Download Estrogen receptor alpha pathway is involved in

Carcinogenesis vol.32 no.4 pp.589–596, 2011
doi:10.1093/carcin/bgq276
Advance Access publication December 20, 2010
Estrogen receptor alpha pathway is involved in leptin-induced ovarian cancer cell
growth
Jung-Hye Choi, Kyung-Tae Lee and Peter C.K.Leung1,
Department of Life and Nanopharmaceutical Science, Kyung Hee University,
Seoul 130-701, Republic of Korea and 1Department of Obstetrics and
Gynecology, Child and Family Research Institute, University of British
Columbia, Vancouver, British Columbia, Canada V6H 3V5
To whom correspondence should be addressed. Tel: 1 604 875 2718;
Fax: 1 604 875 2717;
Email: [email protected]
Correspondence may also be addressed to Jung-Hye Choi. Tel: 82 2 961 2172;
Fax: 82 2 962 0860;
Email:[email protected]
Previously, we demonstrated that leptin, a pleiotropic hormone
produced by adipocytes, stimulates the growth of BG-1 ovarian
cancer cells via the extracellular signal-regulated kinase signaling
pathway. In this study, we further investigated the involvement of
estrogen receptor (ER) pathway in the mechanism of leptininduced ovarian cancer cell growth. Treatment with leptin
(100 ng/ml) resulted in a significant increase in the cell growth
of ERa-transfected OVCAR-3 and A2780 cells, whereas no significant difference was observed in ERb-transfected cells. Downregulation of ERa using small interfering RNA completely
reversed leptin-induced growth of BG-1 cells. Treatment with leptin resulted in ER transcriptional activation, i.e. nuclear localization of ER and increased expression of pS2, an estrogen-dependent
gene. Luciferase reporter assay revealed that treatment of BG-1
cells with leptin (100 ng/ml) stimulated the expression of the reporter gene in the absence of estradiol (E2). To examine an
involvement of Janus kinase 2/signal transducers and activators
of transcription 3 (STAT-3) and phosphatidyl-inositol 3-kinase
(PI3K)/Akt in leptin-induced pathway, we demonstrated that leptin increased phosphorylation of STAT-3 and Akt in BG-1 cells in
a time- and dose-dependent manner. On the other hand, leptininduced cell growth and ER transactivation were effectively blocked
by specific STAT-3 inhibitor AG490 and, to a lesser extent, by PI3K
inhibition. Further study with coimmunoprecipitation assay revealed that stimulation with leptin induced STAT-3 binding to
ERa. Taken together, these results indicate that the stimulation of
ovarian cancer cell growth by leptin involves, at least in part, ER
transcriptional activation via the STAT-3 signaling pathways.
Introduction
Leptin, a product of the obese (ob) gene, is predominantly secreted by
adipocytes and shows a strong positive correlation with total body fat
and body mass index. Although the primary function of leptin is the
regulation of food intake and energy consumption via its effects on the
brain, leptin also acts to promote proliferation in various cell systems,
such as vascular endothelium, lung, gastric mucosa, keratinocytes and
pancreatic b cells. More interestingly, recent studies have demonstrated that this hormone stimulates growth, migration, invasion and
angiogenesis in tumor cell models, suggesting that leptin is capable of
promoting an aggressive cancer phenotype (1,2). Ovarian cancer is the
sixth most common cancer and the fifth leading cause of cancerAbbreviations: ER, estrogen receptor; ERE, estrogen response element; ERK,
extracellular signal-regulated kinase; FBS, fetal bovine serum; GAPDH, glyceraldehyde phosphate dehydrogenase; ICI, ICI 182,680 (Faslodex); IOSE,
immortalized ovarian surface epithelium; JAK2, Janus kinase 2; MTT, 3(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide; PI3K, phosphatidyl-inositol 3-kinase; mRNA, messenger RNA; PCR, polymerase chain
reaction; siRNA, small interfering RNAs; STAT-3, signal transducers and activators of transcription 3.
related deaths among women in developed countries. Due to the lack
of a proven method for early detection, only about one-fourth of the
women have localized disease at the time of diagnosis. Risk factors
for the disorder include age, nulliparity and family history of ovarian
cancer, particularly first-degree relatives. The increasing studies demonstrated that obesity is a risk factor for ovarian cancer. The positive
relation between high body mass index/waist to hip ratio and occurrence of ovarian cancer has been reported (3–5). In addition, a recent
study demonstrated that adipocyte expression and circulating levels of
leptin increase in ovarian, breast and endometrial cancer patients (6).
The effects of leptin are mediated by the transmembrane leptin
receptor (ObR), which belongs to the cytokine receptor superfamily.
