European Journal of Cancer (2014) 50, 3003 3010

Available at www.sciencedirect.com

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journal homepage: www.ejcancer.com

Original Research

Loss of progesterone receptor links to high proliferation

and increases from primary to metastatic endometrial
cancer lesions
Ingvild Lberg Tangen a,b,, Henrica M.J. Werner a,b, Anna Berg a,b, Mari K. Halle a,b,
Kanthida Kusonmano a,b,c, Jone Trovik a,b, Erling A. Hoivik a,b, Gordon B. Mills d,
Camilla Krakstad a,b, Helga B. Salvesen a,b
a

Centre for Cancer Biomarkers, Department of Clinical Science, University of Bergen, Norway
Department of Gynecology and Obstetrics, Haukeland University Hospital, Norway
c
Computational Biology Unit, University of Bergen, Bergen, Norway
d
Department of Systems Biology, University of Texas, MD Anderson Cancer Center, Houston, TX, USA
b

Received 2 June 2014; received in revised form 12 August 2014; accepted 10 September 2014
Available online 30 September 2014

KEYWORDS
Endometrial cancer
Progesterone receptor
Survival
CDK inhibitors

Abstract Objective: In endometrial cancer loss of progesterone receptor (PR, gene name
PGR) is associated with aggressive disease and altered response to hormonal treatment. The
aim of this study was to investigate changes in PR expression level with disease progression,
and explore whether differences in gene expression according to PR status can be linked to
processes involved in cancer development elucidating new therapeutic opportunities.
Methods: 686 primary endometrial cancers and 171 metastatic lesions were investigated for
PR expression in relation to clinical and histopathological data. Protein levels were investigated by immunohistochemistry and reverse phase protein array, and mRNA levels by
DNA oligonucleotide microarray.
Results: PR protein level was signicantly associated with PGR mRNA expression (P < 0.001)
and patient survival (P < 0.001). Loss of PR increased with disease progression, with 23% of
the primary tumours and 76% of metastases demonstrating PR loss. Using a cell cycle progression signature score, PR loss was associated with increased proliferation for both oestrogen
receptor (ER) positive and negative tumours. Through a Connectivity Map search, CDK
inhibitors and other drugs with anti-proliferative effects were suggested in particular for treatment of patients with loss of PR.

Corresponding author at: Department of Clinical Science, Section for Gynecology and Obstetrics, University of Bergen, Jonas Lies Vei 72, 5020
Bergen, Norway. Tel.: +47 55 97 42 00; fax: +47 55 97 49 68.

http://dx.doi.org/10.1016/j.ejca.2014.09.003
0959-8049/ 2014 Elsevier Ltd. All rights reserved.

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I.L. Tangen et al. / European Journal of Cancer 50 (2014) 30033010

Conclusion: Loss of PR in endometrial cancer is associated with increased proliferation, poor

survival, and increases from primary to metastatic lesions. Based on expression proles, CDK
inhibitors may have activity in PR negative tumours, supporting further testing in clinical trials for patients with systemic endometrial cancer dependent on PR status.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
When endometrial cancer is identied and treated at
an early stage there is subsequent good prognosis. However, for patients with systemic disease either recurrent or
metastatic at presentation, the prognosis is poor,
unchanged over the last several decades, and with limited
treatment options [1]. Although endometrial cancer is the
most common gynaecological malignancy in developed
countries and the incidence is increasing [2], the progress
in development of treatment for advanced or recurrent
disease has been slow. Eective new targeted therapies,
combined with robust biomarkers to identify patient subgroups that will benet most from emerging as well as
available treatments will improve patient care.
Progesterone is important for regulation of normal
reproductive function, and is involved in controlling
changes in the uterus and ovaries during the menstrual
cycle. The eect of progesterone is mediated through
progesterone receptor (PR), and PR is expressed in a
variety of human tissues, including the uterus, mammary gland and ovary [3].
In breast cancer progesterone plays a role in controlling tumour promotion [4], whilst in the endometrium
and the ovaries it has a suppressive eect on tumour
development [5,6]. Although the eect of progesterone
diers depending on the target tissue, the PR expression
prole has demonstrated a prognostic value in uterine,
breast and ovarian malignancies, and loss of PR is associated with worse outcome [710].
In the endometrium oestrogen induces proliferation
whilst progesterone suppresses the oestrogen mediated
signals and has a dierentiating eect [6]. Oestrogen
dependent endometrial cancers are thought to arise from
unopposed oestrogen exposure, not balanced by the differentiating eect of progesterone [11]. Currently, both
drugs antagonising oestrogen eects, and progesterone
analogues are used in endometrial cancer treatment. In
advanced or recurrent disease, treatment with progesterone has shown modest response rates [12]. However, in
premenopausal woman with well dierentiated endometrial cancer the response rates are reported to be higher,
allowing fertility preserving treatment [13,14]. Although
response to progesterone therapy is reported to be
dependent on progesterone receptor (PR) status
[12,15], and PR is reported to be a prognostic marker
in endometrial cancer [9,10,16], evaluation of PR expression is not routinely performed in endometrial cancer to
guide treatment decisions.

