Aldo-keto reductase 1C3 expression in MCF-7 cells reveals roles in steroid hormone and prostaglandin metabolism that may explain its over-expression in breast cancer

Article abstract of DOI:10.1016/j.jsbmb.2009.12.009

Aldo-keto reductase (AKR) 1C3 (type 5 17β-hydroxysteroid dehydrogenase and prostaglandin F synthase), may stimulate proliferation via steroid hormone and prostaglandin (PG) metabolism in the breast. Purified recombinant AKR1C3 reduces PGD₂ to 9α,11β-PGF₂, Δ⁴-androstenedione to testosterone, progesterone to 20α-hydroxyprogesterone, and to a lesser extent, estrone to 17β-estradiol. We established MCF-7 cells that stably express AKR1C3 (MCF-7-AKR1C3 cells) to model its over-expression in breast cancer. AKR1C3 expression increased steroid conversion by MCF-7 cells, leading to a pro-estrogenic state. Unexpectedly, estrone was reduced fastest by MCF-7-AKR1C3 cells when compared to other substrates at 0.1 μM. MCF-7-AKR1C3 cells proliferated three times faster than parental cells in response to estrone and 17β-estradiol. AKR1C3 therefore represents a potential target for attenuating estrogen receptor α induced proliferation. MCF-7-AKR1C3 cells also reduced PGD₂, limiting its dehydration to form PGJ₂ products. The AKR1C3 product was confirmed as 9α,11β-PGF₂ and quantified with a stereospecific stable isotope dilution liquid chromatography-mass spectrometry method. This method will allow the examination of the role of AKR1C3 in endogenous prostaglandin formation in response to inflammatory stimuli. Expression of AKR1C3 reduced the anti-proliferative effects of PGD₂ on MCF-7 cells, suggesting that AKR1C3 limits peroxisome proliferator activated receptor γ (PPARγ) signaling by reducing formation of 15-deoxy-Δ^12,14-PGJ₂ (15dPGJ₂).

Full text of DOI:10.1016/j.jsbmb.2009.12.009

Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 177–187
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry and Molecular Biology
journal homepage: www.elsevier.com/locate/jsbmb
Aldo-keto reductase 1C3 expression in MCF-7 cells reveals roles in steroid
hormone and prostaglandin metabolism that may explain its over-expression
ଝ,ଝଝ
in breast cancer
∗
Michael C. Byrns, Ling Duan, Seon Hwa Lee, Ian A. Blair, Trevor M. Penning
Centers of Excellence in Environmental Toxicology and Cancer Pharmacology, Department of Pharmacology, University of Pennsylvania School of Medicine,
130C John Morgan Bldg, 3620 Hamilton Walk, Philadelphia, PA 19104-6084, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 September 2009
Received in revised form
Aldo-keto reductase (AKR) 1C3 (type 5 17␤-hydroxysteroid dehydrogenase and prostaglandin F syn-
thase), may stimulate proliferation via steroid hormone and prostaglandin (PG) metabolism in the breast.
4
Purified recombinant AKR1C3 reduces PGD2 to 9␣,11␤-PGF2, ꢀ -androstenedione to testosterone, pro-
1
5 December 2009
gesterone to 20␣-hydroxyprogesterone, and to a lesser extent, estrone to 17␤-estradiol. We established
MCF-7 cells that stably express AKR1C3 (MCF-7-AKR1C3 cells) to model its over-expression in breast
cancer. AKR1C3 expression increased steroid conversion by MCF-7 cells, leading to a pro-estrogenic
state. Unexpectedly, estrone was reduced fastest by MCF-7-AKR1C3 cells when compared to other sub-
strates at 0.1 ␮M. MCF-7-AKR1C3 cells proliferated three times faster than parental cells in response
to estrone and 17␤-estradiol. AKR1C3 therefore represents a potential target for attenuating estrogen
receptor ␣ induced proliferation. MCF-7-AKR1C3 cells also reduced PGD2, limiting its dehydration to
form PGJ2 products. The AKR1C3 product was confirmed as 9␣,11␤-PGF2 and quantified with a stere-
ospecific stable isotope dilution liquid chromatography–mass spectrometry method. This method will
allow the examination of the role of AKR1C3 in endogenous prostaglandin formation in response to
inflammatory stimuli. Expression of AKR1C3 reduced the anti-proliferative effects of PGD2 on MCF-7
cells, suggesting that AKR1C3 limits peroxisome proliferator activated receptor ␥ (PPAR␥) signaling by
Accepted 17 December 2009
Keywords:
1
7␤-Hydroxysteroid dehydrogenase
Prostaglandin F synthase
Prostaglandin D2
Estrogen receptor
Peroxisome proliferator activated receptor
␥
1
2,14
reducing formation of 15-deoxy-ꢀ
-PGJ2 (15dPGJ2).
©
2010 Elsevier Ltd. All rights reserved.
1
. Introduction
first, the primary enzymes thought to convert estrone to 17␤-
estradiol are 17␤-HSD types 1 and 7 [1,2]. The 17␤-HSD enzyme
thought to convert ꢀ -androstenedione to testosterone in breast
4
In post-menopausal women, proliferation of hormone-
dependent breast cancer is driven by local production of estrogens
1]. The efficacy of aromatase inhibitors against estrogen receptor
(ER␣) positive breast cancer demonstrates that blockade of
is aldo-keto reductase (AKR) 1C3 (also known as type 5 17␤-HSD
or prostaglandin F synthase) [1–3]. Testosterone formed would
then undergo aromatization to 17␤-estradiol. It therefore appears
that reductive 17␤-HSDs, including AKR1C3, represent important
targets for prevention or treatment of estrogen-dependent breast
cancer.
