Progesterone Inhibits Epithelial-to-Mesenchymal
Transition in Endometrial Cancer
Paul H. van der Horst1*, Yongyi Wang1, Ingrid Vandenput2, Liesbeth C. Ku ¨hne1, Patricia C. Ewing3,
Wilfred F. J. van IJcken4, Marten van der Zee1, Frederic Amant2, Curt W. Burger1, Leen J. Blok1
1Department of Obstetrics and Gynaecology, Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands, 2Division Gynecologic Oncology, University
Hospital Gasthuisberg, Catholic University Leuven, Leuven, Belgium, 3Department of Pathology, Erasmus University Medical Center Rotterdam, Rotterdam, The
Netherlands, 4Department of Biomics, Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands
Background: Every year approximately 74,000 women die of endometrial cancer, mainly due to recurrent or metastatic
disease. The presence of tumor infiltrating lymphocytes (TILs) as well as progesterone receptor (PR) positivity has been
correlated with improved prognosis. This study describes two mechanisms by which progesterone inhibits metastatic
spread of endometrial cancer: by stimulating T-cell infiltration and by inhibiting epithelial-to-mesenchymal cell transition
Methodology and Principal Findings: Paraffin sections from patients with (n=9) or without (n=9) progressive endometrial
cancer (recurrent or metastatic disease) were assessed for the presence of CD4+ (helper), CD8+ (cytotoxic) and Foxp3+
(regulatory) T-lymphocytes and PR expression. Progressive disease was observed to be associated with significant loss of
TILs and loss of PR expression. Frozen tumor samples, used for genome-wide expression analysis, showed significant
regulation of pathways involved in immunesurveillance, EMT and metastasis. For a number of genes, such as CXCL14, DKK1,
DKK4, PEG10 and WIF1, quantitive RT-PCR was performed to verify up- or downregulation in progressive disease. To
corroborate the role of progesterone in regulating invasion, Ishikawa(IK) endometrial cancer cell lines stably transfected
with PRA (IKPRA), PRB(IKPRB) and PRA+PRB (IKPRAB) were cultured in presence/absence of progesterone (MPA) and used for
genome-wide expression analysis, Boyden- and wound healing migration assays, and IHC for known EMT markers. IKPRB
and IKPRAB cell lines showed MPA induced inhibition of migration and loss of the mesenchymal marker vimentin at the
invasive front of the wound healing assay. Furthermore, pathway analysis of significantly MPA regulated genes showed
significant down regulation of important pathways involved in EMT, immunesuppression and metastasis: such as IL6-, TGF-b
and Wnt/b-catenin signaling.
Conclusion: Intact progesterone signaling in non-progressive endometrial cancer seems to be an important factor
stimulating immunosurveilance and inhibiting transition from an epithelial to a more mesenchymal, more invasive
Citation: van der Horst PH, Wang Y, Vandenput I, Ku ¨hne LC, Ewing PC, et al. (2012) Progesterone Inhibits Epithelial-to-Mesenchymal Transition in Endometrial
Cancer. PLoS ONE 7(1): e30840. doi:10.1371/journal.pone.0030840
Editor: Irina Agoulnik, Florida International University, United States of America
Received June 23, 2011; Accepted December 22, 2011; Published January 25, 2012
Copyright: ? 2012 van der Horst et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work of LJB and MvdZ is supported by a grant from the Dutch Cancer Society (EMCR 2008-4056). The funders had no role in study design, data
collection and analyses, decision to publish or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Each year, worldwide, more than 287,000 women develop
endometrial cancer making it the most common gynecological
cancer in the world and the fourth most common female malig-
nancy in developed countries . Usually endometrial cancer is
detected in an early stage and surgery is the cornerstone of treat-
ment. Where there is recurrent or metastatic disease, however, the
situation is different. (Neo-)Adjuvant radiation and/or systemic
therapy in combination with surgery is usually indicated and in
general, progressive disease has a poor prognosis accounting for
74,000 deaths worldwide each year [2,3]. Prognostic factors for
recurrent and metastatic endometrial cancer include surgical
FIGO stage, grade of differentiation, histopathological subtype
and myometrial and lymphovascular invasion [2,4,5,6,7].
In several types of cancer, the presence of tumor infiltrating
lymphocytes (TILs) has been correlated with improved prognosis,
and much researchhasbeen
[8,9,10,11,12,13,14,15]. The rationale is that well differentiated
cancer evokes an inflammatory response similar to an acute injury
which, after sequential infiltration of different dendritic cell
populations, eventually results in T-lymphocyte infiltration .
Infiltration of TILs as a positive prognostic factor was first
described in cutaneous melanoma, where the presence of TILs was
predictive for improved survival . Galon et al. in 2006, showed
that infiltration of lymphocytes of the adaptive immune system
into the center and invasive margin of colorectal cancer was
positively correlated with reduced recurrence and improved
survival . In 2009 Kilic et al., showed that high levels of TILs
within non-small-cell lung cancer correlated with reduced
performed on this topic
PLoS ONE | www.plosone.org1 January 2012 | Volume 7 | Issue 1 | e30840
recurrence and enhanced survival . In ovarian cancer, the
presence of intratumoral T-lymphocytes was also positively
correlated with improved survival and delayed recurrence of the
disease . Furthermore, TILs in ovarian cancer were also
associated with increased levels of INF-c, IL2 and chemokines
which indicates T-cell activation and attraction .
The presence of TILs has not been extensively investigated in
endometrial cancer. In endometrial cancer, infiltration of cytotoxic
(CD8+) T-lymphocytes in the area of the lesion has been described
as an independent prognostic factor and is positively correlated to
disease free- and overall survival [17,18]. In addition, a high
cytotoxic T-lymphocyte/regulatory T-lymphocyte (CD8/FOXP3)
ratio has been described to be correlated to improved survival in
type I endometrial cancer .