In human tissues, at least four isoforms of ObR with different COOHterminal cytoplasmic domains exist, including a long form (ObRb)
and a short form (7). The long form of ObR (ObRb) activates classical cytokine Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT-3) pathways (8,9), as well as the ras/
extracellular signal-regulated kinase (ERK) (8,10) and phosphatidylinositol 3-kinase (PI3K) (11) pathways. In contrast, the shorter ObR
isoforms mainly activate the ERK signaling pathway (8,12).
Estrogen receptor (ER)a, a classical form of ER, is expressed in up
to 60% of ovarian epithelial tumors, and its level is generally higher
than that of benign tumors or normal ovaries (13–16). In addition,
a second form of ER (ERb) has been reported in normal and cultured
primary ovarian cells or ovarian cancer cell lines at the messenger
RNA (mRNA) and protein levels by us (17) and others (18,19). It is of
note that a majority of studies suggest an increased ERa/b ratio in
ovarian cancer, implying a mechanism that results in ERa overexpression or a selective growth advantage for ERa-positive cells (19–21).
Treatment with exogenous estrogens resulted in a growth stimulation
of several ER-positive ovarian carcinoma cell lines in vitro (22,23).
Recent studies have suggested that the estrogen and leptin systems are
related with functional cross talk in various tissues. For example, estrogens have been shown to regulate the expression of leptin (24,25) and
its receptor (26) in adipose tissue and brain, respectively. Reciprocally,
leptin has been found to modulate aromatase activity (27,28). More
recently, it has been demonstrated that leptin stimulates functional
activation of ERa (10) and interfere with anti-estrogen ICI 182,680
(Faslodex)-induced growth inhibition in MCF-7 breast cancer cells (1).
We previously demonstrated that both long (ObRb) and short (7)
forms of leptin receptors are expressed in immortalized ovarian surface epithelium (IOSE) cells and ovarian cancer cells. We also found
that treatment with leptin resulted in the growth stimulation of BG-1
cells via the activation of ERK1/2 (29). In the present study, we
further investigated whether ER, especially ERa, is involved in leptininduced cell growth in ovarian cancer cells.
Materials and methods
Materials
Recombinant leptin was purchased from Sigma–Aldrich (Oakville, ON). PD98059
[2-(2-amino-3-methoxyphenyl)-4H-1-benzopy-ran-4-one], a mitogen-activated
protein kinase/ERK kinase (MEK) inhibitor, LY294002 [2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], a specific cell permeable PI3K inhibitor and AG490 [a-cyano-(3,4-dihydroxy)-N-benzyl-cinnamide], a JAK-2/STAT-3
inhibitor were purchased from New England Biolabs (Beverly, MA) and Calbiochem (La Jolla, CA). Antibodies against ERa, ERb, nucleolin and b-actin were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-STAT-3,
total STAT-3, phospho-Akt and total Akt were obtained from Cell Signaling Technology (Beverly, MA). Methyl piperidinopyrazole, a selective ERa antagonist, was
purchased from Tocris Bioscience (Ellisville, MO).
Cell culture
BG-1 cells were maintained in Dulbecco’s modified Eagle’s F12 medium
supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories,
Ó The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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Logan, UT), 100 U/ml penicillin G and 100 g/ml streptomycin (Life Technologies, Rockville, MD) in a humidified atmosphere of 5% CO2–95% air at 37°C.
For hormone induction, the cells were cultured in phenol red-free Dulbecco’s
modified Eagle’s F12 containing 10% charcoal-stripped FBS. Other ovarian
cancer cells (OVCAR-3, A2780 and SKOV-3), IOSE cells (IOSE-7 and IOSE80PC) and breast cancer cells (MCF-7) were cultured in medium RPMI (Sigma–
Aldrich) containing 10% FBS, 100 U/ml penicillin G and 100 g/ml streptomycin
in a humidified atmosphere of 5% CO2–95% air at 37°C. The IOSE cell lines
were generated by transfecting ovarian surface epithelial cells with SV40-T
antigen and generously provided by Drs N.Auersperg (University of British
Columbia, Vancouver, British Columbia, Canada) and A.Godwin (Fox Chase
Cancer Center, Philadelphia, PA). In addition, the BG-1 cell line was kindly
provided by Dr K.S.Korach (National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC).
Cell transfection
Full-length human ERa (pCMV5- ERa) and ERb (pRST7-ERb) expression
plasmids were generously provided by Dr B.S.Katzenellenbogen (Department
of Molecular and Integrate of Physiology, University of Illinois at UrbanaChampaign) and Dr P.P.McDonnell (Department of Pharmacology and Cancer
Biology, Duke University Medical Center, Durham, NC). ERa or ERb vector
(0.5 lg) was transfected into OVCAR-3 cells using FuGENE 6 (Roche Applied
Science, Laval, QC) according to the manufacturer’s suggested protocol at
50% confluence on six-well plates. ERa, ERb and control small interfering
RNAs (siRNAs) were synthesized by Bioneer technology (Daejon, South
Korea). BG-1 cells were transfected with siRNA at a final concentration of
100 nmol/l using lipofectamine (Invitrogen, Carlsbad, CA) according to the
manufacturer’s suggested protocol.