[
␣
local estrogen production is effective for treatment and pre-
vention of breast cancer [1,2]. Production of 17␤-estradiol from
4
ꢀ
-androstenedione requires both aromatase and a reductive
1
7␤-hydroxysteroid dehydrogenase (HSD) [2]. If aromatase acts
Blockade of prostaglandin biosynthesis is another strategy for
prevention of breast cancer. Regular use of non-steroidal anti-
inflammatory drugs (NSAIDs) has been associated with a reduced
risk of breast cancer [4–6]. Ibuprofen and celecoxib effectively pre-
vent mammary tumors in rodents [7]. Celecoxib is in early clinical
trials as an adjuvant for treatment of human breast cancer along
with the aromatase inhibitor, exemestane [8]. NSAIDs act, in part,
by inhibiting prostaglandin-dependent upregulation of aromatase,
thereby limiting local production of estrogen [9]. The AKR1Cs are
also inhibited by NSAIDs [10,11], and these enzymes represent
alternative targets for the anti-cancer effects of these drugs.
ଝ
A preliminary account of some of this work was reported in a review article (M.C.
Byrns, T.M. Penning, Type 5 17␤-hydroxysteroid dehydrogenase/prostaglandin F
synthase (AKR1C3): role in breast cancer and inhibition by non-steroidal anti-
inflammatory drug analogs, Chem. Biol. Interact. 178 (1–3) (2009) 221–227).
ଝଝ
Supported by RO1-CA90744 and P30-ES013508 awarded to T.M.P. and
UO1ES016004 and RO1CA09101 awarded to I.A.B. MCB was funded by NIH training
grants T32-DK007314-25 and T32-HD007305-22.
∗ Corresponding author. Tel.: +1 215 898 9445; fax: +1 215 573 2236.
E-mail address: penning@upenn.edu (T.M. Penning).
0
960-0760/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jsbmb.2009.12.009

1
78
M.C. Byrns et al. / Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 177–187
AKR1C3 is expressed in normal breast tissue, its upregu-
2.2. Stable transfection of MCF-7 cells
lation has been detected in 65–85% of breast cancer tissues,
and its upregulation is associated with poor prognosis [12–14].
Purified recombinant AKR1C3 reduces ꢀ -androstenedione to
Human MCF-7 hormone-dependent breast cancer cells were
maintained in RPMI media supplemented with 10% heat-
inactivated fetal bovine serum (FBS, HyClone Laboratories,
Inc., Logan, UT), 2% l-glutamate, and 100 units/mL penicillin/
streptomycin.
4
testosterone, progesterone to 20␣-hydroxyprogesterone, desoxy-
corticosterone to 20␣-hydroxy-desoxycorticosterone and, to a
lesser extent, estrone to 17␤-estradiol [3,15,16]. These reac-
tions have been observed in HEK293 cells that over-express
AKR1C3 [16,17]. Because of the lower rate of estrone reduction
The cDNA encoding AKR1C3 was excised from a pcDNA-3 plas-
mid [28] with BamHI and ApaI. A pLNCX2 retroviral vector was
linearized with StuI, and the AKR1C3 cDNA was inserted using
a Rapid DNA ligation kit (Roche Diagnostics, Indianapolis, IN).
Escherichia coli DH5␣ cells were transformed with the AKR1C3-
pLNCX vector, and the amplified vector was purified using a Plasmid
Maxi kit (Qiagen, Valencia, CA). This vector was added to PT67 pack-
aging cells using FuGENE 6 (Roche Diagnostics). Media from these
cells was collected and added to MCF-7 cells. Stable transfectants
were selected with media containing geneticin (0.5 mg/mL). Iso-
lated clones were phenotyped by RT-PCR and immunoblot assay.
Cells stably expressing AKR1C3 were maintained in RPMI media
containing 0.25 mg/mL geneticin.
4
relative to ꢀ -androstenedione and progesterone reduction, it
was proposed that for breast cancer, the most important reac-
4
tion catalyzed by AKR1C3 is reduction of ꢀ -androstenedione
to testosterone, which would be a substrate for aromatase to
form 17␤-estradiol [2]. AKR1C3-mediated progesterone reduc-
tion might further contribute to
a pro-estrogenic state in
the breast by increasing the ratio of estrogen to proges-
terone.
In addition to its HSD activities, AKR1C3 is characterized
as human prostaglandin (PG) F synthase. Homogenous recom-
binant AKR1C3 stereospecifically and efficiently converts PGH2
to PGF2␣ and PGD2 to 9␣,11␤-PGF2 [18,19]. Conversion of
PGD2 to a PGF2 isomer by leukemia cells was reduced by the
AKR1C3 inhibitor indomethacin or a shRNA targeting AKR1C3 [20].
PGF2␣, acting through the F prostanoid (FP) receptor, induces
cell proliferation, invasiveness, and angiogenesis in endometrial
cancer [21–23]. Through depletion of PGD2 levels, AKR1C3 pre-
vents its spontaneous dehydration and rearrangement to form
2.3. RT-PCR
Total RNA was isolated from cells grown to ∼90% confluence
using an RNeasy kit (Qiagen, Valencia, CA), according to the manu-
facturer’s instructions, and treated with DNAse I (Qiagen) to remove
genomic DNA. A GeneAmp RNA PCR Core kit (Applied Biosystems,
Foster City, CA) was used to form cDNA from 1 ␮g RNA, according to
the manufacturer’s instructions. PCR amplification was performed
using Taq DNA polymerase as previously described [3], except the
anti-proliferative and anti-inflammatory PGJ isomers, includ-
2
ing 15-deoxy-ꢀ1
2,14
-PGJ2 (15dPGJ ). 15dPGJ2 covalently reacts
2
with a key cysteine residue in PPAR␥, resulting in its activa-
tion [24,25]. 15dPGJ2 also reacts with cysteine residues in other
proteins, including DNA binding domains of nuclear factor ␬B
◦
◦
annealing temperature was set to 68 C for AKR1C1 and 65 C for
AKR1C2 and AKR1C3 and 25 cycles were performed. The primers
used to provide isoform-specific detection of the AKR1Cs were:
(
[
NF-␬B) and ER␣, resulting in the loss of transcriptional activity
26,27].