The aim of this study was to investigate changes in

progesterone receptor expression during disease progression, and to explore if genes dierentially expressed
according to PR status, can be linked to biological processes involved in cancer development. The identied
genetic alterations were explored to search for new
potential drug candidates.
2. Materials and methods
2.1. Patient series
A population based patient series was prospectively
collected from 2001 to 2013, and includes 686 primary
tumours from patients diagnosed with endometrial cancer in Hordaland County (Norway). Patients were surgically staged according to the International Federation of
Gynecology and Obstetrics (FIGO) 2009 criteria. Clinical
data were collected as described earlier [17]. Biopsies from
metastatic tissue were available from 76 patients (in total
171 lesions). When available, fresh frozen tissue was collected in parallel with formalin xed paran embedded
(FFPE) tissue and used for mRNA and protein extraction. Tissue microarrays (TMA) were generated from
FFPE tissue as previously described [18]. The independent endometrial cancer patient series with data for Ki67 and PR has previously been described and published
[19,20]. All parts of the study have been approved according to Norwegian legislation, including the Norwegian
Data Inspectorate, Norwegian Social Sciences Data Services and the Western Regional Committee for Medical
and Health Research Ethics, (NSD15501; REK 052.01).
Participants gave written informed consent.
2.2. Protein detection
TMAs were dewaxed in xylene, and rehydrated in ethanol before microwave antigen retrieval and stained for
PR expression using M3569 (Dako). The staining was
evaluated as previously described [19]. Staining index 0
was considered PR negative, and 19 PR positive. Kappa
value was calculated to be 0.82 for PR in two groups.
Oestrogen receptor (ER) was stained and scored as previously described [21,22]. When evaluating multiple metastatic lesions from the same patient, PR was dened as
lost if any of the metastatic lesions demonstrated loss.
Reverse phase protein array (RPPA) was performed on
358 primary tumours as previously described [23,24].

I.L. Tangen et al. / European Journal of Cancer 50 (2014) 30033010

Briey, fresh frozen patient samples were homogenised

in lysis buer before proteins were denatured using
SDS and serial diluted in lysis buer. Samples were
printed on nitrocellulose coated slides before staining
for PR (ab32085 Abcam) and proliferation cell nuclear
antigen (PCNA) (ab29 Abcam) expression. Relative protein levels were determined by tting each dilution curve
with a logistic model (Supercurve Fitting http://bioinformatics.mdanderson.org/OOMPA).
2.3. Gene expression analyses
RNA was extracted from fresh frozen tissue from 18
hyperplasias, 174 primary tumours and 42 metastases
using the RNeasy Mini Kit (Qiagen). Samples were hybridised to Agilent Whole Human Genome Microarrays 44k
(Cat. No. G4112F), scanned and normalised as previously described [21]. Dierentially expressed genes were
identied by Signicance Analysis of Microarray
(SAM). A False Discovery Rate (FDR) of <0.01 was considered signicant. Gene Set Enrichment Analysis
(GSEA; www.broadinstitute.org/gsea) was used to identify Gene Ontology gene sets enriched in dierent groups.
The cell cycle progression (CCP) signature published by
Cuzick et al. [25] was explored in our gene set. The average expression value of the 31 genes was calculated for
each patient. Genes dierentially expressed according to
PR status were used to search the Connectivity Map database for potential drugs.
2.4. Statistical analyses
Statistical analyses were performed using the software
package SPSS 19.0 (SPSS Inc, Chicago, IL). Probability
of <0.05 was dened as statistically signicant. For categorical variables, the Pearson v2 or Fishers exact test was
used to evaluate correlations between groups, and for
continuous variables the MannWhitney U test or KruskalWallis test was used. Univariate analyses were done
using the KaplanMeier (product-limit) method. Entry
date was the date of primary surgery, and time to death
due to endometrial carcinoma was the endpoint (disease
specic survival). Survival between groups was compared
using the log-rank test (MantelCox). The prognostic
impact of PR adjusted for other prognostic markers
was evaluated by the Cox proportional hazard regression
model. An interaction between FIGO stage and histologic type was suspected, and the Cox analysis was therefore performed for the endometrioid subgroup.
3. Results
3.1. Loss of PR associates with aggressive endometrial
carcinoma
Positive PR staining was predominantly nuclear and
observed both in glandular and stromal tissues, but only

