Progesterone receptor modulates estrogen receptor-α action in
breast cancer
Hisham Mohammed
1
, I. Alasdair Russell
1
, Rory Stark
1
, Oscar M. Rueda
1
, Theresa E.
Hickey
2
, Gerard A. Tarulli
2
, Aurelien A. A. Serandour
1
, Stephen N. Birrell
2
, Alejandra
Bruna
1
, Amel Saadi
1
, Suraj Menon
1
, James Hadfield
1
, Michelle Pugh
1
, Ganesh V. Raj
3
,
Gordon D. Brown
1
, Clive D’Santos
1
, Jessica L. L. Robinson
1
, Grace Silva
4
, Rosalind
Launchbury
1
, Charles M. Perou
4
, John Stingl
1
, Carlos Caldas
1,5,6
, Wayne D. Tilley
2,7
, and
Jason S. Carroll
1,7
1
Cancer Research UK Cambridge Institute, University of Cambridge, Robinson Way, Cambridge,
CB2 0RE, UK.
2
Dame Roma Mitchell Cancer Research Laboratories and the Adelaide Prostate Cancer
Research Centre, School of Medicine, Hanson Institute Building, University of Adelaide, Adelaide,
SA 5005, Australia.
3
Department of Urology, University of Texas, Southwestern Medical Center at Dallas, Dallas,
Texas, 75390, USA.
4
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 450 West
Drive, CB7295, Chapel Hill, NC, 27599, USA.
5
Cambridge Breast Unit, Addenbrooke’s Hospital, Cambridge University Hospital NHS
Foundation Trust and NIHR Cambridge Biomedical Research Centre, Cambridge CB2 2QQ, UK.
6
Cambridge Experimental Cancer Medicine Centre, Cambridge, CB2 0RE.
Summary
Progesterone receptor (PR) expression is employed as a biomarker of estrogen receptor-α (ERα)
function and breast cancer prognosis. We now show that PR is not merely an ERα-induced gene
target, but is also an ERα-associated protein that modulates its behaviour. In the presence of
agonist ligands, PR associates with ERα to direct ERα chromatin binding events within breast
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7
To whom correspondence should be addressed: jason.carroll@cruk.cam.ac.uk or wayne.tilley@adelaide.edu.au.
Author contributions
Experimental work was conducted by H.M, I.A.R, T.E.H, G.A.T, A.A.A.S, A.B, A.S, C.D, J.L.L.R, R.L and G.S. Computational
analysis was conducted by R.S, O.M.R, S.M and G.D.B. Clinical samples and clinical information was obtained from S.N.B, G.V.R,
C.M.P and C.C. In vivo work was conducted by J.S. Genomic work was conducted by J.H and M.P. All experiments were overseen by
W.D.T and J.S.C. The manuscript was written by H.M, I.A.R, T.E.H, W.D.T and J.S.C with help from the other authors.
Data deposition
All microrray data are deposited in GEO accession number GSE68359.
All ChIP-seq data are deposited in GEO accession number GSE68359.
All proteomic data are deposited with the PRIDE database with the accession number PXD002104.
Conflict of interest
None of the authors have any conflicts of interest.
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Published in final edited form as:
Nature. 2015 July 16; 523(7560): 313–317. doi:10.1038/nature14583.
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cancer cells, resulting in a unique gene expression programme that is associated with good clinical
outcome. Progesterone inhibited estrogen-mediated growth of ERα+ cell line xenografts and
primary ERα+ breast tumour explants and had increased anti-proliferative effects when coupled
with an ERα antagonist. Copy number loss of PgR is a common feature in ERα+ breast cancers,
explaining lower PR levels in a subset of cases. Our findings indicate that PR functions as a
molecular rheostat to control ERα chromatin binding and transcriptional activity, which has
important implications for prognosis and therapeutic interventions.