Next to the influx of T-lymphocytes into the tumor area, the
presence of progesterone receptors (PR) is also described as an
important asset in prognosis and treatment of endometrial cancer
[19,20,21]. In well differentiated endometrial cancer PR expres-
sion is usually maintained and treatment with medroxyprogester-
one acetate (MPA), of those patients with well differentiated
disease who chose to preserve fertility, is usually successful [22,23].
Loss of PR, however, is a negative prognostic factor and is
associated with progressive disease in which MPA treatment is
usually only temporally successful in 15–20% of cases .
Recently, our group has studied the mechanism through which
progesterone can induce differentiation during the normal mens-
trual cycle and can inhibit well differentiated endometrial cancer
growth. It was observed that progesterone treatment results in
induction of expression of two important inhibitors of Wnt/b-
catenin signaling: DKK1 and FOXO1 [25,26]. In endometrial
cancer, activation of Wnt/b-catenin signaling is observed in 30–
40% of well differentiated endometrioid carcinomas  and
progesterone induced inhibition of the Wnt signaling pathway is
hypothesized to be an important mechanism to reduce cancer
In this study we aimed to investigate the role of progesterone as
a direct inhibitor of the migratory capacities of endometrial cancer
cells and its role in T-lymphocyte associated inhibition of
Materials and Methods
Primary endometrial carcinoma tissue from women with (n=9)
and without (n=9) a known episode of recurrence or metastasis,
was obtained from patients treated between 1997 and 2006 in the
University Hospital Gasthuisberg, Catholic University Leuven,
Belgium. From this point on, non-recurrent disease is referred as
non-progressive disease and recurrent/metastatic disease as
progressive disease. Histopathological grading, staging and typing
were determined according to the guidelines of the WHO and
FIGO [28,29] and all tumors were revised by a pathologist
experienced in gynaecopathology (PCE). Patients with an
endometrioid type and a FIGO stage I endometrial carcinoma
were included. Patients treated with radio- or chemotherapy prior
to surgery, using hormonal steroids or with a second malignancy
were excluded. Complete clinical history was obtained from all
patients and follow-up was revised to date. Specimens were snap-
frozen in liquid nitrogen for RNA-isolation or fixed in formalin
and embedded in paraffin for immunohistochemistry (IHC). For
microarray analysis, from 4 non-progressive and 4 progressive
patients, snap frozen tumor specimens were used. These were
chosen because they contained .80% tumor tissue and
good quality RNA could be isolated from them. For RT-PCR, 6
non-progressiveand6 progressive snap frozenpatienttissuesamples
were used. For IHC 9 non-progressive and 9 progressive paraffin
embedded patient tissue samples were available. Tissue and clinical
data collection for the current research study was approved by the
Medical Ethical Committeeofthe University HospitalGasthuisberg
and patients gave written informed consent for tissue collection and
clinical data collection for all research purposes.
For all cell line experiments, Ishikawa endometrial cancer cell
lines stably transfected with PRA (IKPRA-1), PRB (IKPRB-1) or
PRA and PRB (IKPRAB-36) (previously described by Smit-
Koopman et al. ) were cultured and maintained in regular
culture medium (DMEM/F12 Glutamax, Invitrogen, Carlsbad,
CA, USA) in the presence of 5% Fetal Calf Serum (Invitrogen)
supplemented with penicillin and streptomycin (Invitrogen).
Neomycin (ICN Biomedicals, Costa Mesa, CA, USA) and
hygromycin (Invitrogen) 1:200 were used to maintain selection.
For all assays, cells were cultured in DMEM/F12 Glutamax
culture medium supplemented with penicillin and streptomycin
(Invitrogen), containing 5% charcoal stripped FCS (Invitrogen)
with addition of hygromycin and neomycin.
IHC studies for CD4 (Sanbio BV, Uden, The Netherlands),
CD8 (Dako, Glostrup, Denmark), FOXP3 (Natutech, Frankfurt
am Main, Germany) and PRA+PRB (Progesterone Receptor Ab-
8, Neomarkers, Fremont, CA, USA) were performed on 4 mm
paraffin sections on Starfrost-slides (Knittel, Braunschweig,
Germany). Prior to incubation with the primary antibody, the
slides were deparaffinized in xylene and rehydrated to 70%
ethanol. For CD4+ and CD8+ T-lymphocyte staining, slides were
microwaved at 850 Watt in Tris/EDTA pH 9.0 for 15 min.
Endogenous peroxidase activity was blocked with 30% H2O2in
PBS for 5 min. Primary antibodies were applied at respectively
1:160 (CD4) and 1:200 (CD8) in Tris/HCl pH 8.0 and incubated
at room temperature for 30 min. After washing with Tris/HCl
pH 8.0, sections were incubated for 30 min. at room temperature
with biotinylated secondary antibody (Dako, 1:400). After washing
with Tris/HCL, the substrate Diaminobenzidine (Dako) was used
for visualization of antigen–antibody reactivity.
For FOXP3, slides were blocked (peroxidase deactivation) for
20 min at room temperature (RT) in 30% H2O2in methanol and
boiled (antigen retrieval) in a citrate-buffer pH 6.0 for 15 min.
