = 0.025) but not breast tumours (Fig. 7D), which was confirmed using Cox multivariate analysis (Table 4B). These results indicate that both CREB and XBP1 are associated with the expression of select oestrogen-responsive genes only in endometrial and not breast tumours. However, only XBP1 was an independent predictor of progression-free survival in endometrial tumours, suggesting that XBP1 may have a unique role in these tumours.

Experiments in cell lines demonstrated that, despite XBP1 knockdown being more efficient in MCF7 cells (Fig. 7E), reduced levels of XBP1 blunted the oestrogen-mediated induction of PGR in Ishikawa but not MCF7 cells (Fig. 7F), supporting our hypothesis that XBP1 has a distinct role in oestrogen signalling in endometrial cancer.

Discussion

We sought to explore the mechanisms that mediate the tissue-specific response of oestrogen, using data

Figure 5ERα-binding events and coregulator expression differentiate between endometrial and breast cancer cell lines but recruitment to target genes is similar. (A) ERα-binding events are significantly enriched in breast cells vs endometrial cells (P < 0.0001, Fisher’s exact test). ERα-binding events in the 602 ERα-pSer118-associated genes identified from breast tumours were investigated in MCF7 cells and binding events in the 293 ERα-pSer118-associated genes identified from endometrial tumours were investigated in Ishikawa cells. Data are shown for at least one ERα binding event present in at least one sample. (B) Unsupervised clustering of the 17 differentially expressed ERα coregulators between endometrial and breast cell lines, as assessed using three statistical approaches (adjusted P < 0.05). Coregulators with an asterisk were also differentially expressed in TCGA tumours. FOXA1 and GATA3 are marked with arrows. Despite their low expression in endometrial cells, ChIP indicates that both (C) FOXA1 and (D) GATA3 are recruited to target genes, and this is promoted by oestrogen at select genes. Mean ± s.e.m. of two independent experiments is shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Welch’s t-test).



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available from TCGA as the basis for conducting more detailed molecular analyses in cell lines; these findings are summarised in Table 5. To date, Ishikawa is the most widely used endometrial cancer cell line. We also used the less frequently studied ERα-positive, endometrioid cell line JHUEM14 and the extensively studied breast cancer cell line MCF7.

Using phosphorylation of ERα (ERα-pSer118) as a marker of ERα transcriptional activation, we demonstrated that ERα-pSer118 was an independent prognostic factor in endometrial but not breast tumours (Fig. 1 and Table 2). Studies in breast cancer have often, but not always, indicated that ERα-pSer118 is associated with improved survival (reviewed in Murphy et al. 2011). However, these studies used immunohistochemistry with different

antibodies and cut-off scores, whilst we used RPPA data, potentially accounting for conflicting results.

We identified genes and pathways associated with ERα-pSer118 in TCGA endometrial and breast tumours. Oestrogen-responsive genes have been identified in the literature using different cell lines, varying time points and concentrations of oestrogen, as well as different cut-offs for fold-change or P values. With such varied methodologies, a search for concordance between different statistical approaches may be more informative. Using three statistical methods, we identified 293 consistently differentially expressed genes between endometrial tumours with high and low ERα-pSer118 levels, and 602 genes in breast tumours, and termed these as ERα-pSer118-associated genes (Supplementary Table 4). These genes were predominantly

Figure 6XBP1 recruitment to target genes is only altered with oestrogen in endometrial cancer cell lines. (A) ChIP reveals that CREB-pSer133 is recruited to target genes in both endometrial and breast cell lines and this is promoted by oestrogen at select genes. (B) ChIP of ERα followed by re-ChIP with CREB-pSer133 indicates that ERα and CREB-pSer133 are co-recruited to target genes in all three cell lines and this is promoted by oestrogen at select genes. (C) ChIP indicates that XBP1 is recruited to target genes in all three cell lines, and this is promoted by oestrogen only in endometrial cells. (D) ChIP of ERα followed by re-ChIP with XBP1 indicates that ERα is co-recruited with XBP1 to target genes in all three cell lines, but this is only promoted by oestrogen in endometrial cells. Mean ± s.e.m. of two independent experiments is shown. *P < 0.05, **P < 0.01, ***P < 0.001 (Welch’s test). ChIP using IgG as a negative control can be seen in Supplementary Fig. 5B.



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unique between tumour types; however, the processes they were involved in exhibited considerable overlap (Table 3), suggesting that it may be more informative to look at pathways as opposed to individual genes.

