The mTOR/AKT inhibitor temsirolimus prevents deep infiltrating endometriosis in mice.

Abstract

Deep infiltrating endometriosis (DIE) is a particular clinical and histological entity of endometriosis responsible for chronic pelvic pain and infertility. Here we characterize the proliferative phenotype of DIE cells, to explore the cellular and molecular mechanisms that could explain their aggressive potential. In addition, the inhibition of mTOR/AKT pathway was tested, as a potential treatment of DIE. Included were 22 patients with DIE and 12 control patients without endometriosis. Epithelial and stromal cells were extracted from biopsies of eutopic endometrium and deep infiltrating endometriotic nodules from patients with DIE. Cell proliferation was determined by thymidine incorporation. Oxidative stress was assayed by spectrofluorometry. The ERK and mTOR/AKT pathways were analyzed in vitro by Western blot and for AKT in vivo in a mouse model of DIE. The proliferation rate of eutopic endometrial cells and of deep infiltrating endometriotic cells from DIE patients was higher than that of endometrial cells from controls. The hyperproliferative phenotype of endometriotic cells was associated with an increase in endogenous oxidative stress, and with activation of the ERK and mTOR/AKT pathways. mTOR/AKT inhibition by temsirolimus decreased endometriotic cell proliferation both in vitro and in vivo in a mouse model of DIE. Blocking the mTOR/AKT pathway offers new prospects for the treatment of DIE.

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Metabolic, Endocrine, and Genitourinary Pathobiology
The mTOR/AKT Inhibitor Temsirolimus Prevents
Deep Infiltrating Endometriosis in Mice
Mahaut Leconte,*†Carole Nicco,* Charlotte Ngô,*‡
Christiane Chéreau,* Sandrine Chouzenoux,*
Wioleta Marut,* Jean Guibourdenche,§
Sylviane Arkwright,¶Bernard Weill,*
Charles Chapron,‡Bertrand Dousset,†and
Frédéric Batteux*
From the Laboratory of Immunology * and the Departments of
Digestive and Endocrine Surgery,†Gynecology Obstetrics II and
Reproductive Medicine,‡Hormonology,§and Pathology,¶Paris
Descartes University, Hospital Cochin, Paris, France
Deep infiltrating endometriosis (DIE) is a particular
clinical and histological entity of endometriosis respon-
sible for chronic pelvic pain and infertility. Here we
characterize the proliferative phenotype of DIE cells, to
explore the cellular and molecular mechanisms that
could explain their aggressive potential. In addition, the
inhibition of mTOR/AKT pathway was tested, as a po-
tential treatment of DIE. Included were 22 patients with
DIE and 12 control patients without endometriosis. Ep-
ithelial and stromal cells were extracted from biopsies
of eutopic endometrium and deep infiltrating endo-
metriotic nodules from patients with DIE. Cell prolifer-
ation was determined by thymidine incorporation. Ox-
idative stress was assayed by spectrofluorometry. The
ERK and mTOR/AKT pathways were analyzed in vitro
byWesternblotandforAKTinvivoinamousemodelof
DIE. The proliferation rate of eutopic endometrial cells
and of deep infiltrating endometriotic cells from DIE
patients was higher than that of endometrial cells from
controls. The hyperproliferative phenotype of endo-
metrioticcellswasassociatedwithanincreaseinendog-
enous oxidative stress, and with activation of the ERK
and mTOR/AKT pathways. mTOR/AKT inhibition by
temsirolimusdecreasedendometrioticcellproliferation
both in vitro and in vivo in a mouse model of DIE.
Blocking the mTOR/AKT pathway offers new prospects
for the treatment of DIE.
(Am J Pathol 2011, 179:880–889;
DOI: 10.1016/j.ajpath.2011.04.020)
Endometriosis, a common disease that affects approxi-
mately 6% to 10% of women of childbearing age,1is
characterized by the presence of endometrial tissue out-
side the uterine cavity. Little is known about the factors
that could explain the clinical and histological heteroge-
neity of endometriosis and, particularly, the development
of deep infiltrating endometriosis (DIE). DIE is an aggres-
sive disease responsible for chronic pelvic pain,2in its
intensity inducing severe disability3and infertility.4Surgi-
cal resection of DIE lesions is the main curative treatment
available,5but surgery often needs to be extensive6and
is associated with significant morbidity.7Effectiveness of
medical treatments, currently based on hormonal therapy
that blocks ovarian function, is only transient. New drugs
have recently been considered for the treatment of en-
dometriosis; the use of antiaromatases,8,9antioxidant
molecules,10or more recently anti-metabolites such as
5-FU11has been suggested. The effectiveness of those
treatments in vitro and in animal models relies on the
modulation of cellular mechanisms responsible for inva-
sion, unrestrained growth, neoangiogenesis, and distant
spreading of endometriotic cells.12In line with those ob-
servations, it has recently been shown that ovarian endo-
metriotic cells display a high endogenous oxidative
stress, with profound alteration of the reactive oxygen
species (ROS) detoxification pathways associated with
increased cellular proliferation and activation of the MAP
kinase ERK 1/2 pathway.10Activation of the ERK pathway
has been found in eutopic endometrium and in ovarian
endometrioma of a large number of women with ovarian
endometriosis.13,14
Little is known about the role of other potentially propro-
liferative pathways in endometriosis. The PI3K/mTOR/AKT
pathway has been found to be activated in ovarian endo-
metriosis15–17and has been implicated in the pathogenesis
of ovarian cancer.17To date, however, no study has ex-
plored this pathway in DIE, and the involvement of this
pathway has never been explored in vivo.
