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Department of Biology, Hormonal Regulatory Mechanisms Laboratory, University of Western Ontario, London, Ontario, Canada N6A 5B7

(Requests for offprints should be addressed to J P Wiebe; Email: jwiebe{at}uwo.ca)

	Abstract

Top Abstract Introduction Progesterone metabolism in... Cancer-related actions of the... Receptors for progesterone... Effect of the progesterone... Implications of changes in... Implications of progesterone... Summary, significance, and... References

 
In the 70 years since progesterone (P) was identified in corpus luteum extracts, its metabolism has been examined extensively in many tissues and cell lines from numerous species. In addition to the reproductive tissues and adrenals, every other tissue that has been investigated appears to have one or more P-metabolizing enzyme, each of which is specific for a particular site on the P molecule. In the past, the actions of the P metabolizing enzymes generally have been equated to a means of reducing the P concentration in the tissue microenvironment, and the products have been dismissed as inactive waste metabolites. In human breast tissues and cell lines, the following P-metabolizing enzymes have been identified: 5-reductase, 3-hydroxysteroid oxidoreductase (3-HSO), 3ß-HSO, 20-HSO, and 6-hydroxylase. Rather than providing diverse pathways for inactivating and controlling the concentration of P in breast tissue microenvironments, it is proposed that the enzymes act directly on P to produce two types of autocrines/paracrines with opposing regulatory roles in breast cancer. Evidence is reviewed which shows that P is directly converted to the 4-pregnenes, 3-hydroxy-4-pregnen-20-one (3-dihydroprogesterone; 3HP) and 20-dihydroprogesterone (20HP), by the actions of 3-HSO and 20-HSO respectively and to the 5-pregnane, 5-pregnane-3,20-dione(5-dihydroprogesterone; 5P), by the irreversible action of 5-reductase. In vitro studies on a number of breast cell lines indicate that 3HP promotes normalcy by downregulating cell proliferation and detachment, whereas 5P promotes mitogenesis and metastasis by stimulating cell proliferation and detachment. The hormones bind to novel, separate, and specific plasma membrane-based receptors and influence opposing actions on mitosis, apoptosis, and cytoskeletal and adhesion plaque molecules via cell signaling pathways. In normal tissue, the ratio of 4-pregnenes:5-pregnanes is high because of high P 3- and 20-HSO activities/expression and low P 5-reductase activity/expression. In breast tumor tissue and tumorigenic cell lines, the ratio is reversed in favor of the 5-pregnanes because of altered P-metabolizing enzyme activities/expression. The evidence suggests that the promotion of breast cancer is related to changes in in situ concentrations of cancer-inhibiting and -promoting P metabolites. Current estrogen-based theories and therapies apply to only a fraction of all breast cancers; the majority (about two-thirds) of breast cancer cases are estrogen-insensitive and have lacked endocrine explanations. As the P metabolites, 5P and 3HP, have been shown to act with equal efficacy on all breast cell lines tested, regardless of their tumorigenicity, estrogen sensitivity, and estrogen receptor/progesterone receptor status, it is proposed that they offer a new hormonal basis for all forms of breast cancer. New diagnostic and therapeutic possibilities for breast cancer progression, control, regression, and prevention are suggested.

	Introduction

Top Abstract Introduction Progesterone metabolism in... Cancer-related actions of the... Receptors for progesterone... Effect of the progesterone... Implications of changes in... Implications of progesterone... Summary, significance, and... References

 
The name ‘progesterone’ was first adopted in 1935 (Allen 1970), shortly after it was isolated from corpus luteum extracts (Allen & Corner 1929), purified (Allen 1930, Slotta et al. 1934), and structurally identified (Butenandt 1934). In the 70 years since its discovery, nearly 100 000 papers have been published dealing with progesterone P; (4-pregnene-3,20-dione) on many levels. As the name implies, its main actions have been linked primarily to human female reproductive aspects involving the uterine changes associated with the menstrual cycle and gestation. However, P is now known to influence (directly or indirectly) many other tissues and facets of regulatory physiology and endocrinology, including those of the mammary glands. Soon after its discovery, the metabolism of P began to be investigated, primarily with the aim of determining its route of inactivation. It has become apparent that many tissues have P-metabolizing enzymes, which can modify different parts of the molecule. Although the resulting metabolites have been shown, in some tissues, to be active molecules in and of themselves, for the most part there has been a reluctance to accept them as anything other than waste products, with their formation as a means of decreasing the local P concentrations.

PR has long been linked to the proliferative changes in the normal breast, but its role in breast cancer is unclear. Recent studies have provided evidence that P metabolites formed in breast tissue have regulatory functions with respect to breast cancer that may previously have been attributed to P. We first suggested (Wiebe et al. 2000) that the P metabolites produced within breast tissues might be independently active hormones functioning as cancer-promoting or -inhibiting regulatory agents. By this hypothesis, the maintenance of normalcy or progression to neoplasia would depend on the ratios of pro- to anti-cancer P metabolites in the local breast tissue microenvironment.