Immunoblot and coimmunoprecipitation assay
The cells were seeded at a density of 2 105 cells in 35 mm culture dishes and
cultured for at least 2 days. The cells were washed once with medium, and
serum starved for at least 4 h prior to treatments with leptin. The cells were
washed twice with ice-cold phosphate-buffered saline and lysed in ice-cold
radioimmunoprecipitation assay buffer [150 mM NaCl, 1% nonidet P-40, 0.5%
deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris (pH 7.5), 1 mM
phenylmethylsulfonyl fluoride, 10 g/ml leupeptin and 100 lg/ml aprotinin].
The extracts were placed on ice for 15 min and centrifuged to remove cellular
debris. Nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (PierceBiotech., Rockford, IL) according to the
manufacture’s suggested protocol. The protein concentration of supernatants
was determined using the Bradford assay (Bio-Rad Laboratories, Hercules,
CA). Thirty micrograms of total protein was run on 10% sodium dodecyl
sulfate–polyacrylamide gels and electrotransferred to a nitrocellulose membrane (Amersham Pharmacia Biotech.). The membrane was immunoblotted
using specific primary antibodies at 4°C overnight. After washing, the signals
were detected with horseradish peroxidase-conjugated secondary antibody for
1 h and visualized using the ECL chemiluminescent system (Amersham Pharmacia Biotech.). To determine whether ERa directly interacts with STAT-3,
immunoprecipitation was carried out at 4°C for 1 h using an anti-ERa antibody
and protein A magnetic beads (New England Biolabs). Immunoprecipitates
were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis
followed by western blotting with a phospho-STAT-3 antibody. The reverse
immunoprecipitation was also performed using anti-p-STAT-3 antibody
followed by immunoblot analysis using anti-ERa and ERb.
Reverse transcription–polymerase chain reaction
Total RNA was prepared using the TRIzol reagent (Invitrogen, Burlington,
Ontario, Canada), according to the manufacturer’s instructions. Total RNA
(2.5 lg) was reverse transcribed into first-strand complementary DNA
(Amersham Pharmacia Biotech, Oakville, Ontario, Canada) following the
manufacturer’s procedure. The synthesized complementary DNA was used
as a template for polymerase chain reaction (PCR) amplification. A conventional semi-quantitative PCR amplification was carried out with denaturing for
1 min at 94°C, annealing for 60 s at 58°C, extension for 60 s at 70°C and a final
extension for 15 min at 72°C using a thermal cycler (DNA Thermal Cycler;
Perkin-Elmer, Norwalk, CT). The primers were designed to amplify pS2
mRNA based on the published sequences of human pS2 (10). In addition,
amplification of human glyceraldehyde phosphate dehydrogenase (GAPDH)
was performed using specific primers to rule out the possibility of RNA degradation and was used to control the variation in mRNA amount in PCR
reaction. The primers of pS2 are composed of sense, 5#-TTCTATCCTAATACCATCGACG-3#, and antisense, 5#-TTTGAGTAGTCAAAGTCAGAGC-3#.
The sequences of GAPDH amplification are sense, 5#-ATGTTCGTCATGGGTGTGAACCA-3#, and antisense, 5#-TGGCAGGTTTTTCTAGACGGCAG3#. Real-time PCR was performed using Thermal Cycler Dice Real Time PCR
System (Takara, Japan). The primers used for SYBR Green real-time reverse
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transcription–polymerase chain reaction were as follows: for ERa, sense
primer, 5#-AGCACCCTGAAGTCTCTGGA-3#, and antisense primer, 5#-GATGTGGGAGAGGATGAGGA-3#; for ERb, sense primer, 5#-CAGTTATCACATCTGTATGCGG-3#, and antisense primer, 5#-ACTCCATAGTGATATCCCGA-3#; for GAPDH, sense primer, 5#-GAGTCAACGGATTTGGTCGT-3#,
and antisense primer, 5#-TTGATTTTGGAGGGATCTCG-3#. A dissociation
curve analysis of ERa, ERb and GAPDH showed a single peak. PCRs were
carried out for 40 cycles using the following conditions: denaturation at 95°C
for 5 s, annealing at 57°C for 5 s and elongation at 72°C for 5 s. Mean Ct of the
gene of interest was calculated from triplicate measurements and normalized
with the mean Ct of a control gene, GAPDH.
