Progestins and Breast Cancer

By Pasqualini, Jorge R

Abstract Progestins exert their progestational activity by binding to the progesterone receptor (form A, the most active and form B, the less active) and may also interact with other steroid receptors (androgen, glucocorticoid, mineralocorticoid, estrogen). They can have important effects in other tissues besides the endometrium, including the breast, liver, bone and brain. The biological responses of progestins cover a very large domain: lipids, carbohydrates, proteins, water and electrolyte regulation, hemostasis, fibrinolysis, and cardiovascular and immmunological systems. At present, more than 200 progestin compounds have been synthesized, but the biological response could be different from one to another depending on their structure, metabolism, receptor affinity, experimental conditions, target tissue or cell line, as well as the biological response considered.

There is substantial evidence that mammary cancer tissue contains all the enzymes responsible for the local biosynthesis of estradiol (E^sub 2^) from circulating precursors. Two principal pathways are implicated in the final steps of E^sub 2^ formation in breast cancer tissue: the ‘aromatase pathway’, which transforms androgens into estrogens, and the ‘sulfatase pathway’, which converts estrone sulfate (E^sub 1^S) into estrone (E^sub 1^) via estrone sulfatase. The final step is the conversion of weak E^sub 1^ to the potent biologically active E^sub 2^ via reductive 17beta-hydroxysteroid dehydrogenase type 1 activity. It is also well established that steroid sulfotransferases, which convert estrogens into their sulfates, are present in breast cancer tissues.

It has been demonstrated that various progestins (e.g. nomegestrol acetate, medrogestone, promegestone) as well as tibolone and their metabolites can block the enzymes involved in E^sub 2^ bioformation (sulfatase, 17beta-hydroxysteroid dehydrogenase) in breast cancer cells. These substances can also stimulate the sulfotransferase activity which converts estrogens into the biologically inactive sulfates. The action of progestins in breast cancer is very controversial; some studies indicate an increase in breast cancer incidence, others show no difference and still others a significant decrease. Progestin action can also be a function of combination with other molecules (e.g. estrogens). In order to clarify and better understand the response of progestins in breast cancer (incidence, mortality), as well as in hormone replacement therapy or endocrine dysfunction, new clinical trials are needed studying other progestins as a function of the dose and period of treatment.

Keywords: Progestins, breast cancer, estrogens, enzymes, hormone replacement therapy

Introduction

Progesterone can have multiple and important biological effects in a variety of tissues, such as the endometrium, breast, liver, bone and brain. These actions cover a wide domain including lipid, carbohydrate, protein, water and electrolyte regulation, hemostasis and fibrinolysis, as well as the cardiovascular and immunological systems. At present, more than 200 progestin compounds have been synthesized, but they differ from one to another in terms of their structure, metabolism, receptor affinity, target tissues, biological effects, dosage required and use of cell clone. In this review we summarize the actions of the various progestins in breast tissue, including their effects on the enzymes involved in the formation and transformation of estrogens and on cell proliferation, as well as recent developments and clinical implications of progestins in breast cancer.

Classification of progestins

The progestin closest to natural progesterone is retroprogesterone, followed by the pregnane (17- hydroxyprogesterone, C21) derivatives and the 19-norprogesterone (19- norpregnane, C20) derivatives. A clinically important group, and the basis for the success of hormonal contraception, is the 19- nortestosterone derivatives, which can be subdivided into estranes (C18) and gonanes (C17). A spirolactone derivative has also recently been developed for clinical use. Some of these compounds are pro- drugs, which are metabolized to the active compounds in the liver (e.g. promegestone is converted to trimegestone, desogestrel to 3- ketodesogestrel, and norgestimate to norgestrel) [1-4]. Table I summarizes the different structural groups of progestins and their members.

Receptor binding is important for the biological response of progestins. The affinity for the receptor varies widely between the various progestins; Table II shows the relative affinity of these molecules, considering that of progesterone as 100% [5-7].