The aim of this review is to summarize observations which indicate that most (if not all) tissues/cells may have some capacity to convert P and that mammary tissue in particular has enzymes which catalyze the direct conversion of P to two classes of active metabolites. Evidence is reviewed that these P metabolites function as independent pro-or anti-cancer autocrine/paracrine hormones that regulate cell proliferation, adhesion, apoptosis and cytoskeletal, and other cell status molecules via novel receptors located in the cell membrane and intrinsically linked to cell signaling pathways. Current endocrine therapies are based on suppressing estrogen levels or inhibiting its actions. Unfortunately, only a fraction of all breast cancer patients respond to this estrogen-based therapy and the response is only temporary (McGuire 1987). As the breast tissue P metabolites act on breast cell lines regardless of their tumorigenicity, estrogen sensitivity and estrogen receptor (ER) and progesterone receptor (PR) status, they are suggested to provide a new endocrine-based explanation for progression to the various forms of breast cancer as well as for the maintenance of normalcy in breast tissues. Based on the findings, it is proposed that in breast tissue P serves as a precursor for active steroid hormones whose relative concentrations determine the levels of mitogenic, apoptotic, and metastatic activities locally within the tissue.

Progesterone is metabolized by many tissues

Soon after its identification, a large number of studies followed to determine the metabolism of P. In the early decades, many workers in the field identified and measured urinary metabolites of P with the aim of ascertaining how the body inactivated this progestagen. By 1954, almost 100 naturally occurring steroids had been isolated from tissue and urinary sources (Dorfman 1954). The urinary P derivatives were assumed to result from metabolism in the liver and included 5ß-pregnanes such as pregnanediol (5ß-pregnane-3,20-diol) and pregnanolone (5ß-pregnan-3-ol-20-one) as well as the 5-pregnanes, 5-pregnane-3,20-dione (5P), 5-pregnan-3-ol-20-one, 5-pregnan-3ß-ol-20-one, and 5-pregnan3-3(ß), 20-diols (Atherden 1959). The rapid metabolism of intravenously administered [¹⁴C]progesterone by eviscerated rats (Berliner & Wiest 1956, Wiest 1956) in which tissues such as liver, spleen, gut, and adrenals had been removed, showed that P conversion was also occurring extrahepatically. It then soon became apparent that P serves as the precursor for the major steroid hormones (androgens, estrogens, corticosteroids) produced by the gonadal and adrenal cortical tissues.

A large number of metabolism studies on a variety of reproductive tissue from various species and physiological states showed that P is not only converted to the well-known steroid hormones such as estradiol and testosterone, but also to various 21-carbon derivatives for which there were no well-defined functions (Fig. 1). Studies on uterine tissues from rats (Marrone & Karavolas 1981, 1982, Redmond & Pepe 1986), guinea pigs (Glasier et al. 1994, Hobkirk et al. 1997), and humans (Bryson & Sweat 1967, 1969, Pollow et al. 1975, Milewich et al. 1977, Arici et al. 1999), as well as placentae from humans (Little et al. 1959) and goats (Sheldrick et al. 1981), showed the presence of numerous P-converting enzymes. Similarly, incubations with ovarian tissues (especially granulosa cells) from rat (Zmigrod et al. 1972, Lacy et al. 1976, Nimrod 1977, de la Llosa-Hermier et al. 1983, Moon et al. 1986, 1987, Wiebe et al. 1994a), human (Sweat et al. 1960), and chicken (Marrone 1986, Wiebe et al. 1990), as well as incubations with testicular cells or homogenates from trout (Andersson & Rafter 1990), frog (Canosa et al. 1998), mouse (Kuwata et al. 1976), rat (Slaunwhite & Samuels 1956, Wiebe 1978, Wiebe & Tilbe 1979, Wiebe et al. 1980, Tilbe & Wiebe 1981), rabbit (Matsumoto et al. 1976), and human (Savard et al. 1956, Stegner & Lisboa 1984), have shown the presence in these tissues of a number of enzymes capable of converting P to a variety of products.

View larger version (9K): [in this window] [in a new window]   Figure 1 The structure of progesterone and the major classes of steroids resulting from its metabolism. P, the 21-carbon precursor of all the major steroid hormones produced in the gonads and adrenals, is also directly altered by enzymes within many, if not all, other tissues. The enzymes are specific for particular sites on the molecule (examples indicated by the arrows) and their actions lead directly to the 5-pregnane, 5ß-pregnane, and 4-pregnene metabolites of P and indirectly to the corticosteroids, androgens, and estrogens. In this review, evidence is presented that in human breast tissue 5-pregnanes and 4-pregnenes are hormones whose actions may determine normalcy or progression to breast cancer.

Thus, many metabolism studies from a large number of tissues and various species had indicated that, in addition to the gonads and adrenals, perhaps most, if not all, tissues have some capacity to convert P to other products. The studies had demonstrated the presence in tissues and cells of a number of enzymes capable of acting on various sites in the P molecule, leading to the formation of various classes of 21-carbon steroids, in addition to the known hormones, as illustrated in Fig. 1. These P-metabolizing enzymes included 5-reductase, 5ß-reductase, 3-hydroxysteroid oxido-reductase (3-HSO), 3ß-HSO, 20-HSO, 20ß-HSO, 6(ß)-, 11ß-, 17-, and 21-hydroxylase, and C_17–20-lyase. In spite of this large number of enzymes capable of local transformation of P, the 21-carbon P metabolites were for the most part considered to be waste products and the P-metabolizing enzymes as a means of controlling the local (in tissue) concentrations of P.