[3H]thymidine incorporation assay
[3H]thymidine incorporation assay was performed to analyze the proliferative
effect of leptin in BG-1 cell line (17). The cells were plated in 24-well plates at
2 104 cells per well in 0.5 ml medium 199:MCDB105 supplemented with
10% FBS and antibiotics and incubated for 48 h. Before treatment with leptin,
the cells were starved in serum-free media for at least 4 h. After starvation, the
cells were incubated with leptin (100 ng/ml) or estradiol (E2; 10 7 M) in
serum-free media for 24 h. One lCi[3H]thymidine (5.0 Ci/mmol; Amersham
Pharmacia Biotech.) was added at 4 h prior to the termination of the experiments. At the end of the incubation period, the culture medium was removed,
and the cells were washed three times with phosphate-buffered saline and
incubated in 10 % trichloroacetic acid for 20 min at 4°C. The precipitate
was washed twice with methanol and solubilized in 0.1 N sodium hydroxide
and the incorporated radioactivity was measured with a Tri-Carb Liquid Scintillation Analyzer (Model 2100TR; Packard Instrument, Meriden, CT).
3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay
Cell viability was estimated using the 3-(4,5-dimethylthiazole-2-yl)-2,5diphenyl tetrazolium bromide (MTT; Sigma–Aldrich) assay. Cells were seeded
in 96-well plates and incubated for 24 h. To examine the growth stimulatory
effect of leptin, BG-1 cells were treated with leptin for 24 h. On the day of
collection, 50 ll of MTT solution (2 mg/ml in phosphate-buffered saline) was
added to the medium and the cells were incubated at 37°C with for 4 h.
The MTT-containing medium was removed and the cells were solubilized in
dimethyl sulfoxide (100 ll) for 30 min. The optical density at 490 nm
was determined using a microplate spectrophotometer (Fisher Scientific Ltd,
Ottawa, Ontario, Canada).
Luciferase assays
The estrogen response element (ERE)2-tk109-Luc reporter plasmid was provided by Dr J.L.Jameson (Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Medical School, Chicago, IL). One
micrograms of ERE2-luciferease vector was transfected into BG-1 cells using
FuGENE 6 (Roche Applied Science) according to the manufacturer’s suggested protocol when the cells were 50% confluent in six-well plates. After
transfection for 24 h, the cells were treated with leptin for 6 h and extracts were
prepared with luciferase cell lysis buffer (Promega, Madison, WI). Luciferase
activity was measured in the extracts from triplicate samples using the luciferase assay kit (Promega). b-Galactosidase activity was measured using the
b-Galactosidase Enzyme Assay System (Promega). Promoter activity was calculated as luciferase activity/b-galactosidase activity.
Data analysis
Data are shown as the mean ± SD of three individual experiments performed in
triplicate and presented as the mean. Statistical analysis was performed by oneway analysis of variance followed by Dunnett’s test. P , 0.05 was considered
statistically significant.
Results
Inhibitory effect of ER antagonist ICI 182,780 on leptin-induced cell
growth in BG-1 cells
To examine the possible contribution of ER to leptin-stimulated cell
growth of BG-1 cells, we evaluated the effect of ICI 182,780, a pure
ER antagonist, on leptin-induced proliferation by thymidine incorporation and MTT assays. As illustrated in Figure 1A, treatment with
leptin (100 ng/ml) or E2 (10 7 M) induced approximately a 2-fold
increase in cell proliferation as determined by thymidine incorporation assay. Pretreatment with ICI 182,780 completely abolished the
proliferative effects of leptin or E2 (Figure 1A), suggesting that ER
mediates the proliferative effect of leptin in BG-1 cells. The inhibitory
effect of ICI 182,780 on leptin- or E2-induced cell growth was also
Ligand-independent ERa activation by leptin in ovarian cancer
Fig. 1. Effect of ICI 182,780 on leptin-induced cell growth in BG-1 cells. The cells were treated for 24 h with estradiol (E2; 10 7 M) or leptin (100 ng/ml) in the
absence or presence of ICI 182,780 (10 nM). (A) Proliferative index was measured using thymidine incorporation assay. (B) Cell growth was measured using MTT
assay. Data are derived from three experiments and are presented as the mean ± SD; P , 0.05.
confirmed by measuring cellular viability using the MTT assay
(Figure 1B).
Effect of ERa and ERb expression on the cell proliferative effect of
leptin
In the previous study, we demonstrated that leptin can stimulate cell
growth in BG-1 cells but not in other tested ovarian cancer cells,
including OVCAR-3 cells (29). As shown in Figure 2, BG-1 cells
were found to have a substantially higher expression level of ERa
and ERb than other ovarian cancer cells (OVCAR-3 and A2780-3)
and IOSE cells (IOSE-7 and IOSE-80PC).