3005

staining in the glandular tissue was scored. Loss of PR

was signicantly associated with markers for poor
outcome (Table 1) and poor survival in all
patients (Fig. 1A) and in the endometrioid subgroup
(Supplementary Fig. 1A). In a multivariate analysis of
the endometrioid subgroup adjusting for age, FIGO
stage and histologic grade, PR had an independent
prognostic impact (hazard ratio (HR): 2.0, 95% condence interval (CI): 1.054.0, P = 0.036) (Supplementary
Table 1). Loss of PR was not associated with survival in
the non-endometrioid patients (Supplementary Fig. 1B).
mRNA expression of PGR was evaluated in 174
overlapping samples. Protein level measured by
immunohistochemistry (IHC) was signicantly associated with mRNA expression of PGR (Fig. 1B). In line
with the protein results from IHC, PGR expression was
signicantly associated with several markers for poor
outcome (Supplementary Table 2). The strong
association between PR protein level, assessed by IHC,
and markers for outcome was also demonstrated when
protein level was assessed by RPPA in 358 overlapping
primary tumours (Supplementary Table 2). These data
support that PR protein level and mRNA expression
are highly correlated, and that PR loss is signicantly
correlated with aggressive disease.
3.2. Metastatic lesions demonstrate the highest proportion
of PR loss
A gradual decrease in PR expression was observed
with disease progression. Of 686 primary tumours investigated, 23% had lost expression of PR. Considering
endometrioid tumours, 14% demonstrated loss of PR
compared with 65% in non-endometrioid tumours.
The percentage of metastases with loss of PR further
increased to 76% (Fig. 1C). In metastases from endometrioid tumours PR was lost in 64%, whilst metastases
from non-endometrioid tumours showed loss of PR in
94%. For patients with multiple metastatic lesions 75%
of the patients with PR loss in the metastatic setting
had a homogenous expression and demonstrated PR
loss in all metastatic lesions, whilst 25% had multiple
metastases with heterogeneous expression pattern,
where at least one demonstrated loss.
3.3. Loss of PR associates with enhanced cell proliferation
in malignant tissue
Potential biological processes related to the observed
hormone receptor loss were further explored by investigating related transcriptional alterations. SAM analysis
revealed that 1158 genes were dierentially expressed
according to PR status and 495 genes were dierentially
expressed according to ER status, of which 324 genes
overlapped. Several of the genes, signicantly up-regulated in PR negative tumours, are known to be involved

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I.L. Tangen et al. / European Journal of Cancer 50 (2014) 30033010

Table 1
Clinico-pathological variables related to progesterone receptor (PR) status (protein level evaluated by
IHC).
Variable

PR positive n (%)

PR negative n (%)

Age
<66
P66

290 (81)
237 (73)

69 (19)
90 (27)

