Activity and expression of progesterone metabolizing 5α-reductase, 20α-hydroxysteroid oxidoreductase and 3α(β)-hydroxysteroid oxidoreductases in tumorigenic (MCF-7, MDA-MB-231, T-47D) and nontumorigenic (MCF-10A) human breast cancer cells

Background Recent observations indicate that human tumorous breast tissue metabolizes progesterone differently than nontumorous breast tissue. Specifically, 5α-reduced metabolites (5α-pregnanes, shown to stimulate cell proliferation and detachment) are produced at a significantly higher rate in tumorous tissue, indicating increased 5α-reductase (5αR) activity. Conversely, the activities of 3α-hydroxysteroid oxidoreductase (3α-HSO) and 20α-HSO enzymes appeared to be higher in normal tissues. The elevated conversion to 5α-pregnanes occurred regardless of estrogen (ER) or progesterone (PR) receptor levels. To gain insight into these differences, the activities and expression of these progesterone converting enzymes were investigated in a nontumorigenic cell line, MCF-10A (ER- and PR-negative), and the three tumorigenic cell lines, MDA-MB-231 (ER- and PR-negative), MCF-7 and T-47D (ER- and PR-positive). Methods For the enzyme activity studies, either whole cells were incubated with [14C]progesterone for 2, 4, 8, and 24 hours, or the microsomal/cytosolic fraction was incubated for 15–60 minutes with [3H]progesterone, and the metabolites were identified and quantified. Semi-quantitative RT-PCR was employed to determine the relative levels of expression of 5αR type1 (SRD5A1), 5αR type 2 (SRD5A2), 20α-HSO (AKR1C1), 3α-HSO type 2 (AKR1C3), 3α-HSO type 3 (AKR1C2) and 3β-HSO (HSD3B1/HSD3B2) in the four cell lines using 18S rRNA as an internal control. Results The relative 5α-reductase activity, when considered as a ratio of 5α-pregnanes/4-pregnenes, was 4.21 (± 0.49) for MCF-7 cells, 6.24 (± 1.14) for MDA-MB-231 cells, 4.62 (± 0.43) for T-47D cells and 0.65 (± 0.07) for MCF-10A cells, constituting approximately 6.5-fold, 9.6-fold and 7.1 fold higher conversion to 5α-pregnanes in the tumorigenic cells, respectively, than in the nontumorigenic MCF-10A cells. Conversely, the 20α-HSO and 3α-HSO activities were significantly higher (p < 0.001) in MCF-10A cells than in the other three cell types. In the MCF-10A cells, 20α-HSO activity was 8-14-fold higher and the 3α-HSO activity was 2.5-5.4-fold higher than in the other three cell types. The values of 5αR:20α-HSO ratios were 16.9 – 32.6-fold greater and the 5αR:3α-HSO ratios were 5.2 – 10.5-fold greater in MCF-7, MDA-MB-231 and T-47D cells than in MCF-10A cells. RT-PCR showed significantly higher expression of 5αR1 (p < 0.001), and lower expression of 20α-HSO (p < 0.001), 3α-HSO2 (p < 0.001), 3α-HSO3 (p < 0.001) in MCF-7, MDA-MB-231 and T-47D cells than in MCF-10A cells. Conclusion The findings provide the first evidence that the 5αR activity (leading to the conversion of progesterone to the cancer promoting 5α-pregnanes) is significantly higher in the tumorigenic MCF-7, MDA-MB-231 and T-47D breast cell lines than in the nontumorigenic MCF-10A cell line. The higher 5αR activity coincides with significantly greater expression of 5αR1. On the other hand, the activities of 20α-HSO and 3α-HSO are higher in the MCF-10A cells than in MCF-7, MDA-MB-231 and T-47D cells; these differences in activity correlate with significantly higher expression of 20α-HSO, 3α-HSO2 and 3α-HSO3 in MCF-10A cells. Changes in progesterone metabolizing enzyme expression (resulting in enzyme activity changes) may be responsible for stimulating breast cancer by increased production of tumor-promoting 5α-pregnanes and decreased production of anti-cancer 20α – and 3α-4-pregnenes.