Transient transfection of OVCAR-3 and A2780 cells with ERa or
ERb was performed to evaluate which isoforms of ER is involved in
the response to leptin. OVCAR-3 and A2780 cells were used because
they have low endogenous ER levels and lack a proliferative response
to leptin or E2 in our cell culture conditions. Transfected cells
were incubated with leptin (100 ng/ml) for 24 h and then assayed
for thymidine incorporation. Leptin and E2 induced a significant increase in the proliferation of ERa-transfected OVCAR-3 and A2780
cells (Figure 3A and B). In addition, the effects of leptin in ERaoverexpressing cells were suppressed by ICI 182,780. To further confirm a direct involvement of ERa in the cell growth, we decreased the
ERa levels in BG-1 cells using ERa-specific siRNA. As expected,
downregulation of ERa, but not ERb, significantly reversed leptininduced growth of BG-1 cells. Similar data were obtained with
a specific ERa antagonist methyl piperidinopyrazole (supplementary Figure 1 is available at Carcinogenesis Online). These results
indicate that leptin-induced cell growth is ERa-dependent in ovarian
cancer cells.
Functional cross talk between leptin and estrogen had been shown
in breast cancer, including that leptin enhanced the estrogen-induced
cell growth (30). In this regard, we examined their interaction on cell
growth in BG-1 ovarian cancer cells. BG-1 cells were treated with
vehicle, leptin, E2 or both for 24 h, and the cell growth was measured.
As shown in Figure 4B, either leptin alone or E2 alone stimulated the
cell growth as shown earlier; moreover, leptin together with E2 had
further stimulation. The result indicated that leptin and E2 did not
completely share the intracellular signal pathway and their cooperation stimulated further cell growth in ovarian cancer cells.
Effect of leptin on the activation of ERa
To test whether leptin can mimic classical ER activation by E2, semiquantitative reverse transcription–polymerase chain reaction was performed to measure the mRNA expression of the E2-responsive pS2
Fig. 2. Expression of ERa and ERb in various ovarian normal and cancer
cells. The mRNA expression of ERa and ERb was determined by real-time
reverse transcription–polymerase chain reaction. IOSE-7 and IOSE-80PC are
IOSE cell line. A2780, SKOV-3, OVCAR-3 and BG-1 cells are ovarian
cancer cell line. MCF-7 is breast cancer cell line.
gene. Stimulation of BG-1 cells with leptin or E2 increased pS2
mRNA levels in a time-dependent manner (Figure 5A). In addition,
we examined whether leptin stimulates the transactivation potential of
ER in BG-1 cells by utilizing the ERE2-luciferase reporter gene construct. As shown in Figure 5B, ERE2-luciferase activity in BG-1 cells
was substantially increased following treatment with leptin. We further evaluated the abundance of cytoplasmic and nuclear ERa in BG-1
cells treated with leptin (100 ng/ml) or E2 (10 7 M). As with E2,
treatment with leptin for 6 h increased the nuclear expression of ERa
and decreased its cytoplasmic levels (Figure 5C).
Effect of STAT-3 activation on the cell proliferative effect of leptin
In the previous study, we demonstrated that leptin increased ERK
activation resulting in enhanced proliferation, whereas inhibition of
the ERK pathway by PD98059 abolished leptin-induced proliferation
of BG-1 cells (29). Considering that leptin exerts its biological function in various tissues through JAK/STAT-3 and PI3K/Akt as well
as ERK (31–34), we further examined the phosphorylation of
STAT-3 and Akt following treatment with leptin in a time-dependent
(100 ng/ml for 5–90 min) and dose-dependent manner (10, 100 and
1000 ng/ml for 30 min). Immunoblot analysis was performed with
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Fig. 3. Effect of overexpression of ERa and ERb on cell growth in OVCAR-3 (A) and A2780 (B) cells. OVCAR-3 and A2780 cells were transfected with ERa
or ERb expression vector using FuGENE 6 reagent to produce ERa or ERb overexpressing cells, respectively. Empty vector transfected and ER-transfected
cells were treated with 10 7 M E2 or 100 ng/ml leptin for 24 h. A proliferative index was measured using thymidine incorporation assay. Data are presented as
the mean ± SD of three experiments; P , 0.05.