Breast cancer evolution

Initially most breast cancers (approximately 95%) are hormone- dependent, where the estrogen estradiol (E^sub 2^) plays an important role in the development and progression of the tumor [8- 10]. The hormoneestrogen receptor (ER) complex can mediate the activation of proto-oncogenes and oncogenes (e.g. c-fos, c-myc) and nuclear proteins, as well as other target genes. After a period that may last several years, the tumor becomes hormone-independent by a mechanism which, although not yet fully elucidated, is currently under scrutiny. One explanation for the progression toward hormone independence may be the presence of ER mutants [11,12]. In hormonedependent cells, the interaction of the hormone with the receptor molecule is the basic step for eliciting a hormone response. As the cancer cell evolves, mutations, deletions and truncations appear in the receptor gene [13,14]; the ER becomes ‘nonfunctional ‘ and, despite the estrogen binding, the cell fails to respond to the hormone. Figure 1 describes the progression of normal mammary cells toward a hormone-independent carcinoma. A ‘nonfunctional’ ER might explain why 35-40% of patients with ER- positive tumors fail to respond to antiestrogen therapy [15,16].

Table I. Classification of progestins [1-4].

Table II. Relative binding affinities of progesterone and synthetic progestins to steroid receptors and serum binding proteins [5-7].

Progesterone is another major, although controversial, player in mammary gland biology. This ovarian steroidal hormone also acts, in conjunction with estrogen, through its specific receptor (PgR) in the normal epithelium to regulate breast development. The effects of these hormones on the proliferative activity of the breast, which are indispensable for its normal growth and development, have long been, and still are, the subject of heated controversy [17]. The remaining 5% of breast cancers, denoted BRCA-1, are considered hereditary [18].

Figure 1. Evolution of the transformation of breast cells from normal to cancerous (ER^sup +^, estrogen receptor-positive, i.e. detectable and functional; ER mutants, estrogen receptor detectable but non-functional; ER , estrogen receptor-negative, i.e. not detectable).

The mechanism behind the different steps from normal breast to invasive ductal or lobular carcinoma is still unclear. However, in recent years, new methodology including DNA amplification [19], tissue microdissection [20] and genome and transcriptome analyses [21] has provided very interesting information on the molecular evolution of breast cancer. At present, we can accept that breast cancer is a heterogeneous disease encompassing a wide variety of pathological activities. It is expected that the statistical data on these new molecular aspects will provide useful clinical applications.

Concentration of estrogens in normal and cancerous breast

Estrogen sulfates are quantitatively one of the most important forms of circulating estrogens. High concentrations of these conjugates are found in the fetal and maternal compartments of humans and several animal species [22]. During the menstrual cycle and in postmenopausal women, estrone sulfate (E^sub 1^S) levels are five to ten times those of unconjugated estrogens (estrone, estradiol and estriol) [23,24].

Most authors agree that plasma levels of unconjugated estrone (E^sub 1^) and E^sub 2^ are similar in normal women and in breast cancer patients, both before and after the menopause. However, the E^sub 1^S level is significantly higher in the follicular phase of premenopausal breast cancer patients than in normal women [25].

Breast cancer tissues have a high capacity to accumulate various estrogens, particularly in postmenopausal women. In addition, comparative studies indicate that the levels of estrogen in cancer tissues are significantly higher than in the area of the breast considered as normal [26]. Table III gives the tissue concentrations of various estrogens in pre- and postmenopausal breast cancer patients [27-32].

Enzymes involved in the formation and transformation of estrogens in breast tissue and their control by progestins

Different studies in various laboratories have clearly established that breast tissues have the capacity for local bioformation of estrogens from circulating precursors. Two principal pathways are implicated in the final steps of E^sub 2^ formation in breast cancer tissues: the ‘aromatase pathway’, which transforms androgens into estrogens [33-35], and the ‘sulfatase pathway’, which converts E^sub 1^S to E^sub 1^ via estrone sulfatase (EC: 3.1.6.2) [36-40]. The final step of steroidogenesis is the conversion of the weak E^sub 1^ to the potent, biologically active E^sub 2^ by the action of a reductive 17beta-hydroxysteroid dehydrogenase type 1 activity (EC: 1.1.1.62) [41,42]. Quantitative evaluation indicates that, in human breast tumors, E^sub 1^S ‘via sulfatase’ is a much more likely precursor for E^sub 2^ than are androgens ‘via aromatase’ [25,43]. It is also well established that steroid sulfotransferases, which convert estrogens into their sulfates, are also present in breast cancer tissues [44,45]. Figure 2 gives a general view of estrogen formation and transformation in human breast cancer.