ꢀ
ꢀ
AKR1C1 (forward) 5 -GTA AAG CTT TAG AGG CCA C-3 and (reverse)
ꢀ
ꢀ
ꢀ
5
-CAC CCA TGG TTC TTC TCG G-3 ; AKR1C2 (forward) 5 -GTA AAG
Given its over-expression in breast cancer and potential to
ꢀ
ꢀ
CTC TAG AGG CCG T-3 and (reverse) 5 -CAC CCA TGG TTC TTC TCG
regulate ligand access to estrogen and progesterone receptors
and alter prostaglandin signaling, we hypothesized that AKR1C3
might play an important role in hormone-dependent breast cancer.
To test this hypothesis, we established MCF-7 cells that sta-
bly express the AKR1C3 protein. The expression of AKR1C3 in
ꢀ
ꢀ
ꢀ
A-3 ; AKR1C3 (forward) 5 -GTA AAG CTT TGG AGG TCA C-3 and
ꢀ
ꢀ
(
reverse) 5 -CAC CCA TCG TTT GTC TCG T-3 .
2.4. Immunoblot analysis
4
MCF-7 cells resulted in the reduction of ꢀ -androstenedione to
Cells were lysed in protease inhibitor-containing RIPA buffer
testosterone, progesterone to 20␣-hydroxyprogesterone, estrone
by sonication. Samples were centrifuged at 9000 × g and the pro-
tein concentration in the supernatant was determined using the
Bradford reagent (Bio-Rad Hercules, CA) according to the manufac-
turer’s instructions. Each sample, containing 30 ␮g total protein,
was separated by 10% SDS/PAGE and transferred onto a nitrocellu-
lose membrane. AKR1C3 was detected with a mouse monoclonal
antibody against AKR1C3 [13] at 1:1000 dilution, visualized with
an horse radish peroxidase-conjugated sheep anti-mouse antibody
to 17␤-estradiol and PGD2 to 9␣,11␤-PGF . Unexpectedly, at
2
low concentrations estrone reduction was the fastest of these
reactions. Furthermore, formation of 17␤-estradiol by AKR1C3
significantly increased MCF-7 cell proliferation, demonstrating
that AKR1C3 likely plays an important role in the development
of estrogen-dependent breast cancer. AKR1C3 expression also
reduced the anti-proliferative effects of PGD , which suggests that
2
it produces tumor promoting signals independent of ER␣ signal-
ing.
(1:2000) using the ECL reagent (GE Healthcare, Piscataway, NJ).
2.5. Radiometric determination of steroid metabolism
2
. Materials and methods
For determination of estrone reduction by purified recombinant
AKR1C3, reaction mixtures containing 0.2 ␮Ci [2,4,6,7- H]-estrone,
varied unlabeled estrone to obtain a final concentration of
3
2.1. Chemicals and reagents
3
.75–30 ␮M, 1 mM NADPH, and 4% ethanol in 100 mM potassium
Unlabeled estrone and 17␤-estradiol were from ICN Biomedical
Inc. (Aurora, OH); other unlabeled steroids were from Steraloids
Newport, RI). Unlabeled and deuterium-labeled prostaglandins
were from Cayman Chemical (Ann Arbor, MI). Radiolabeled steroids
and prostaglandins were from PerkinElmer Life Sciences (Waltham,
MA). Media and cell culture reagents were from Invitrogen (Carls-
bad, CA) except as noted. All organic solvents were from Fisher
Scientific (Fair Lawn, NJ). Homogenous recombinant AKR1C3 was
purified as previously described [3].
phosphate buffer (pH 7.0, 200 ␮L total volume) were incubated at
3
◦
7 C for 1 h. Reactions were initiated with the addition of NADPH
(
and were terminated with the addition of 1 mL of cold water-
saturated ethyl acetate. The organic fraction was evaporated to
dryness under reduced pressure, and redissolved in 50 ␮L ethyl
acetate. The details of substrate and product separation are given
below.
For determination of radiolabeled steroid metabolism by MCF-
and MCF-7-AKR1C3 cells, 1 × 106 cells were plated in 6 well
7

M.C. Byrns et al. / Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 177–187
179
dishes and incubated overnight. In order to limit background
estrogen signaling, media was replaced with 2 mL phenol-free
RPMI with 1% charcoal-stripped FBS (HyClone Laboratories, Inc.).
dards (1 ng each of PGD -d , PGE -d , 15dPGJ -d , PGF -d , and
2 4 2 4 2 4 2␣ 4
9␣,11␤-PGF -d ) were added, media was acidified with 5 ␮L formic
2
4
acid (20%), and extracted twice with 1 mL ethyl ether. Organic
fractions were combined and dried under nitrogen. Eicosanoids
were derivatized with pentafluorobenzyl bromide and analyzed
using liquid chromatography-electron capture atmospheric pres-
sure chemical ionization/mass spectrometry (LC-ECAPCI/MS) as
previously described [29]. Multiple reaction monitoring (MRM)
was performed on the following transitions: m/z 351 → m/z 271
(PGD and PGE ), m/z 355 → m/z 275 (PGD -d and PGE -d ), m/z
14
After a 30 min equilibration, [4- C]-estrone (0.013 ␮Ci, 0.1 ␮M
14
final concentration), [4- C]-progesterone (0.010 ␮Ci, 0.1 ␮M), or
14
4
[
4- C]-ꢀ -androstenedione (0.096 ␮Ci, 0.1 ␮M) and unlabeled
steroid (if necessary) in DMSO were added to attain a final concen-
tration of 0.1 or 5 ␮M steroid and 0.25% DMSO. After incubation for
0, 4, 6, 24, or 48 h, 1 mL media was removed from cells and extracted
twice with 2 mL cold ethyl acetate. Organic fractions were pooled,
dried under reduced pressure, redissolved in 100 ␮L ethyl acetate.
