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Endocrinology |
Blood & Cancer Research Program, The Hospital for Sick Children [A. E., M. L.], Division of Reproductive Science, The Samuel Lunenfeld Research Institute, Mt. Sinai Hospital [T. J. B.], and Department of Zoology [A. E., T. J. B.], Institute of Medical Sciences [S. K. J.], Department of Obstetrics and Gynecology [T. J. B., M. L.], and Department of Immunology [M. L.], University of Toronto, Toronto, Ontario M5G 1X8, Canada
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-dihydrotestosterone (DHT), but not
estradiol-17ß or progesterone, for 60 h down-regulated the
expression of mRNA for TGF-ß receptors I and II (TßR-I and
TßR-II), betaglycan, and endoglin but had no effect on
TGF-ß1 mRNA levels. Androgen receptor (AR) mRNA expression in HEY
cells was compared to other ovarian cancer cell lines. OVCAR-3 cells
expressed AR mRNA levels similar to that of androgen-responsive LNCaP
prostate cancer cells, whereas SKOV-3 and HEY cells expressed only 3
and 0.01%, respectively. Western blot analysis and saturation binding
assays confirmed the expression of AR protein in these three cell
lines, but at the limit of detection in SKOV-3 and HEY cells. Treatment
of SKOV-3 and HEY cells for 24 h with 1–50 nM DHT
resulted in a dose-dependent down-regulation of TßR-II mRNA. The AR
antagonist hydroxyflutamide did not reverse the effect of DHT on SKOV-3
cells but by itself down-regulated TßR-II mRNA. This apparent
androgen-mimetic action of hydroxyflutamide and the ability of SKOV-3
and HEY cells to respond to DHT may be due to their expression of
AR-associating protein 70, an AR co-activator reported to amplify AR
transactivation and to result in agonist activity of AR antagonists.
DHT was able to reverse TGF-ß1 growth-inhibitory action in SKOV-3
cells and in a primary culture of ovarian cancer cells derived from
ascites. Thus, androgens may promote ovarian cancer progression in part
by decreasing TGF-ß receptor levels, thereby allowing ovarian cancer
cells to escape TGF-ß1 growth inhibition. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A mechanism by which steroid hormones might stimulate the growth and/or progression of ovarian cancer is by blocking the action of TGF-ß,4 a potent inhibitor of ovarian epithelial cell growth in culture (10 , 11) . Development of resistance to TGF-ß has been observed in many cancers (12) and can occur gradually during the multistep process of tumor progression (12 , 13) .
TGF-ßs (TGF-ß1, -ß2, and -ß3) mediate their effects on target cells by forming a heteromeric complex with transmembrane serine/threonine kinase receptors, TßR-II and TßR-I (14) . Binding of TGF-ß to constitutively active TßR-II leads to recruitment, interaction, and phosphorylation of TßR-I, which then propagates the signal to downstream Smad proteins (14) . In addition, two other proteins can be found in TGF-ß receptor complexes, betaglycan, and endoglin. Betaglycan binds TGF-ß1, -ß2, and -ß3 directly and is referred to as type III receptor; it presents ligand to TßR-II but does not have a recognizable signaling domain (15) . Endoglin is a dimeric transmembrane glycoprotein that binds TGF-ß1 and -ß3 via its association with TßR-II (16 , 17) . Overexpression of endoglin has been shown to modulate cellular responses to TGF-ß1 (18) . Thus, the down-regulation or loss of functional expression of receptors and/or signaling mediators for TGF-ß would allow cancer cells to become resistant to this potent growth inhibitor.
In the present study, we tested the effects of steroid hormones on the expression of TGF-ß and its receptors in ovarian cancer cells. As steroid hormones regulate ovarian function, we hypothesize that an imbalance in their action could promote ovarian cancer progression by decreasing the action of TGF-ß.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All cell cultures were maintained at 37°C in a humidified 5% CO2 atmosphere. sFBS was substituted for normal FBS 48 h before beginning experiments. DHT, estradiol-17ß, and progesterone (Sigma Chemical Co., St. Louis, MO) and OH-FLUT (Schering-Plow, Madison, NJ) were dissolved in ethanol before addition to culture medium. The final ethanol concentration was 0.001%. TGF-ß1 (R&D Systems, Minneapolis, MN) was diluted in culture media and stored at 4°C before addition to DHT-treated cultures.
