AKR1C1 and AKR1C3 may determine progesterone and estrogen ratios in endometrial cancer

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Abstract

Endometrial cancer is the most common malignancy of the female genital tract. Its incidence correlates with prolonged estrogen stimulation unopposed by progesterone or synthetic progestins. Estrogen and progestin action is regulated at the pre-receptor level, by interconversion of active hormones (estradiol (E2), progesterone (P)) with their inactive counterparts (estrone (E1), 20α-hydroxyprogesterone (20α-OHP)) in target tissues. Expression of enzymes that control the ratio of E2 and P may thus play role in the disease process. We first confirmed that AKR1C1 (human 20α-hydroxysteroid dehydrogenase) in a cellular context inactivates P by forming 20α-OHP but does not catalyze the reverse reaction. We next examined the expression of AKR1C1 and AKR1C3 (type 5 17β-hydroxysteroid dehdyrogenase) in 16 paired specimens of endometrial cancer and adjacent normal endometrium. Quantification by isoform specific real-time PCR revealed higher expression of AKR1C1 in nine specimens and higher expression of AKR1C3 in four specimens of endometrial cancer. Importantly, upregulation of both enzymes in the same specimen was observed. Since AKR1C1 inactivates P its elevated expression in diseased endometrium may contribute to diminished protection by P, while eleveated expression of AKR1C3 which forms E2 in vivo, may contribute to the enhanced estrogen action. It is suggested that the expression of AKR1C1 and AKR1C3 in endometrial cancer will govern the ratio of P:E2

Introduction

Endometrial cancer is the most common malignancy of the female genital tract, with the majority of cases arising in postmenopausal women (Persson, 2000). It is the fourth most common cancer in women in USA, Western Europe and also in Slovenia (Persson, 2000, American Cancer Society, 2005, Institute of Oncology, 2005, International Agency for Research on Cancer, 2005). In Slovenia, the incidence of endometrial cancer (29.1 cases per 100,000 for 2005) surmounts the one reported for USA and Western Europe (22.9 cases and 22.5 cases per 100,000, respectively) indicating significance of the research on endometrial cancer for this country (Persson, 2000, American Cancer Society, 2005, Institute of Oncology, 2005, International Agency for Research on Cancer, 2005). Endometrial cancer is classified into type 1, an estrogen dependent type, that represents more than 80% of all cancer cases and type 2 that is considered estrogen independent (10–20% cases) (Inoue, 2001). Type 1 develops mainly through simple, complex and atypical hyperplasia, while type 2 occurs de novo or through simple and atypical metaplasia (Inoue, 2001).

In the course of the normal menstrual cycle, the human endometrium demonstrates a regular sequence of proliferation, differentiation and degeneration in response to cyclial changes in circulating concentrations of estrogens and progestins (Mote et al., 1999). Estrogens induce proliferation of glandular epithelia and stroma, while P affects differentiation of endometrial stromal and epithelial cells and limits cell proliferation (Mote et al., 1999, Yang et al., 2001). The exposure to estrogens unopposed by P or synthetic progestins increases the mitotic activity of endometrial cells and number of DNA replication errors followed by somatic mutations that can lead to hyperplasia and endometrial cancer (Akhmedkhanov et al., 2001, Henderson and Feigelson, 2000, Inoue, 2001). P counteracts the effect of estrogens by inducing differentiation of the endometrium (Sasaki et al., 2001). Epidemiologic evidence provides indirect support for this hypothesis. Women taking unopposed estrogen replacement therapy have a higher risk of endometrial cancer, whereas women taking combined estrogen–progesterone replacement therapy experience risk reduction or no increase in risk (Akhmedkhanov et al., 2001, Flötotto et al., 2001). In addition, miscarriage late in reproductive life, which is a marker for P deficiency, is associated with increased risk of endometrial cancer (Akhmedkhanov et al., 2001). On the other hand, the use of P derivatives (medroxyprogesterone acetate, megestrol and others) to successfully treat young patients with atypical endometrial hyperplasia as well as differentiated endometrioid adenocarcinoma, corroborates the protective role of P (Sasano et al., 2000, Kaku et al., 2001).

Estrogen and progestin action is regulated at the receptor level by regulation of expression of estrogen and progesterone receptors (ERα/ERβ and PRa/PRb), as well as at the pre-receptor level, by the interconversion of active hormones (E2, P) with their inactive counterparts (E1, 20α-OHP). The enzymes that are responsible for these interconversions are 17β-hydroxysteroid dehydrogenases (17β-HSDs) and 20α-hydroxysteroid dehydrogenases (20α-HSDs). These enzymes belong either to the short-chain dehydrogenase/reductase superfamily (SDR) or to the aldo-keto reductase superfamily (AKR) (Penning, 1997, Penning, 2003).