0.010

International Federation of Gynecology and Obstetrics (FIGO)-09 stage

III
467 (81)
109 (19)
IIIIV
60 (55)
50 (45)
Histologic type
Endometrioid
483 (86)
79 (14)
Non-endometrioid
44 (36)
80 (64)
Histologic gradea
Grade 1
230 (94)
15 (6)
Grade 2
185 (88)
26 (12)
Grade 3
58 (61)
37 (39)
Metastatic nodes
Negative
395 (79)
106 (21)
Positive
36 (55)
29 (45)
Ploidy
Diploid
280 (80)
69 (20)
Aneuploid
55 (58)
40 (42)
ERa
Positive
452 (90)
52 (10)
Negative
70 (41)
103 (59)
Myometrial inltration
<50%
353 (83)
73 (17)
P50%
173 (67)
84 (33)
a

P-value

<0.001

<0.001

<0.001

<0.001

<0.001

<0.001

<0.001

Only endometrioid.

in cell proliferation. We continued to explore the PR

related genes, excluding genes found in both the PR
and the ER list, in GSEA. Gene sets associated with cell
cycle regulation were signicantly enriched in PR negative compared to positive tumours (Supplementary
Table 3).
Cuzick et al. have published a cell cycle progression
(CCP) gene signature [25]. In our dataset this CCP score
increased with disease progression (Supplementary
Fig. 2A), and high CCP score was also reected in significantly worse survival (Supplementary Fig. 2B). We
investigated if hormone receptor status also reected
changes in CCP score. Both in the whole population
(Fig. 2A) and in subgroups according to ERa status
(Fig. 2B) high CCP score was signicantly associated with
loss of PR. In contrast, we found no association between
ERa status and CCP score within the PR positive or PR
negative subgroups (Fig. 2B). PCNA levels, assessed by
RPPA, were also found to be signicantly associated with
PR status within subgroups of both ERa positive and
negative cases, contrasting the lack of association
between ERa and PCNA levels within subgroups of PR
positive or PR negative cases (Fig. 2C). We also reexamined an additional independent endometrial cancer series
previously published with data available for PR status
and the alternative proliferation marker Ki-67 [19,20]
conrming the pattern for a signicant increase in

proliferation related to PR loss in ER positive tumours

(Supplementary Fig. 3).
To explore if transcriptional alterations related to PR
loss in the tumours could suggest new targets for treatment, Connectivity Map [26] was queried for drug signatures negatively correlated with the gene expression
prole of PR negative tumours. Amongst the top scoring
drugs were two targeting the phosphoinositide 3-kinase
(PI3K) signalling pathway (Sirolimus and LY294002).
The observation that mammalian target of rapamycin
(mTOR) inhibitors are active in endometrial cancer [27]
supports the utility of the Connectivity Map analysis.
Interestingly, several of the top ranked compounds are
known to be anti-proliferative, including cyclindependent kinase 2 (CDK2) inhibitors (Table 2 and
Supplementary Table 4) suggesting a potential new
treatment opportunity in systemic endometrial cancer.
4. Discussion
In this study we investigate PR protein level with
related transcriptional alterations in a large and unique
collection of extensively annotated primary and metastatic endometrial cancer cases. We nd, as already well
documented from earlier studies [9,10,16], that loss of
PR is associated with markers for aggressive disease
and predicts poor survival. In addition, we nd a

I.L. Tangen et al. / European Journal of Cancer 50 (2014) 30033010

1.0

PR positive 479/42

0.8
0.6

PR negative 151/47

0.4
0.2
0

P<0.001
0

36
12
24
40
Length follow up (months)

Lesions with PR loss (%)

11.0
10.0
9.0
8.0
7.0
6.0
5.0 P<0.001
PR positive PR negative

60

C
100

B
PGR mRNA (log 2 transformed)

Disease specific survival

3007

P=0.1

80

P<0.001

60
P<0.001
40
20

P=0.02

Gr 1

Gr 2

Gr 3

NE

Met

Endometrioid
Fig. 1. Loss of progesterone receptor (PR) is signicantly associated with poor survival (A) and there is a signicant association between protein
level and mRNA expression of PGR (B). The proportion of cases with loss of PR increases signicantly with dedierentiation and disease
progression, and the highest proportion of cases with PR loss is found in metastatic lesions (76% with PR loss) (C). Examples of PR staining in
primary tumours and metastases, (D) PR positive primary tumour and corresponding (E) PR positive metastases, (F) PR positive primary tumour
and corresponding (G) PR negative metastases, (H) PR negative primary tumour and corresponding (I) PR negative metastases. (Abbreviations:
NE: non-endometrioid, Gr: grade. Met: metastases).