In terms of neoplasia, the presence of P-metabolizing enzymes had been demonstrated in rat testicular interstitial cell tumors (Chatani et al. 1990), androblastoma (Sertoli-Leydig cell tumor) (Stegner & Lisboa 1984), dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors (Mori et al. 1978, Mori & Tamaoki 1980, Eechaute et al. 1983), human endometrial carcinoma (Collins & Jewkes 1974, Pollow et al. 1975), human breast tissues (Lloyd 1979, Miller 1990), modified breast cancer cell lines (T47Dco) (Fennessey et al. 1986, Horwitz et al. 1986), and virally transformed adrenocortical cells (Wiebe et al. 1987). Although selective differences in P-metabolizing enzyme activities between normal and tumor tissues were noted in some of these studies, they were not linked to any potential effects of the metabolites themselves on cancer induction or promotion prior to our studies (Wiebe et al. 2000).

	Progesterone metabolism in breast tissues and breast cell lines

Top Abstract Introduction Progesterone metabolism in... Cancer-related actions of the... Receptors for progesterone... Effect of the progesterone... Implications of changes in... Implications of progesterone... Summary, significance, and... References

 
P was known to be involved in normal breast development as well as in the proliferative changes that occur during the menstrual cycle, pregnancy, and lactation (Going et al. 1988, Potten et al. 1988). However, its direct role in mammary cancer was not clear (McGuire & Horwitz 1977, King 1993) and a number of studies provided conflicting results. Some reports indicated stimulation (Anderson et al. 1989), while others observed regression of, or no effect on, human tumors (Horwitz et al. 1985, Santen et al. 1990) resulting from treatment with P or synthetic progestins. Similarly, in other species such as rodents (Jabara 1967, Welsh 1982, Luo et al. 1997) and dogs (Segaloff 1975, Mol et al. 1996), progestins were shown to either stimulate or inhibit tumor growth. In vitro studies of the effects of progestins on human breast cancer cell lines likewise showed either stimulation or inhibition of cell proliferation and cell cycle progression (Braunsberg et al. 1987, Clark & Sutherland 1990, Cappelletti et al. 1995, King 1993, Pike et al. 1993, Musgrove & Sutherland 1994, Clarke et al. 1994, Groshong et al. 1997).

The conflicting results regarding the role of P in breast cancer, in addition to the lack of evidence that tumor progression could be substantially related to changes in in situ P levels, led us to speculate about the potential importance of further metabolism of steroids occurring locally within the tumor and its adjacent host tissue. This led us to hypothesize that P may be converted within breast tissue into several types of metabolites, some of which stimulate while others inhibit cell proliferation and tumorigenesis. By this hypothesis, P would serve as a precursor (or pro-hormone) and the metabolites as the active hormones in regulating breast cancer. The state or progression of mammary tumors could then depend on the ratio of cancer-promoting to cancer-inhibiting steroid compounds. If such P metabolites could be shown to exist, they might provide an alternate or additional endocrine explanation for the estrogen-sensitive and -insensitive breast carcinomas as well as for normalcy of breast tissues.

Breast tissues and breast cell lines convert progesterone to 5-pregnanes and 4-pregnenes

To test the hypothesis, studies were conducted to determine the capacity of tumor and surrounding normal (nontumorous) breast tissues to metabolize [¹⁴C]P. The paired tissue specimens came from premenopausal, menopausal and postmenopausal women with various subtypes and grades of infiltrating duct carcinomas and included tissues that were estrogen-receptor (ER) and progesterone-receptor (P) negative and/or positive (Wiebe et al. 2000). All the breast biopsies examined converted [¹⁴C]P into at least ten different metabolites that could be grouped into two structurally different classes of steroids (illustrated in Fig. 2): those with a delta-4 double bond in ring A (the 4-pregnenes) and those that are 5-reduced (the 5-pregnanes). Reduction of P to 5-pregnanes is catalyzed by 5-reductase and the direct 5-reduced metabolite of P is 5-pregnane-3,20-dione (5P). The 5-reductase reaction is irreversible, but 5P can in turn be altered to 3- and 20-hydroxy pregnanes by the reversible actions of 3-HSO, 3ß-HSO, and 20-HSO (Fig. 2).

View larger version (9K): [in this window] [in a new window]   Figure 2 Progesterone conversion to 4-pregnene and 5-pregnane metabolites by human breast tissues and cell lines. Note that 5-reductase reaction is not reversible (see text for details; modified from Wiebe et al. 2005).

Changes in progesterone metabolite ratios and metabolizing enzyme activities

Although both normal (nontumorous) and tumorous breast tissues converted P to the two classes of metabolites, there were significant quantitative differences. In normal breast tissue, conversion to 4-pregnenes greatly exceeded the conversion to 5-pregnanes, whereas in tumorous tissue, conversion to 5-pregnanes greatly exceeded that to 4-pregnenes (Fig. 3a). The differences in amounts of 5-pregnanes and 4-pregnenes were mainly due to changes in the amounts of 5P and 3HP (Fig. 3b) and the ratio of 5P:3HP was nearly 30-fold higher in tumorous than in normal breast tissues. The results indicated that P 5-reductase activity is significantly higher, whereas P 3-HSO and 20-HSO activities are significantly lower in tumor than in normal tissues (Wiebe et al. 2000). Earlier studies with cell-free homogenates of breast tissues (Lloyd 1979, Miller 1990) and chemically induced rat mammary tumors (Mori et al. 1978) had also shown higher 5-reductase and lower 20-HSO activities in tumors than in normal glands.