The details of substrate and product separation are given below.
For both recombinant enzyme and cell experiments, samples
were applied to LK6D Silica thin layer chromatography (TLC) plates
2
2
2
4
2
4
315 → m/z 271 (15dPGJ ), m/z 319 → m/z 275 (15dPGJ -d ), m/z
2
2
4
353 → m/z 193 (PGF and 9␣,11␤-PGF ), and m/z 357 → m/z 197
2␣
2
(PGF -d and 9␣,11␤-PGF -d ).
2␣
4
2
4
Individual eicosanoids were quantitated by comparing the areas
of the peaks corresponding to the unlabeled compounds to the
areas of the peaks for the deuterated internal standards. Calibration
curves were established by adding known concentrations of unla-
beled prostanoids and a set amount (1 ng) of deuterated standards
to the media minus cells.
(
Whatman Inc., Clifton, NJ). TLC plates were developed using a
methylene chloride/ethyl acetate (80:20, v/v) solution for estrone
or progesterone metabolites or a methylene chloride/ethyl ether
4
(
110:10, v/v) solution for ꢀ -androstenedione metabolites and
counted with a Bioscan System 200 plate reader (Washington, DC).
Identification of products was determined by co-migration on TLC
with nonradioactive reference steroids (25 ␮g each) visualized with
an acetic acid/sulfuric acid/anisaldehyde (100:2:1, v/v/v) solution
and heating. Peaks were integrated as a percent of total radioactiv-
ity, assuming that each steroid was recovered with equal efficiency.
The specific radioactivity of the substrate was used to calculate
nmoles of steroid product formed. Independent measurements
showed that the recovery of the steroids from media following
extraction was >95%.
2.8. Cell proliferation
Proliferation in response to estrogens was determined with the
3
-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) assay. MCF-7 and MCF-7-AKR1C3 cells were maintained in
phenol-free RPMI containing 5% charcoal-stripped FBS for 72 h.
4
Cells (1 × 10 ) were then plated into 96 well plates containing
2
00 ␮L phenol-free RPMI with 5% FBS and grown overnight.
Estrone and 17␤-estradiol were added in ethanol to obtain the
indicated concentration of estrogen and 0.25% ethanol. Cells were
incubated for 6 days then stopped with the addition of 0.1 mg
MTT reagent (Molecular Probes, Eugene, OR) in 20 ␮L phosphate
buffered saline. Cells were incubated 4 h, prior to removal of
supernatant. Crystals were dissolved with 150 ␮L DMSO and read
at 570 nm, with 630 nm as a reference using a Synergy 2 plate
reader (Biotek, Winooski, VT). Cell numbers were normalized to
ethanol-treated control cells and statistics were performed using
a two-sided Student’s t-test.
2
.6. Radiometric determination of PGD2 metabolism
For determination of PGD2 metabolism by MCF-7 and MCF-
6
7
-AKR1C3 cells, 1 × 10 cells were plated in 6 well dishes and
incubated overnight. Because initial prostaglandin metabolism
experiments were not successful in the presence of serum, and
MCF-7 cells required either serum or phenol red (an estrogen) for
survival, media was replaced with 2 mL serum-free RPMI (with phe-
3
nol red) containing [5,6,8,9,12,14,15- H]-PGD2 (0.17 ␮Ci, 0.5 nM)
Proliferation in response to prostaglandins was measured by
bromo-deoxyuridine (BrDU) incorporation with a bioluminescent
detection kit (Roche Diagnostics). The BrDU assay was used in place
of the MTT assay because in the absence of serum the latter assay
lacks sufficient sensitivity to detect proliferation of MCF-7 cells. It
was necessary to perform these assays in the absence of serum since
and unlabeled PGD2 to attain a final concentration of 0.1 or 5 ␮M
along with 0.1% ethanol. After incubation for 0, 3, 6, or 24 h, 1 mL
media was removed from cells and acidified with 5 ␮L of 20% formic
acid. Twenty microliters of acidified media was added to 5 mL
of UltimaGold scintillation fluid (PerkinElmer Life Sciences). The
remaining media was extracted twice with 2 mL cold ethyl acetate
and organic fractions were pooled and dried under reduced pres-
sure. Twenty microliters of the aqueous fraction was added to 5 mL
scintillation fluid and the initial media and aqueous fractions were
analyzed with a TriCarb 2100 (Packard Instrument, PerkinElmer
Life Sciences) scintillation counter. Dried extracts were redissolved
in 200 ␮L of ethyl acetate and applied to LK6D Silica TLC plates.
TLC plates were developed using the organic layer from an ethyl
acetate/2,2,4-trimethyl pentane/water/acetic acid (110:50:100:20,
v/v/v/v) solution and were counted with the Bioscan System 200
plate reader. Identification of products was determined through
inclusion of nonradioactive reference prostaglandins visualized as
4
serum limits the uptake of PGD2 into these cells. Cells (1 × 10 )
were maintained in normal RPMI media with 10% FBS and pas-
saged into 96 well plates containing 200 ␮L of the same media.
Twenty-four hours after passaging, cells were washed twice with
phosphate buffered saline and media was replaced with serum-free
normal RPMI media. After an additional 24 h, cells were treated
with PGD , 9␣,11␤-PGF , or 15dPGJ in 0.1% DMSO. Cells were
2
2
2
incubated for 3 days, BrDU labeling reagent was added and cells
were incubated an additional 24 h. Cell numbers were determined
using luminescence on the Synergy 2 plate reader according to
the manufacturer’s instructions. Cell numbers were normalized to
ethanol-treated control cells and statistics were performed using a
two-sided Student’s t-test.
above. This method did not separate 9␣,11␤-PGF2 from PGF2␣
.