Primary ovarian cancer cells (OVCAS-16) were isolated from ascites of a patient diagnosed with papillary adenocarcinoma of the ovary, as described by Hirte et al. (19) . Cells were propagated in DMEM/F12 media (without phenol red) supplemented with 3% sFBS, 5 µg/ml insulin, 5 x 10-5 M ethanolamine, 5 ng/ml EGF, 5 x 10-5 M phosphoethanolamine, 10 µg/ml transferrin, penicillin-streptomycin, and fungizone. The cells were shown by flow cytometry to express the epithelial ovarian cancer antigens 2G3 and M2A (19 , 20) . These monoclonal antibodies were provided by Dr. A. Marks. OVCAS-16 cells were maintained in the presence or absence of 10 nM DHT added daily from the time of isolation. Experiments were performed within 2 weeks and after one passage.
RNA Preparation, cDNA Synthesis, and Quantitative Analysis of
mRNA by PCR
Cell cultures were washed in ice-cold PBS or serum-free medium, and
total cellular RNA was isolated by the guanidinium thiocyanate
extraction method (21)
. RNA concentration in all samples
was measured in two independent determinations and at two dilutions, as
its accuracy is essential for quantitative RT-PCR. cDNAs for TGF-ß
receptors, AR, ARA70, and ß-actin were generated using oligo(dT)
primer, whereas an internal primer was used to generate TGF-ß1 cDNA
to increase the specificity of the reaction (22)
.
Quantitative analysis of PCR products was performed according to Murphy et al. (23) . Selected primers for amplification of TGF-ß1, TGF-ß receptors, and ß-actin have been described previously (22) . Three sets of AR-specific primers spanning exons 1–4, 4–7, and 1–7 selected from the cDNA sequence (24) were originally tested in the AR-positive prostate cancer cell line LNCaP. As identical results were obtained with all three primer sets, forward primer 5'-GTCAAAAGCGAAATGGGCCCC-3' and reverse primer 5'-CTTGGTGAGCTGGTAGAAGCG-3', which yielded a 1031-bp PCR product, were chosen for the current study. Primers for ARA70, selected from the cDNA sequence (25) , were forward primer 5'-GCTTTGCAGAGTGTGTGTGTG-3' and reverse primer 5'-GGGGAAGTTATGTTCCTCCTG-3', which yielded a product of 509 bp. PCR amplification conditions were as described previously (22) with an annealing temperature of 58°C for all except AR (64°C), and 28 cycles for all except betaglycan and AR (30 cycles). Aliquots (10 or 20 µl) of PCR products were fractionated on 4–20% gradient polyacrylamide gels in 0.5% Tris-borate/EDTA buffer (Helixx Technologies, Scarborough, Ontario, Canada) or on 1% agarose gels. Gels were transferred to nylon membranes and hybridized with specific 32P-dATP-labeled or 3' end-labeled digoxigenin-11-ddUTP internal probes. The probes were as described previously (18 , 22) with the addition of the internal probe, 5'-GCATCCTGGAGTTGACATTGG-3', for AR and 5'-TGATAAGCCACTCCGACAAGG-3' for ARA70. The blots were analyzed by computer-assisted densitometry as reported previously (18 , 22) . Data were plotted as log of densitometric units versus log of equivalent RNA concentration to identify a linear range of exponential amplification. Linear regressions were obtained using SigmaPlot scientific graphing software (version 2.00, Jandel Corp., San Rafael, CA). For TGF-ß receptor mRNA expression, the amount of total RNA estimated from the linear range of amplification after steroid treatment was expressed as a percentage of that measured in cells treated with ethanol vehicle (control). All measurements were corrected for ß-actin expression.
Cytosol Preparation and Analysis of AR Binding and Protein
Expression
Cytosol extracts were prepared as described previously
(26)
. For Western blot analysis, aliquots of cytosol
protein (1–50 µg) were fractionated by 8% SDS-PAGE and transferred
to Immobilon polyvinylidene difluoride membrane (Millipore Corp.,
Bedford, MA). The membranes were preincubated for 1 h in blocking
solution (50 mM Tris-HCl, 0.15 M NaCl, pH 7.4,
0.2% (v/v) Tween 20, and 5% powdered skim milk) followed by 1 h
with 1 µg/ml affinity-purified polyclonal antibody to AR, PAR-1
(27)
. Immunoreactive proteins were visualized with
horseradish peroxidase-labeled goat anti-rabbit antisera (Amersham
Pharmacia Biotech, Oakville, Ontario, Canada) at 1:10,000
dilution in blocking solution for 30 min at room temperature and
detected by enhanced chemiluminescence (Amersham Pharmacia Biotech) on
Kodak X-OMAT X-ray film.
