Previous
in vitro metabolism studies have shown that mammary tissue from mouse [
18], rat [
19‐
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‐
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‐
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 [
14C]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:4-pregnenes. 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. Differences in biochemical [
33] and pharmacological properties [
34] and tissue pattern of expression [
28,
33], and affinities for substrates [
33,
35], have suggested distinct physiological functions for 5αR1 and 5αR2. As in the case of 5α-reductase activity, the presence or absence of ER and PR do not appear to be related to 5α-reductase expression.
The role of 5α-reduction of androgens in prostatic hyperplasia [
36] and carcinoma [
37] as it relates to 5α-dihydrotestosterone production [
38] and 5α-reductase gene expression [
39] 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.
The 20α – and 3α-(3β)-Hydroxysteroid Oxidoreductases
The formation of progesterone metabolites by breast tissue also involves the activities of 3α-HSO, 20α-HSO and 3β-HSO, as evidenced by the formation of the metabolites: 20α-hydoxy-4-pregnen-3-one (20αDHP), 20α-hydroxy-5α-pregnan-3-one, 3α-hydroxy-4-pregnen-20-one (3αHP), 3α-hydroxy-5α-pregnan-20-one, 3β-hydroxy-5α-pregnan-20-one, 4-pregnene-3α,20α-diol, and 5α-pregnane-3α (β),20α-diol.
20α-HSO is responsible for the reductive/oxidative interconversions of progesterone and 20αDHP, 3αHP and 4-pregnene-3α,20α-diol, 5α-pregnane-3,20-dione and 20α-hydroxy-5α-pregnan-3-one, and 5α-pregnan-3α (β)-ol-20-one and 5α-pregnane-3α (β),20α-diol. Our report shows that the 20α-HSO activity was significantly higher in MCF-10A cells than in the three other cell lines and that this activity resulted primarily in 4-pregnene-20α-hydroxy steroids (20αDHP and 4-pregnene-3α,20α-diol). Higher levels of 20α-HSO activity have been reported in normal human [
1] and rat [
19] mammary gland tissue than in respective tumorous tissues. Human 20α-HSO is mainly associated with microsomes [
40]. Its characterization from human skin [
41] showed that human 20α-HSO preferentially catalyzes the reduction of progesterone to 20αDHP with relatively low reverse (oxidative) activity.
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).
Human 3α-HSO is responsible for the interconversions of 3-oxo and 3α-hydroxy steroids and in the case of progesterone metabolism, results in interconversions between progesterone and 3αHP, 20αDHP and 4-pregnene-3α,20α-diol, 5α-pregnane-3,20-dione and 3α-hydroxy-5α-pregnan-20-one, 20α-hydroxy-5α-pregnan-3-one and 5α-pregnan-3α,20α-diol. The 3α-HSO activities for MCF-7, MDA-MB-231 and T-47D cells (5–10 pmoles/10
6 cells/hour) were about 2.5–5.5-fold lower than the 3α-HSO activity of MCF-10A cells. Interestingly, in matched breast tissue samples [
1] the 3α-reductive activity was 2.9- to 3.3-fold higher in normal (nontumorous) than in tumorous breast tissues [
1]. Using RT-PCR we showed that the three isoforms
AKR1C1‐
AKR1C3 are expressed in each of the four cell lines examined and that expression of each was highest in MCF-10A cells. In human, all three isoforms have been shown to be able to act as NAD(P)(H)-dependent 3α-oxidoreductases, with
AKR1C2 and
AKR1C3 predominating in 3α-HSO activity [
43,
45] in prostate and mammary glands [
43]. A fourth isoform,
AKR1C4 (3α-HSO type 1), was not expressed in any of the four cell lines, agreeing with previous findings using human breast tissue [
42].
3β-HSO activities have been noted in human breast tissue [
1] and in MCF-7 [
46], T-47Dco [
47] and ZR-75-1 [
48] human breast cell lines. Our data also indicates 3β-HSO activity and mRNA expression of
HSO3B1 (3β-HSO type 1) in MCF-7, MDA-MB-231, T-47D and MCF-10A breast cell lines but no significant differences between the cell types.
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 hormone-related breast cancers, and "hormone independence" implies "estradiol independence". Our previous studies [
1‐
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 inhibitory – progesterone 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.