Primary antibody was applied at 1:25 and incubated at 4uC
overnight. After washing with PBS, slides were incubated for
30 min. with a secondary rabbit-anti-rat antibody (DAKO, 1:150)
and incubated for 30 min. with AB-complex (Dako). The substrate
Diaminobenzidine (Dako) was used for visualization of antigen–
For PRA+PRB staining, endogenous peroxidase activity was
blocked for 5 min at RT in a 10% H2O2in methanol solution and
the slides were microwaved (antigen retrieval) in a microwave-
oven at 850 Watt in 10 nM citric acid buffer pH 6.0 (DAKO) for
15 min. After cooling to room temperature slides were washed
with PBS and blocked for 30 min with 0.3% BSA/PBS. Primary
antibody was applied at 1:50 and incubated at 4uC overnight.
After washing with PBS, slides were incubated for 30 minutes with
a biotinylated secondary goat-anti-mouse antibody (Dako, 1:400).
After the second wash the slides were incubated for 30 min with
AB-complex (Dako, 1:1:50). The substrate Diaminobenzidine
(Dako) was used for visualization of reactivity. All slides were
counterstained with hematoxylin for 30 s, then dehydrated and
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For Vimentin staining, a wound-healing assay was performed in
2-well chamber slides (Lab-Tek, Thermo Fisher Scientific,
Waltham, MA, USA), in the presence and absence of 1 nM
medroxy-progesterone acetate (MPA), and terminated after 48 hr.
The cells were washed three times with PBS, fixed with 4%
formaldehyde/PBS for 15 minutes and permeabilized with 0,3%
Triton100/PBS for 5 minutes. After washing, endogenous perox-
idase activity was blocked with 10% H2O2 in methanol for
5 minutes. Slides were washed and then blocked for 30 minutes
with 0.3% BSA/PBS. The anti-vimentin antibody (Invitrogen) was
applied at 1:50 and the slides were incubated for 30 minutes at
room temperature. After washing with PBS, slides were incubated
with a GFP-fluorescent goat-anti-mouse secondary antibody
(Invitrogen) at 1:500. After washing, the slides were incubated
for 5 minutes with DAPI Nucleic Acid Staining Solution
(Invitrogen) for nuclear staining. After termination of the reaction
with dH2O, the slides were mounted and fluorescent images were
taken with the Axioplan 2 Imaging Fluorescent Microscope (Carl
Zeiss AG, Jena, Germany).
After staining, the slides were scanned with the NDP slide
scanner (Hamamatsu, Hamamatsu City, Japan) and CD4, CD8
and FOXP3 positive tumor infiltrating lymphocytes (TILs) were
counted using Image J software (National Institutes of Health,
Bethesda, MD, USA). The number of TILs was determined inside
the tumor (Intratumoral), at the tumor edge (Tumor Edge) and at
the endometrial/myometrial border (EM). The complete tumor
edge and endometrial/myometrial border were evaluated for
the presence of TILs. The intratumoral count was performed
by counting the TILs in 10 different randomly picked areas
(1170 mm6932 mm) chosen by an independent investigator,
thereby eradicating observer bias.
For the WST1 proliferation assay, IKPRA-1, IKPRB-1 and
IKPRAB-36 cell lines were cultured in the absence or presence of
MPA in a 96 well plate (Corning Costar, Cambridge, MA, USA).
At time 0, the cells were incubated with cell proliferation reagent
WST1 (Roche, Basel, Switzerland) for 3 hours at 37uC and
absorbance was measured with the Microplate Reader (BIORAD,
model 550, Hercules, CA, USA). After baseline measurement the
cell lines were cultured in the presence and absence of 1 nM MPA
for 96 hours and at 96 hours, the WST1 assay was repeated.
For the wound-healing assay, IKPRA-1, IKPRB-1 and
IKPRAB-36 cell lines were cultured in a 6-well plate (Corning
Costar). After inducing the wound, cells were incubated with
1 nM MPA for 96 hours. Wound healing was verified every 24 hr
by photography, and analyzed by measuring closure of the wound.
For the modified Boydon assay, cells were seeded in the upper
well of a modified Boydon chamber (Transwell, 8 mm pores,
24 mm inserts, 6 well plate, Corning Costar) at 2.56105cells per
well in the presence or absence of 1 nM MPA. Furthermore as a
control, cells were cultured in a Boyden chamber in the presence
or absence of 1 nM MPA in combination with 100 nM of the anti-
progestagin Org31489 (Organon, Oss, The Netherlands). After
96 hours, cells that had migrated through the filter into the lower
well or to the bottom of the insert were trypsinized and counted
under the microscope.
IKPRA-1, IKPRB-1, IKPRAB-36 and IKLV-8 cell lines were
cultured in the absence or presence of 1 nM MPA for 96 hrs and
subsequently lysed at 0uC in Cell Lysis Buffer (Cell Signaling
Technology, Danvers, MA, USA) for 5 minutes. Then the cells
were scraped, centrifuged at 14.000 rpm for 10 minutes and the
supernatant was removed. The protein concentration was
calculated using the Protein Assay Kit (Pierce, Thermo Scientific,
Rockford, IL, USA) and of each sample 4.5 mg protein in 30 mL
lysisbuffer+BSA was loaded on a 10% SDS-PAGE gel. Western
blotting was performed according to standard procedures. The
blotting paper was blocked for 30 minutes at RT with Blocking
Buffer (LI-COR Biotechnology, Lincoln, NE, USA) and then
incubated overnight at 4uC using rabbit polyclonal anti-hFOXO1
antibody (1:5000, Bethyl Laboratories, Montgomery, TX, USA) in
Blocking Buffer (LI-COR Biotechnology). Next, the blotting
membrane was incubated with the secondary goat-anti-rabbit
IgG (IRDye 800CW, 1:5000, LI-COR Biotechnology) for
30 minutes at RT and washed. As a loading control, the
membrane was incubated for 30 minutes with the monoclonal
anti-b-actin (1:1000, Sigma-Aldrich, Saint Louis, MO, USA),
washed with PBS and incubated for 30 minutes with the
secondary goat-anti-mouse IgG (IRDye 680CW, 1:5000, LI-
COR Biotechnology). The specific protein bands were detected
using the Odyssey Scanning System (LI-COR Biotechnology).