In the present study, we explored the proliferative phe-
notype of deep infiltrating endometriotic cells and high-
Supported by grants from Paris Descartes University.
Accepted for publication April 29, 2011.
B.D. and F.B. contributed equally to this work.
Address reprint requests to Frédéric Batteux, M.D., Ph.D., Laboratoire
d’immunologie, Hôpital Cochin, 75679 Paris cedex 14, France. E-mail:
frederic.batteux@cch.aphp.fr.
The American Journal of Pathology, Vol. 179, No. 2, August 2011
Copyright © 2011 American Society for Investigative Pathology.
Published by Elsevier Inc. All rights reserved.
DOI: 10.1016/j.ajpath.2011.04.020
880

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lighted the cellular and molecular mechanisms that could
explain their aggressive potential. Based on our in vitro
results showing a constitutive activation of AKT/mTOR
pathway in deep infiltrating endometriotic cells, we eval-
uated the therapeutic potential of an mTOR inhibitor in
vitro and in vivo in DIE.
Materials and Methods
Sample Collection
Biopsies of eutopic endometrium and deep infiltrating
endometriotic nodules were obtained from 22 patients
undergoing surgical treatment for DIE with rectal involve-
ment defined by muscularis involvement.18Low rectal
endometriosis was defined preoperatively, based on the
following clinical and endoscopic ultrasonographic crite-
ria: rectal invasion of the infraperitoneal rectum (located
within 8 cm of the dentate line, reachable on rectal ex-
amination) and full-thickness invasion of the muscular
layer (?15 mm on rectal endoscopic ultrasonography).7
DIE was confirmed in all cases by a pathologist experi-
enced in endometriosis (S.A.). All of the patients had
been treated by luteinizing-hormone releasing hormone
(LHRH) agonists for a minimum of 1 month before sur-
gery. Control endometrial specimens were obtained from
12 patients without macroscopic endometriosis undergo-
ing laparoscopy for other reasons (tubal obstruction in-
fertility, nonendometriotic ovarian cyst, myoma). Ethics
approval for the present study was obtained from the
ethics committee at Cochin Hospital (no. 05-2006 “gé-
nomique et protéomique de l’endométriose”). Written in-
formed consent was obtained from each patient and con-
trol subject.
Specimens were collected under sterile conditions and
immediately transported to the laboratory in Dulbecco’s
modified Eagle’s medium (Gibco Invitrogen, Cergy Pon-
toise, France) with 10% fetal calf serum. The addition of
10% fetal calf serum affected neither the ERK nor the
ARK pathways (Velarde et al19and unpublished data).
No steroid hormones were added in cell culture. Estrogen
(e2) and progesterone (p4) were undetectable in cell
culture supernatants as determined by an immunodiag-
nostic system (Advia Centaur XP immunoassay system;
Siemens Healthcare Diagnostics, Saint-Denis, France).
The absence of steroid hormones in culture supernatants
reproduces clinical conditions in patients who receive
treatment with LHRH agonists. The time that elapsed
between the biopsy and the procedure of cell isolation
never exceeded 1 hour. The same culture medium was
used throughout the study.
Cell Isolation and Culture
Primary endometrial and deep endometriotic cell cultures
were prepared from biopsies using an adaptation of the
method described by Velarde et al.19Biopsy specimens
were rinsed and minced into small pieces, then digested
with dispase 5‰ and collagenase (2 mg/mL, Gibco In-
vitrogen, Cergy Pontoise, France) for 1 hour at 37°C and
separated using serial filtration. Red blood cells were
removed by hypotonic lysis (0.15 mol/L NH4Cl, 1 mmol/L
KHCO3, 0.1 mmol/L Na2EDTA). Debris was removed by
filtration through 100-?m aperture sieves. Epithelial cells
were retained on 40-?m aperture sieves, with stromal
cells remaining in the filtrate. Both types of cell were
plated onto Primaria flasks (Becton Dickinson Labware,
Le Pont de Claix, France) and were cultured in Dulbec-
co’s modified Eagle’s medium (Gibco Invitrogen) with
10% fetal calf serum.14For each patient with DIE, four cell
populations were obtained: eutopic endometrial stromal
cells (Es), eutopic endometrial epithelial cells (Ee), deep
infiltrating endometriotic stromal cells (Ds), and deep in-
filtrating endometriotic epithelial cells (De). For each con-
trol, we used two cell populations: control endometrial
stromal cells (Cs) and control endometrial epithelial cells
(Ce).
The purification of stromal and epithelial cells was as-
sessed by staining with 1:100 fluorescein isothiocyanate-
labeled anti-cytokeratin and Cy3-labeled anti-vimentin
antibodies (Sigma-Aldrich, St. Louis, MO). Fluorescence
was analyzed using an Olympus fluorescent microscope
(Hamburg, Germany) and images were captured using
an Olympus cell imaging station. Both populations were
negative for CD3 (T cell), CD45 (leukocytes), and CD11b
(monocytes and granulocytes) staining. All experiments
were performed on primary cultures of each cell popula-
tion, and the various tests were performed in triplicate.
Cell Proliferation
The cells that reached confluence were harvested after
trypsin treatment. Then cells were counted with a Trypan
Blue method and 104cells well were seeded onto 96-well
plates (Nunc, Roskilde, Denmark). Cellular viability was
approximately 95% to 100%, regardless of cell type.