dramatic change in proportion of patients with PR loss

in metastatic compared to primary endometrial cancer
lesions. Importantly, the eect of treatment with
synthetic progesterone is reported to be dependent on
PR status [12,15], yet assessment of receptor status is
not routinely performed before treatment initiation. In
a review of randomised clinical trials investigating the
eect of hormonal therapy in advanced or recurrent
endometrial cancer, information regarding hormone
receptor status was not included in patient stratication
in ve of six trials [28], suggesting that hormonal
therapy may have a yet not fully explored potential both
in the primary setting, and for systemic disease.
In the primary lesions we observe loss of PR in 23% of
patients, and many tumours lose PR expression from primary to their metastatic counterpart, demonstrated by
76% PR loss for metastases. It is interesting to note that
the proportion of the poor-prognosis non-endometrioid
patients with intact expression of PR in the primary setting is relatively high (36%) and it is tempting to speculate
that this patient group could benet from hormonal treatment if biomarker status was implemented. Likewise, the

response rate for hormonal treatment is currently

reported to be low in advanced disease [28] where, at present, often the endometrioid subtype is urging the clinician
to consider hormonal therapy. Determination of hormone receptor status in recurrent disease after re-biopsy
from the metastatic lesion would give a valid presentation
of PR as target in the systemic disease setting in endometrial cancers and would better tailor the treatment to
patients with suspected benets. This suggests that the
eect of PR targeted therapy if restricted to patients with
PR positivity in tumours may be higher, warranting
development of trials to test this hypothesis. The nding
that expression of PR changes during endometrial cancer
progression is in line with a recent study in breast cancer
indicating that levels of ER, PR and HER-2 are unstable
throughout tumour progression [29]. Investigation of
biopsies from metastatic lesions is also recommended in
this study as it may have important implications for therapeutic strategy.
In this study we have investigated total progesterone
receptor expression in endometrial tissue. There are
however two principal isoforms of progesterone

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I.L. Tangen et al. / European Journal of Cancer 50 (2014) 30033010

Cell cycle progression signature score

13
12
11
10
9
8
7

P=0.001
PR positive

Cell cycle progression signature score

PR negative

P=0.98
13

P=0.31
12
11
10
9
8

P=0.053

P=0.015

PRPR+
ER positive

PR+
PRER negative

P=0.45

1.5
P=0.83

PCNA (RPPA)

1.0

0.5
0.0

-0.5

-1.0

P=0.001

P=0.023

PRPR+
ER positive

PR+
PRER negative

Fig. 2. Loss of progesterone receptor (PR) is signicantly associated

with markers for high proliferation demonstrated by the correlation
with high level of the mRNA cell cycle progression score, both in the
whole population (A) and in subgroups of ERa positive and ERa
negative samples (B). High proliferation documented by assessing
protein level for the proliferation cell nuclear antigen (PCNA) in cases
with PR loss is validated, both within ERa positive and ERa negative
lesions (C).

receptor, PRA and PRB, and these are known to play

dierent roles in cell physiology [6,30]. PRB is known
to be a stronger transcriptional activator compared to