The results from the previous studies [1][2][3] imply important cancer regulating functions for autocrine/paracrine progesterone metabolites and suggest regulatory roles for 5αR, 20α -and 3α(β)-HSO activities in human breast cancer progression. In order to further study the role of these enzymes in breast cancer it was of interest to determine if breast cell lines exhibit these activities and if gene expression levels can be correlated to the levels of activity. For comparisons we chose a breast epithelial cell line (MCF-10A) known to be nontumorigenic [4] and three human breast cancer cell lines (MCF-7, MDA-MB-231 and T-47D) known to be tumorigenic [5,6] in athymic nude mice. The MCF-10A cell line was derived from human fibrocystic mammary tissue and is estrogen (ER-) and progesterone (PR-) receptor negative [4]. The MCF-7, MDA-MB-231 and T-47D cell lines were derived from human breast adenocarcinoma, MCF-7 and T-47D cells being ER + and PR + [7] and estrogen-dependent for tumorigenicity [5,6], and MDA-MB-231 cells being ER-and PR- [8] and will form tumors with or without supplemental estrogen [5]. Therefore, the objectives of the current studies were (a) to compare the activities of 5αR, 20α-HSO, 3α-HSO and 3β-HSO in one nontumorigenic and three tumorigenic human breast cell lines, and (b) to determine if differences in level of enzyme activities might be related to differences in specific mRNA expression.

Enzyme Activities -Whole Cells in Culture
For the in vivo enzyme activity studies cells were seeded in 35 mm plastic (Falcon) dishes at approximately 750 K cells per dish and allowed to attach overnight. Some dishes were used to determine cell numbers by hemocytometer at 0 and 24 hours after the start of incubations. Medium was removed and replaced with fresh medium containing [ 14 C]progesterone (0.2 µCi). Cell numbers were determined by hemocytometer in parallel dishes incubated without [ 14 C]progesterone for 0 or 24 hours. Additional controls consisted of incubations of medium containing [ 14 C]progesterone in the absence of cells. Each treatment consisted of 3-4 dishes and the progesterone metabolism of each cell type was examined in at least two separate experiments. Incubations were terminated at 0 (control), 2, 4, 8 and 24 hours by removing cells and media to siliconized glass extraction tubes on ice and adding 2.0 ml ice cold 0.05 M NaOH and 6.0 ml ether:chloroform (4:1). Known amounts of unlabeled steroid standards were added for recovery calculations and identification purposes.
The [ 14 C]progesterone metabolites were extracted three times with the ether:chloroform mixture, the extracts were delipidated, run two-dimensionally on TLC, identified by TLC-autoradiograpy, high performance liquid chromatography (HPLC), derivatization, capillary gas chromatography-mass spectrometry and quantified by scintillation spectrometry as previously described [1,12,13]. The amount of each metabolite produced was determined as percent of the total, and, following corrections for procedural losses, was calculated as ng per hour per 10 6 cells.
Because these values had begun to decrease somewhat for the main metabolites at 24 hours, the values given in this report are those determined from the 4-and 8-hour incubations.

RNA Isolation and Reverse Transcription
Total RNA was extracted from the cells using TRIzol ® reagent (Invitrogen) following the manufacturer's protocol. RNA yields were measured by spectrophotometry and RNA integrity was analysed by agarose gel electrophoresis. Prior to reverse transcription, RNA was treated with DNase I for 30 minutes at 37°C using a DNA-free™ kit (Ambion). Complementary DNA (cDNA) was obtained from 2.0 µg of DNase treated total RNA using 200 U Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Invitrogen) and random hexamer primers (Invitrogen) in a total volume of 20 µl following the manufacturers protocol. To exclude amplification of genomic DNA, experiments included conditions in which the reverse transcriptase enzyme was omitted.