Fig. 4. Effect of knockdown of ERa and ERb on cell growth in BG-1 cells. BG-1 cells were transfected with ERa or ERb siRNA using Lipofectamin. (A) Control
siRNA or ER siRNA-transfected BG-1 cells were treated with 100 ng/ml leptin for 24 h. (B) After transfection with siRNA, cells were treated with 10 7 M
estradiol (E2), 100 ng/ml leptin or 10 7 M E2 plus 100 ng/ml leptin for 24 h. Cell growth was measured using MTT assay. Data are presented as the mean ± SD of
three experiments; P , 0.05.
specific antibodies targeting the phosphorylated and total forms of
STAT-3 and Akt. As shown in Figure 6A, treatment with leptin
induced a significant increase in phosphorylated STAT-3 and Akt in
both a dose- and time-dependent manner in BG-1 cells. The involvement of STAT-3 and PI3K in leptin-induced proliferation of BG-1
cells was investigated using a specific STAT-3 inhibitor AG490 and
a PI3K inhibitor LY294002. We observed that treatment with AG490
completely reduced leptin-induced cell growth (Figure 6B), whereas
LY294002 caused a partial inhibition. In addition, we used the inhibitors to test whether leptin-stimulated transcriptional activation of ER
is mediated by the JAK2/STAT-3 and/or PI3K/Akt pathway. Pretreatment with AG490 completely abolished leptin-induced ERE2 luciferase activity in BG-1 cells (Fig. 6C). In contrast, LY294002 partially
inhibited leptin-induced luciferase activity. These results indicate that
leptin-induced growth stimulation and ER transcriptional activation
are mainly mediated by STAT-3 but only partially mediated by PI3K.
Effect of leptin on the interaction between STAT-3 and ERa
To analyze the molecular basis of leptin-induced activation of the STAT3 and ERa signaling pathways, we employed a coimmunoprecipitation
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technique to test for an interaction between ERa and STAT-3. BG-1 cells
were treated with leptin for 30 min, immunoprecipitated with an ERaspecific antibody and the immunoprecipitates were immunoblotted with
a phospho-STAT-3-specific antibody. As shown in Figure 7A, treatment
with leptin increased the amount of phospho-STAT-3 protein coimmunoprecipitated with ERa. An association of ERa and STAT-3 was also
apparent in a reverse immuporecipitation employing STAT-3 antibody
followed by immunoblotting with ERa antibody (Figure 7B). In addition, no significant binding between ERb and STAT-3 was observed.
These results indicate that leptin may induce a direct and functional
protein–protein interaction between ERa and STAT-3 in these cells.
Discussion
Over the past few decades, mounting epidemiological findings have
suggested the influence of obesity on the risk of death from cancer
(35). In addition to its essential role in metabolism and cardiovascular
and renal function, there is convincing evidence that leptin functions
as a mitogen in various cancers including breast, prostate and gastrointestinal cancer (36). Despite these observations, whether leptin
Ligand-independent ERa activation by leptin in ovarian cancer
Fig. 5. Effect of leptin on functional activation of ERa in BG-1 cells. (A) The cells were treated with 10 7 M E2 or 100 ng/ml leptin and the expression of an
estrogen-dependent gene, pS2, was determined by reverse transcription–polymerase chain reaction. The data are representative of four separate experiments. (B)
The cells were transfected with a luciferase vector containing an ERE, treated with E2 (10 7 M) or leptin (100 ng/ml) for 6 h and luciferase activity was quantified.
(C) Effect of leptin on nuclear abundance of ERa in BG-1 cells. The cells were treated with 10 7 M estradiol (E2) or 100 ng/ml leptin for 6 h and the cytoplasmic
and nuclear levels of ERa were determined by western blot analysis. The expression of b-actin and nucleolin was assessed as a control of protein loading.
Fig. 6. Effect of STAT-3 and Akt activation on leptin-induced proliferation and ER transactivation in BG-1 cells. (A) Effect of leptin on the activation of STAT-3
and Akt in BG-1 cells. Western blot analysis was performed following treatment with leptin (100 ng/ml) in a time-dependent manner (5, 15, 30, 60 and 90 min)
or increasing doses of leptin (10, 100 and 1000 ng/ml) for 30 min. Total STAT3 (T-STAT-3) and total Akt were used to normalize the level of phosphorylated
STAT-3 (P-STAT-3) and Akt, respectively. Each blot is representative of at least three independent experiments. (B) Inhibitory effects of LY294002 and AG490 on
leptin-induced proliferation. Following pretreatments with LY294002 (10 lM) or AG490 (100 lM) for 30 min, the cells were treated with leptin (100 ng/ml)
and thymidine incorporation assay was performed. (C) Inhibitory effects of LY294002 and AG490 on leptin-induced ER transcriptional activation. Following
pretreatments with LY294002 (10 lM) or AG490 (100 lM) for 30 min, the cells were treated with leptin (100 ng/ml) and luciferase assay was performed. Data are
presented as the mean ± SD of three experiments; P , 0.05.
plays a role in ovarian cancer remains to be elucidated and the exact
mechanism of the response to leptin is poorly understood. Recently,
we demonstrated that both short and long isoforms of leptin receptors
are expressed in IOSE and its neoplastc counterpart ovarian cancer
cells and that treatment with leptin stimulated the growth of BG-1
cells (29). Moreover, the stimulatory effect of leptin was significantly
abolished in the presence of an ERK inhibitor. In this study, we
demonstrated, for the first time, that ERa is responsible for mediating
leptin-induced cell proliferation in a ligand-independent manner. Furthermore, we showed that leptin-stimulated proliferation is dependent
on the activation of STAT-3 as well as ERK.