Effect of progestins on estrone sulfatase activity

Estrone sulfatase activity in mammary tumors is significantly higher in postmenopausal than in premenopausal women, and this activity is increased in the tumor tissue compared with the surrounding area or in areas considered as ‘normal’ [25,26]. No correlation has been found between sulfatase activity levels and ER status [46,47]. However, in intact breast cancer cells in culture, a marked difference in sulfatase activity exists between hormone- dependent (MCF-7, T-47D) and hormone-independent (MDA-MB-231, MDA- MB-468) cells. The former possess high activity, whereas the latter have little effect on the hydrolysis of E^sub 1^S [28,48]. However, when these hormone-independent cells are homogenized, the sulfatase activity is restored [49,50]. The data regarding the mechanism involved in sulfate hydrolysis suggest that ‘stimulatory factors’ necessary for enzyme activity are present in the hormone-dependent cells, but could be absent in the hormone-independent cells.

Table III. Concentrations of unconjugated estrogens and their sulfates in malignant breast tissues (pmol/g tissue).

Figure 2. Enzymatic mechanism involved in the formation and transformation of estrogens in human breast cancer: the sulfatase pathway (A) is quantitatively 100-500 times greater than the aromatase pathway (B) (17beta-HSD-1, 17beta-hydroxysteroid dehydrogenase type 1).

The effect of progestins on sulfatase activity in breast cancer has, to date, been explored only in in vitro studies. In breast tumors, progestins such as demegestone or chlormadinone acetate at concentrations of 10 ^sup -5^ M inhibit sulfatase activity by 25- 50% [51,52]. Other progestins such as medroxyprogesterone acetate (MPA) and norgestrel, at the same concentration, moderately stimulate this activity [52]. In breast cancer cells (MCF-7, T- 47D), promegestone (R-5020), nomegestrol acetate, medrogestone and norethisterone, as well as danazol, at a range of concentrations between 5 x 10^sup -7^ and 5 x 10^sup -6^ M, decrease sulfatase activity by 40-70% [53-58]. Tibolone and its metabolites (Org 4094, Org 30126, Org OM38) at low doses (5 x 10^sup -8^ M) are very potent anti-sulfatase agents as they inhibit the activity of this enzyme by 80-90% in the same cell lines [59]. R-5020 is a competitive inhibitor of the sulfatase enzyme in homogenates of breast cancer cells (MCF-7, T-47D) [60]. This progestin can also reduce the expression of sulfatase mRNA in both cells, an effect that is correlated with the reduction in enzymatic activity [61]. It has also been demonstrated that dydrogesterone (Duphaston(R)) and its 20-dihydro derivatives are potent inhibitors of estrone sulfatase in MCF-7 and T-47D breast cancer cells [62]. The relative inhibitory activity of progestins and tibolone and its metabolites is shown in Table IV [55,59,63-65].

Progestins can also block sulfatase activity in total breast tissues. It has been demonstrated that nomegestrol acetate at 5 x 10^sup -7^ M can inhibit sulfatase activity (conversion of E^sub 1^S to E^sub 2^) by 23% in normal breast tissue and 32% in cancer tissue [66]. In an interesting study, Miyoshi and colleagues [67] suggested that sulfatase mRNA levels can serve as a significant, independent prognostic factor in ER-positive breast cancer and that high levels of sulfatase mRNA are associated with a poor prognosis.

Table IV. Comparative effect of various progestins, tibolone and its metabolites, and estradiol on estrone sulfatase inhibition in T- 47D breast cancer cells [55,59,63-65].

Effect of progestins on 17beta-hydroxysteroid dehydrogenase activity

17beta-Hydroxysteroid dehydrogenase (17beta-HSD) is the enzyme that inter-converts the 17-oxo- and 17beta-hydroxy steroids. At present, 14 types of this enzyme have been characterized. In breast cells, the most important with regard to estrogens is 17beta-HSD type 1, which converts E^sub 1^ into E^sub 2^, and 17beta-HSD type 2, which transforms E^sub 2^ into E^sub 1^.

In epithelial cells from normal breast, the progestin promegestone has been shown to increase 17beta-HSD activity in the oxidative (E^sub 2^ to E^sub 1^) direction (type 2). This stimulatory effect of progestins depends on preliminary sensitization by estrogens [68,69].