View larger version (14K): [in this window] [in a new window]   Figure 3 Differences in P metabolism between paired nontumorous (normal) and tumorous (tumor) human breast tissues ((a) and (b)) and between nontumorigenic (MCF-10A) and tumorigenic (MCF-7) human breast cell lines (c). In (a) and (c), the conversion to 4-pregnenes and 5-pregnanes is presented as percentage of total P metabolism. In (b) the amounts of the specific metabolites, 3HP and 5P, formed from P by tissues is given as nanogram/milligram protein. *, significantly different from normal at P < 0.05; ***, significantly different from normal ((a) and (b)) or from MCF-10A (c) at P < 0.001. (Modified from Wiebe et al. 2000, Wiebe & Lewis 2003.)

View this table: [in this window] [in a new window]   Table 1 Levels of 5P, 3HP, and progesterone in breast nipple aspirate fluid samples from tumorous breasts

Overall, the metabolism studies showed that the altered direction in P metabolism, and hence in metabolite ratios, was due to significantly elevated 5-reductase and depressed 3- and 20-HSO activities in breast tumor tissues and tumorigenic cells. It appeared, therefore, that changes in P-metabolizing enzyme activities might be related to the shift toward mammary cell tumorigenicity and neoplasia. The changes in enzyme activity might reasonably be expected to be due to changes in expression of the enzyme genes.

Changes in expression of progesterone-metabolizing enzymes

The above metabolic studies and in vitro enzyme kinetics studies showed that the activity of 5-reductase is higher, whereas that of the 3(20)-HSOs is lower in tumor tissue and tumorigenic breast cell lines than in normal breast tissue and cell lines. Several factors can account for changes in enzyme activity. In vivo, changes in enzyme activity can result from changes in levels of the enzyme due to changes in expression of the mRNA coding for the enzyme, or from changes in the milieu in which the enzyme operates (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 identical between incubations, and therefore, observed differences can be more easily ascribed to differences in enzyme amounts.

To determine if the differences in P-metabolizing enzyme activities between normal and carcinoma tissues/cells could be attributed to changes in enzyme mRNA expression, reverse transcriptase (RT)-PCR studies were carried out on breast tissues and cells lines. RT-PCR analyses on tissues from 38 patients showed significantly higher levels of expression of 5-reductase type 1 (SRD5A1) and 5-reductase type 2 (SRD5A2) mRNA and significantly lower levels of expression of the 3-HSO type 2 (AKR1C3), 3-HSO type 3 (AKR1C2) and 20-HSO (AKR1C1) mRNAs in the tumor tissues than in the normal tissues (Lewis et al. 2004) (Fig. 4a). These results were similar to those from enzyme mRNA expression studies on breast cell lines (Wiebe & Lewis 2003), which showed higher 5-reductase and lower HSO gene expressions in tumorigenic than in nontumorigenic cell lines (Fig. 4b). Other reports also indicate lower HSO mRNA expression levels in tumor than in normal portions of breast (Ji et al. 2004) and prostate tissues (Ji et al. 2003).

View larger version (25K): [in this window] [in a new window]   Figure 4 Examples of differences in progesterone-metabolizing enzyme expression levels (a) between paired normal and tumor breast tissues and (b) between nontumorigenic (MCF-10A) and tumorigenic (MCF-7) cell lines. For simplicity, only expression levels for 5-reductase type 1 (5-R1) and 3-HSO type 3 (3-HSO3) are shown; expression levels of the other enzyme genes (5-R2, 3-HSO2, and 20-HSO) showed similar differences. Insets show representative RT-PCR results (gels) for (a) one tumor (T) and adjacent normal (N) breast tissue sample, and (b) MCF-10A (10A) and MCF-7 (M7) cells. Differences between normal and tumor and between MCF-10A and MCF-7 are significant at P < 0.001. (See Wiebe & Lewis 2003, Lewis et al. 2004 for details.)

The studies cited above provided evidence of selective changes in levels of enzyme activities/expression and in P metabolites formed in breast carcinoma, but there was as yet no evidence that P metabolites exhibited regulatory functions related to cancer. Some of the same P metabolites had been identified as active regulatory molecules in other tissues and with respect to other processes. For example, 5-pregnanes such as 5P (Selye 1942), 5-pregnan-3-ol-20-one (Majewska et al. 1986, Kavaliers & Wiebe 1987), and 3HP (Wiebe & Kavaliers 1988) elicited marked anesthetic or analgesic effects via mechanisms involving calcium channels, the -aminobutyric acid(GABA)–benzodiazepine–chloride complex and endogenous opioid systems. 20HP elevated serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels in rats (Gilles & Karavolas 1981), whereas 3HP selectively suppressed basal and LH-releasing hormone-stimulated FSH secretion from primary cultures of anterior pituitary cells (Wood & Wiebe 1989) by nongenomic mechanisms at the level of the gonadotrope membrane, protein kinase C cell signaling pathway, and intracellular Ca²⁺ mobilization (Dhanvantari & Wiebe 1994, Wiebe et al. 1994b, Beck et al. 1997). The next step was to test the P metabolites for possible effects on mitogenic and metastatic parameters.