Cells were incubated for 48 hours in Dulbecco’s modified
Eagle’s medium with 10% fetal calf serum alone or with
the addition of 6.4 mmol/L N-acetyl-L-cysteine (NAC), or
200 ?mol/L ERK inhibitor (A77-1726), or various concen-
trations of the mTOR/AKT inhibitor temsirolimus (0.3 to 24
?mol/L) at 37°C under 5% CO2. Cell proliferation was
determined by pulsing the cells with [3H]thymidine (Am-
ersham, 1 ?Ci per well; GE Healthcare) during the last 18
hours of culture20and measuring the radioactivity incor-
porated by liquid scintillation counting. Results are ex-
pressed as counts per minute (cpm).
Cellular Production of ROS
Cells (104per well) were seeded into 96-well plates
(Nunc, Roskilde, Denmark) and were incubated for 18
hours alone or with 6.4 mmol/L NAC. Cellular levels of
O2
etry using 125 ?mol/L dihydroethidium (DHE; Interchim,
Montluçon, France), 100 ?mol/L 2=,7=-dichlorohydrofluo-
rescein diacetate (H2DCFDA; Molecular Probes, Eu-
gene, OR), and 4=, 5=-diaminofluorescein diacetate
(DAF2DA; Sigma-Aldrich), respectively, during 5 hours.
The levels of O2
each sample as follows. The ROS rate in arbitrary units
per minute was calculated by subtracting initial fluores-
●?, H2O2, and NO were assessed by spectrofluorom-
●?, H2O2, and NO were calculated in
mTOR/AKT Pathway and Deep Endometriosis
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cence intensity from final fluorescence intensity and di-
viding the difference by the final number of minutes.
Conditions in the present study were 300 minutes and
104cells per well.
Immunoblotting of Cell Lysates
Cells were lysed in ice-cold radioimmunoprecipitation as-
say buffer (10 mmol/L TrisHCl, pH 7.5, 5 mol/L NaCl, 1%
Triton X-100, 0.1% SDS) supplemented with 25 mmol/L
sodium fluoride, 0.5 mmol/L sodium orthovanadate, and
anti-protease 1%. Equal amounts of protein (30 ?g) were
loaded and separated by 10% SDS-PAGE. Transfer and
blocking were performed. For ERK/pERK staining, poly-
vinylidene difluoride membrane was saturated with 5%
skim milk for 1 hour at room temperature, then incubated
with rabbit anti-human ERK IgG antibodies or with rabbit
anti-human pERK IgG antibodies (Santa Cruz Biotechnol-
ogy, Santa Cruz, CA) overnight at 4°C. For AKT/pAKT
staining, polyvinylidene difluoride membrane was satu-
rated with TBSA (BSA-TBST: 1? Tris-buffered saline,
0.1% Tween-20 with 5% bovine serum albumin) overnight
at 4°C with rabbit anti-human AKT IgG antibodies and
rabbit anti-human pAKT IgG antibodies (Cell Signaling
Technology, Danvers, MA) for 1 hour at room tempera-
ture. For phospho-p70S6K (serine 371/threonine 389)
staining, polyvinylidene difluoride membrane was satu-
rated with TBSA (BSA-TBST: 1? Tris-buffered saline,
0.1% Tween-20 with 5% bovine serum albumin) for 1 hour
at room temperature, then incubated with rabbit anti-
human phospho-p70S6K IgG antibodies (Ozyme, Saint-
Quentin-en-Yvelines, France) overnight at 4°C. ?-Actin
was used as housekeeping protein. Specific antibodies
were detected using horseradish peroxidase-conjugated
goat anti-rabbit IgG Ab and visualized by an enhanced
chemoluminescence system (Pierce ECL; Perbio Sci-
ences, Berbières, France; Thermo Fisher Scientific,
Rockford, IL).21
IHC Analysis of pERK and pAKT
Paraffin-embedded tissue sections (4-?m thick) were
deparaffinized and antigen retrieval was performed by
microwave boiling of the sections in 10 mmol/L citrate
buffer (pH 6.0) twice for 10 minutes each. Sections were
incubated in 20% (v/v) normal goat serum in PBS with 4%
(w/v) bovine serum albumin for 30 minutes at room tem-
perature, and then the tissue sections were stained over-
night at 4°C with antibodies against phospho-p44/42
MAP kinase (clone 20G11, diluted 1:100; Cell Signaling
Technology) or with antibodies against phospho-AKT
(clone 736E11, diluted 1:25; Cell Signaling Technology).
Horseradish peroxidase-conjugated secondary antibod-
ies were detected using the 3,3=-diaminobenzidine per-
oxidase substrate kit (Vector Laboratories, Burlingame,
CA). Cells were considered positive only when a clear
staining of the cell nucleus and/or the cytoplasm was
observed.
Mouse Model
Nude mice were grafted with two fragments of a nodule
from one patient with DIE. Female nude mice between 6
and 8 weeks of age weighing 20 to 22 g were used.
Animals received humane care in compliance with insti-
tutional guidelines. Mice were anesthetized with intraper-
itoneal injections of tribromoethanol. An incision was
made on the ventral midline and two fragments of human
endometriotic nodules were sutured to the parietal peri-
toneum with 7/0 prolene. Each fragment was measured
using a rule caliper. An additional fragment was fixed with
10% formaldehyde and set in paraffin for initial histolog-
ical analysis. The abdominal wall was sutured with a 6/0
nylon thread.22A subcutaneous injection of 0.5 ?g ?-es-
tradiol was performed on day 1 and day 2 to facilitate the
implantation of endometriotic nodules.23
On day 7 after implantation, surgery was performed on
the mice to confirm viability of the implant. Treatment
started on day 7: the treated group received intraperito-
Table 1.
Clinical Characteristics of DIE and Control Patients
DIEControl
Sample size
Age (years)*
Treatments before surgery (no.)