PRA [31,32], whilst PRA has an inhibitory eect on steroid hormone receptors, including ER [33,34]. Therefore, in future studies, investigating the expression
level of the two dierent PR isoforms during cancer progression, and their association with clinical outcome will
be of interest to better understand the response to hormone treatment in endometrial cancer.
Abnormal proliferation and cell cycle dysregulation
are common in all cancer types. We show that PR loss
is associated with increased proliferation, measured by
PCNA and Ki-67. PCNA has been reported as a valid
marker for proliferation and has prognostic value
[35,36]. Ki-67 has however been suggested to be more
specic [37]. The clinical utility of Ki-67 versus PCNA
as proliferation marker in endometrial cancer is not
yet established. However, we identify a similar pattern
of signicant increase in proliferation estimated by both
PCNA and Ki-67. This may support that use of both
proliferation markers detects the increase in proliferation related to PR loss in endometrial cancer. Progesterone is known to inhibit proliferation through opposing
the proliferative eect of oestrogen in the normal endometrium [38], through inhibition of ER gene expression,
enhanced degradation of ER and possibly by opposing
ER-mediated gene regulatory events (reviewed in [39]).
Furthermore, progesterone induces dierentiation of
endometrial cells rendering them less sensitive to the
eect of ER as well as other growth factors [40]. However, progesterone has also been shown to inhibit cell
growth in ER negative tumours suggesting that paracrine interactions may be important. Furthermore, Dai
et al. showed that in an ER negative endometrial cancer
cell line, progesterone limited cell growth through induction of the cyclin-dependent kinase inhibitors p21 and
p27 [41]. Due to its involvement in processes that inhibit
tumour development and progression, progesterone has
been referred to as the ultimate endometrial tumour suppressor [6], and losing PR can be compared to losing the
brake in processes inhibited by progesterone. Our results
clearly support such anti-proliferative eect of PR,
underscoring its clinical relevance.
The primary treatment for patients with endometrial
cancer is surgery, and for the intermediate to high-risk
patients, adjuvant treatment with radiation and/or chemotherapy is widely used, although with an uncertain
survival benet [42]. The response to conventional systemic treatment for patients with advanced or recurrent
disease is limited. As patients with retained expression
of hormone receptors benet most from progesterone
treatment [12,15], this appears to be reasonable if PR
status is conrmed in metastatic lesion(s). For tumours
with PR loss, our data suggest that drugs inhibiting
proliferation such as CDK inhibitors and in particular
CDK2 inhibitors may be particularly relevant for
future analysis in clinical trials. Development of drugs
that inhibit CDKs has been an area of research for

I.L. Tangen et al. / European Journal of Cancer 50 (2014) 30033010

3009

Table 2
Selected identied compounds with anti-proliferative eect that negatively correlate to the gene signature from PR negative endometrial
cancers.
Ranka

Name of compound

Known target/function

5
7
10
18
20

Sirolimus
LY-294002
GW-8510
01750290000
Alsterpaullone

Mammalian target of rapamycin (mTOR) inhibitor

Phosphoinositide 3-kinase (PI3K) inhibitor
CDK2 inhibitor
CDK inhibitor
CDK inhibitor

44
61
4
3
3

<0.00001
<0.00001
0.0002
0.002
0.002

N: number of instances the compound were tested in Connectivity Map.

a
Complete list given in Supplementary Table 4.

many years. The rst generation of CDK inhibitors

demonstrated only modest eect [43]. One of these,
avopiridol, which is a weak pan CDK inhibitor, was
tested as second line treatment in a phase II study on
patients with recurrent or persistent endometrial cancer, however in this population avopiridol as a single
agent had minimal eect [44]. New small molecule
CDK inhibitors such as the dual CDK4/6 inhibitor palbociclib show more encouraging results with prolongation of progression free survival in combination with
letrozole in breast cancer patients [45]. Whether PR
negative endometrial cancer patients will benet from
selective CDK1/2 or CDK4/6 inhibitors warrants both
preclinical and clinical evaluations. Our results suggest
CDK inhibitors as potential therapy in particular for
PR negative endometrial cancer patients, supporting
dierent clinical trial strategies being tested for PR
positive and PR negative endometrial carcinoma
patients.
Financial support
This study was supported by Helse Vest, the University of Bergen, the Norwegian Cancer Society (Harald
Andersen Legat), the Research Council of Norway
and Bergen Medisinske Forskningsstiftelse.
Conict of interest statement
None declared.
Acknowledgements
We thank Ellen Valen, Britt Edvardsen, Kadri
Madissoo and Bendik Nordanger for technical assistance. G.B.M. was supported by P50 CA098258. RPPA
analysis was supported by MD Anderson Cancer Center
Support Grant (CCSG) P30 CA016672 from the
National Cancer Institute.
Appendix A. Supplementary data
Supplementary data associated with this article can
be found, in the online version, at http://dx.doi.org/
10.1016/j.ejca.2014.09.003.

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