RNA-isolation, gene expression analyses and quantitative
Patient tissue samples were sectioned (5 mm, cryostat) and every
10thsection was HE stained and revised by the pathologist (PCE)
to assess tumor load. Only sections containing .80% tumor were
lysed in Trizol (Invitrogen) and sonified for 1 min. The PRA and
PRB expressing Ishikawa cell line (IKPRAB-36) was cultured for
48 h in the absence or presence of 1 nM MPA (n=3), placed on
ice and lysed in Trizol (Invitrogen).
Phase separation was accomplished with 0.2 ml chloroform and
centrifugation for 15 min. The supernatant was transferred and
isopropanol was added for RNA precipitation. The precipitated
RNA was washed with 75% ethanol. All RNA was cleaned with
the Rneasy Minelute cleanup kit (Qiagen, Venlo, The Nether-
lands). Amount and quality of the RNA was assessed by using the
Nanodrop (Nanodrop, Wilmington, DE, USA) and Bio-analyzer
(Aligent, Santa Clara, CA, USA).
RNA isolated from patient and cell line material was labeled
according to Affymetrix labeling protocols and labeled RNA was
applied to genome-wide expression arrays (Affymetrix U133plus2
GeneChips containing 54,614 probe sets, representing approxi-
mately 47.000 transcripts (Affymetrix, Santa Clara, CA, USA)).
Using RMA (Robust Multi-array Analysis ), normalization of
raw data was performed to be able to produce gene lists and
eventually calculate significantly regulated genes using SAM
(Stanford University, Stanford, CA, USA ). Lists of SAM
regulated genes (1.25 fold or more; delta-values resembling
p,0.05) were loaded in the Ingenuity pathway assist software to
assess the involvement of different biological pathways (Ingenuity,
Redwood City, CA, USA). For the patient materials raw lists of
regulated genes (1.25 fold or more) were loaded in Ingenuity.
All micro-array data is MIAME compliant and raw data has
been deposited in the MIAME compliant GEO database under
series: GSE29437 (consisting of GSE29435: cell line data; and
GSE29436: patient data).
Genes for quantitative real-time RT-PCR were identified by
micro-array analysis and pathway analysis. RNA was transcribed
into cDNA with the use of the Affymetrix one-cycle cDNA
synthesis kit (Affymetrix). For identified genes, primers were
ordered and tested (a list of primers is included in Table S1). The
housekeeping gene b-actin was used as a reference gene. RT-PCR
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was performed and analyzed using the CFX RT-PCR system (Bio-
Rad, Veenendaal, The Netherlands).
For the statistical analyses of the CD4+, CD8+ and FOXP3+
cell counts, modified Boyden chamber assay data, WST1 assay
data and RT-PCR data, SPSS 15.0 was used (IBM, Armonk, NY,
USA). For normal distributed data a t-test and for skewed data a
Mann-Whitney U-test was performed to assess P-values. A P-
value,0.05 was considered statistically significant. To calculate
the p-value of regulated pathways, Ingenuity pathway assist soft-
ware uses a Fisher’s exact test.
Patient characteristics (Table 1)
Patients with (n=9) and without (n=9) progressive endometrial
abdominal- or laparoscopically assisted vaginal hysterectomy and a
bilateral salpingo-oophorectomy combined with lymph node re-
moval. None of the women received chemotherapy and only one
woman in the progressive disease group was given radiotherapy
after surgery. Histopathological subtypes were endometrioid
(n=17) and mixed endometrioid/mucinoid (n=1). Tumor grades
were 1 (n=7), 2 (n=4) and 3 (n=7) and FIGO stages were Ia
(n=11) and Ib (n=7). In the progressive disease group all 9 patients
had one or more episodes of local recurrence and 4 patients
developed one or multiple distant metastases. Recurrences were
vaginal, pelvic or (retro)peritoneal, and metastatic sites were the
lungs (n=3), liver (n=1), spleen (n=1) and brain (n=1). Clinical
follow-up to date was available for all patients. In the non-
progressive group 8 patients are currently free of disease and 1
patient died in follow-up. In the progressive disease group 3 patients
are free of disease and 6 patients died from their endometrial cancer
related disease. Patient characteristics are detailed in Table 1.
Progesterone receptor status and detection of CD4+ T-
helper, CD8+ cytotoxic T-cells and FOXP3+ regulatory T-
cells in non- progressive and progressive disease
The presence of tumor infiltrating lymphocytes has been
correlated to prolonged survival in endometrial cancer [17,18].
Furthermore, loss of progesterone receptor (PR) expression in
Table 1. Clinical characteristics of the included patients.
Non-progressive (n=9) Progressive (n=9)
Patients 1–9Patients 10–18
Age - yearp=0,606
Endometrioid9 (100) 8 (88,9)
Mixed 0 (–)1 (11,1)
FIGO stage no. (%)
Ia4 (44,4)7 (77,8)
Ib 5 (55,6)2 (22,2)
Tumor gradeno. (%)
1 2 (22,2) 5 (55,6)
2 3 (33,3)1 (11,1)
3 4 (44,5) 3 (33,3)
Current statusno. (%)
NED 8 (88,9)3 (33,3)
DOD 1 (11,1)6 (66,7)
Recurrence no. (%)
No 9 (100)0 (–)
Yes 0 (–) 9 (100)
No9 (100)5 (55,6)
Yes 0 (–)4 (44,4)
Table 1 shows the characteristics of the patients included in the study. A p-value of ,0.05 was considered as statistically significant. BMI=body mass index; NED=no
evidence of disease; DOD=death of disease.