None
Estroprogestative agents
LHRH agonists
Duration of treatment (months)†
Infertility
Duration of symptoms (months)†
Gynecologic symptoms
Bowel symptoms
Cyclic exacerbation of symptoms
C-reactive protein (mg/L)*
Biopsy
Endometrium
Deep lesion
n ? 22
n ? 12
32 ? 1.5 (21–47) 31.9 ? 1.09 (19–36)
2
9
1
1
6 (2–32)
3
–
–
–
–
–
22
0
–
22
11 (4–60)
8
48 (12–168)
22
22
20
7.55 ? 2.78 (0.09–36.7)
20
21
12
–
*Data are presented as mean ? SD (range).
†Data are presented as median (range).
882
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neal injections of temsirolimus (3 mg/kg in 100 ?L PBS, 5
days per week for 2 weeks) and the control group re-
ceived intraperitoneal injections of 100 ?L PBS under the
same conditions. After 2 weeks of treatment, animals
were sacrificed by cervical dislocation. Implants were
extracted and fixed with 10% formaldehyde and set in
paraffin. Serial 4-?m sections were prepared and stained
with H&E and were examined histologically by patholo-
gists experienced in endometriosis (S.A., F.B., and M.L.).
Pathology scores were on a scale of 0 (receded lesion
with stromal fibrosis, hemosiderin and absence of glan-
dular structure) to 3 (active lesion with fresh blood, pro-
fuse stromal cellular infiltration and developed glandular
organization).10Scoring was performed by three different
scientists unaware of the treatments received in each
group (S.A., F.B., and M.L.).
Statistical Analysis
Data are reported as means of independent triplicate
experiments for each cell population. Means were com-
pared by Student’s t-test. A level of P ? 0.05 was ac-
cepted as significant.
Results
Biopsies and Characteristics of Population
The study population comprised 22 patients. Twenty bi-
opsies of eutopic endometrium and 21 biopsies of deep
infiltrating endometriotic nodule were obtained from
those patients. Initially, 20 Ee and 20 Es cell lines were
extracted from 20 samples of eutopic endometrium, and
20 De and 21 Ds cell lines were extracted from 21 sam-
ples from deep infiltrating endometriotic nodules. Nine
cell lines were eliminated for failure of culture (n ? 6) or
bacterial contamination (n ? 3). A final total of 18 Ee, 18
Es, 17 De, and 18 Ds cell lines were included for study.
As control subjects, 12 women were included, and 12
biopsies of healthy endometrium were collected. Two of
the control subjects had a preoperative hormonal treat-
ment (estroprogestatives in one case for contraception
and LHRH agonists to reduce operative bleeding during
uterine myoma resection in the other case). The 12 sam-
ples of endometrium provided 12 epithelial and 12 stro-
mal control endometrial cell lines. Of these, 3 cell lines
were eliminated for failure of culture, yielding a final total
of 11 Ce and 10 Cs cell lines. The clinical characteristics
of the population are shown in Table 1.
Purification of Endometrial and Endometriotic
Cells
The cells were checked by optical microscopy and the
patterns of expression of cytokeratin and vimentin were
characterized by direct immunofluorescence using spe-
cific antibodies (Figure 1A). The epithelial cells showed
positive staining for cytokeratin and negative staining for
vimentin. The stromal cells showed positive staining for
vimentin and negative staining for cytokeratin.
Proliferation of Deep Infiltrating Endometriotic
Cells
There were no significant differences among De, Ee, and
Ce cells in terms of proliferative rates. The proliferative
rate of Es cells was not higher than that of Cs cells;
however, the proliferative rate of Ds cells was greater by
39% than that of Es cells (P ? 0.01) and by 68% than that
of Cs cells (P ? 0.001) (Figure 1B).
Within deep nodules, the proliferative rate of Ds cells
was increased by 26% compared with De cells (P ? 0.05)
(Figure 1B).
There were no differences in terms of cellular prolifer-
ation between the group of fertile patients (n ? 14) and
the group of infertile patients (n ? 8) (data not shown),
nor between the group with C-reactive protein of ?5 mg/L
Figure 1. Purification and proliferative rates of endometriotic cells. A: Eu-
topic endometrial epithelial cells (Ee), eutopic endometrial stromal cells (Es),
deep infiltrating endometriotic epithelial cells (De), and deep infiltrating
endometriotic stromal cells (Ds) examined by contrast phase microscopy
(top row), immunofluorescence with fluorescein isothiocyanate anti-cytok-
eratin antibodies (middle row), or Cy3 anti-vimentin antibodies (bottom
row). Original magnification, ?100. B: Basal proliferative rate was assessed
in the various cell lines: control endometrial epithelial cell lines (Ce), n ? 11;
Ee cell lines, n ? 18; De cell lines, n ? 17; control endometrial stromal cell
lines (Cs), n ? 10; Es cell lines, n ? 18; and Ds cell lines, n ? 18. Cell
proliferation was determined by thymidine incorporation. Results are ex-
pressed as counts per minute (cpm). *P ? 0.001, Es versus Ce cells;†P ? 0.01,
De versus Ee cells;‡P ? 0.05, stromal versus epithelial cells within the same
endometrial eutopic or deep infiltrating lesion.
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(n ? 13) and the group with C-reactive protein of ?5
mg/L (n ? 9) (data not shown).
Cellular Production of ROS
Production of Superoxide Anions
The spontaneous production of superoxide anions
(O2
Ce cells (P ? 0.05). The production of O2
was not increased significantly compared with Ee cells,
but was increased by 36% compared with Ce cells (P ?