There is compelling evidence that inclusion of a progestogen as part of hormone
replacement therapy (HRT) increases risk of breast cancer, implying that PR signalling can
contribute towards tumour formation
1
. However, the increased risk of breast cancer
associated with progestogen-containing HRT is mainly attributed to specific synthetic
progestins, in particular medroxyprogesterone acetate (MPA), which is known to also have
androgenic properties
2
. The relative risk is not significant when native progesterone is
used
3
. In ERα+ breast cancers, PR is often used as a positive prognostic marker of disease
outcome
4
, but the functional role of PR signalling remains unclear. While activation of PR
may promote breast cancer in some women and in some model systems, progesterone
treatment has been shown to be antiproliferative in ERα+ PR+ breast cancer cell lines
5-7
and
progestogens have been shown to oppose estrogen-stimulated growth of an ERα+ PR+
patient-derived xenograft
8
. In addition, exogenous expression of PR in ERα+ breast cancer
cells blocks estrogen-mediated proliferation and ERα transcriptional activity
9
. Furthermore,
in ERα+ breast cancer patients, PR is an independent predictor of response to adjuvant
tamoxifen
10
, high levels of PR correlate with decreased metastatic events in early stage
disease
11
and administration of a progesterone injection prior to surgery can provide
improved clinical benefit
12
. These observations imply that PR activation in the context of
estrogen-driven, ERα+ breast cancer, can have an anti-tumourigenic effect. In support of
this, PR agonists can exert clinical benefit in ERα+ breast cancer patients that have relapsed
on ERα antagonists
13
.
Breast cancers are typically assessed for ERα, PR and HER2 expression to define
histological subtype and guide treatment options. PR is an ERα-induced gene
14
and ERα+
PR+ HER2- tumours tend to have the best clinical outcome because PR positivity is thought
to reflect a tumour that is driven by an active ERα complex and therefore likely to respond
to endocrine agents such as tamoxifen or aromatase inhibitors
10,15
. While ERα+ PR+
tumours have a better clinical outcome than ERα+ PR− tumours
4
, clinical response to ERα
antagonists can vary, even among tumours with similar ERα and PR status
15,16
and recent
evidence suggests that PR may be prognostic, but not predictive
17
. Some ERα+ PR−
tumours that are resistant to one class of ERα antagonists gain clinical benefit from another
class, suggesting that in a subset of ERα+ PR− breast cancers, the lack of PR expression
does not reflect a nonfunctional ERα complex. It has been proposed that the non-functional
ERα complex theory cannot completely explain PR negativity
18
. An alternative hypothesis
is that other factors contribute to the loss of PR expression, which consequently influences
breast tumour responses to ERα target therapies.
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PR is recruited to the ERα complex
Given the controversial and complex interplay between the ERα and PR pathways in breast
cancer, we explored the possible functional crosstalk between these two transcription factors
and the implications for clinical prognosis in ERα+ disease. Ligand-activated ERα and PR
protein complexes were purified to ascertain interplay between these two transcription
factors. Asynchronous ERα+ PR+ MCF-7 and T-47D breast cancer cells were grown in
SILAC-labelled growth media, which contains sufficient estrogen to elicit maximal ERα
binding to chromatin
19
. Estrogen treatment is required to induce detectable levels of PR in
MCF-7 cells but not T-47D cells
20
. The two cell lines were subsequently treated with
vehicle or one of two progestogens: native progesterone or the synthetic progestin R5020.
Cells were cross-linked following hormone treatment and endogenous PR was
immunopurified followed by mass spectrometry, using a technique we recently developed
called RIME
21
. Under estrogenic conditions, progesterone treatment significantly induced
an interaction between PR and ERα in the MCF-7 and T-47D cell lines, in support of
previous findings showing a physical interaction between these two nuclear receptors
22
. In
addition to ERα, progesterone treatment induced interactions between PR and known ERα-
associated co-factors, including NRIP1, GATA3 and TLE3
21
in both cell lines (Figure 1a).
As expected, treatment with natural ligand decreased interaction between PR and
chaperone/co-chaperone proteins such as HSP90 and FKBP4/5 (Figure 1a). The same
findings were observed when R5020 was used as a synthetic ligand (Extended data figure 1).