Semi-Quantitative RT-PCR
Prior to semi-quantitative RT-PCR, primers for the following commonly used housekeeping genes were tested for variability of expression between cell lines: 18S rRNA, βactin, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and cyclophilin. Based on least variation of expression between cell lines, 18S was chosen as the control for the enzyme expression studies. The PCR primers were purchased from Invitrogen with sequences given in table 1 [15][16][17]43]. PCR conditions for the primer sets were as follows: 95°C denature for 4 minutes followed by cycling, each cycle with a 20 second denature at 94°C, a 30 second anneal at 62°C, and a 30 second extension at 72°C. Cycling was followed by a final extension for 4 minutes at 72°C. For each reaction an 2 µl aliquot of cDNA product was amplified in a 25 µl total volume using 1.25 U of Platinum Taq DNA Polymerase (Invitrogen), 2 mM MgCl, 0.2 mM dNTP's, 2 mM each primer. The same cycling conditions and PCR reagent concentrations were used for each set of primers. The kinetics of the PCR reactions for each primer set was determined by varying the amount of template and the number of cycles. All PCR product identities were confirmed by separation on a 1.5% agarose gel and sequencing at the Robarts Research Institute Sequencing Facility (London, ON).
For each cDNA sample, aliquots were amplified for 13 cycles with the 18S rRNA primers, 25 cycles with the 5αR1 primers, 33 cycles with the 5αR2 primers and 22 and 25 cycles with the 20α-HSD, 3α-HSD2 and 3α-HSD3 primers respectively. Products were separated on 9% polyacrylamide gels and the intensities of the appropriate bands were quantified by Quantity One 4.2.1 Gel Doc Software (BioRad Laboratories) and expressed as total pixel value, based on intensity and number of pixels per band. Results are expressed as ratios of the total pixel value of the band of interest to the total pixel value of the 18S rRNA band. Each ratio represents the average of duplicate PCRs performed for each gene from 5-7 replicate experiments.

Enzyme Activities in Microsomal/Cytosolic Fractions
Incubating whole cells in their normal medium in the presence of [ 14 C]progesterone results in both reductive and oxidative reactions promoted by the endogenous cofactors. In a cell-free system it is possible to determine more precisely the reductive or oxidative reaction velocities by supplying the corresponding cofactors and controlling the pH. More direct measurements of reductive enzyme activities were obtained by incubating microsomal/cytosolic fractions of cells with [ 3 H]progesterone (at pH 7.0) in the presence of NADH and NADPH. The results, presented in Table 2 as the mean 5αR, 20α-HSO and 3α-HSO activities (pg/hour/mg protein or as percent of total enzyme activities) for duplicate incubations, in general are in line with the findings in cultured cells. The ac-

Figure 2
Relative 5α-reductase (5αR) activities in MCF-7, MDA-MB-231, T-47D, and MCF-10A cells, calculated (mean ± SEM; n = 3-6) as (a) percent of total metabolites, (b) ratio of 5α-pregnanes:4-pregnenes, and (c) pmoles per hour per 10 6 cells of 5α-reduced metabolites produced. In (a) the mean levels of 4-pregnenes are significantly different from the mean levels of 5α-pregnanes at p < 0.001. In (b) and (c), *, **, and *** indicate significant differences from MCF-10A cells at p < 0.05, p < 0.01, and p < 0.001, respectively.  Table 2). The oxidative 3α-HSO activity (determined in MCF-7, T-47D and MCF-10A microsomal/cytosolic fractions using the substrate, [ 3 H]3αHP, and cofactors, NAD and NADP, at pH 8.0) was about 8-and 18-fold higher in MCF-10A than in MCF-7 and T-47D cells, respectively (results not shown).  is amplified. When less than 1.0 µl equivalent amounts of template were amplified, the bands were invisible or barely visible and the software tended to underestimate the pixel values. The results showed that quantification by this procedure is linear over an 8-fold range for any single cycle number. Based on these findings we determined that 18S could be quantified in the 4 cell types by amplifying for 13 cycles, 5αR1 could be quantified by amplifying for 25 cycles, and 5αR2 could be quantified by amplifying for 33 cycles. Due to a greater variability of expression be-tween cell types, 20α-HSD, 3α-HSD2 and 3α-HSD3 were amplified for 22 and 25 cycles to cover a greater range of expression.

Expression of 5αR1 and 5αR2
Expression of 5αR1 mRNA, shown in Figure   3β-HSD Type 1 (HSD3B1) was expressed in all cells but the levels were inconsistent for reliable quantification (results not shown). 3β-HSO type 2 expression was not detected in any of the four cell types.

Discussion
Previous in vitro metabolism studies have shown that mammary tissue from mouse [18], rat [19][20][21] and human [1,22,23] can convert progesterone to metabolites whose formation would require the action of the enzymes, 5αR, 3α-HSO, 3β-HSO and 20α-HSO. The results of the current investigation show that MCF-7, MDA-MB-231, T-47D and MCF-10A human breast cell lines retain the activities of these progesterone metabolizing enzymes. An outline of the major metabolic pathways and the positions of the enzymes in the breast cell lines is presented in Figure 6. Of interest is that these pathways appear to be the same as those found in breast tissues [1]. Moreover, differences between tumorous and nontumorous breast tissue in terms of relative activities of enzymes such as 5αR and 20α-HSO [1,18,19,23] were observed between tumorigenic and nontumorigenic cell lines in the present study.