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endometrial cancer has been found to be a risk factor for pro-
gressive disease . In order to substantiate the relationship
between intact PR signaling and the presence of infiltrating
lymphocytes in non-progressive disease, immunohistochemical
staining and, when appropriate, quantitative measurements were
As exemplified in Fig. 1A, in progressive disease immunohis-
tochemical staining for CD4+, CD8+ and FOXP3+ T-lympho-
cytes seems reduced as compared to staining in non-progressive
disease. Quantification of the number of CD4+, CD8+ and
FOXP3+ T-lymphocytes in progressive disease indeed confirmed
a lower number of positive cells located on the endometrial-
myometrial border (Fig. 1B, EM), at the edge of the tumor (Fig. 1B,
Tumor Edge) and within the tumor (Fig. 1B, Intratumoral).
Whether the reduced cell counts were significantly different
between the non-progressive and progressive endometrial cancer
tissues is indicated in the Figure (Fig. 1B).
Furthermore, reviewing consecutive sections in non-progressive
disease for expression of progesterone receptors (PR) revealed that
the presence of CD4+ and CD8+ T-lymphocytes was positively
correlated with the presence of PR staining (Fig. 1C and 1D).
Genome-wide expression analyses of primary
endometrial carcinoma tissue
To investigate whether the correlation between PR signaling
and the presence of tumor infiltrating lymphocytes could indicate
a causative relationship, a genome-wide mRNA expression
analysis on snap-frozen primary endometrial carcinoma specimens
from 4 patients without and 4 patients with progressive disease was
performed. At the individual gene level it was observed that a
marked number of chemokines and cytokines were differentially
regulated between non-progressive and progressive disease (Table
S2). For example, the chemokines CCL21 (21.5x), CXCL9
(22.9x), CXCL10 (22.1x) and CXCL14 (three data sets present:
233.0x; 220.5x; 26.4x, respectively) were all down regulated in
progressive disease while the cytokines IL8 (2.0x; 5.7x; 9.5x) and
IL32 (1.9x) were up-regulated in progressive disease (Table S2).
Furthermore, earlier work from our group has indicated activation
of Wnt/b catenin signaling in progressive disease  and in
agreement with this a number of Wnt/b-catenin inhibitory- and
target genes were lost from progressive disease (DKK1, DKK4
and WIF1) (Table S2).
Interestingly, a number of the above mentioned genes which
were down-regulated in progressive disease, have been described
in literature to be up-regulated by progesterone (CXCL14 ,
DKK1 , MMP7  and SFRP4 ). This is in agreement
with the finding that PR expression (at protein and mRNA
expression level (Fig. 1C and 1D and Table S2) is down regulated
in progressive disease.
Upon reviewing pathways regulated between non-progressive
and progressive disease, regulation of a number of pathways
involved in carcinogenesis and invasive disease and involved in
immunosurveillance was found to be significantly regulated:
Integrin Signaling, Molecular Mechanisms of Cancer, Antigen
Presentation Pathway, Non-Small Cell Lung Cancer Signaling,
IGF-1 Signaling, Role of Tissue Factor in Cancer, Leukocyte
Extravasation Signaling, ERK/MAPK Signaling, Colorectal
Cancer Metastasis Signaling (which includes Wnt/b catenin
signaling), FGF Signaling, FAK Signaling, etc (the complete list
of regulated pathways and their consecutive p-values can be
accessed from Table S3).
For a number of genes (CXCL14, DKK1, DKK4, PEG10 and
WIF1) a quantitative real-time RT-PCR was performed in order
to verify regulation (Fig. 2).
Effect of progesterone on migration of the Ishikawa
endometrial cancer cell lines
In order to further corroborate the possible role for progester-
one in regulating invasion, Ishikawa endometrial carcinoma cell
lines stably transfected with PRA, PRB, or PRA and PRB 
were cultured in the presence or absence of MPA for varying
periods of time and used in two different experiments measuring
cell migration. To verify cell proliferation during the different
experiments a WST1 proliferation test was performed which
showed that within the indicated timeframe no significant
differences in proliferation could be detected between cells
incubated with or without MPA.
In Figure 3, different Ishikawa cell lines were subjected to a
wound-healing assay in the presence or absence of MPA (1 nM)
for up to 96 h. It was observed that, in the stably PRB expressing
(IKPRB-1) and PRA+PRB expressing (IKPRAB-36) Ishikawa cell
lines, MPA inhibited closure of the manually inflicted wound
(Fig. 3A–D). Furthermore, when we stained the edge of the wound
for the mesenchymal marker vimentin, it was observed that in the
presence of MPA vimentin expression was clearly reduced
(Fig. 3E). Next to this detail on expression of vimentin, the overall
vimentin levels were decreased in IKPRB-1 and IKPRAB-36 cell
lines incubated with 1 nM MPA. It was also observed that in the
stably PRA expressing (IKPRA-1) Ishikawa cell line, neither
wound healing nor vimentin expression was affected by MPA
(Fig. 3A and 3E).
In Figure 4, another approach was used to study the migratory
capacity of different Ishikawa cell lines in the presence or absence
of progesterone. It was observed that for IKPRB-1 as well as
IKPRAB-36 cells, migration in a modified Boyden chamber was
inhibited in the presence of progesterone. Furthermore, for the
IKPRA-1 cell line such a differential regulation of migration under
the influence of MPA was not observed.