Peaks on the radiochromatogram were integrated and quantitated
as described above, except that the results were adjusted to account
for the percentage of radioactivity that remained in the aqueous
fraction following extraction.
3. Results
3.1. Steady state kinetic parameters for the reduction of estrone
catalyzed by recombinant AKR1C3
2.7. LC–MS determination of PGD2 metabolism
MCF-7 and MCF-7-AKR1C3 cells were treated with 0.1 ␮M PGD2
as described above, except that only unlabeled PGD2 was used.
After 6 h, 1 mL media was removed, deuterated internal stan-
We have previously reported kinetic constants for the reduc-
4
tion of ꢀ -androstenedione to testosterone (Km 6.6 ␮M; kcat
−
1
−1 −1
and kcat/Km = 24 min mM ) [10] and the reduc-
0.16 min

1
80
M.C. Byrns et al. / Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 177–187
to 5␣-androstanedione, with testosterone and androsterone
formed as minor metabolites (Fig. 2). The MCF-7-AKR1C3 trans-
fectants formed much higher levels of testosterone from both
4
concentrations of ꢀ -androstenedione. The testosterone peak
accounted for 23 and 11% of total radioactivity at 24 h with 0.1
4
and 5 ␮M ꢀ -androstenedione, respectively. 5␣-DHT was also
observed as a product in MCF-7-AKR1C3 cells, accounting for 5% of
4
the total radioactivity by 24 h when 0.1 ␮M ꢀ -androstenedione
was used as precursor. 5␣-DHT would be formed through the
sequential action of AKR1C3 and 5␣-reductase.
When MCF-7 cells were incubated with 0.1 ␮M [14C]-
progesterone, there was substantial endogenous metabolism,
with 54% of the parent compound metabolized by 24 h (Fig. 3).
The primary products formed were polar compounds, possibly
glucuronides, that did not migrate on the TLC plate. 5␣-
Dihydroprogesterone and 5␣-pregnane-20␣-ol-3-one were also
detected and reflect significant 5␣-reductase activity in these
cells. At 5 ␮M [14C]-progesterone, these products were formed by
parental cells at much lower levels, and 20␣-hydroxyprogesterone
was detected as a very minor metabolite. The MCF-7-AKR1C3
cells metabolized both concentrations of progesterone at a much
higher rate. With 0.1 ␮M progesterone, 49% was reduced by 6 h
and 64% of 5 ␮M progesterone was reduced by 24 h. The initial
primary product formed by MCF-7-AKR1C3 transfectants was 20␣-
hydroxyprogesterone, which underwent further metabolism to 3␣-
Fig. 1. Expression of AKR1C isoforms in parental and AKR1C3 transfected MCF-
cells. (A) Measurement of AKR1C isoform expression by RT-PCR using primers
7
specific for AKR1C1, AKR1C2, AKR1C3 in MCF-7 parental cells and eight cell lines
stably expressing AKR1C3. Left panel shows lack of AKR1C expression in the parental
cells. The right panel shows expression of AKR1C3, but not AKR1C1 or AKR1C2, in
the stably transfected cell lines. The last three lanes show the detection of cDNA
from plasmids containing AKR1C1, AKR1C2, and AKR1C3, respectively, as positive
controls. GAPDH was also amplified in each of the cell lines for normalization. (B)
Detection of AKR1C3 protein by immunoblot with an isoform-specific monoclonal
antibody in parental MCF-7 cells and AKR1C3-expressing cells. An antibody against
and 5␣-reduced products.
When [14C]-estrone was incubated with parental MCF-7 cells,
substantial endogenous 17-keto-steroid reductase activity was
observed. This activity resulted in 53% conversion of 0.1 ␮M estrone
to 17␤-estradiol by 24 h (Fig. 4). No other metabolic pathways were
observed. MCF-7-AKR1C3 cells converted 0.1 ␮M estrone to 17␤-
estradiol at a much faster rate. By 6 h, 82% of the estrone had been
converted into 17␤-estradiol by MCF-7-AKR1C3 cells. However,
when reactions were performed with 5 ␮M estrone, expression of
AKR1C3 had a more modest impact on 17␤-estradiol formation,
increasing the amount of estradiol at 24 h from 10% of the total to
␤
-actin was used as a loading control.
tion of progesterone to 20␣-hydroxyprogesterone (Km = 2.8 ␮M;
−1
−1
kcat = 1.04 and kcat/Km = 370 min mM ) [16]. However, kinetic
constants for the reduction of estrone to 17␤-estradiol have not
been previously reported due to the low turnover number and
the difficulty in obtaining reliable steady state kinetic constants.
In the present study, we were able to circumvent these prob-
1
9%.
−
1
lems and measure a Km = 9.0 ␮M and a kcat = 0.068 min yielding
−1
−1
a kcat/Km = 7.6 min mM . These measurements predict that in
vitro the conversion of estrone to 17␤-estradiol is the least favor-
able of the reactions monitored. By contrast, we and others have
shown that 11-keto reduction of prostaglandin D2 is a highly
3.4. AKR1C3 reduces PGD2 to 11ˇ-PGF2 in MCF-7 cells
3
When incubated in cell-free media, [ H]-PGD2 spontaneously
dehydrated to form PGJ2 and 15dPGJ , which accounted for 60 and
2
−
1
favoredreactionwitha Km = 1.1 ␮Mand a kcat = 1.4 min , yieldinga
8
% of the total radioactivity by 24 h, respectively (data not shown).
−1
−1
kcat/Km = 1270 min mM [18]. To determine whether these reac-
tions could occur in a breast cancer cell environment and whether
there would be consequences on cell phenotype, a stably trans-
fected cell line was sought.