Interestingly, when ERα was purified under the same treatment conditions, PR was the only
differentially recruited protein in both cell lines (Figure 1b). Moreover, the interaction
between ERα and known ERα-associated co-factors was not differentially affected by
progesterone treatment. A list of all interacting proteins under all experimental conditions is
provided in Supplementary table 1. The progesterone-induced ERα-PR interaction was
confirmed by standard co-IP experiments in both MCF-7 and T-47D cells (Figure 1c). We
conclude that activation of PR results in a robust association between PR and the ERα
complex. However, it remains unclear what impact this may have on ERα/PR DNA binding
or what the primary DNA tethering mechanism may be (Figure 1d).
Progesterone reprograms ER
α binding events
Since PR is a transcription factor, we hypothesised that the progestogen-induced interaction
between PR and ERα alters chromatin binding properties of the ERα-co-factor complex.
MCF-7 and T-47D cell lines grown in complete (estrogenic) media were stimulated with
progesterone, R5020 or vehicle control for 3 or 24 hours, followed by analysis of genome-
wide ERα and PR and the co-activator p300 profiles by ChIP-seq. p300 deposits the
H3K27Ac mark, which is indicative of functional enhancers
23
. We found comparable ERα,
PR and p300 binding at 3 hours and 24 hours (Extended data figure 1 and data not shown)
and chose 3hr for the remaining experiments. All ChIP-seq experiments were subsequently
repeated in triplicate following 3 hours of treatment (sample clustering is provided in
Extended data figure 2). Whereas robust ERα binding (29,149 sites in MCF-7 cells and
8,438 sites in T-47D cells) was observed in estrogenic conditions, limited PR binding events
were seen under these conditions (Supplementary table 2). Treatment with progesterone or
the synthetic progestin R5020 in estrogen-rich media resulted in robust PR recruitment to
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chromatin, with 46,191 PR binding events observed in T-47D cells with both ligands and
29,554 PR binding events in MCF-7 cells. Using DiffBind to identify differential peaks
24
that occurred following treatment of cells with progesterone or R5020, a rapid and robust
redistribution of ERα binding to novel genomic loci was observed (Figure 2a and 2b). In
T-47D cells, 14,223 ERα binding events were reproducibly gained following 3 hours of
treatment with progesterone, a finding that was similar following stimulation with R5020,
confirming a predictable redistribution of ERα chromatin binding following stimulation of
the PR pathway. A similar rapid redirection of ERα was observed in MCF-7 cells after
progesterone or R5020 treatment (Extended data figure 1). The ERα sites reprogrammed by
progesterone are likely to be functional, as indicated by the global recruitment of the co-
activator p300 (Figure 2b and Extended data figure 2). These ERα binding events appear to
be mediated by PR, since 99% of the gained ERα binding sites in T-47D cells overlapped
with a PR binding event (Figure 2c) (the overlap in MCF-7 cells was 94%) and motif
analysis of the ERα gained sites revealed the presence of progesterone responsive elements
(PREs), but not estrogen responsive elements (EREs) (Figure 2b). This suggests that PR
mediates the interaction between the ERα/PR/p300 complex and DNA. ERα ChIP-seq was
repeated in MCF-7 and T-47D cells following hormone deprivation and subsequent
treatment with vehicle, estrogen alone or progesterone alone. Single hormone treatments did
not induce the ERα binding events observed under dual hormone conditions (Extended data
figure 3), confirming that the ERα reprogramming is dependent on having both receptors
activated simultaneously. In addition, Forkhead motifs were enriched at the ERα gained
sites and 49% of the gained ERα/PR binding events were shown to overlap with FoxA1
binding (Extended data figure 4), consistent with previous findings showing that PR binding
involves the pioneer factor FoxA1
25
. In keeping with the anti-proliferative effects of
progestogens in ERα+ breast cancer cells, analysis of the genes bound by ERα following
progesterone treatment revealed enrichment for cell death, apoptosis and differentiation
pathways (Supplementary table 3).