Using GSEA and Ingenuity Pathway Analysis, ERα-pSer118-associated genes in breast tumours were recognised to be involved in known oestrogen responses (Table 3) and, as expected, ERα was a predicted upstream regulator (Fig. 2B). However, neither oestrogen response nor ERα was identified to be significantly involved with ERα-pSer118-associated genes in endometrial tumours. Nevertheless, using publicly available ChIP-seq data from tumours and cell lines, the majority of ERα-pSer118-

associated genes were indicated to have at least one ERα binding event in either tumour type, although these were significantly enriched in breast cancer (Figs 2A and 5A). Taken together, these results suggest that ERα has a key role in both tumour types; however, non-classical ERα signalling may be more prevalent in endometrial tumours. Additionally, oestrogen-responsive and ERα target genes listed in databases have been identified predominantly from breast cancer and our analyses and others (eg. Droog et al. 2017) indicate that the oestrogen-associated transcriptome is tissue specific. We suggest that current databases need to recognise this tissue specificity and be updated accordingly.

Figure 7XBP1 has a distinct role in endometrial cancer. (A) The expression of select oestrogen-responsive genes is significantly altered in TCGA endometrial tumours (left) when comparing (A) high CREB (quartile 4) to low CREB (quartile 1) expression and (B) high XBP1 (quartile 4) to low XBP1 (quartile 1) expression, but not in breast tumours (right). Mean ± s.e.m.; *P < 0.05, **P < 0.01, ****P < 0.0001 (Mann–Whitney U test). (C) High CREB expression (>Median) is not associated with altered survival compared to low CREB expression (<Median) in either tumour type. (D) High XBP1 expression (>Median) is associated with significantly improved progression-free survival in endometrial tumours (left) compared to low XBP1 expression (<Median). There is no difference in breast tumours (right). (E) XBP1 protein levels are reduced after knockdown in Ishikawa and MCF7 cells as demonstrated by Western blot (left) and band quantification (right). Spliced XBP1 (54 kD) is marked with an arrow, the unspliced variant (29 kD) is only present in MCF7 cells. GAPDH is a loading control. (F) At the RNA level, XBP1 knockdown blunts the oestrogen-mediated induction of PGR, an oestrogen-responsive gene in both cancer types, only in Ishikawa and not MCF7 cells. Results are shown as fold-change normalised to the shNS–E2 condition. Mean ± s.e.m. of two independent experiments is shown. **q < 0.01, ****q < 0.0001.



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Pathway analysis of ERα-pSer118-associated genes predicted that SMAD3 was a significant upstream regulator in both tumour types (Fig. 2B). Oestrogen-regulated networks in MCF7 cells have previously been demonstrated to target downstream genes via SMAD3 (Cicatiello et al. 2010). We found that there is cross-over between oestrogen and TGFβ signalling in both endometrial and breast cancer cells (Supplementary Fig. 2C), which has previously been shown in MCF7 cells (Ito et al. 2010). Specifically, SMAD3 was co-recruited with ERα to target genes, and this recruitment was increased with oestrogen to select genes, particularly in Ishikawa cells (Fig. 3C). Consistent with SMAD3 playing a role in oestrogen signalling, alterations in SMAD3 expression were associated with altered expression of select oestrogen-responsive genes in both tumour types (Fig. 4A). SMAD3 may, however, have a distinct role in endometrial cancer as it was an independent predictor of worse progression-free survival (Table 4A).

Contrary to previous reports, we found that ERα recruitment to TFF1 was not consistently significantly increased with oestrogen in MCF7 cells, whereas it was at other loci (Fig. 3A). ERα recruitment to TFF1 cycles rapidly, potentially accounting for inconsistent results between independent assays (Métivier et al. 2003). Tissue-specific responses of ERα may depend on different ERα coregulator expression or usage (Thenot et al. 1999, Shang & Brown 2002). We showed differential expression of the majority of known ERα coregulators between endometrial and breast tumours, with the classic co-activators FOXA1 and GATA3 being the most differentially expressed (Fig. 2C). FOXA1 is a pioneer factor for ERα in breast cancer (Carroll et al. 2005, Laganière et al. 2005). GATA3 motifs are commonly enriched around ERα-binding sites in MCF7 cells (Kong et al. 2011). FOXA1 and GATA3 expression have been shown to differentiate ERα-positive

breast cancers from other cancers, including endometrial cancer (Davis et al. 2016). Nonetheless, FOXA1 signalling was implicated as a major hub in hormonal endometrial cancer (Cancer Genome Atlas Research Network et al. 2013, Berger et al. 2018). Using ChIP analyses, we found that both FOXA1 and GATA3 were recruited to target genes in endometrial cells similar to MCF7 cells (Fig. 5C and D). Altered expression of either cofactor was associated with the altered expression of select oestrogen-responsive genes in both tumour types (Supplementary Fig. 4A and B), and this has also been demonstrated in cell lines (Hurtado et al. 2011, Theodorou et al. 2013, Wang et al. 2014). It therefore follows that the reduced expression levels of either co-activator cannot be extrapolated to a reduced functional role in endometrial cancer and serves as a warning not to deduce mechanistic consequences simply from lower expression levels.