0.05). The production of O2
creased significantly compared with Cs cells. In contrast,
the production of O2
compared with Es cells (P ? 0.001) and by 43% com-
pared with Cs cells (P ? 0.001) (Figure 2A).
Within deep nodules, the production of O2
differ significantly between Ds and De cells (Figure 2A).
●?) in Ee cells was increased by 28% compared with
●?in De cells
●?in Es cells was not in-
●?in Ds cells was increased by 35%
●?did not
Production of Hydrogen Peroxide
There were no significant differences among De, Ee,
and Ce cells in terms of H2O2production. The production
of H2O2in Es cells was not increased significantly com-
pared with Cs cells. In contrast, the production of H2O2in
Ds cells was increased by 39% compared with Es cells
(P ? 0.01) and by 39% compared with Cs cells (P ? 0.01)
(Figure 2B).
Within deep nodules, the production of H2O2in Ds
cells was increased by 23% compared with De cells (P ?
0.01) (Figure 2B).
Production of Nitric Oxide
The production of NO in Ee cells was increased by
36% compared with Ce cells (P ? 0.01). In contrast, the
production of NO in De cells did not differ significantly
from that in Ee and Ce cells. The production of NO in Es
cells was increased by 46% compared with Cs cells (P ?
0.05). The production of NO in Ds cells was not increased
significantly compared with Es cells, but was increased
by 30% compared with Cs cells (P ? 0.05) (Figure 2C).
Within deep nodules, production of NO did not differ
significantly between Ds and De cells (Figure 2C).
Cellular Detoxification of H2O2by
N-Acetyl-L-Cysteine
Treatment of Ce, Ee, and De cells with 6.4 mmol/L NAC
decreased their production of H2O2by 64%, 48%, and
38%, respectively, compared with untreated cells (P ?
0.001 for all comparisons). Treatment of Cs, Es, and Ds
cells with 6.4 mmol/L NAC decreased the production of
H2O2by 56%, 42%, and 47% compared with untreated
cells (P ? 0.001 for all comparisons) (Figure 2D).
Treatment of Ce and Cs cells with 6.4 mmol/L NAC did
not decrease their proliferative rates, but for Ee and De
cells the same treatment decreased their proliferative
rates by 65% and by 36% compared with untreated cells
(P ? 0.001 for both comparisons). Treatment of Es and
Ds cells with 6.4 mmol/L NAC decreased their prolifera-
tive rates by 77% and by 39% compared with untreated
cells (P ? 0.001 for both comparisons) (Figure 2E).
Figure 2. A–C: Cellular production of ROS and detoxification of H2O2by N-acetyl-L-cysteine (NAC). Basal intracellular levels of O2●?(A), H2O2(B), and NO (C)
were assessed by spectrofluorometry in the various cell lines (11 Ce, 18 Ee, 17 De, 10 Cs, 18 Es, and 18 Ds). U.A., arbitrary fluorescence intensity units. *P ? 0.05,
**P ? 0.01, and ***P ? 0.001, Es versus Ce cells.††P ? 0.01,†††P ? 0.001, De versus Ee cells.‡‡P ? 0.01, stromal versus epithelial cells within the same endometrial
eutopic or deep infiltrating lesion. D and E: Intracellular levels of H2O2(D) and proliferative rate (E) were assessed in cell lines treated with 6.4 mmol/L NAC versus
untreated cells. The level in untreated cells is indicated by a horizontal dashed line. **P ? 0.01, ***P ? 0.001.
884
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Exploration of the ERK Pathway
The ERK protein was equally present in the epithelial and
stromal cell lines derived from control endometrium (Ce,
Cs), eutopic endometrium (Ee, Es), and deep infiltrating
endometriotic nodules (De, Ds). Levels of pERK, the
phosphorylated activated form of ERK, were low in the Ce
and Cs cell lines; however, pERK was expressed in the
epithelial and stromal cell lines in both eutopic endome-
trium (Ee, Es) and deep infiltrating endometriotic nodules
(De, Ds) (Figure 3A).
ERK activation, reflected by an elevated pERK/ERK
ratio, was increased by 69% in Ee cells compared with
Ce cells (P ? 0.05). ERK activation in De cells was not
increased compared with Ee cells, but was increased by
73% compared with Ce cells (P ? 0.01) (Figure 3B).
ERK activation in Es cells was not increased signifi-
cantly compared with Cs cells, nor in Ds cells compared
with Es cells, but was increased by 68% compared with
Cs cells (P ? 0.05) (Figure 3B).
Within deep nodules, ERK activation did not differ sig-
nificantly between Ds and De cells (Figure 3B).
Activation of the ERK pathway was also demonstrated
by immunohistochemistry (IHC) on deep infiltrating tissue
(Figure 3C).
Inhibition of the ERK Pathway by the Selective
Protein Tyrosine Kinase Inhibitor A77-1726
Treatment of Ce cells with 200 ?mol/L of the selective
ERK inhibitor A77-1726 did not significantly decrease
their proliferative rate. Treatment of Ee and De cells with
200 ?mol/L of the selective ERK inhibitor A77-1726 de-
creased their proliferative rates by 87% and by 93%,
respectively, compared with untreated cells (P ? 0.001
for both comparisons) (Figure 3D).
Treatment of Cs cells with 200 ?mol/L of the selective
ERK inhibitor A77-1726 did not significantly decrease
their proliferative rate. Treatment of Es and Ds cells with
200 ?mol/L A77-1726 decreased their proliferative rate
by 95% and 89%, respectively, compared with untreated
cells (P ? 0.001 for both comparisons) (Figure 3D).