In our previous study, we found that leptin exerts its proliferative
action only in E2-sensitive BG-1 cells but not in other ovarian cancer
cells including OVCAR-3 cells which are less sensitive to E2 with
respect to cell growth. In addition, recent studies have suggested
a functional cross talk between leptin and E2/ER system
(26–28,37,38). In this regard, we tested the hypothesis that the estrogen/ER system is implicated in the proliferative effect of leptin in
estrogen-responsive ovarian cancer cells. This postulate was supported by our data that ICI 182,780, a pure ER antagonist, blocked
leptin-induced cell growth in BG-1 cells. The involvement of ER in
leptin-induced signaling is in agreement with recent studies on breast
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Fig. 7. Interaction between p-STAT-3 and ERa. BG-1 cells were treated with
leptin (100 ng/ml) in a time-dependent manner (30, 60, 90 and 120 min).
(A) Cell extracts were immunoprecipitated with an antibody against ERa
separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis
and immunoblotted with a phosphorylated STAT-3 (p-STAT-3) antibody.
(B) In the reverse immunoprecipitation, ERa and ERb were detected by
immunoblot analysis following immunoprecipitation of p-STAT-3.
cancer cells (1). Similarly, Catalano et al. reported that leptin can
amplify E2 signaling via a direct functional activation of ERa and
enhance in situ E2 production in breast cancer cells (10,27).
The two ER isoforms (ERa and ERb) show tissue-dependent expression pattern in normal OSE, benign tumors and malignant ovarian
cancers. ERa is mainly expressed in malignant cancer cells, whereas
ERb is the dominant form in OSE, or benign tumors, suggesting that
the ERa/ERb ratio seems to increase in ovarian cancer as in prostate
and breast cancers (39). In the present study, cell growth was significantly increased in ERa-overexpressing, but not in ERb-overexpressing,
OVCAR-3 and A2780 cells following leptin treatment. In addition,
downregulation of ERa using a specific siRNA completely inhibited
leptin-induced cell growth in BG-1 cells. This phenomenon can explain our previous observations that leptin did not show any proliferative effect in OVCAR-3 with low expression of ERa or SKOV-3
cells bearing mutated ERa. Furthermore, it is noteworthy that overexpression of ERb decreased basal proliferation of OVCAR-3 cells.
Since it has been suggested that ERb plays a protective role antagonizing ERa mitogenic activity, further study will be required to explore whether ERb exerts a similar effect on leptin/ERa-induced
proliferation.
Interestingly, the mechanism by which leptin activates ERa appears
to be ligand independent. Both transcriptional activation of ERa and
leptin-induced cell proliferation were observed in BG-1 cells even
though all the experiments were performed in phenol red-free medium
containing charcoal–dextran-treated serum to avoid an estrogenic effect. Moreover, to rule out the possibility that leptin induces E2 production in BG-1 cells, we measured estrogen levels in the culture
medium following treatment with leptin. Although a recent report
suggested stimulatory effect of leptin on aromatase activity in breast
cancer (27), no significant change in E2 level was observed in leptintreated BG-1 cells when compared with control cells (data not
shown). Considering the accumulating evidence of ER activation in
a ligand-independent manner, these results indicate that leptin may
exert its action on BG-1 cell growth via activation of ERa in a ligandindependent manner. Indeed, ligand-independent ER activation has
previously been reported in ovarian cancers. For example, an interaction between a membrane receptor CD44 and ERa in the absence of
E2 has been suggested. CD44 interaction with hyaluronan recruits
IQGAP1 and induces activation of ERK2 leading to the phosphorylation of both Elk-1 and ERa (40). ERK2-mediated Elk-1/ERa phos-
594
phorylation increases Elk-1/ERE-mediated transcription, as well as
tumor migration via F-actin binding. In addition, in MCF-7 breast
cancer cells, it has been demonstrated that leptin stimulates functional
activation of ERa via ERK1/2 activation (10).
The JAK/STAT signaling pathway regulates diverse biological responses and is associated with many hormones, cytokines and growth
factors. STAT-3, a well-known downstream signaling molecule of
leptin in various tissues, has been shown to regulate the expression
of several genes related to cell cycle, proliferation and apoptosis.