17β-HSDs either reduce 17-ketosteroids or oxidize 17β-hydroxysteroids and thereby regulate the levels of active androgens and estrogens in target tissues (Fig. 1). Estrogenic 17β-HSDs catalyse the reduction of estrone (E1; a weak estrogen) to 17β-estradiol (E2: a potent estrogen) while other isoforms catalyze the oxidation of E2 back to E1. To date eleven different human 17β-HSD isoenzymes have been characterized and cloned (Peltoketo et al., 1999, Adamski and Jakob, 2001, Midnich et al., 2004; Lukacik, Workshop on 11β- and 17β-HSDs: role in human disease, Elmau 2005). Except type 5 17β-HSD they all belong to the SDR superfamily. Types 1 and 7 act as E1 reductases and the isoenzymes 2, 4, 8, 10, 11 and 13 act as E2 oxidases (Midnich et al., 2004; Lukacik, Workshop on 11β- and 17β-HSDs: role in human disease, Elmau 2005). Type 2 17β-HSD was detected in normal human endometrium, endometrial hyperplasia and endometrial cancer, while the expression of type 4 17β-HSD was examined only in normal endometrium (Husen et al., 2000, Sasano et al., 2000, Utsunomiya et al., 2001, Utsunomiya et al., 2003). The expression of other isoforms has not yet been examined in human endometrium.

20α-HSDs catalyze the reduction of P, a potent progestin, to 20α-OHP, a weak progestin or the oxidation of 20α-OHP to form P (Fig. 1). 20α-HSD activity has been reported for different 17β-HSD isozymes, type 1 and type 2 17β-HSD from the SDR superfamily and isozymes AKR1C1–AKR1C4 from the AKR superfamily (Penning et al., 2000). Among the SDR enzymes: type 1 17β-HSD is a reductase and inactivates P, and type 2 17β-HSD acts as an oxidase and converts inactive 20α-OHP back to the active P. Among these two enzymes only type 2 17β-HSD is known to be expressed in normal endometrium (Sasano et al., 2000, Utsunomiya et al., 2001, Utsunomiya et al., 2003).

HSDs from the AKR superfamily function as 3-keto, 17-keto and 20-ketosteroid reductases or as 3α, 3β, 17β and 20α-hydroxysteroid oxidases to varying degrees (Penning et al., 2000, Penning, 2003, Steckelbroeck et al., 2004). Of the four human AKR1C isozymes, AKR1C1–AKR1C3 are expressed in uterus (Penning et al., 2000). It is not known whether these isozymes are differentially expressed in diseased tissue and whether they are implicated in the development of hyperplasia and endometrial cancer. We focused our attention especially on AKR1C1 since in vitro this isozyme possesses the highest 20α-HSD activity and is regarded as a dominant form of human 20α-HSD (Penning et al., 2000) and on AKR1C3 (also known as type 5 17β-HSD) that possesses the highest 17β-HSD activity (Penning et al., 2000).

We first confirmed that within a cellular context AKR1C1 will reduce P to its inactive metabolite 20α-OHP but that it was not capable of performing the reverse reaction. We next examined the expression of AKR1C1, AKR1C3 and other 20α-HSD and 17β-HSD isozymes in normal and diseased endometrium in paired samples. AKR1C1 was upregulated in 9/16 and AKR1C3 was upregulated in 4/16 specimens. Importantly, the up regulation of both enzymes was observed within the same specimens. The elevated expression of AKR1C1 in endometrial cancer may lower P levels and contribute to diminished protection by P. The partial upregulation of AKR1C3 may also lead to a higher concentration of E2 and may thus contribute to enhanced estrogen action.

Section snippets

Tissues

Seven specimens of normal endometrium (Table 1) and 16 specimens of endometrial cancer and adjacent normal endometrium (Table 2) were obtained from patients undergoing hysterectomy. The study was approved by the Ethical Committee at Medical Faculty, University of Ljubljana, Slovenia. Histopathological classification was done according to the WHO histological grading system for endometrial adenocarcinoma. Specimens were immediately placed into (Qiagene) RNA stabilization solution and kept at −20 

AKR1C1 acts as a reductase or an oxidase in vitro

We confirmed that homogenous recombinant AKR1C1 converted P to 20α-OHP and 20α-OHP back to P in the presence of NADPH or NAD+, respectively (Fig. 2). The final specific activity for this reaction was 11.5 nmoles P reduced/min/mg and 3.7 nmol 20α-OHP oxidized/min/mg, respectively. Next, we followed the directionality of AKR1C1 in cell lysates. We transiently transfected AKR1C1 (pcDNA3-AKR1C1) and an empty pcDNA3 vector into monkey COS-1 kidney cells. We examined the ability of COS-1 mock and

Discussion

AKR1C1 acts as a reductase or an oxidase in vitro, in the presence of the major reductive (NADPH) or oxidative (NAD+) coenzymes. In intact COS-1-AKR1C1 cells, the enzyme preferentially catalyzes the reduction of P and similar results were seen upon transfection into human embryonic kidney (HEK-293) cells (Zhang et al., 2000). We have also shown that the directionality of dual pyridine nucleotide specific HSDs in a cellular context is governed by the NAD+/NADPH ratio (Lanišnik Rižner et al., 2003

Conclusions

Progesterone action in the endometrium can be modulated at the pre-receptor level by 20α-HSDs. These enzymes can deprive the PR of its ligand P by reducting P to the less active 20α-OHP. This sequence may regulate the occupancy and attenuate trans-activation of the PR and lead to unopposed estrogen action. Our results indicate that among the enzymes that could eliminate P in endometrium the level of type 1 17β-HSD is very low, while transcripts for AKR1C1–AKR1C3 are present in normal and

Acknowledgements

Supported by MESS L3-6225, SLO-USA/05-06/004 and Dr. J. Cholew Foundation to T.L.R., and R01-CA90744 awarded by National Institutes of Health to T.M.P.

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