	Cancer-related actions of the progesterone metabolites

Top Abstract Introduction Progesterone metabolism in... Cancer-related actions of the... Receptors for progesterone... Effect of the progesterone... Implications of changes in... Implications of progesterone... Summary, significance, and... References

 
Transformation of normal human cells into malignant cancers involves changes in, or deregulation of, a number of cell characteristics and processes (Hanahan & Weinberg 2000). Cardinal among these are: (a) proliferation rates, (b) cell-to-cell and cell-to-substrate adhesion, (c) cytoskeletal and adhesion molecules, (d) receptors that transduce growth regulating signals, and (e) mitogenic growth signaling pathways. A summary of the effects of the P metabolites on these parameters follows.

Effects of progesterone metabolites on cell proliferation, mitosis, and apoptosis

Uncontrolled cell proliferation is one of the hallmarks of cancer, and factors which affect cell proliferation rates are known to affect cancer rates (Cohen & Ellwein 1990, Pike et al. 1993, Hanahan & Weinberg 2000). Initial studies conducted on MCF-7 cells showed significant, but opposite, effects on cell proliferation; 3HP inhibited whereas 5P-stimulated proliferation dose-dependently between 10^–9 and 10^–6 M (Fig. 5a). In this concentration range, estradiol resulted in weak stimulation at 10^–8 M and either no effects or slight inhibition at higher concentrations (Fig. 5a). Stimulation in cell numbers was also observed when cells were treated with other 5-pregnanes, such as 5-pregnan-3-ol-20-one, 5-pregnan-20-ol-3-one, and 5-pregnane-3,20-diol, whereas other 4-pregnenes such as 20-HP and 4-pregnene-3,20-diol resulted in suppression of cell proliferation similar to that of 3HP (Wiebe et al. 2000). Stimulation of cell proliferation with 5P and inhibition with 3HP were also observed in all other breast cell lines examined, whether ER/P-negative (MCF-10A, MDA-MB-231) or ER/P-positive (T47D, ZR-75-1) and whether requiring estrogen for tumorigenicity (MCF-7, T47D) or not (MDA-MB-231), or whether they are nontumorigenic (MCF-10A) (Wiebe et al. 2000, Pawlak et al. 2005, G Zhang & J P Wiebe, unpublished results).

View larger version (13K): [in this window] [in a new window]   Figure 5 Effects of 5P and 3HP exposure (72 h) on proliferation (cell number) and adhesion (detachment) of MCF-7 and MCF-10A human breast cell lines. Values are presented as percent in relation to controls (0, C). In (a) and (b) cells were exposed to 0 or 10–9–10–6 M 5P or 3HP and results show highly significant dose-dependent effects. In (a) MCF-7 cells were also exposed to estradiol for comparison purposes. The effect of combining 5P and 3HP (each at 10–7 M) on proliferation and detachment of MCF-7 cells are shown in (c); different letters (a, b, and c) on bars denote significant differences (P < 0.05) within proliferation or detachment results. (Modified from Wiebe et al. 2000, Wiebe & Muzia 2001, Pawlak et al. 2005.)

Effects of progesterone metabolites on cell adhesion

Cellular adhesion is a critical aspect of cancer biology. In culture conditions, normal cells of mesenchymal or epithelial origin generally depend on anchoring to a solid substratum for cell division. This dependence on support by solid substrates for cell proliferation is lessened as cells become neoplastic and metastatic (Raz 1988). Some time during the development of most types of human cancer, pioneer cells are spawned that are capable of moving out of the primary tumor masses and of traveling to distant sites where they may succeed in founding new colonies. It is these distant settlements of tumor cells — metastases — that are the cause of about 90% of human cancer deaths (Sporn 1996). The capability of escaping the primary tumor mass and colonizing new terrain involves a number of cellular changes, not the least of which are cell–cell and cell–substrate adhesion characteristics. To allow the initial escape, adhesion must be decreased and attachment severed.

To determine whether P metabolites might play a role in the acquisition of metastatic potential, their effects on cell adhesion were examined (Wiebe et al. 2000, Wiebe & Muzia 2001) by quantitative cell–substrate attachment and detachment assays that had been developed earlier (Dinsdale et al. 1992) for baby hamster kidney cells. The first tests were on MCF-7 cells and the results showed that 5P caused significant dose-dependent decreases in attachment to, and increases in detachment from, the substratum (Fig. 5b). The opposite effect was observed with 3HP, which promoted cell attachment and decreased cell detachment (Fig. 5b). Similar effects have also been demonstrated recently in MCF-10A, T47D, and MDA-MB-231 cells (Wiebe et al. 2004, Pawlak et al. 2005). The opposing actions of 5P and 3HP on both cell anchorage and proliferation strengthen the hypothesis that the direction of P metabolism in vivo toward higher 5-pregnane and lower 4-pregnene concentrations could promote breast neoplasia and lead to malignancy.

Proof of principle

Confirmation of the hypothesis that the move from normalcy to neoplasia in breast cells is influenced by the in situ increase in the 5-pregnane:4-pregnene ratio requires studies in which 5-reductase activity is blocked, as well as paradigms where various concentrations of a 5-pregnane and a 4-pregnene are used in combination and in various temporal sequences. We have used the 4-azasteroid dutasteride, a known inhibitor of 5-reductase types 1 and 2 (Bramson et al. 1997) that has been employed in trials to inhibit the 5-reduction of testosterone to DHT in men with benign prostate hyperplasia (Brown & Nuttal 2003, Clark et al. 2004) and prostate cancer (Andriole et al. 2004, Iczkowski et al. 2005). First, we demonstrated that in MCF-7 cells dutasteride at 10^–6 M inhibited P conversion to 5-pregnanes by >95% and at the same time increased 4-pregnene production. Next, it was demonstrated that treatment of cells with P alone, without medium change for 72 h, resulted in significant conversion to 5-pregnanes and concomitant increases in cell proliferation and detachment. These increases in proliferation and detachment were blocked in cells incubated with P plus dutasteride. In turn, the suppression by dutasteride was overridden by the addition of 5P. The results are seen as providing proof of the principle that the effects on proliferation and adhesion were not due to P, but due to the 5-reduced metabolites (Wiebe et al. 2006).