Progestins can induce 17beta-HSD type 1 activity, with an increase in both the 1.3-kb mRNA species and the enzyme protein, in hormone-dependent T-47D breast cancer cells [70,77]. Coldham and James [72] found that MPA stimulated the reductive (E^sub 1^ to E^sub 2^) activity of MCF-7 cells when phenol red was excluded from the tissue culture media. They suggested that this could be the way in which progestins increase cell proliferation in vivo. On the other hand, Couture and associates [73] observed that when hormone- dependent ZR-75-1 breast cancer cells were incubated with MPA, the oxidative (E^sub 2^ to E^sub 1^) direction was predominant; this effect appears to implicate the androgen receptor. Other progestins, such as progesterone, levonorgestrel and norethisterone, increased both the oxidative and reductive 17beta-HSD activity in MCF-7 cells [74], whereas promegestone had no significant effect on the reductive activity of 17beta-HSD [75] but increased the oxidative (E^sub 2^ to E^sub 1^) activity in T-47D cells [76].

Effect of progestins on sulfotransferase activity

Comparative studies on the formation of estrogen sulfates after the incubation of E^sub 1^ with hormonedependent (MCF-7, T-47D) and hormone-independent (MDA-MB-231) breast cancer cells show significandy higher sulfotransferases in the former [77,78].

Medrogestone is a synthetic pregnane derivative used in the treatment of pathological deficiency of natural progesterone. It has secretory activity in the estrogen-primed uterus, is thermogenic, and acts as an antiestrogen and anti-gonadotropin. Its effect on sulfotransferase activity in MCF7 and T-47D breast cancer cells is biphasic: at a low concentration (5 x 10^sup -8^ M) it stimulates the formation of estrogen sulfates in both cell lines, whereas at a high concentration (5 x 10^sup -5^ M) sulfotransferase activity is not modified in MCF-7 cells or is inhibited in T-47D cells [79]. A similar dual effect on sulfotransferase was seen with the progestins nomegestrol acetate and promegestone [78,80] (Table V). The effects on sulfotransferase activity of tibolone and its metabolites 3alpha- hydroxytibolone (Org 4094), 3beta-hydroxytibolone (Org 30126) and the 4-ene isomer (Org OM 38) have been explored in MCF-7 and T-47D breast cancer cells. These compounds also provoke a dual effect on sulfotransferase activity: stimulatory at low doses (5 x 10^sup – 8^ M) but inhibitory at higher doses (5 x 10^sup -5^ M). The 3beta- hydroxy derivative is the most potent compound with regard to stimulatory effects on sulfotransferases [77]. Table V shows the comparative effects of various progestins, as well as that of tibolone and its metabolites, on sulfotransferase activity in T-47D breast cancer cells [77-81].

Table V. Comparative effect of various progestins, tibolone and its metabolites on sulfotransferase activity in T-47D breast cancer cells [77-81].

As some progestins, as well as tibolone, can stimulate sulfotransferase activity, and as these compounds can inhibit the growth of breast cancer cells or the size of transplanted tumors, it was hypothesized that a correlation exists between cell proliferation and sulfotransferase activity [82,83]. Using reverse transcriptase-polymerase chain reaction amplification, the expression of estrogen sulfotransferase mRNA was detected in hormone- dependent MCF-7 and T-47D, as well as in hormone-independent MDA-MB- 231 and MDAMB-468, human breast cancer cells. An interesting correlation with the relative sulfotransferase type 1 mRNA expression was found in the various breast cancer cells studied [78].

A study of the effects of promegestone on the activity of type 1 human estrogen sulfotransferase and its mRNA in MCF-7 and T-47D cells showed that, at low doses, there was a significant increase in mRNA levels, which correlated with enzyme activity. However, at high doses, an inhibitory effect was observed on mRNA and enzyme activity [78]. Falany and co-workers [84] suggested that loss of estrogen sulfotransferase expression in the transformation of normal breast tissue to breast cancer may be an important factor in increasing the growth responsiveness of pre-neoplastic cells to estrogen stimulation.

Effect of progestins on aromatase activity

Although levels of aromatase activity are relatively low in the normal or cancerous breast [26,43], this local production of estrogens ‘on site’ can contribute to the pathogenesis of estrogen- dependent breast cancers. The role of progestins on aromatase activity in the breast is very limited. Perel and co-workers [85] observed that progestins can inhibit the aromatase activity in cultured breast carcinoma cells. In another series of studies, it was demonstrated that nomegestrol acetate can inhibit aromatase activity in breast cancer cells [86].