Exploration of the mTOR/AKT Pathway
The AKT protein and its phosphorylated activated form,
pAKT, were present in all cell lines, but were present in
higher amounts in the epithelial and stromal cell lines
derived from eutopic endometrium (Ee, Es) and deep
infiltrating endometriotic nodules (De, Ds) than in the
epithelial and stromal cell lines derived from control
endometrium (Ce, Cs). The phosphorylated form of
p70S6K (serine 371/threonine 389) was present mostly
in the epithelial and stromal cell lines derived from
deep infiltrating endometriotic nodules (De, Ds), as
opposed to the epithelial and stromal cell lines derived
from eutopic (Ee, Es) and control endometrium (Ce,
Cs) (Figure 4A).
AKT expression in Ee cells was increased by 33%
compared with Ce cells. AKT expression in De cells was
not increased compared with Ee cells, but was increased
Figure 3. Exploration of the ERK pathway. A: Determination of ERK and
pERK by Western blot in cell lysates. Western blotting of ERK and pERK
were performed on lysates of the various cell lines (11 Ce, 18 Ee, 17 De,
10 Cs, 18 Es, and 18 Ds) using specific anti-ERK and anti-pERK antibodies.
A representative Western blot is shown, obtained with all cell lines
extracted from one patient and from one control. B: Quantitative analysis
of ERK and pERK. Quantitative analysis of ERK and pERK was performed
in the various cell lines (11 Ce, 18 Ee, 17 De, 10 Cs, 18 Es, and 18 Ds) by
Western blot analysis. The mean optical density ratio for pERK/ERK was
calculated in each endometriotic cell type and compared with control
endometrial cells: *P ? 0.05, **P ? 0.01. C: IHC expression of pERK on
deep infiltrating endometriotic tissue. A representative specimen is
shown, from one of three patients. pERK-positive cells appear black.
Original magnification, ?400. D: Effect of A77-1726 on endometriotic cell
proliferation. Proliferative rates were determined by thymidine incorpo-
ration in the various cell lines (11 Ce, 18 Ee, 17 De, 10 Cs, 18 Es, and 18
Ds) treated with 200 ?mol/L A77-1726 and were compared with untreated
cells. The level in untreated cells is indicated by a horizontal dashed line.
***P ? 0.001.
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by 33% compared with Ce cells (Figure 4B). AKT expres-
sion in Es cells was increased by 46% compared with Cs
cells (P ? 0.05), and AKT expression in Ds cells was
increased by 52% compared with Cs cells (P ? 0.001)
(Figure 4B). Within deep nodules, AKT expression in Ds
cells was increased by 55% compared with De cells (P ?
0.05) (Figure 4B).
The expression of pAKT in Ee cells was increased by
33% compared with Ce cells and pAKT expression in De
cells was increased by 37% compared with Ce cells
(Figure 4C). The expression of pAKT in Es cells was
increased by 52% compared with Cs cells (P ? 0.05) and
pAKT expression in Ds cells was increased by 60% com-
pared with Cs cells (P ? 0.05) (Figure 4C). Within deep
nodules, the expression of pAKT in Ds cells was in-
creased by 61% compared with De cells (P ? 0.05)
(Figure 4C).
The expression of phospho-p70S6K in Ee cells was
increased by 81% compared with Ce cells and phospho-
p70S6K expression in De cells was increased by 92%
compared with Ce cells (P ? 0.05) (Figure 4D). The
expression of phospho-p70S6K in Es cells was increased
by 92% compared with Cs cells (P ? 0.05), and phospho-
p70S6K expression in Ds cells was increased by 95%
compared with Cs cells (P ? 0.05) (Figure 4D). Within
deep nodules, the expression of phospho-p70S6K in Ds
cells was increased by 48% compared with De cells (P ?
0.05) (Figure 4D).
Activation of the AKT pathway was also demonstrated
by IHC on deep infiltrating tissue (Figure 4E).
Inhibition of the AKT Pathway by the mTOR
Inhibitor Temsirolimus
With 24 ?mol/L temsirolimus, the proliferative rate of Es
and Ds cells was decreased by 94% and by 98% respec-
tively (P ? 0.001 for both comparisons). The proliferative
rate was dose-dependently decreased and significant for
all concentrations. The proliferative rate of Cs cells was
dose-dependently decreased and significant from 3
?mol/L (Figure 4F).
Mouse Model
Twelve nude mice were grafted with two fragments of
nodules from two patients with DIE. Two mice died on day
8, which was 1 day after laparotomy to confirm the via-
bility of the implant. The control group comprised 4 mice
with 8 implants and the treated group comprised 6 mice
with 12 implants. On day 0, the appearance of the im-
planted deep infiltrating endometriotic tissue was typical,
with endometriotic glands, simple endometrial epithelium
with stroma surrounded by fibrosis, and hyperplastic
smooth muscle fibers. At 3 weeks after the implantation of
deep endometriotic infiltrating endometriotic tissue and
only 2 weeks after the beginning of the treatment, the 8
control implants showed persistence of active lesions
with glandular organization (score 2.44 ? 0.18), whereas
the 12 treated implants displayed fibrotic and avascular
lesions with hemosiderin (score 1.19 ? 0.25, P ? 0.01)
(Figure 5, A and B).