Androgen saturation binding assays were performed as reported previously (26) . All samples contained 1 µM triamcinolone acetonide to block possible binding of [3H]R1881 to progesterone or glucocorticoid receptor (28) .
Cell Proliferation Assays
SKOV-3 proliferation was determined by the MTT (Sigma) reduction method
as described previously (26)
. Cells were treated with 10
nM DHT or vehicle daily beginning 24 h prior to
seeding into 96-well microtiter plates at 2500 cells/well. At time 0
(24 h after seeding) and at 48 h, 5 pM TGF-ß1
(optimal dose for SKOV-3 inhibition) or vehicle was added to each well.
MTT reduction was quantified using 570 nm absorbance with an ELISA
microplate reader (µQuant Biomolecular Spectrophotometer, BIO-TEK
Instruments, Winooski, VT). Results were expressed as the
mean ± SE of 6–8 replicates.
Proliferation of the primary ovarian cancer cells (OVCAS-16) was assessed by counting the number of viable cells, as these cells did not reduce the MTT reagent. Cells maintained in the presence or absence of 10 nM DHT or vehicle were seeded at 15,000 cells/well in 24-well plates. TGF-ß1 (25 pM, optimal dose for OVCAS-16 inhibition) and/or 10 nM DHT was added every 24 h during proliferation assays. Cell counts were obtained using a hemacytometer, with four replicates per treatment group.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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However, the autoradiograms from a representative
experiment revealed that DHT down-regulated mRNA for TßR-I, -RII,
betaglycan, and endoglin, whereas estradiol-17ß and progesterone did
not (Fig. 1B)
. The expression of ß-actin mRNA was
unaffected by treatment with steroids, suggesting that a concentration
of 500 nM had no nonspecific inhibitory effects
on mRNA synthesis.
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. TGF-ß receptor mRNA levels within the exponential
range were quantified for each treatment and expressed as percentage of
that measured in control cells. Expression of steady-state mRNA levels
of TßR-I, TßR-II, betaglycan, and endoglin were markedly reduced to
9–18% that of control cells. Treatment of HEY cells with
estradiol-17ß or progesterone did not affect mRNA levels for any of
the TGF-ß receptors (Fig. 1C)
.
Variable Expression of AR in Ovarian Cancer Cell Lines
Because androgenic responses should be mediated by AR, we examined its
expression in HEY cells, as well as in several other ovarian cancer
cell lines. RNA was extracted from HEY, SKOV-3, OVCAR-3, HOC-7, and
OVCA-429 ovarian cancer cell lines, and AR mRNA was detected by RT-PCR.
The AR-positive LNCaP and AR-negative DU-145 prostate cancer cell lines
were included as positive and negative controls, respectively. A
1031-bp PCR product, corresponding to exons 1–7 of AR, was amplified
in all of the ovarian cancer cell lines tested (Fig. 2A);
however, the apparent levels of expression were variable.
High levels of AR mRNA were observed in LNCaP and OVCAR-3 cells, and
intermediate levels were found in SKOV-3 cells. HEY, HOC-7, and
OVCA-429 cells gave markedly less intense signals and required 100 ng
instead of 20 ng per lane for detection (Fig. 2A)
. As
expected, no AR mRNA expression was detected in DU-145 cells (Fig. 2A)
.
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. A linear range of exponential
amplification was obtained with less than 0.4 ng of equivalent RNA for
both LNCaP and OVCAR-3 cells, whereas the linear range of amplification
for SKOV-3 cells was 0.8–20 ng of equivalent RNA. In comparison, HEY
cells had barely detectable AR expression (Fig. 2B)
. By
determining the relative amount of RNA needed to obtain similar
densitometric units, SKOV-3 and HEY cells were found to express 3 and
0.01%, respectively, of the level of AR mRNA measured in OVCAR-3 cells
(Fig. 2C)
. OVCAR-3 cells expressed levels of AR mRNA similar
to that expressed by LNCaP cells. DU-145 cells did not express
detectable AR mRNA. ß-Actin levels were comparable for all cell
lines, indicating an equivalent amount of RNA was amplified (Fig. 2C)
.
Cytosol extracts from ovarian cancer cell lines were analyzed for AR
protein by Western blot analysis and by saturation androgen binding
assay (Fig. 3
and Table 1
). LNCaP and DU-145 cells were included as positive and negative
controls, respectively. Western blot analysis revealed a 110-kDa
immunoreactive protein in OVCAR-3 and LNCaP cells, whereas no AR
immunoreactive protein was detected in DU-145, HOC-7, or OVCA-429
cells, even at higher protein concentration (Fig. 3)
. A 110-kDa
AR-immunoreactive protein was detected in SKOV-3 and HEY cells with 50
µg of cytosol protein (Fig. 3)
.