Genome-wide expression analysis of Ishikawa
endometrial cancer cell line
To further document progesterone-induced inhibition of
cellular migration and to investigate the involvement of proges-
terone signaling in T-lymphocyte infiltration, IKPRAB-36 cells
were cultured for 48 h in the presence or absence of 1 nM MPA
and used for genome-wide expression analysis. It was observed
that 1616 genes were significantly regulated by progesterone in the
IKPRAB-36 cell line (1029 up-regulated, 587 down-regulated,
Using Ingenuity pathway analysis of significantly regulated
genes, the following pathways were observed to be regulated by
progesterone (the complete list of regulated pathways and their
consecutive p-values can be accessed from Table S5): IGF-1
signaling, Neuregulin signaling, TNFR1 signaling, P13K signaling
in B-lymphocytes, VDR/RXR signaling, Acute Phase Response
signaling, Hepatic Fibrosis/Hepatic Stellate Cell activation,
Molecular Mechanisms of Cancer (which includes Wnt/b-catenin
and TGF-b signaling), TGF-b signaling, Axonal Guidance
Signaling etc. Interestingly, it was noted that 41/67 pathways
observed to be significantly regulated by progesterone in the cell
line were also found to be significantly regulated between non-
progressive and progressive disease (see Table S6). Furthermore, it
was also noted that a number of pathways specifically involved in
transition from a epithelial state to a mesenchymal state (EMT)
was significantly regulated by progesterone and in the endometrial
cancer samples: EGF signaling (p=0.029), IGF-1 signaling
(p=0.0000006), IL-6 signaling (0.013), ILK signaling (p=0.018),
PDGF signaling (p=0.03), TGF-b (p=0.003), VEGF signaling
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Figure 1. Expression and histological distribution of PRA+ +PRB and CD4+ +, CD8+ + and Foxp3+ + T-lymphocytes in primary endometrial
carcinoma specimens. A: Overview of immunohistochemical staining for CD4, CD8 and FOXP3 in primary endometrial cancer specimens in non-
progressive disease (n=9) compared to progressive disease (n=9) (magnification 0,4x, inlay 10x). Non-progressive disease shows pronounced
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(p=0.022) and Wnt/b-catenin signaling (p=0.036). In Figure 5A
and B, MPA-induced gene regulation in Wnt/b-catenin and
TGF-b signaling is shown. Next to this, a heat map confirmed a
major overlap between gene regulation by MPA and differential
gene expression between non-progressive and progressive disease
Regulation of the Wnt signaling pathway was further confirmed
by showing progesterone induction of the Wnt inhibitor FOXO1
at the protein level (Fig. 5C).
In general, patients with endometrial cancer have a good
prognosis since early diagnosis is frequent and the disease has
usually not spread beyond the uterus. However, the prognosis for
recurrent or metastatic endometrial cancer remains poor and in
order to improve therapy it is vital to understand the processes
which inhibit and stimulate cancer progression.
Infiltration of T-lymphocytes into the region of the lesion, for
example, is an anticancer signal which helps to confine a tumor
until cancer-induced T-cell death establishes tumor immune
tolerance opening the road to progression. The transition of an
epithelial phenotype towards a more mesenchymal phenotype is a
subsequent step which leads to further progression to invasive
disease. Central to this epithelial to mesenchymal transition (EMT)
is the activation of important signaling pathways such as Wnt/b-
catenin and TGF-b . Activation of these pathways results in
induction of Snail1/2 induced transcription, eventually causing
degradation of the basement membrane by induction of matrix
metalloproteinases, loss of epithelial markers such as E-cadherin
and gain of mesenchymal markers such as vimentin .
In the current investigations non-progressive and progressive
primary endometrial cancer tissues were compared and it was
observed that progression of disease was characterized by 1. Loss
of progesterone signaling, 2. Loss of CD4, CD8 and FOXP3 T-
lymphocytes driven immunosuppression and 3. Modulation of
genes and pathways reminiscent of EMT. The aim of the present
investigations was to assess the role of decreased progesterone
signaling in progressive disease, and more particularly in relation
to loss of immunosuppression and transition from an epithelial
phenotype to a more invasive mesenchymal phenotype.
Loss of PR expression correlates with loss of
immunosupression and increased EMT in progressive
Measuring tumor infiltrating lymphocytes (TILs) in primary
endometrial cancer tissues from non-progressive and progressive
disease indicated that in patients with non-progressive endometrial
cancer, TILs were abundantly present. This is in agreement with
studies by Kondratiev et al. in 2004  and De Jong et al. in
2009 , which showed that high levels of CD8+ T-lymphocytes
were associated with improved disease free survival. Furthermore,
the presence of several chemokines (CCL21, CXCL9, CXCL10,
CXCL14, IL8 and IL32) indicated that there is an active process
which directs TILs to the site of the lesion . Interestingly, a
number of these chemokines are up-regulated during the secretory
phase of the menstrual cycle when progesterone levels are in-
creased (CCL21: 1.5-fold up, CXCL10: 1.3-fold up and CXCL14:
staining, whereas progressive disease shows reduced staining. The scale-bar represents 10 mm. B: Quantification of CD4, CD8 and FOXP3 cell counts
on the tumor edge (Tumor Edge), in the tumor (Intratumoral) and on the endometrial-myometrial border (EM border). *indicates a p-value,0.05
(Mann-Whitney U-test). C and D: Representative non-progressive (C) and progressive (D) patient tissues were stained for CD4, CD8 and PRA+PRB and
show a positive correlation between the presence of TILs and the expression of PR. Magnification is 5x and the scale-bar represents 1 mm. Patients 6
and 11 were both included in the micro-array analyses. Furthermore patient 11 had only recurrent disease, while patient 12 had recurrent and
Figure 2. RT-PCR results of genes of interest in the patient samples. CXCL14, DKK1, DKK4, WIF1 and PEG10 were selected from the micro-
array results and verified with real time RT-PCR. Significance was calculated using a Mann-Whitney U-test. A p-value of 0.05 was considered to be
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Figure 3. Progesterone induced inhibition of migration in a wound-healing assay. IKPRA-1 (A), IKPRB-1 (B) and IKPRAB-36 (C) cells were
cultured in the absence (white bullets) or presence (black bullets) of 1 nM MPA and used for a wound-healing assay (n=3) and closure of the wound
was measured as a percentage of total closure (100% means the wound is open, 0% means the wound has closed). D shows representative images of
the process of wound-healing with in red the wound. E shows IF for nuclei (DAPI) and vimentin expression on the invasive front of the manually
inflicted wound. In this figure, the wound was always situated on the right side.