To identify the transcriptional targets of the progestogen-induced ERα binding events, we
treated estrogen-stimulated MCF-7 and T-47D cells with progesterone, R5020 or vehicle for
3 hours and performed eight replicates of RNA-seq. In total, 470 genes were differentially
regulated by dual treatment with estrogen and progesterone or R5020 compared to estrogen
alone in both T-47D and MCF7 cell lines (Extended data figure 4). GSEA analysis revealed
a pronounced enrichment of progestogen-induced ERα binding events near genes up-
regulated by progestogen treatment in the presence of estrogen (Figure 2d). Collectively,
these findings suggest that the progestogen-mediated changes in ERα binding events are
functionally significant, since they co-recruit p300 and lead to new gene expression profiles
(Figure 2d and Extended data figure 4). Importantly, increased expression of a gene
signature that results from progesterone-stimulated ERα binding confers good prognosis in a
cohort of 1,959 breast cancer patients (Extended data figure 5).
The relative degree of ERα reprogramming and gene expression changes following
progesterone treatment was higher in T-47D cells compared to MCF-7 cells (Supplementary
table 2), possibly due to the differences in PR levels between these two cell lines
20
. We
assessed the PR gene (PgR) in these cell lines, which revealed copy number gain of the PgR
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gene in T-47D cells and a heterozygous loss of the PgR gene in MCF-7 cells (Extended data
figure 5). Exogenous expression of PR (both isoforms) in the MCF-7 cell line resulted in
growth inhibition (Extended data figure 6), confirming an anti-proliferative role for PR, via
modulation of ERα transcriptional activity
9
.
Progesterone blocks ER
α+ tumour growth
To explore the hypothesis that progesterone stimulation could have beneficial effects on
ERα+ tumour growth in vivo, we established MCF-7-Luciferase xenografts in NOD/SCID/
IL2Rg-/- (NSG) mice and exposed the mice to control (i.e. no hormone), slow release
estrogen pellets or slow release estrogen plus standard high concentration progesterone
pellets
26
. Ten tumours for each condition were implanted (two tumours per mouse and five
mice per condition) and tumour formation was monitored using bioluminescent imaging.
After 25 days, in the absence of any hormone, tumours did not grow, but stimulation with
estrogen alone resulted in tumour growth. Co-treatment with estrogen plus progesterone
resulted in a significant decrease in tumour volume, as compared to estrogen alone, when
measured by bioluminescence (Figure 3a and 3b) (p = 0.0021) or tumour volume (p = 0.019)
(Extended data figure 6). Immunohistochemistry confirmed that PR expression was induced
under both estrogen and estrogen plus progesterone conditions (Extended data figure 7), but
importantly, PR will only be active under dual hormone conditions. We repeated the MCF-7
xenograft experiment in ovariectomised mice, in order to eliminate any confounding issues
related to endogenous mouse hormones. Assessment of tumour growth (Extended data
figure 7) confirmed the previous finding (Figure 3b) that progesterone inhibited tumour
formation. We performed ERα and PR ChIP-seq on six randomly selected sets of matched
estrogen or estrogen plus progesterone stimulated xenograft tumours (taken from the final
time point) and identified differentially bound ERα binding events. The major variable
driving clustering of ERα binding events within tumour xenografts was the treatment
condition (Extended data figure 7). As observed in the short-term cell line experiments
(Figure 2), estrogen-stimulated ERα binding in the xenograft tumours was substantially
altered by progesterone treatment. We observed 3,603 differentially regulated ERα binding
events in xenograft tumours from estrogen plus progesterone conditions, when compared to
estrogen-only conditions (Figure 3c). As such, progesterone induced a global
reprogramming of ERα binding events in vivo, even following long-term hormonal
treatment.
To extend our findings into primary tumours, we employed a novel ex vivo primary tumour
culture system
27,28
to cultivate ERα+ PR+ primary tumours for short time periods, in order
to study the effect of hormonal treatment on cell growth. Fourteen independent ERα+ PR+
primary tumours were used for the analysis. Each tumour was cut into small pieces and
randomised onto gelatine sponges half-submersed in media to sustain tissue architecture and
viability. Tumour explants were then cultivated under hormone-deprived conditions for 36
hours, followed by a 48 hour treatment with vehicle control, estrogen, the synthetic
progestin R5020 or estrogen plus R5020. Tumours retained normal cellular and
morphological features after treatment in the ex vivo context (Extended data figure 8). Fixed
tissues were stained for Ki67 to assess changes in proliferation. Most of the tumours
responded to estrogen with a coincident increase in the percentage of cells that expressed
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