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

Abstract

Aldo-keto reductase (AKR) 1C3 (type 5 17beta-hydroxysteroid dehydrogenase and prostaglandin F synthase), may stimulate proliferation via steroid hormone and prostaglandin (PG) metabolism in the breast. Purified recombinant AKR1C3 reduces PGD(2) to 9alpha,11beta-PGF(2), Delta(4)-androstenedione to testosterone, progesterone to 20alpha-hydroxyprogesterone, and to a lesser extent, estrone to 17beta-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 0.1muM. MCF-7-AKR1C3 cells proliferated three times faster than parental cells in response to estrone and 17beta-estradiol. AKR1C3 therefore represents a potential target for attenuating estrogen receptor alpha induced proliferation. MCF-7-AKR1C3 cells also reduced PGD(2), limiting its dehydration to form PGJ(2) products. The AKR1C3 product was confirmed as 9alpha,11beta-PGF(2) 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 PGD(2) on MCF-7 cells, suggesting that AKR1C3 limits peroxisome proliferator activated receptor gamma (PPARgamma) signaling by reducing formation of 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)).

Keywords: 17β-Hydroxysteroid dehydrogenase; estrogen receptor; peroxisome proliferator activated receptor γ; prostaglandin D2; prostaglandin F synthase.

Fig. 1

Expression of AKR1C isoforms in parental and AKR1C3 transfected MCF-7 cells. (A) Measurement of AKR1C isoform expression by RT-PCR using primers 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 β-actin was used as a loading control.

Fig. 2

Expression of AKR1C3 increases conversion of Δ⁴-androstenedione to testosterone by MCF-7 cells. (A) Representative metabolic profiles at 24 h demonstrating metabolism of 0.1 µM [¹⁴C]-Δ⁴-androstenedione by MCF-7 and MCF-7-AKR1C3 cells. Time courses of metabolism of (B) 0.1 µM and (C) 5 µM [¹⁴C]-Δ⁴-androstenedione by MCF-7 and MCF-7-AKR1C3 cells. Results are from three independent experiments performed in triplicate. Abbreviations used: T, testosterone; 3α,5α-A-17-one, androsterone; DHT, 5α-dihydrotestosterone; Δ⁴-AD, Δ⁴-androstenedione; 5α-AD, 5α-androstanedione.

Fig. 3

Expression of AKR1C3 increases conversion of progesterone to 20α-hydroxyprogesterone by MCF-7 cells. (A) Metabolic profiles at 24 h demonstrating metabolism of 0.1 µM [¹⁴C]-progesterone by MCF-7 and MCF-7-AKR1C3 cells. Time courses of metabolism of (B) 0.1 µM and (C) 5 µM [¹⁴C]-progesterone by MCF-7 and MCF-7-AKR1C3 cells. Results are from three independent experiments performed in triplicate. Abbreviations used: P, progesterone; 20α,5α-P, 5α-pregnan-20α-ol-3-one; 20α-P, 20α-hydroxyprogesterone; 5α-P, 5α-dihydroprogesterone.

Fig. 4

Expression of AKR1C3 increases conversion of estrone to 17β-estradiol by MCF-7 cells. (A) Metabolic profiles at 24 h demonstrating metabolism of 0.1 µM [¹⁴C]-estrone by MCF-7 and MCF-7-AKR1C3 cells. Time courses of metabolism of (B) 0.1 µM and (C) 5 µM [¹⁴C]-estrone by MCF-7 and MCF-7-AKR1C3 cells. Results are from three independent experiments performed in triplicate. Abbreviations used: E₁, estrone; E₂, 17β-estradiol.

Fig. 5

Expression of AKR1C3 increases conversion of PGD₂ to 9α,11β-PGF₂ by MCF-7 cells. (A) Metabolic profiles at 24 h demonstrating metabolism of 0.1 µM [³H]-PGD₂ by MCF-7 and MCF-7-AKR1C3 cells. Time courses of metabolism of (B) 0.1 µM and (C) 5 µM [³H]-PGD₂ by MCF-7 and MCF-7-AKR1C3 cells. Results are from three independent experiments performed in at least duplicate.

Fig. 6

LC/MS analysis confirmed that AKR1C3 stereospecifically generates 11β-PGF₂ from PGD₂ in MCF-7 cells. Extracts of media from cells incubated with PGD₂ for 6h were monitored for the detection of pentafluorobenzyl derivatives of (A) PGE₂ and PGD₂, (B) 15dPGJ₂, and (C) 9α,11β-PGF₂ and PGF_2α. Deuterated standards (dashed lines) for each prostaglandin were added prior to extraction.

Fig. 7

Expression of AKR1C3 modifies proliferation of MCF-7 cells in response to estrogens and prostaglandins. (A) AKR1C3 expression increased proliferation of MCF-7 cells in response to estrone and 17β-estradiol as measured with the MTT assay. (B-D) Proliferation of MCF-7 and MCF-7-1C3 cells in response to (B) PGD₂, (C) 15dPGJ₂, and (D) 9α,11β-PGF₂ as measured by BrDU incorporation. Results are from at least three independent experiments performed in triplicate. * p < 0.05; ** p < 0.01.