Progesterone Inhibits EMT in Endometrial Cancer
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90-fold up; ). Furthermore, CXCL14 has also been described
by other groups to be a progesterone induced gene in the
endometrium involved in chemo-attraction of uterine natural killer
cells to the epithelial glands . In summary, this indicates a
putative role for progesterone signaling in attracting TILs in non-
progressive endometrial cancer.
In the patient tissues which were used in the current investi-
gations, progesterone receptor expression was lost from progres-
sive disease. The fact that hormonal control of a tissue is lost upon
progressive malignant transformation is not a new finding and
besides loss of PR expression in endometrial cancer  this has
also been described for other cancer types like breast cancer (loss
of estrogen signaling ) and prostate cancer (loss of androgen
signaling ) as well.
According to previous work from our group, besides stimulating
TILs, progesterone can inhibit Wnt/b-catenin signaling and loss
of progesterone signaling may be involved in tumor onset and
progression towards a more invasive disease [21,25,42,43]. Inter-
estingly, upon reviewing gene expression profiles obtained from
progressive and non-progressive endometrial cancer, a number of
inhibitors of Wnt/b-catenin signaling were indeed found to be
down-regulated in progressive disease (DKK1, DKK4 and WIF1).
These findings are in accordance with the hypothesis that Wnt/b-
catenin signaling becomes activated through loss of PR signaling,
thus accommodating progressive disease . Down-regulation of
the Wnt/b-catenin signaling inhibitor WIF1, in this respect, is of
interest because down regulation of WIF-1 in prostate cancer cells
was observed to be associated with an increased capacity for cell
migration and invasion . In keeping with this, in colorectal
cancer, overexpression of activated nuclear b-catenin (the
hallmark of activated Wnt/b-catenin signaling) is located at the
invasive front of the tumor  and in colorectal cancer cell lines,
activation of b-catenin directly induces EMT .
PEG10 was found to be significantly up regulated in progressive
disease. Interestingly, PEG10 is a biomarker for progressive
development and invasion of hepatocellular carcinoma, gallblad-
der adenocarcinoma and acute lymphoid leukemia and is found to
be regulated by androgens [47,48,49,50]. Next to this, PEG10 and
IL10 expression is activated by ligation of CCL10-CCR7 and
CXCL13-CXCR5 in B-cell acute lymphatic leukemia, and
PEG10 contributes to the up-regulation of IL10, which can lead
to impairment of the cytotoxicity of CD8+ T-lymphocytes . It
was observed that CXCL13 (3,17x) and PEG10 (9,38x and 4,38x,
p=0,05) were both up-regulated in progressive disease and pos-
sibly this up-regulation can contribute to impairment of the T-
lymphocyte mediated anti-tumor response in progressive disease.
Upon reviewing other pathways which were differentially
expressed between non-progressive and progressive endometrial
cancer, significant up-regulation of a number of pathways involved
in progression towards a more mesenchymal phenotype was noted
(Table S3). IL8 signaling is one of those regulated pathways and
IL8 itself was found to be up regulated 9.5-fold in progressive
Figure 4. Invasion of PR positive Ishikawa EC cell lines. IKPRA-1, IKPRB-1 and IKPRAB-36 cells were cultured in the absence (black dots) or
presence (white dots) of 1 nM MPA in a modified Boyden chamber. After 96 hours, cells that had migrated through the pores of the upper well were
counted. The figure represents three independent experiments performed in triplicate. *indicates a p-value of ,0.05 (Mann-Whitney U-test).
Progesterone Inhibits EMT in Endometrial Cancer
PLoS ONE | www.plosone.org9 January 2012 | Volume 7 | Issue 1 | e30840
disease. These data are in line with literature showing that IL8 is a
progesterone down-regulated gene  and that high levels of IL8
correlate with endometrial metastatic disease .
MPA inhibits EMT in the Ishikawa endometrial cancer cell
In order to further substantiate the above finding that loss of
progesterone signaling in progressive disease may play a role in
diminished T-cell infiltration and induction of EMT, progesterone
signaling in the well differentiated Ishikawa endometrial cancer
cell line was investigated.
Although both PRA and PRB can activate transcription of
target genes in response to progesterone, PRA and PRB have
different transcriptional activities . It has been documented
that PRB is a stronger activator of transcription than PRA and
PRA is thought to be a dominant repressor of PRB . Next to
this, the difference in transcriptional activity is further explained
by the recruitment of different cofactors by PRA and PRB [56,57].
In the present study, it was observed that culture of the IKPRB-
1 and IKPRAB-36 endometrial cancer cell line, but not IKPRA-1,
in the presence of MPA resulted in inhibition of migration and
down regulation of the mesenchymal marker vimentin at the edge
of a manually inflicted wound.