To confirm the hypothesis that the ratio of 5-pregnanes:4-pregnenes is a determinant of the degree of cell proliferation and adhesion, detailed studies will need to be carried out using various concentrations of 3HP and 5P in combination and in various temporal sequences. Similar studies could also determine if the progression toward neoplasia can be impeded or even reversed by high 3HP:5P ratios, i.e. ratios of P metabolites that favor the 4-pregnenes. Data from studies in which cells were treated simultaneously with both 3HP and 5P show that the independent effects of the individual hormones on proliferation and adhesion are cancelled out when present in equal concentrations (Fig. 5c) (Pawlak et al. 2005) and support the view that the overall effects may depend on the relative concentration of each in the milieu.

Effects of progesterone metabolites on cytoskeletal and adhesion complexes

The transformations in morphology, replication, and adhesion during the transition from normal to cancerous cell have been shown to be accompanied by rearrangements of cytoskeletal and adhesion structures. The cytoskeletal organization differs between normal and cancerous cells (Ben-Ze’ev 1985, Holme 1990, Holth et al. 1998) and between high- and low-metastatic cells (Suzuki et al. 1998). For example, the level of organization of the actin cytoskeleton observed in normal cells (Bershadsky et al. 1995, Helige et al. 1997) is characterized by higher levels of polymerized actin, whereas transformation to the metastatic condition may be accompanied by disruption and/or visible disappearance of actin filaments (Suzuki et al. 1998). Similarly, vinculin, a protein that is associated with cell-to-cell and cell-to-substrate adhesion sites (Wilkins & Lin 1982, Luna & Hitt 1992, Humphries & Newham 1998), may show alterations. In normal cells, vinculin may be readily detected, while in highly malignant cell lines its organization may be significantly altered (Schliwa et al. 1984) or it may not be detected at all (Sadano et al. 1992), suggesting that depolymerization or suppression of vinculin expression may be closely related to progression of malignancy.

To determine the cellular sites of action of the proliferation- and detachment-promoting P metabolite, 5P, its effects on MCF-7 cell morphology, F-actin expression, polymerization, and filament distribution, as well as vinculin expression and vinculin-containing adhesion plaque numbers, were examined by immunohistochemistry, morphometry, and western blotting (Wiebe & Muzia 2001). Figure 6a shows typical distribution of polymerized actin filaments and terminal vinculin molecules in a normal cell. Treatment of cells with 5P resulted in dose-dependent decreases in vinculin-containing adhesion plaques and vinculin expression (Fig. 6b), as well as in polymerized actin stress fibers (Fig. 6c). Similar results were observed with MCF-10A, MDA-MB-231, and T47D breast cell lines (Wiebe et al. 2004), again confirming that the P metabolites appear to be able to target a variety of human breast cells. The results suggest that the observed decreases in adhesion and increases in cell proliferation following 5P-treatment may be related to depolymerization of actin and decreased expression of vinculin.

View larger version (22K): [in this window] [in a new window]   Figure 6 Examples of effects of 5P on cytoskeletal and adhesion complex molecules in MCF-7 cells. (a) The arrangement of vinculin-containing adhesion plaques (red) and polymerized F-actin fibers (green) is shown in a control cell. The effects of 5P (10–6 M) treatment result in marked reduction in vinculin expression (inset) and in number of vinculin-containing adhesion plaques (b), and depolymerization (insoluble to soluble change) of F-actin (c). (From Wiebe & Muzia, 2001 and unpublished results).

	Receptors for progesterone metabolites in human breast cells

Top Abstract Introduction Progesterone metabolism in... Cancer-related actions of the... Receptors for progesterone... Effect of the progesterone... Implications of changes in... Implications of progesterone... Summary, significance, and... References

The actions of hormonal steroids are considered to generally require complexing with specific-binding sites (receptors) on target cells. Therefore, an important step in elucidating the mechanisms of action of a regulatory hormone is the identification of such receptors. To identify potential binding sites for P metabolites in mammary cells, competition radioreceptor assays were conducted on nuclear, cytosolic, and membrane fractions of MCF-7 and MCF-10A breast cell lines using [³H]-labeled 5P and 3HP (Weiler & Wiebe 2000, Pawlak et al. 2005). The studies showed that binding of 5P or 3HP occurs in the plasma membrane fractions, but not in the nuclear or cytosolic compartments (Fig. 7a). Saturation and Scatchard analyses indicated separate high-specificity, high-affinity, low- capacity receptors for 5P and 3HP that are distinct from each other and from the well-studied nuclear/cytosolic P, estrogen, and androgen and corticosteroid receptors; binding of [³H]5P or [³H]3HP was not displaced by 200 to 500-fold concentrations of P, estradiol, androgens, corticosteroids, and other P metabolites. In turn, binding of [³H]P or [³H]estradiol to cytosolic or nuclear fractions was not displaced by excess 5P or 3HP. The binding studies showed that the criteria of high affinity, specificity, saturability, and association and dissociation kinetics required of receptor designation (Laduron 1984, Limbird 1996) were met. The studies thus provided the first demonstration of the existence of specific P metabolite receptors. Identifying the presence of distinct and separate receptors for 3HP (3HPR) and 5P (5PR) in human breast cells is important in light of the findings that the two P metabolites exert opposing actions with respect to cell proliferation and adhesion.