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. The mean androgen binding capacity in
OVCAR-3 cells was approximately 7% of that measured in the AR-positive
LNCaP cells (Table 1)
. SKOV-3 and HEY cells had much lower levels of
binding activity, near the limit of sensitivity of the assay (Table 1)
.
Low-Dose DHT Down-Regulates TßR-II mRNA Expression in Several
Ovarian Cancer Cell Lines
Because TßR-II is the ligand-binding receptor and its expression and
function are more frequently altered in many human cancers
(12)
, we examined the effect of DHT treatment on its
expression in ovarian cancer cell lines. OVCAR-3, SKOV-3, and HEY cells
were treated with ethanol vehicle or with 1, 10, or 50
nM DHT, and TßR-II mRNA levels were compared by
quantitative RT-PCR. LNCaP and DU-145 prostate cancer cells were
included as controls. Autoradiograms from a representative experiment,
performed after 24 h of DHT treatment, are shown in Fig. 4
. A linear range of exponential amplification for TßR-II mRNA
expression was detected between 0.08 and 10 ng of equivalent RNA per
lane (Fig. 4)
. Reduction in TßR-II mRNA levels was observed in SKOV-3
and HEY cells with 1 and 10 nM DHT (Fig. 4B)
.
With OVCAR-3 cells, the effect was less apparent, whereas with DU-145
cells, no effect was noted, as expected (Fig. 4B)
. LNCaP
cells did not express detectable TßR-II mRNA (Fig. 4A)
, in
agreement with previous reports (29)
. Androgen treatment
did not alter ß-actin mRNA levels in the different cell lines tested,
confirming the specificity of the TßR-II down-regulation (Fig. 4, A and B)
.
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. DHT down-regulated the expression of TßR-II mRNA in a dose-dependent
manner. Treatment with 1, 10, or 50 nM DHT down-regulated
the expression of TßR-II mRNA levels by 30, 80, and 90% in HEY cells
and by 70, 90, and 99%, respectively in SKOV-3 cells measured 24 h after addition of DHT to the culture medium (Fig. 5)
. With OVCAR-3
cells, a decrease in TßR-II mRNA expression was only noted after
treatment with 50 nM DHT; however, this did not attain
statistical significance (Fig. 5)
. By 48 h after addition of DHT
to the culture medium, the suppression of TßR-II mRNA levels was no
longer observed, as shown for SKOV-3 cells (Fig. 5)
.
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, suggesting that OH-FLUT may act as an AR agonist in SKOV-3 cells.
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Expression of ARA70 mRNA was detected by RT-PCR as a 509-bp product in
all ovarian cancer cell lines and in the two prostate cancer cell lines
tested (Fig. 7A).
A linear range of amplification was attained between 3.2
and 400 pg of equivalent RNA with all cell lines, which expressed
similar levels of ARA70 (Fig. 7, B and C)
.
ß-Actin mRNA levels were comparable for all cell lines (Fig. 7C)
.
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Maintenance of SKOV-3 cells in 10
nM DHT alone had no effect on growth but
prevented the inhibitory effect of TGF-ß1.
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. These values were significantly different from the
vehicle control group (P < 0.05).
Maintenance of these cells in 10 nM DHT did not
have a significant effect on growth; however, it prevented the
inhibition by TGF-ß1 at all time points, resulting in cell numbers
that were not significantly different from control (Fig. 8B)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ability of DHT to down-regulate TßR-II mRNA levels in SKOV-3 and HEY cells was somewhat surprising given the near-nondetectable levels of AR expressed by these cells. Our findings that high levels of estradiol or progesterone did not inhibit expression of TGF-ß receptor mRNAs in HEY cells indicate that the effect of DHT is specific. Although we were unable to block DHT-induced down-regulation of TßR-II mRNA expression with OH-FLUT, this cannot be taken as evidence of an AR-independent mechanism, as treatment of cells with the AR antagonist alone also resulted in decreased TßR-II expression.