These findings suggest that progesterone, in vitro, can inhibit
cancer cell migration due to inhibition of EMT. Assessment of
pathways involved in EMT showed progesterone modulated down
regulation of EGF, IGF-1, IL-6, Integrin/ILK, PDGF, TGF-b,
VEGF and Wnt/b-catenin signaling. Interestingly, all of these
pathways were also observed to be modulated in progressive
disease (Table S6). As shown, many of the observed altered
signaling pathways in the patient samples (Table S3) were also
significantly altered in the Ishikawa cell line, when incubated with
or without progesterone (Table S5). In the Ishikawa culture
obviously no tumor infiltrating lymphocytes are present and it is
only progesterone signaling that contributes to these changes in
signaling. Therefore we conclude that regulation of signaling
pathways in patient samples can not only be attributed to the
presence or absence of tumor infiltrating lymphocytes, but also to
changes in progesterone receptor signaling.
Progesterone inhibition of TGF-b signaling and induction of
TGF-b signaling in progesterone insensitive progressive disease is
an interesting finding because enhanced TGF-b signaling has
been shown to be a very potent immunosuppressant signal used in
transplantation medicine. Several agents inhibiting TGF-b
signaling (anti-TGF-beta antibodies, small molecule inhibitors of
TGF-beta, Smad inhibitors) are in the early stages of development
aiming to alleviate immunosuppression during carcinogenesis .
Furthermore, neutralizing TGF-b resulted in a CD8+ T-
lymphocyte anti-tumor immune response in mouse models .
Enhanced TGF-b signaling is also of interest because it has been
described as an important major driving force of EMT. Reviewing
the pathway in more detail revealed for example up regulation of
cell adhesion molecule L1CAM. For L1CAM, regulation of
transcription by TGF-b signaling has been described , but,
interestingly, in colorectal cancer L1CAM has also been shown to
be a target gene of Wnt/b-catenin signaling and expression of
L1CAM was found to co-localize with b-catenin in the invasive
front of the tumor . Recently, for endometrial cancer similar
observations have been described confirming promoter-binding
sites for the Wnt/b-catenin inducing transcription factor LEF-1
and, interestingly, also for the EMT inducing transcription factors
SNAI1 and SNAI2 .
In summary, intact progesterone signaling in non-progressive
endometrial cancer seems to be an important factor stimulating
Figure 5. MPA induced regulation of TGF-b and Wnt/b-catenin signaling in the IKPRAB-36 cell line. A and B: In these pathways a green
color represents down regulation by MPA and a red color represents up regulation by MPA. Signaling pathways were provided by Ingenuity Pathway
Assist Software? and individual gene expression levels are available in Table S4. C: Western blot showing FOXO1 expression in the IKPRA-1, IKPRB-1,
IKPRAB-36 and IKLV-8 cell lines cultured in the absence (control) or presence (MPA) of 1 nM MPA. *indicates significant regulation in the micro-array
analysis (Table S4).
Progesterone Inhibits EMT in Endometrial Cancer
PLoS ONE | www.plosone.org10 January 2012 | Volume 7 | Issue 1 | e30840
immunosuppression and inhibiting transition from an epithelial to
a more mesenchymal, more invasive phenotype.
in endometrial carcinoma samples. List of used primers for
the q-PCR experiments.
Primers of genes of interest used for RT-PCR
endometrial carcinoma patient samples. Differentially
regulated genes between non-progressive (n=4) and progressive
(n=4) endometrial cancer samples. Negative values indicate down
regulation in progressive disease, positive values indicate up
regulation in progressive disease. A fold chance of +/21.25 was
used as a cutoff point.
List of differentially expressed genes in
progressive versus non-progressive endometrial cancer
patients. A list of differentially regulated genes in the progressive
group was entered in Ingenuity pathway analysis software. A fold
chance of +/21.25 was used as a cutoff point. P-values were
calculated with a Fishers exact test and a p-value,0.05 was
considered statistically significant.
List of differentially regulated pathways in
the IKPRAB-36 endometrial cancer cell line. List of
significantly MPA regulated genes in the Ishikawa IKPRAB-36
cell line (n=3). Negative values indicate down regulation by MPA,
positive values indicate up regulation by MPA. A fold chance of
+/21.25 was used as a cutoff point and the delta value was 0.53,
which resembles p,0.05.
List of significantly MPA regulated genes in
IKPRAB-36 endometrial cancer cell line. A list of
significantly MPA regulated genes was entered in Ingenuity
pathway analysis software. A fold change of +/21.25 was used as
cutoff point. Ingenuity uses a Fishers exact test for calculate
significance and a p-value of ,0.05 was considered statistically
List of MPA regulated pathways in the
PRAB-36 cell line and in endometrial cancer patient
samples. A p-value of ,0.05 was considered to be statistically
significant. A grey colored pathway resembles a known EMT
Pathways significantly regulated in the IK-
the IKPRAB-36 cell line and in endometrial cancer
patient samples. List of genes both regulated by MPA in the
Ishikawa IKPRAB-36 cell line (n=3) and differentially regulated
between non-progressive versus progressive disease. Negative
values indicate down regulation by MPA in IKPRAB-36 cells
and in non-progressive as compared to progressive disease,
positive values indicate up regulation by MPA in IKPRAB-36
cells and in non-progressive as compared to progressive disease. A
fold chance of +/21.25 was used as a cutoff point.
List and heat map of genes both regulated in
Conceived and designed the experiments: PHvd LJB WFJvIJ MvdZ FA
CWB. Performed the experiments: PHvd IV YW LCK WFJvIJ. Analyzed
the data: PHvd PCE. Wrote the paper: PHvd LJB. Commented on the
manuscript with important intellectual contributions: WFJvIJ MvdZ FA
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PLoS ONE | www.plosone.org12 January 2012 | Volume 7 | Issue 1 | e30840