3
In parental MCF-7 cells, [ H]-PGD2 disappeared at a similar rate,
but only very low levels of PGJ products were detected, with most
2
of the radioactivity staying in the aqueous phase after extraction
(
Fig. 5). These results were consistent with conjugation of PGJ and
2
1
5dPGJ2 to glutathione or other cellular nucleophiles. No 9␣,11␤-
3
.2. Development of an MCF-7 cell line that stably expresses
PGF formation was observed in parental cells or cell-free media. In
incubations with MCF-7-AKR1C3 cells, [ H]-PGD2 disappeared at a
2
AKR1C3
3
faster rate, which corresponded to formation of 9␣,11␤-PGF2 as a
major metabolite, accounting for 35 and 18% of total radioactivity at
A pLNCX retroviral vector was used to stably express AKR1C3
in MCF-7 cells. RT-PCR using isoform-specific primers indicated
that these new MCF-7 cell lines expressed AKR1C3, but not AKR1C1
or AKR1C2 (Fig. 1A). Expression of AKR1C3 protein was observed
in each of eight transfected clonal lines by Western blot analysis
using an isoform-specific monoclonal antibody (Fig. 1B). Based on
a standard curve for recombinant AKR1C3, AKR1C3 accounted for
approximately 0.01–0.1% of total protein in the lysate. Clone #5,
which exhibited high AKR1C3 expression, was selected for in depth
study (henceforth referred to as MCF-7-AKR1C3 cells).
2
4 h with 0.1 and 5 ␮M PGD as substrate, respectively. Lower levels
2
of PGJ2 and aqueous metabolites were formed by MCF-7-AKR1C3
cells relative to parental cells at both concentrations of substrate.
Because our TLC conditions did not separate 9␣,11␤-PGF2
from PGF2␣, a method utilizing pentafluorobenzyl derivatiza-
tion of prostaglandins followed by chiral normal phase HPLC
and analysis with ECAPCI-MRM/MS was used to identify and
quantify the products formed from 0.1 ␮M PGD2 by MCF-7 and
MCF-7-AKR1C3 cells (Fig. 6). As expected, MCF-7-AKR1C3 cells
stereospecifically reduced PGD to 9␣,11␤-PGF with no detectable
2
2
3.3. AKR1C3 reduces keto-steroids in MCF-7 cells
PGF2␣. Quantification of PGD , 15dPGJ , and the PGF isomers was
2 2 2
obtained through inclusion of deuterium-labeled internal stan-
dards. Standard curves were established using cell-free media that
demonstrated no interference from media to the assay and a linear
In parental MCF-7 cells, 0.1 and 5 ␮M [14C]-ꢀ4-
androstenedione was primarily metabolized by 5␣-reductase

M.C. Byrns et al. / Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 177–187
181
4
Fig. 2. Expression of AKR1C3 increases conversion of ꢀ -androstenedione to testosterone by MCF-7 cells. (A) Representative metabolic profiles at 24 h demonstrating
1
4
4
14
4
metabolism of 0.1 ␮M [ C]-ꢀ -androstenedione by MCF-7 and MCF-7-AKR1C3 cells. Time courses of metabolism of (B) 0.1 ␮M and (C) 5 ␮M [ C]-ꢀ -androstenedione
by MCF-7 and MCF-7-AKR1C3 cells. Results are from three independent experiments performed in triplicate. Abbreviations: T, testosterone; 3␣,5␣-A-17-one, androsterone;
4
4
DHT, 5␣-dihydrotestosterone; ꢀ -AD, ꢀ -androstenedione; and 5␣-AD, 5␣-androstanedione.
2
relationship (R > .995) between the observed and predicted ratios
of labeled to unlabeled prostaglandins across the concentrations
tested. The concentrations of products detected by LC–MS were
consistent with those observed with radiolabeled substrate.
this equilibrium is established by reductive (whose expression is
type 7 > type 1 > type 12) and oxidative (primarily type 4) 17␤-HSD
isoforms present in MCF-7 cells [30,31]. Selective inhibitors of 17␤-
HSDs 1, 7, and 12 all inhibit the reduction of estrone by parental
MCF-7 cells by about 30%, suggesting that all of these enzymes play
a role [30]. In transfected cells, AKR1C3 (type 5) provides an addi-
tional reductive 17␤-HSD that could alter the equilibrium in favor
of 17␤-estradiol, thus stimulating proliferation.
3
.5. Expression of AKR1C3 increases estrogen-dependent cell
proliferation in MCF-7 cells
We next explored whether the conversion of estrone to 17␤-
estradiol catalyzed by AKR1C3 would alter proliferation of MCF-7
cells. Treatment with picomolar concentrations of estrone and 17␤-
estradiol increased the number of parental MCF-7 cells by up to
3.6. Expression of AKR1C3 reduces the anti-proliferative effects of
prostaglandins
2
.2-fold over ethanol-treated controls at 6 days. Expression of
We next explored how AKR1C3 expression would affect cell
AKR1C3 significantly increased the responsiveness of the cells to
both estrogens, with up to a 7-fold increase in the number of the
MCF-7-AKR1C3 cells following estrogen treatment as compared to
ethanol-treated controls (Fig. 7A). Estrone was almost as effective
as 17␤-estradiol at stimulating proliferation in both parental and
AKR1C3-expressing MCF-7 cells. However, the effect of both estro-
gens was consistently much greater in the AKR1C3 transfectants.