Figure 4. Exploration of the mTOR/AKT pathway. A: Determination of AKT, pAKT, and phospo-p70S6K (serine 371/threonine 389) by Western blot in cell
lysates. Western blotting of AKT, pAKT, phospo-p70S6K, and ?-actin was performed on lysates of the various cell lines (11 Ce, 18 Ee, 17 De, 10 Cs, 18 Es, and
18 Ds) using specific anti-AKT, anti-pAKT, anti-phospo-p70S6K, and anti-?-actin antibodies. The figure represents one representative Western blot obtained with
all cell lines extracted from one patient and from one control. B and C: Quantitative analysis of AKT (B) and pAKT (C) was performed in the various cell lines
(11 Ce, 18 Ee, 17 De, 10 Cs, 18 Es, and 18 Ds) by Western blot analysis. The mean optical density ratios AKT/?-actin and pAKT/?-actin were calculated in the
various endometriotic cell lines. *P ? 0.05 and **P ? 0.001, endometriotic versus control cells;†P ? 0.05, stromal versus epithelial cells within the same endometrial
eutopic or deep infiltrating lesion. D: Quantitative analysis of phospo-p70S6K was performed in the various cell lines (11 Ce, 18 Ee, 17 De, 10 Cs, 18 Es, and 18
Ds) by Western blot analysis. The mean optical density ratio phospo-p70S6K/?-actin was calculated in the various endometriotic cell lines. *P ? 0.05,
endometriotic versus control cells. Stromal versus epithelial cells within the same endometrial eutopic or deep infiltrating lesion:†P ? 0.05. E: IHC expression of
pAKT on deep infiltrating endometriotic tissue. A representative specimen is shown, from one of three patients. pAKT-positive cells appear black. Original
magnification, ?400. F: Effect of temsirolimus on proliferation. Proliferative rate was determined by thymidine incorporation for the various stromal cell lines (10
Cs, 18 Es, and 18 Ds) treated in vitro with increasing concentrations (0.3 to 24 ?mol/L) of temsirolimus. The basal level of proliferation of untreated endometriotic
cells is indicated by a horizontal dashed line. *P ? 0.05, treated versus untreated cells.
886
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Discussion
Here, we show for the first time the hyperproliferative
phenotype of deep infiltrating endometriotic cells and the
direct relationship between an increased endogenous
oxidative stress and the activation of the ERK and the
mTOR/AKT pathway. Moreover, we demonstrate that the
pharmacological inhibition of the mTOR/AKT pathway ab-
rogates the proliferation of endometriotic cells both in vitro
and in vivo and is therefore a potential new treatment for
endometriosis in humans.
The population of patients studied was homogeneous
for symptoms. In our institution, all DIE patients are pre-
operatively treated with LHRH agonists. Indeed, this
treatment is not only effective on the rAFS score,24,25but
also blocks the menstrual cycle, so that all patients can
be compared in terms of hormonal menstrual variation.
Even if LHRH agonists modulate endogenous oxidative
stress or the ERK and AKT pathways as they do for
IL-8,26eutopic endometrium clearly differs from ectopic
endometrium in each patient in terms of activation of
these processes. This observation suggests that our data
can be interpreted independently of LHRH treatment.
Even though two control subjects had hormonal treat-
ment, the control group was homogeneous for all of the
experiments.
The yield of the isolation procedure was satisfactory, in
that more than 17 lines of each cell type were derived
from endometrial and deep infiltrating endometriotic bi-
opsies (?75% success). Although cell-cell interactions
between epithelial and stromal cells may play a role in the
initiation and development of endometriosis,27we chose
to culture and test the two cell types separately, to de-
termine the specific molecular abnormalities in each of
them.
We first investigated the relationship between the cell
proliferative rate and alterations in intracellular oxidative
stress. In contrast to De cells, Ds cells displayed an
increased proliferative rate and oxidative stress. Ovarian
endometriotic cells are also characterized by a hyperpro-
liferative rate in association with an up-regulated oxida-
tive stress.10However, the disorders predominantly
affect epithelial cells in ovarian endometriosis and pre-
dominantly affect stromal cells in DIE, suggesting that
ovarian endometriosis and deep infiltrating endometriosis
result from the dysregulation of two different cell types.
The increase in oxidative stress results from the imbal-
ance between production of ROS and their detoxification
by antioxidant systems.28Reinforcing catalase and glu-
tathione reductase activities using the antioxidant mole-
cule NAC significantly decreased the intracellular con-
centration of H2O2. The subsequent decrease in the
cellular proliferative rate is reminiscent of our previous
observations in cancer cells.20Although the effects of
NAC on the intracellular concentration of H2O2were sim-
ilar in both eutopic and deep infiltrating epithelial and
stromal cells, the inhibition of proliferation predomi-
nated in eutopic epithelial and stromal cells. These
results suggest that other mechanisms or amplified
reactions act within deep infiltrating lesions to maintain
the cells in a hyperproliferative state. Moreover, de-
creasing the amounts of H2O2generated in control
endometrial epithelial and stromal cells NAC does not
decrease their proliferation rates. Thus, in normal cells,
the levels of such pro-oxidative molecules as O2
H2O2are not high enough to down-regulate cell prolif-
eration as the ERK pathway does. In those cells, there-
fore, the inhibition of basal level of H2O2has no influ-
ence on the proliferation rate. Taken together, those
results suggest that antioxidant molecules could be
used as a new treatment for DIE.
Oxidative stress, particularly the overproduction of
O2
ERK 1/2 pathway in several cell types.29Indeed, we
found the ERK pathway activated through the phosphor-
ylation of ERK, especially in deep infiltrating endometri-
otic stromal cells. This is in agreement with the known role
of the ERK pathway in survival and proliferation of endo-
metriotic cells,30namely through expression of c-Fos and
c-Jun.31Those data have been confirmed by observa-
tions that the ERK pathway was hyperactivated10,13,19
and the related genes were overexpressed32–34in pa-
tients with endometriosis. Furthermore, a relationship be-
tween activation of the ERK pathway and proliferation of
deep infiltrating endometriotic cells could be established
using a specific inhibitor of phosphorylation of the protein
tyrosine kinase ERK (A77-1726).