View larger version (11K): [in this window] [in a new window]   Figure 7 Binding sites (receptors) for progesterone metabolites in MCF-7 and MCF-10A human breast cell lines. Specific, saturable-binding sites (receptors) for 5P and 3HP are located only in the membrane fraction (a). 5P receptors (5PR) are significantly, dose-dependently and additively up-regulated by estradiol and 5P (b) and down-regulated by 3HP and 20HP (c). The increases and decreases in 5PR numbers due to 5P and 3HP respectively are abolished when cells are treated simultaneously with both hormones (d). (From Weiler & Wiebe, 2000 and Pawlak et al. 2005.)

Since the action mechanisms of hormonal steroids are generally initiated by the binding to specific receptors, the level of cellular response to steroids is limited not only by the local concentration of the hormone, but also by the receptor number (Vanderbilt et al. 1987, Webb et al. 1992). Due to the potential importance of 5P in promoting breast cancer via the binding to its membrane-based receptors, the role of mitogenic (estradiol, 5P) and anti-mitogenic (3HP, 20HP) endogenous steroids on 5PR levels in a tumorigenic (MCF-7) and a nontumorigenic (MCF-10A) breast cell line were explored (Pawlak et al. 2005). Exposure of MCF-7 cells for 24 h to estradiol or 5P resulted in significant dose-dependent increases in 5PR levels (Fig. 7b), whereas 3HP or 20HP resulted in significant dose-dependent decreases in 5PR levels (Fig. 7c). Treatment with two mitogenic (estradiol or 5P) or two anti-mitogenic (3HP or 20HP) hormones resulted in additive effects on 5PR numbers (Fig. 7b and c), whereas treatment with one mitogenic and one anti-mitogenic hormone abolished the mitogen-induced increases (Fig. 7d). In addition, preliminary experiments in which MCF-7 cells were exposed to 1.0 nM estradiol for 24 h showed a 60% decrease in 3HPR numbers (Weiler & Wiebe 2000).

The nontumorigenic breast cell line, MCF-10A, was also shown to posses specific, high-affinity plasma membrane receptors for 5P that are up-regulated by estradiol and 5P and down-regulated by 3HP (Pawlak et al. 2005). Estradiol binding was demonstrated in MCF-10A cell membrane fractions and may explain the estradiol action in these cells, which reportedly lack intracellular ER. In both MCF-7 and MCF-10A cells, the increases in 5PR due to estradiol or 5P and decreases due to 3HP or 20HP correlated with respective increases and decreases in cell proliferation as well as detachment (Pawlak et al. 2005), indicating the functional relevance of alterations in 5PR concentrations. Together, the receptor results suggest that the putative tumorigenic actions of 5P may be significantly augmented by the estradiol-induced increases in 5P binding and decreases in 3HP binding.

Role of progesterone metabolites in regulating ER levels

Estradiol can influence mitogenicity of ER-positive mammary cells and therefore the regulation of ER levels may be important for the progression of estrogen-dependent mammary neoplasias. Estradiol and P are known to play a role in modulating ER concentrations (Shyamala et al. 2002). To determine if P metabolites affect ER levels, MCF-7 cells were exposed for 24 h to 5P, 3HP, 20HP, and estradiol, or combinations of these steroids, and ER concentrations were determined in cytosolic and nuclear fractions by specific-binding of [³H]estradiol (Pawlak & Wiebe 2005). Estradiol and 5P resulted in significant dose-dependent increases, whereas 3HP and 20HP each resulted in dose-dependent decreases in total ER as well as inhibition of estradiol- or 5P-induced ER levels. In combination, estradiol + 5P or 3HP + 20HP resulted in additive increases or decreases respectively in ER numbers.

The results are the first to show that the pro- and anti-cancer P metabolites have also marked selective (up or down) regulatory effects on ER levels in ER-positive MCF-7 breast cancer cells. The suggested implications for breast cancer are that the stimulatory and inhibitory effects of 5P and 3HP respectively on cell replication and cell detachment might be significantly modified by exposure to estradiol, 4-pregnenes, and 5-pregnanes and, in turn, that the P metabolites may significantly affect ER response in estrogen-targeted cells.