These results suggest that a rapid equilibrium between estrone
and 17␤-estradiol was reached in these cells. In parental cells,
proliferation in the presence of PGD , 9␣,11␤-PGF , and 15dPGJ
2
2
2
(Fig. 7B–D). Each prostaglandin had little effect on proliferation
of parental and AKR1C3-expressing MCF-7 cells at concentrations
below 1 ␮M, suggesting that they lack cell surface receptors for
these prostaglandins. Consistent with this hypothesis, we were
unable to detect the FP receptor by Western blot (data not shown)
or RT-PCR analyses (Supplemental Figure). 15dPGJ2 inhibited pro-
liferation of parental MCF-7 cells at concentrations of 1 ␮M and
higher. PGD2 inhibited proliferation starting at a concentration

1
82
M.C. Byrns et al. / Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 177–187
Fig. 3. Expression of AKR1C3 increases conversion of progesterone to 20␣-hydroxyprogesterone by MCF-7 cells. (A) Metabolic profiles at 24 h demonstrating metabolism
of 0.1 ␮M [14C]-progesterone by MCF-7 and MCF-7-AKR1C3 cells. Time courses of metabolism of (B) 0.1 ␮M and (C) 5 ␮M [ C]-progesterone by MCF-7 and MCF-
14
7
2
-AKR1C3 cells. Results are from three independent experiments performed in triplicate. Abbreviations: P, progesterone; 20␣,5␣-P, 5␣-pregnan-20␣-ol-3-one; 20␣-P,
0␣-hydroxyprogesterone; and 5␣-P, 5␣-dihydroprogesterone.
of 2.5 ␮M. Expression of AKR1C3 eliminated the anti-proliferative
effect of 2.5 ␮M PGD2 and had a small, but significant effect at
rangement to reactive and anti-proliferative products of the PGJ2
series. These data provide a functional correlate to over-expression
of AKR1C3 in breast cancer patients and its association with poor
prognosis [12–14,32].
1
0 ␮M PGD . Surprisingly, AKR1C3 also significantly limited the
2
inhibition of proliferation by 2.5 ␮M 15dPGJ . Micromolar concen-
2
trations of 9␣,11␤-PGF2 stimulated mild MCF-7 cell proliferation,
which was not affected by AKR1C3 expression.
Unexpectedly, in cultured MCF-7-AKR1C3 cells, expression of
AKR1C3 has the most significant effect on reduction of estrone to
1
7␤-estradiol. In AKR1C3-expressing cells, estrone was reduced the
4
. Discussion
fastest at the more physiologically relevant substrate concentra-
tion (0.1 ␮M). Based on this observation and the lack of an effect
at 5 ␮M estrone, we predict that AKR1C3 is a high affinity, low
capacity enzyme towards estrone in vivo. Reduction of estrone
by MCF-7 cells expressing AKR1C3 was much higher than what
was expected based on the activity of homogenous recombinant
AKR1C3. Our observation of rapid 0.1 ␮M estrone reduction by
AKR1C3-expressing MCF-7 cells is in contrast to what was observed
in HEK-293 cells by Dufort et al. [17]. These investigators found
that estrone reduction occurred at a much slower rate than reduc-
We demonstrate that AKR1C3 reduces steroid hormones and
PGD2 when expressed in MCF-7 breast cancer cells. This study
allows a comparison of the roles of AKR1C3 in these metabolic path-
ways in a relevant breast cancer cell model and allows examination
of its effects on cell proliferation. We made the unexpected finding
that AKR1C3 expression has a dramatic effect on the rate of estrone
conversion to 17␤-estradiol, with consequences for cell prolifera-
tion. As predicted, we also observed that AKR1C3 expression led
4
to metabolism of PGD2 to 9␣,11␤-PGF , which prevented its rear-
tion of ꢀ -androstenedione and progesterone. This discrepancy
2

M.C. Byrns et al. / Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 177–187
183
Fig. 4. Expression of AKR1C3 increases conversion of estrone to 17␤-estradiol by MCF-7 cells. (A) Metabolic profiles at 24 h demonstrating metabolism of 0.1 ␮M [14C]-
1
4
estrone by MCF-7 and MCF-7-AKR1C3 cells. Time courses of metabolism of (B) 0.1 ␮M and (C) 5 ␮M [ C]-estrone by MCF-7 and MCF-7-AKR1C3 cells. Results are from three
independent experiments performed in triplicate. Abbreviations: E1, estrone and E2, 17␤-estradiol.
could be explained by the presence of an oxidative 17␤-HSD in
their HEK-293 cells that would catalyze the conversion of 17␤-
estradiol back to estrone and limit the effect of AKR1C3. Consistent
with this hypothesis, we have observed higher rates of oxidation
of 17␤-estradiol to estrone in HEK-293 cells relative to MCF-7 cells
as aromatase, Her-2/neu, COX-2 and matrix metalloproteinases
that could promote development of breast tumors in vivo [34,35].
4
While AKR1C3-mediated rates of ꢀ -androstenedione reduc-
tion were low relative to other substrates, this is still likely an
important AKR1C3-mediated reaction, as it is the primary 17␤-
HSD expressed in breast that catalyzes this reaction [36]. Due to the
(
Duan and Penning, unpublished data). The importance of estrone
4
to 17␤-estradiol conversion due to AKR1C3 expression is further
supported by the ability of this reaction to confer a proliferative
phenotype to estrogen-dependent breast cancer cells. Stimulation
of estrogen-dependent cell proliferation provides an explanation
for AKR1C3 over-expression in hormone-dependent breast can-
cer.
Progesterone is also an important substrate for AKR1C3
in breast. At 5 ␮M, progesterone was the best substrate
assayed, and there was also high activity at the lower con-
centration. Wiebe et al. have reported that progesterone and
lack of aromatase activity [37], reduction of ꢀ -androstenedione
to testosterone would not have a significant effect on MCF-7 cell
proliferation in vitro, but will likely be important to proliferation
of breast cancer in vivo. One study recently detected higher levels
of expression of AKR1C3 in stromal cells, particularly undifferenti-
ated fibroblasts, surrounding breast tumors, rather than in tumor
cells themselves [38]. This study also found that levels of AKR1C3 in
isolated primary fibroblasts were upregulated by MCF-7 cell condi-
tioned media. These undifferentiated fibroblasts exhibit the highest
levels of aromatase expression in breast. Therefore, in mammary
4
2
7
0␣-hydroxyprogesterone similarly inhibit proliferation of MCF-
cells [33], which we have also observed (data not shown).
fibroblasts conversion of ꢀ -androstenedione to testosterone by
AKR1C3 will allow subsequent conversion by aromatase to 17␤-
estradiol, which could stimulate proliferation of adjacent tumor
cells.