Binding sites for several members of the CREB/ATF family of transcription factors were significantly enriched in the ERα-pSer118-associated genes specifically in endometrial tumours (Fig. 2D). This could be a significant basis for the different responses to oestrogen of the two tumour types. CREB/ATF family members bind to the cAMP response element (CRE). CREB has previously been postulated to be recruited to EREs by ERα (Lazennec et al. 2001). Additionally, TCGA had predicted one of the family members, XBP1, to be an important part of a signalling hub in hormonal endometrial-like tumours (Cancer Genome Atlas Research Network et al. 2013), which was validated in a subsequent study (Berger et al. 2018). ChIP assays indicated that recruitment of the phosphorylated form of CREB, CREB-pSer133, which is required for transcriptional activity (Fig. 6A; Mayr & Montminy 2001), as well as co-recruitment with ERα (Fig. 6B) was increased with oestrogen at target genes in

Table 5 Summary of similarities and differences between the predictions made using TCGA data and subsequent analyses in cell lines.

Predicted using TCGA data Analyses in cell lines

ERα binding events are significantly enriched in breast tumours (Fig. 2A)

ERα binding events are significantly enriched in breast cells (Fig. 5A)

SMAD3 predicted to be an activated upstream regulator in both endometrial and breast tumours (Fig. 2B)

Crossover between oestrogen and TGFβ signalling in both endometrial and breast cancer cell lines (Supplementary Fig. 2C). Additionally, ERα and SMAD3 are co-recruited to target genes in both cell types (Fig. 3C)

ERα coregulators, notably FOXA1 and GATA3, are differentially expressed between endometrial and breast tumours (Fig. 2C and Supplementary Fig. 1E)

ERα coregulators, including FOXA1 and GATA3, are differentially expressed between endometrial and breast cancer cell lines (Fig. 5A); however, FOXA1 (Fig. 5B) and GATA3 (Fig. 5C) recruitment to target genes are similar in all cell lines

CREB/ATF binding sites are enriched in ERα-pSer118-associated genes in endometrial tumours (Fig. 2D)

ERα and CREB-pSer133 are co-recruited to target genes in both endometrial and breast cancer cell lines and this is increased by oestrogen in both cell types (Fig. 6B)ERα and XBP1 are co-recruited to target genes in both cell types but this is only increased by oestrogen in endometrial cell lines (Fig. 6D)



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all three cell lines. However, differences in response to oestrogen were seen for the recruitment of XBP1; XBP1 recruitment on its own (Fig. 6C) and co-recruitment with ERα (Fig. 6D) were increased with oestrogen at target genes only in endometrial and not breast cancer cells. In TCGA tumours, altered expression of either CREB or XBP1 was associated with altered expression of select oestrogen-responsive genes only in endometrial tumours (Fig. 7A and C), but only XBP1 was an independent prognostic factor (Table 4B). In-line with the analyses done by TCGA (Cancer Genome Atlas Research Network et al. 2013, Berger et al. 2018), this would therefore suggest that XBP1 plays a distinct role in oestrogen signalling in endometrial cancer. Knockdown of XBP1 in cell lines supported this hypothesis as the oestrogen-mediated induction of PGR, an oestrogen-responsive gene in both cancer types, was reduced only in endometrial and not breast cancer cells (Fig. 7E and F).

The lack of similarity in oestrogen response between ERα-positive endometrial and breast cancers is undeniable at the molecular level. We have shown that activated ERα (ERα-pSer118) is associated with the expression of predominantly different genes in endometrial and breast tumours, which in turn are associated with tumour type-specific factors. SMAD3, FOXA1, GATA3 and CREB-pSer133 are involved in oestrogen signalling in both endometrial and breast cancer, but XBP1 consistently plays a distinct role in oestrogen signalling in endometrial cancer.

Supplementary dataThis is linked to the online version of the paper at https://doi.org/10.1530/ERC-17-0563.

Declaration of interestThe authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

FundingThis work was supported by a grant from The Weekend To End Women’s Cancers.

AcknowledgementsThe authors would like to thank Tim Bruxner, Angelika Christ and Nicole Cloonan for assistance with RNAseq; Pam Pollock for sharing resources and staff in the QIMR Berghofer animal facility, particularly David McNeilly. They would also like to acknowledge everyone who contributed to the TCGA datasets, including the specimen donors, clinicians and research groups. F Gannon and D J Brennan: Joint last authorship.

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Received in final form 20 July 2018Accepted 13 August 2018Accepted Preprint published online 18 August 2018



via free access

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