	Effect of the progesterone metabolite, 5P, on cell signaling pathways

Top Abstract Introduction Progesterone metabolism in... Cancer-related actions of the... Receptors for progesterone... Effect of the progesterone... Implications of changes in... Implications of progesterone... Summary, significance, and... References

 
The location of the receptors for 5P and 3HP on the cell membrane suggests involvement of nongenomic mechanisms of action via cell signaling pathways. Modes of action via plasma membrane-based binding sites and cell signaling pathways have been suggested for estradiol (Watson et al. 1999, Keshamouni et al. 2002, Purves-Tyson & Keast 2004, Simoncini et al. 2004), corticosteroids (Wehling 1997, Croxtall et al. 2000), 3HP (Dhanvantari & Wiebe 1994, Beck et al. 1997, Wiebe 1997), and neurosteroids such as the P metabolite, 5-pregnan-3-ol-20-one (allopregnanolone) (Majewska et al. 1986). Signaling pathways that control cell proliferation and adhesion involve the mitogen-activated protein kinase (MAPK) pathway and, in turn, deregulation of this Ras-Raf-MEK-MAPK cascade plays a central role in human cancer (Chang & Karin 2001, Pearson et al. 2001, Santen et al. 2002). Studies on serum-starved MCF-7 cells showed that treatment with 5P for as briefly as 5 min resulted in significant, dose-dependent increases in activated (phosphorylated) MAPK (Erk1/2) (Wiebe et al. 2005, Cialucu & J P Wiebe, unpublished results). Treatment with the MEK inhibitor, PD98059, resulted in significant suppression of the 5P-induced MAPK activation. Similarly, in concomitant cell proliferation ([³H]thymidine uptake) and detachment assays, 5P resulted in significant increases in cell proliferation and detachment, whereas PD98059 significantly suppressed the 5P-induced increases. The data suggest that the action of 5P on breast cancer cells involves modulation of the MAPK signaling pathway. Whether other cell signaling pathways are involved or 5P and 3HP act via different pathways in promoting or inhibiting neoplasia in breast cells remain to be explored.

	Implications of changes in progesterone 5-reductase activity for androgen action in breast cancer

Top Abstract Introduction Progesterone metabolism in... Cancer-related actions of the... Receptors for progesterone... Effect of the progesterone... Implications of changes in... Implications of progesterone... Summary, significance, and... References

 
Although the majority of primary human breast cancers express androgen receptors, no direct association with any androgen and breast cancer growth and progression has been convincingly established (Bradlow & Sepkovic 2004). How might the up-regulation in 5-reductase in neoplastic breast tissue influence androgen metabolism in the breast and in turn affect the role of transformed androgens in breast cancer?

Suzuki et al.(2001) have suggested that increased conversion of testosterone to DHT resulting from increased 5-reductase activity should inhibit cancer cell proliferation in human breast carcinoma. However, studies with ZR-75-1 (Poulin et al. 1988, Birrell et al. 1995, Kandouz et al. 1999), T47-D (Birrell et al. 1995, Ortmann et al. 2002), MDA-MB-231 (Di Monaco et al. 1995, Ortmann et al. 2002), MFM-223 (Hackenberg et al. 1991), and CAMA-1 cells (Lapointe & Labrie 2001) and with DMBA-induced rat mammary tumors (Boccuzzi et al. 1995) have shown that both testosterone and DHT inhibit cell growth more or less to the same extent. This is in marked contrast to the actions of P metabolites, where the 5-pregnanes stimulate and the 4-pregnenes inhibit cell proliferation. Also, 5-reductase type 2 (SRD5A2), which catalyzes reduction of testosterone to DHT in androgen-dependent tissues such as the prostate, is present in very low levels in breast tissue (Ji et al. 2004, Lewis et al. 2004) and human breast cancer cell lines (Wiebe & Lewis 2003). In breast tissue, 5-reductase type 1 (SRD5A1) is predominant and it may be that P is a better substrate than testosterone for this isoenzyme. Overall, current evidence does not appear to support the notion that increased 5-reductase activity/ expression might significantly alter androgen influences on breast tumor growth.

	Implications of progesterone-metabolizing enzymes for synthetic progestin-based contraceptives and hormone-replacement therapy drugs

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The synthetic progestins used for contraception and hormone replacement therapy (HRT) do not behave like P in terms of their metabolism and probably not with respect to their actions at the level of the breast tissue microenvironment. As different formulations may exhibit marked differences in chemical structure, metabolism, and pharmacodynamic actions, it is not possible to generalize about them. The effects of the drugs at the level of the breast tissue will be governed by the molecular form and bioavailability, but unfortunately these are areas that remain unexplored. At the outset, the level of metabolism may vary greatly, depending on whether the route of administration is oral, transdermal, subcutaneous, or intravaginally (Fotherby 1996). When taken orally, many drugs are readily metabolized in the gastrointestinal tract and/or liver and the degree and site of metabolism varies substantially between different compounds. Some contraceptive and HRT drugs (for example, desogestrel, norgestimate, mestranol, norethisterone acetate, and ethinylestradiol-3-methyl ether) are in fact pro-drugs and are converted into their active metabolites when taken orally (Fotherby 1996, Henzl 2001). On the other hand, compounds like Nestorone must be administered parenterally due to their rapid hepatic metabolism and apparent inactivation (Sitruk-Ware 2004). The different formulations also exhibit great variation in level of binding to serum proteins (Kuhl 1996, Hammond et al. 2003), potential action via estrogen, androgen, P, and corticosteroid receptor binding and consequent androgenic, estrogenic, and progestational potency, and actions on enzymes (Kuhl 1996). To ascertain the possible role of the contraceptive and HRT drugs in breast cancer regulation via the P metabolites, it will be necessary to measure their levels and composition in the breast microenvironment to determine their effects on P metabolism in breast tissue and/or cell lines and to establish whether the P-metabolizing enzymes can further alter the drugs to pro- or anti-cancer moieties.

	Summary, significance, and future prospects

Top Abstract Introduction Progesterone metabolism in... Cancer-related actions of the... Receptors for progesterone... Effect of the progesterone... Implications of changes in...