We have demonstrated that AKR1C3 catalyzes 11-keto-
prostaglandin reduction in MCF-7 cells. This is the first time that
Although 20␣-hydroxyprogesterone is anti-proliferative, it does
not stimulate progesterone receptor dependent gene transcrip-
tion. By reducing nuclear progesterone receptor trans-activation,
AKR1C3 activity is expected to increase expression of proteins such

1
84
M.C. Byrns et al. / Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 177–187
3
Fig. 5. Expression of AKR1C3 increases conversion of PGD2 to 9␣,11␤-PGF2 by MCF-7 cells. (A) Metabolic profiles at 24 h demonstrating metabolism of 0.1 ␮M [ H]-PGD2 by
3
MCF-7 and MCF-7-AKR1C3 cells. Time courses of metabolism of (B) 0.1 ␮M and (C) 5 ␮M [ H]-PGD2 by MCF-7 and MCF-7-AKR1C3 cells. Results are from three independent
experiments performed in at least duplicate.
keto-steroid and keto-prostaglandin reduction by AKR1C3 has been
directly compared within a cell using quantitative mass-balance
measurements. Given the high rate of PGD2 reduction by puri-
fied recombinant AKR1C3 relative to steroid hormone reduction
ing. Because this method allows the separate detection of PGF2␣
and 9␣,11␤-PGF , it definitively shows the stereospecificity of
2
PGD2 reduction in these cells. Because PGF2␣ is the stereospecific
product of PGH2 reduction by AKR1C3, its absence in MCF-7-
AKR1C3 cells suggests that there was not enough endogenous
PGH2 in these cells for that reaction to occur. Detection of PGF2
metabolites by LC–MS has several advantages over other method-
ologies. The addition of radiotracers does not permit detection of
eicosanoids formed endogenously under physiological conditions.
Antibody-based approaches to prostaglandin detection are lim-
ited by difficulties in obtaining specificity to a single prostanoid
in a biological matrix and generally overestimate prostaglandin
levels. Utilizing this LC–MS method, we can examine how expres-
sion of AKR1C3 modifies endogenous metabolism of prostaglandins
formed in response to inflammatory stimuli.
[
18,19], conversion of PGD2 to 9␣,11␤-PGF2 was slower than
expected. At 0.1 ␮M, PGD2 reduction by MCF-7-AKR1C3 cells was
4
faster than ꢀ -androstenedione reduction, but slower than reduc-
tion of either estrone or progesterone. The lower than predicted
rate of PGD2 metabolism may be a consequence of its negative
charge, which would limit diffusion into cells. A two-step model for
metabolism of prostaglandins by 15-hydroxyprostaglandin dehy-
drogenase has been proposed, in which transport into cells via
the prostaglandin transporter can be rate limiting [39]. A simi-
lar situation likely occurs with PGD2 metabolism by AKR1C3, and
expression of the prostaglandin transporter could be a determining
factor in the kinetics of this reaction in MCF-7-AKR1C3 cells.
The stereospecific LC–MS method for detecting PGF2 isomer
formation by AKR1C3 provides an important tool for studying
the contribution of AKR1C3 to endogenous eicosanoid signal-
AKR1C3 expression limited the anti-proliferative effects of PGD2
on MCF-7 cells. This observation is consistent with our hypoth-
esis that AKR1C3 reduces PGD2 concentrations and prevents its
spontaneous dehydration to form PGJ2 products. Surprisingly,

M.C. Byrns et al. / Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 177–187
185
Fig. 6. LC/MS analysis confirmed that AKR1C3 stereospecifically generates 11␤-PGF2 from PGD2 in MCF-7 cells. Extracts of media from cells incubated with PGD2 for 6 h
were monitored for the detection of pentafluorobenzyl derivatives of (A) PGE2 and PGD2, (B) 15dPGJ2, and (C) 9␣,11␤-PGF2 and PGF2␣. Deuterated standards (dashed lines)
for each prostaglandin were added prior to extraction.
AKR1C3 expression also reduced the anti-proliferative effects of
.5 ␮M 15dPGJ . This could suggest that AKR1C3 limits the anti-
proliferative effects of PGD2 through an additional mechanism.
with 15dPGJ . However, there are no cysteines located near the
2
+
2
prostaglandin binding pocket in the AKR1C3·PGD ·NADP struc-
2
2
ture (PDB 1RY0). Although the mechanism is not entirely clear, the
prevention of the anti-proliferative effects of PGD2 and 15dPGJ2
suggests that AKR1C3 acts as a regulator of PPAR␥ in cells.
AKR1C3-mediated formation of PGF2 isomers from PGH2 and
PGD2 could play an important role in cancer progression. PGF2␣
Given the similar structures of PGD2 and 15dPGJ , AKR1C3 will
2
likely bind 15dPGJ , which could reduce its available concentration
2
and keep it from reaching its molecular targets. AKR1C3 also has
several cysteine residues located on its surface, which could react

1
86
M.C. Byrns et al. / Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 177–187
Given the effects of AKR1C3 expression on steroid hormone
metabolism, its inhibition might compliment aromatase inhibitors
by further shutting down estrogen signaling in breast. Interestingly,
celecoxib, which has shown promise as an adjuvant to exemestane
treatment, inhibits AKR1C3 at the plasma concentrations attained
in vivo [11,42]. The anti-cancer effects of celecoxib are in part due to
COX-2 inhibition, but there is evidence for COX-independent mech-
anisms [43], which could include inhibition of AKR1C3-mediated
steroid hormone and prostaglandin reduction.
Acknowledgement
The authors would like to acknowledge the contribution of
Yumin Shen, who constructed the AKR1C3-pLNCX2 vector.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jsbmb.2009.12.009.
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