Aromatase inhibition with anti-aromatase agents is widely used, with very positive results, in the treatment of patients with breast cancer [87-90]. These inhibitors include steroidal and non- steroidal compounds, the most useful being 4- hydroxyandrostenedione, anastrazole (Arimidex(R)), examestane (Aromasin(R)) and letrozole (Femara(R)). A series of reviews has recently been published on the biological effects and clinical applications of these anti-aromatases [91-93]. Effects of progestins in breast cancer: clinical consequences

The use of progestins alone or in combination with estrogens in hormone replacement therapy (HRT) is controversial. In a series of studies in which the effect of MPA or norethisterone acetate was assessed in a large number of women, a significant increase in breast cancer incidence was observed [94-96]. However, another recent study [97] in 10 000 Japanese patients showed that HRT can decrease the relative risk of breast cancer.

The Million Women Study contained multiple errors, sufficient to raise doubts about the care with which the study was carried out and reviewed. Shapiro [98] concluded that it is not possible to distinguish between bias and causation as alternative explanations for the observed associations, and the conclusion from this study that HRT increases the risk of breast cancer is not justified.

Some aspects of the relationship between HRT and tumor type remain unclarified. It has been observed that the incidence of breast cancer in ER-positive patients was higher than in ER- negative patients [99]. In addition, it has been suggested that HRT use increases the incidence of lobular carcinoma more than that of ductal carcinoma [100].

Another important aspect is the effect of progestins on breast proliferation and, as was indicated previously, these data are also controversial since the various progestins can block, stimulate or have no effect on cell growth.

Figure 3. Hydroxyl pathways of estrogens in human breast cancer (2-OH-E^sub 1^, 2-hydroxyestrone; 2-OH-E^sub 2^. 2- hydroxyesrradiol; 4-OH-E^sub 1^, 4-hydroxyestrone; 4-OH-E^sub 2^, 4- hydroxyestradiol; 16alpha-OH-E^sub 1^, 16alpha-hydroxyestrone; 17beta-HSD, 17beta-hydroxysteroid dehydrogenase).

Conclusions

Some studies show that HRT can increase the relative risk of breast cancer; however, it is notable that very few progestins have been tested. As mentioned previously, it is important to understand that not all progestins are alike in structure and biological function. The Million Women Study, which has had a drastic impact on the treatment of postmenopausal women using progestins, has provoked a series of comments raising serious and important criticisms. Heikkinen [101] considered that an apparently small increase in breast cancer risk with combined hormone therapy must be balanced against a reduction in the significant morbidity and mortality associated with fractures, as well as improved well-being, quality of life, and potential protection against colorectal cancer and cardiovascular disease.

An interesting property of progestins is their action on enzymes involved in the formation and transformation of estrogens, as described above in the section on progestins and breast cancer proliferation. A series of studies has shown that various progestins can have a beneficial effect by blocking sulfatase and 17beta-HSD type 1 (the main enzymes involved in the biosynthesis of estradiol in breast cancer cells or total tissue). These progestins can also stimulate sulfotransferase that transforms estrogens into inactive sulfates.

Another aspect to consider is the metabolic transformation of progesterone and progestins. In this regard, it is interesting to note that conversion of progesterone to 5alpha-pregnanes is increased, while conversion to 4-pregnanes is decreased, in breast carcinoma tissue. The 5alpha-pregnane 5a-pregnane-3, 20-dione stimulates cell proliferation, whereas the 4-pregnane 3alpha- hydroxy-4-pregnen-20-one inhibits it [102].

In breast and other tissues, E^sub 2^ and E^sub 1^ are transformed to hydroxylated derivatives: the 2-hydroxy metabolite is considered to be an antiproliferative agent and the 4-hydroxy derivative to be a carcinogenic substance [103-106]. Figure 3 summarizes these metabolic transformations in breast cancer.

In conclusion, as the findings regarding HRT use and breast cancer incidence are contradictory, further information is needed on new administration routes and new regimens (dose and period of administration) in order to evaluate the impact of HRT on breast cancer risk and mortality.

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JORGE R. PASQUALINI

Hormones and Cancer Research Unit, Institut de Puericulture et de Perinatalogie, Paris, France

(Received 17 May 2007; accepted 5 September 2007)

Correspondence: J. R. Pasqualini, Hormones and Cancer Research Unit, Instirut de Puericulture et de Perinatalogie, 26 Boulevard Brune, F-75014 Paris, France. Tel: 33 1 45 42 41 21/45 39 91 09. Fax: 33 1 45 42 61 21. E-mail: Jorge.Pasqualini@wanadoo.fr

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