5α-Reductase
The enzyme responsible for the conversion of 4-ene steroids to 5α-reduced steroids is 4-ene-steroid 5α-reductase, known commonly as 5α-reductase (5αR; EC 1.3.99.5) [24]. There are two known isoforms of the human 5α-reductase, namely type 1 (5αR1) and type 2 (5αR2) [24][25][26]. 5αR1, which is encoded by the SRD5A1 gene and is composed of 259 amino acids, has an optimum pH of 6-9, whereas 5αR2, encoded by the SRD5A2 gene, and composed of 254 amino acids, has an optimum pH of 5.5 [27]. 5αR1 has been detected in various androgen-independent organs, such as the liver and brain [28]. 5αR2 has been found predominantly in androgen-dependent organs, such as epididymis and prostate [24,28] and its role in prostate cancer has been extensively studied. Recently 5αR1 and 5αR2 were located in human breast carcinoma and were studied in relation to 5α-reduction of testosterone [29]. Although several studies have recently examined SRD5A2 polymorphisms in association with breast cancer [30][31][32], to date no study has addressed the relative expression of 5αR1 and 5αR2 in tumorous and normal breast tissues or breast cell lines.
Tumorous mammary gland tissue has a greater ability to convert progesterone to 5α-reduced metabolites (5α-pregnanes) than nontumorous tissue [1,18,19,23]. The results of the present study provide the first demonstration that MCF-7, MDA-MB-231 and T-47D human breast cell lines have significantly greater ability to convert progesterone to 5α-pregnanes than the MCF-10A human breast cell line. Conversion of progesterone to 5α-pregnanes is the result of 5α-reductase activity. The quantitative differences in 5α-reductase activities between cell lines do not appear to be related to the presence or absence of ER or PR, since significantly higher (1.9-3.3-fold) 5α-reductase activity was evident in MCF-7 and T-47D (ER-positive and PR-positive) as well as MDA-MB-231 (ER-negative and PR-negative) cells than in the ER/PR-negative MCF-10A cells. The one consistent difference is that MCF-7, MDA-MB231 and T-47D cells will form tumors [5,6], whereas MCF-10A will not form tumors [4] in immunodeficient mice. These results from cell lines are consistent with results from matched breast tissues of patients in which tumorous tissue exhibited significantly higher progesterone 5α-reductase activity than nontumorous tissue, regardless of presence or absence of ER and/or PR [1]. The 5α-pregnanes:4-pregnenes ratio was about 8-fold higher in tumorous than in nontumorous breast tissue after an 8-hour incubation with [ 14 C]progesterone [1]. Studies with breast cell lines, showing that 5α-pregnanes stimulate proliferation and decrease attachment of cells [1,2] prompted us to suggest that neoplasia in human breast may be promoted by increases in ratio of 5α-pregnanes:4pregnenes. In the current studies, the ratio of 5α-pregnanes:4-pregnenes in the tumorigenic cells was 6.5-fold higher for MCF-7 cells, 9.6-fold higher for MDA-MB-231 cells, and 7.1-fold higher for T-47D cells than for the nontumorigenic MCF-10A cells. Therefore, both tissue and breast cell line studies suggest that an elevated level of progesterone 5α-reductase activity may be an indicator of breast tumorigenesis, regardless of presence or absence of ER and/or PR. However, it should be noted that only a single nontumorous cell line was examined and it will be necessary to study other "normal" breast cell lines before making generalizations regarding the relationship between changes in steroid enzyme activity and tumorigenicity in human breast cell lines.
Several factors can account for increases in 5α-reductase activity. In vivo, increases in enzyme activity can result from increased synthesis of enzyme due to increased expression of the mRNA coding for the enzyme, or from changes in the milieu in which the enzymes operate (such as temperature and pH, and concentrations of cofactors, substrates, products, competitors, ions, phospholipids and other molecules). In in vitro experiments, the milieu is carefully controlled to be constant for all the incubations and therefore observed differences can be more easily ascribed to differences in enzyme amounts resulting from increased expression. The results of the RT-PCR studies show that the expression of 5αR1 is significantly greater in MCF-7, MDA-MB-231 and T-47D cells than in MCF-10A cells. Although 5αR2 is expressed approximately equally in the four cell types, the abundance of 5αR1 mRNA transcripts greatly exceeds that of the 5αR2 transcripts. Using identical PCR conditions it required 8-10 more PCR cycles to amplify a 5αR2 band to the intensity of a 