Consequently, changes in the activity of STAT-3 have been implicated
in cell transformation and cancer progression (41,42). In the present
study, we evaluated whether STAT-3 is involved in leptin-induced
signaling. As in other cell systems, leptin stimulated the activation
of STAT-3 in a time- and dose-dependent manner, and a specific STAT3 inhibitor (AG490) significantly reduced leptin-stimulated proliferation of BG-1 ovarian cancer cells. These results suggest that STAT-3
may play a role in leptin/ERa-mediated proliferation of BG-1 cells.
Our observation that leptin can increase the interaction between
ERa and STAT-3 in BG-1 cells is in accordance with other findings
that activated ERa can physically interact with the STATs family of
transcription factors in various cell systems (43,44). Several lines of
evidences have also demonstrated an indirect cross talk between
STAT-3 and ER signaling via other transcription factors (44,45). In
the present study, treatment with leptin resulted in an increase in cell
growth equal to that of E2. However, the increase in transcriptional
activation of an ERE by leptin was only about 2-fold, whereas E2
stimulated luciferase activity up to 3-fold. Together these results suggest that, in addition to a pathway involving ERE activation, alternative
mechanism(s) may be required to fully mediate the effects of leptin.
One such mechanism may be the regulation of STAT-3-responsive
genes via the interferon-gamma activation site promoter. Although further experimentation is required to confirm this hypothesis, leptininduced phosphorylation of STAT-3 and its increased binding to ERa
may be involved in this type of regulation. Indeed, in neuroblastoma
cells, increased interaction between STAT-3 and ERa induced activation of STAT-3-dependent transcription via interferon-gamma activation site promoter (45). Wessler et al. recently found ERa binding to
STAT-3 in breast cancer cells and demonstrated that expression of ERa
increases leptin-induced STAT-3 activity (46).
The leptin signaling is thought to be transmitted by multiple pathways, including STAT-3 (8,9), ERK (8,10,27) and PI3K (11) pathways. The concomitant activations of the multiple pathways by leptin
were observed in various cancer cells, including acute myelogenous
leukemia and endometrial, colon and liver cancer (47–50). For example, leptin increases the proliferation and mobility capabilities of renal
cell carcinoma cells Caki-2 via upregulating the activation of both
ERK and STAT3, suggesting that the cross talking of JAK-STAT3 and
ERK1/2 pathways in renal cell carcinoma cells (49). In hepatocellular
carcinoma cells, it has been demonstrated that concomitant activation
of the STAT-3, PI3K and ERK signaling is involved in the leptinmediated promotion of invasion and migration (48). In our previous
study (29) and the present study, we found that ERK, STAT-3 and
PI3K pathway are fully or partially required for leptin-induced ER
transactivation and proliferation in ovarian cancer cells. Possible cross
talk between ERK, STAT-3 and PI3K signaling pathways remains to
be further investigated.
Despite ER status is recognized as a prognostic factor for breast
cancer and a critical reference for breast cancer therapy, no clear
relationship between ER expression and tumor characteristics has
been noted in ovarian epithelial cancers. Considering that ER is expressed in up to 60% of ovarian epithelial tumors and complex factors
are associated with ER signaling, the identification of regulatory factors for estrogen/ER system in ovarian cancer and the subsequent
characterization of the molecular mechanisms underlying the action
of the factors are required. The present study, for the first time, suggests that leptin abundance environment can be a stimulating factor of
cell growth in ER-positive ovarian cancer. Thus, this finding may
provide advanced information regarding better attention to the ERa
status in ovarian cancer patients with obesity.
Ligand-independent ERa activation by leptin in ovarian cancer
Supplementary material
Supplementary Figure 1 can be found at http://carcin.oxfordjournals.org/
Funding
Canadian Institutes for Health Research (to P.C.K.L.); National Research Foundation of Korea (NRF) grant (to J.H.C.) funded by the
Korea government (MEST) (No. 2009-0068979).
Acknowledgements
The expression vectors for human ERa and ERb were generously provided by
Dr B.S.Katzenellenbogen (Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign) and Dr P.P.McDonnell
(Department of Pharmacology and Cancer Biology, Duke University Medical
Center, Durham, NC), respectively. The ERE luciferase expression vector was
provided by Dr J.L.Jameson (Division of Endocrinology, Metabolism and
Molecular Medicine, Northwestern University Medical School, Chicago, IL).
We are thankful to Dr Auersperg (University of British Columbia, Vancouver,
British Columbia) and Dr Andrew Godwin (Fox Chase Cancer Center, Philadelphia, PA) for providing IOSE-80 and IOSE-80PC cell lines. We thank Dr
K.S.Korach (National Institute of Environmental Health Sciences, National
Institutes of Health, Research Triangle Park, NC) for providing the BG-1 cell
line.
Conflict of Interest Statement: None declared.
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Received June 30, 2010; revised November 30, 2010;
accepted December 12, 2010