The ability of HEY and SKOV-3 cells to respond to DHT, and the apparent androgen-mimetic action of OH-FLUT on TßR-II mRNA expression, may involve the actions of ARA70. This putative coactivator has a predilection for AR (25) and is highly expressed in ovarian cancer cell lines tested here and in ovarian cancer tissue.5 Expression of ARA70 in prostate cancer cells has been shown to increase AR transactivation potential by approximately 10-fold (25) , although the magnitude of this effect is controversial (33 , 34) . Thus, in ovarian cancer cell lines expressing low AR levels, ARA70 could increase their sensitivity to physiologically relevant levels of androgens. Furthermore, the observation that OH-FLUT acts as an androgen agonist in ovarian cancer cells is consistent with the finding by Miyamoto et al. (30) that ARA70 promotes agonist activity by antiandrogens when overexpressed in transfected DU-145 prostate cancer cells. These data raise the possibility that modulation of TßR-II mRNA by androgens may include potentiation of transcription involving ARA70, leading to increased androgen sensitivity of ovarian cancer cells.
Our findings of low levels of AR expression in ovarian cancer cell lines are compatible with those of Lau et al. (35) , who recently reported little or no AR mRNA expression in ovarian cancer cells by semiquantitative RT-PCR. We detected AR mRNA in all five ovarian cancer cell lines tested, likely because our quantitative RT-PCR method is more sensitive, due to detection of PCR products by hybridization with labeled AR probes. Two of our cell lines, HOC-7 and OVCA-429, expressed very low levels of AR mRNA and barely detectable levels of receptor protein, at the limit of sensitivity of both Western blot and binding assays.
Whereas normal ovarian surface epithelium cells respond to TGF-ß by growth inhibition, many immortalized ovarian cancer cell lines are relatively resistant (10 , 32) . The growth inhibition of SKOV-3 cells in this study was not observed until 96 h after initiation of TGF-ß1 treatment. In contrast, growth inhibition by TGF-ß1 was observed after 24 h in ovarian cancer cells isolated from patient ascites. Hurteau et al. (11) have shown that 19 of 20 primary ovarian cancer cells isolated from ascites fluid of patients were growth inhibited by TGF-ß1. These findings were confirmed by Havrilesky et al. (32) , who reported that 10 of 10 primary cultures were growth inhibited by TGF-ß1. TGF-ß has been shown to be present in ascites derived from patients with epithelial ovarian carcinoma (36) . Furthermore, androgens are produced and secreted by ovarian adenocarcinomas into the ascites fluid (37) and may decrease the expression of TGF-ß receptors on tumor cells and alter their responses to TGF-ß. The down-regulation of TßR-II mRNA by androgens requires sustained levels of DHT, as indicated by return to control levels 48 h after addition of DHT to SKOV-3 cell cultures. Thus, once removed from the ascites and cultured in the absence of androgens, ovarian cancer cells may regain their sensitivity to TGF-ß.
In summary, our data provide a novel mechanism whereby androgens may act to promote ovarian cancer cell growth and progression. The androgen-induced down-regulation of TGF-ß receptors occurred in ovarian cancer cells that expressed very low AR levels. Further studies are therefore warranted to define the role of AR co-activators, such as ARA70, in modulating the sensitivity of ovarian cancer cells to DHT.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from the
National Cancer Institute of Canada and by a grant from the Natural
Sciences and Engineering Research Council of Canada. A. E. was
supported by a GENESIS Foundation Studentship and by an Educational
Grant from the Division of Gynecological Oncology, the Princess
Hospital Margaret Hospital University Health Network. M. L. is a Terry
Fox Research Scientist of the National Cancer Institute of Canada. 
2 Present address: Department of Obstetrics and
Gynecology & Women’s Health, New Jersey Medical School, University of
Medicine and Dentistry of New Jersey, 185 South Orange Avenue,
Newark, NJ 07103.
3 To whom requests for reprints should be
addressed, at Blood and Cancer Research Program, The Hospital for Sick
Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada.
Phone: (416) 813-6258; Fax: (416) 813-6255; E-mail: mablab{at}sickkids.on.ca
4 The abbreviations used are: TGF-ß,
transforming growth factor ß; AR, androgen receptor; ARA70,
AR-associating protein 70; DHT, 5-dihydrotestosterone; FBS, fetal
bovine serum; sFBS, charcoal-stripped FBS; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
OH-FLUT, hydroxyflutamide; RT-PCR, reverse transcription-PCR; TßR-I,
TGF-ß receptor I.
5 P. V. C. Rittenberg, P. A. Shaw, and T. S.
Brown. Activation of androgen receptor associated protein (ARA 70)
expression in ovarian cancer, manuscript in preparation.
Received 7/ 7/99. Accepted 12/13/99.
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