5αR1 band in each of the cell types. Since each cycle theoretically results in a doubling of PCR product, 5αR1 mRNA appears to be present at levels in the range of about 250-1000 fold higher than the 5αR2 mRNA. Although this can only be considered a qualitative observation, it appears reasonable to conclude that 5αR1 mRNA represents the predominant 5αR mRNA present in each of the four cell types tested here. Further, the differences in expression of 5αR1 (Fig. 5a) between cell types (high in MCF-7, MDA-MB-231 and T-47D cells and low in MCF-10A cells) in general parallel the differences in total 5α-reductase activities (Fig. 2). It was also recently demonstrated by immunohistochemistry and RT-PCR that 5αR1 is the main isoform expressed in human breast carcinomas [29] and that 5αR2 may not be associated with risk of breast cancer [30-32]. These observations provide strong evidence that 5αR1 is the primary 5α-reductase expressed in these cell lines and that the differences in 5α-pregnane production between the cells is due primarily to a difference in 5αR1 expression. This does not suggest, however, that 5αR2 may not play any role in progesterone metabolism in these cells under certain limiting conditions. are well recognized. The potential importance of 5α-reduction of progesterone in breast cancer has only recently received attention [1]. The studies presented here on breast cell lines further suggest a link between tumorigenicity and increased 5α-reductase activity. If the 5α-pregnanes resulting from progesterone 5α-reductase activity are involved in the risk and development of breast cancer and if local 5α-reduction of progesterone can influence or be influenced by the behavior of tumors, then changes in expression of 5α-reductase may have important implications for the prevention, therapy and biological understanding of breast cancer. Moreover, tumorigenic and nontumorigenic breast cell lines may provide a useful in vitro system for studying the control of 5α-reductase activity and expression in breast cancer.
Human 20α-HSO is a member of the aldo-keto reductase (AKR) gene superfamily [42,43]. Expression of 20α-HSO mRNA has been demonstrated in a number of tissues, including mammary gland [41]. AKRs are monomeric 37 kDa proteins and are NAD(P)(H)-dependent [44]. The isoform AKR1C1 is predominantly a 20α-HSO [42] although the isoforms AKR1C2-AKR1C4 are also able to reduce progesterone to 20αDHP and oxidize 20αDHP to progesterone [43]. The relative abundance of these isoforms in MCF-7, MDA-MB-231, T-47D and MCF-10A cells was investigated in our studies by RT-PCR and showed significantly higher expression of AKR1C1, AKR1C2 and AKR1C3 mRNA in MCF-10A than in the other cells. These higher levels of expression suggest that the higher 20α-HSO activity observed in the MCF-10A cell progesterone metabolism studies is due to higher levels of AKR1C1 (and perhaps the other AKR isozyme) mRNA(s).
It has been suggested [49] that in hormone-related cancers (breast, prostate, endometrium, testis, ovary, thyroid and osteosarcoma) hormones, both endogenous and exogenous, provide the stimulus along the cancer progression pathway. Therefore anti-hormone therapies (such as tamoxifen) are effective in stopping the progression and thereby increase the time to recurrence and death. Unfortunately hormone-related cancers invariably become what has been called "hormone independent" and thereupon no longer respond to the anti-hormone therapies. Despite a large number of studies, "hormone independence" in cancer is poorly understood. Estradiol17β is considered the active hormone in most so-called hormonerelated breast cancers, and "hormone independence" implies "estradiol independence". Our previous studies [1][2][3] and the current report on the differences in progesterone metabolism between tumorous and normal breast tissue, tumorigenic and nontumorigenic breast cell lines and significant actions of the progesterone metabolites on breast cell lines, regardless of ER and PR status, lead us to suggest that during the breast cancer progression pathway, change in progesterone metabolizing enzyme expression, and hence enzyme activity profile, occur in affected breast tissues. The resulting increases in tumor-and metastasis promoting -and concomitant decreases in inhibitoryprogesterone metabolites may provide the stimulus for progression and malignancy of the tumor. Aspects of the hypothesis could be tested in vitro with breast cell lines and in vivo with immunodefient mice, using specific stimulatory and inhibitory agents for progesterone metabolizing enzyme activity and expression.