This is the first time that the activation of the ERK
pathway has been shown in DIE and that a relationship
has been established between up-regulation of oxidative
●?and
●?and of H2O2, is known to activate the MAP kinase
Figure 5. Effect of temsirolimus on deep infiltrating endometriotic tissue in
a mouse model. A: Pathology scores were calculated on day 21 on untreated
and treated mice: *P ? 0.01. B: Histological views of deep infiltrating implant
on day 21 in untreated mice (left) and in treated mice (right). In untreated
mice, the appearance of the implanted deep infiltrating endometriotic tissue
was typical, with endometriotic glands (black arrow) and with stroma
(white arrow) surrounded by fibrosis and hyperplastic smooth muscle
fibers (dotted black arrow). By contrast, treated mice (right) displayed
fibrotic and avascular lesions (black arrow) with hemosiderin (white
arrow).
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stress and increased cell proliferation. Although the same
observations have been made in ovarian endometriosis,
the distribution of the molecular abnormalities differs be-
tween ovarian endometriosis and DIE, in that they pre-
dominate in epithelial cells in the ovarian endometriosis
and in stromal cells in DIE. This suggests that ovarian
endometriosis is an epithelial disease and DIE, a stromal
disease. On the other hand, oxidative stress is probably
not the only factor responsible for activation of the ERK
pathway. Because proinflammatory cytokines such as
IL-1? and tumor necrosis factor-? are also increased in
the endometrium of endometriosis patients.35–37Thus,
abrogating the activation of the ERK pathway in deep
endometriotic cells using inhibitors of protein tyrosine
kinase can be considered for a new therapeutic ap-
proach.
On the other hand, the phosphatidylinositol-3 kinases
(PI3Ks) as well as the mammalian target of rapamycin
(mTOR) pathways are two key cellular signaling path-
ways that affect broad aspects of cellular functions, in-
cluding metabolism, growth, and survival.38,39Although
initially viewed as two separate pathways, the PI3K and
mTOR pathways are connected via the serine/threonine
kinase AKT.40AKT, also termed PKB (protein kinase B),
is involved in metabolism regulation, growth, proliferation,
and apoptosis through PI3K and mTOR.41The PI3K/
mTOR pathway is activated by a broad array of different
stimuli via specific receptors, including antigen recep-
tors, cytokine receptors, insulin receptor, or the insulin-
like growth factor I receptor. After the engagement of the
receptors, PI3K phosphorylates phosphatidylinositol 4,5-
bisphosphate (PIP2) to generate phosphatidylinositol-
3,4,5-trisphosphate (PIP3) as a second messenger to
recruit and activate downstream targets, including AKT.
One main effector of PI3K and AKT is the high molecular
weight kinase mTOR.42mTOR controls protein synthesis
through phosphorylation and inactivation of the repressor
of mRNA translation, eukaryotic initiation factor 4E-bind-
ing protein 1, and through phosphorylation and activation
of S6 kinase.43The loss of mTOR function leads to an
arrest in the G1 phase of the cell cycle, along with a
severe reduction in protein synthesis.
Several observations have pointed to the potential role
of the mTOR/AKT pathway in endometriosis. First, the
P3KCA mutation responsible for the constitutive activa-
tion of AKT is the most frequently encountered mutation in
ovarian clear cell carcinoma,44a tumor that is associated
with endometriosis.12,45Moreover, AKT activity is higher
in ovarian endometriosis than in normal endometrium,46
and it has been postulated that estrogens might be one of
the factors responsible for the high AKT activation in
endometriotic cells.17However, if estrogens activate AKT
in epithelial endometriotic cells,17this phenomenon is
less clear in stromal endometriotic cells.15Indeed, in the
stromal cells, a specific estrogen receptor antagonist
fails to antagonize the effects of estradiol and exerts a
stimulatory effect on AKT phosphorylation.15We found
that AKT is hyperactivated in endometriotic lesions from
patients with DIE, as it is in ovarian endometriosis; how-
ever, ERK and AKT activation predominates in endo-
metriotic stromal cells in DIE, but in endometriotic epithe-
lial cells in ovarian endometriosis.
Because patients with DIE had been treated with an
LHRH agonist, and because no estrogen could be de-
tected in culture supernatants of endometriotic cells, our
results suggest a constitutive estrogen-independent
mechanism of AKT activation in DIE. This hypothesis is in
line with the predominant involvement of endometriotic
stromal cells in DIE. Moreover, the constitutive activation
of the AKT pathway in endometriotic cells could be ex-
plained by the overproduction of endogenous ROS, as
already observed in cancer cell lines for AKT activation
and in endometriotic cells for ERK activation.47,48Be-
cause AKT affects cell proliferation and survival, the phe-
notype of stromal cells in DIE may be linked to increased
AKT activation. The decrease in the proliferative rate of
eutopic endometrial epithelial cells and deep infiltrating
endometriotic stromal cells after treatment with temsiroli-
mus, a selective inhibitor of mTOR, establishes for the
first time a link between activation of the AKT pathway
and the hyperproliferative phenotype of those cells.
Moreover, the inhibitory effect of temsirolimus on the sur-
vival and development of endometriotic implants in a
mouse model of DIE suggests this molecule for treatment
of patients with DIE, as in patients with kidney cancer.49
This molecule could be rapidly tested in women with DIE.
Acknowledgment
We thank Ms. Agnes Colle for editing the manuscript.
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