Pregnancy, progesterone and progestins in relation to breast cancer risk

In the last two decades the prevailing opinion, supported by the “estrogen augmented by progesterone” hypothesis, has been that progesterone contributes to the development of breast cancer (BC). Support for this opinion was provided by the finding that some synthetic progestins, when added to estrogen in hormone replacement therapy (HRT) for menopausal complaints, increase the BC risk more than estrogen alone. However, recent findings suggest that both the production of progesterone during pregnancy and the progesterone endogenously produced or exogenously administered outside pregnancy, does not increase BC risk, and could even be protective. The increased BC risk found with the addition of synthetic progestins to estrogen in HRT seems in all likehood due to the fact that these progestins (medroxyprogesterone acetate and 19-nortestosterone-derivatives) are endowed with some non-progesterone-like effects which can potentiate the proliferative action of estrogens. The use of progestational agents in pregnancy, for example to prevent preterm birth, does not cause concern in relation to BC risk.     

Endogenous estrogen, testosterone and progesterone levels in relation to breast cancer risk

Multiple lines of evidence support a central role of hormones in the etiology of breast cancer. In epidemiologic studies, considerable effort has focused on delineating the role of endogenous hormones in risk of breast cancer among postmenopausal women. Recently, substantial additional data has accrued from prospective studies where endogenous hormones are measured in study subjects prior to disease diagnosis. In this review, the epidemiologic evidence linking sex steroids—estrogens, testosterone, and progesterone, specifically—with subsequent risk of breast cancer in both premenopausal and postmenopausal women is summarized. Overall, a strong positive association between breast cancer risk and circulating levels of both estrogens and testosterone has now been well confirmed among postmenopausal women; women with hormone levels in the top 20% of the distribution (versus bottom 20%) have a two- to three-fold higher risk of breast cancer. Evidence among premenopausal women is more limited, though increased risk associated with higher levels of testosterone is consistent. However, both positive and null associations have been observed with estrogens and progesterone and clearly more evaluation is needed.     

Estriol in human breast cyst fluid

Although cystic lesions of the breast are not precancerous per se, statistical studies have indicated that this condition predisposes a 2- to 4-fold greater risk for breast cancer. Seeking a hormonal etiology to this correlation, investigators have analyzed breast cyst fluid (BCF) for steroids and have compared the levels to those in the blood. The 17-ketosteroids-androsterone, dehydroisoandrosterone and their sulfates are elevated in BCF. The same is true for estrone sulfate and estradiol-3-sulfate. We have found the most dramatic differences with estriol-3-sulfate (E₃-3S), the concentration of which ranged from 187–6134 pg/ml in over 40 specimens analyzed, whereas in 12 serum specimens from normal women, E₃-3S was barely detectable. The origin of E₃-3S is not known. [³H]E₃-3S is not concentrated in BCF following its injection into an arm vein. The blood half-life of [³H]E₃-3S is much lower than that of estrone sulfate. Samples of breast nipple aspirates from normal women were also analyzed for E₃-3S. None could be detected. The best explanation of the data accumulated thus far is that E₃-3S is synthesized at the epithelial lining of the cyst and released into the BCF, from which its efflux is inefficient.     

Cytokines in human breast cyst fluid

Gross cystic breast disease is a common benign disorder in which palpable cysts occur in the breast and are normally treated by aspiration of the contents. The cysts are classified as either Type 1, containing a high level of potassium ions and a low level of sodium ions, or as Type 2, with low potassium and high sodium ion concentrations. Steroid sulphatase activity in MDA-MB-231 and MCF-7 cell lines is regulated by exogenous breast cyst fluid (BCF), possibly because of cytokines in the BCF. A screening method was used to determine the range of cytokines in eight BCFs, four of each type. This was an array system, which uses antibodies immobilised on a membrane to qualitatively detect 79 different cytokines or growth factors. Nine cytokines were detected well above background levels: all were found in both types of BCF, but only epidermal growth factor (EGF) was higher in Type 1. All the other factors were higher in Type 2 BCF. Two of these cytokines, IL-6 and EGF, have previously been suggested to affect steroid sulphatase expression and several (MIP-1β, IL-8, NAP-2) are known to affect MCF-7 cell chemotaxis. In addition two cytokines were measured by ELISA in 57 BCFs, and both IL-1β and IL-13 were found in BCF, with significantly higher amounts of IL-1β in Type 1 than Type 2 BCF (35.5 ± 4.4 pg/ml versus 9.9 ± 2.9 pg/ml).     

Steroid sulphatase inhibitors for breast cancer therapy

In contrast to aromatase inhibitors, which are now in clinical use, the development of steroid sulphatase (STS) inhibitors for breast cancer therapy is still at an early stage. STS regulates the formation of oestrone from oestrone sulphate (E1S) but also controls the hydrolysis of dehydroepiandrosterone sulphate (DHEA-S). DHEA can be reduced to 5-androstenediol (Adiol), a steroid with potent oestrogenic properties. The active pharmacophore for potent STS inhibitors has now been identified, i.e. a sulphamate ester group linked to an aryl ring. This has led to the development of a number of STS inhibitors, some of which are due to enter Phase I trials in the near future. Such first generation inhibitors include the tricyclic coumarin-based 667 COUMATE. Aryl sulphamates, such as 667 COUMATE, are taken up by red blood cells (rbc), binding to carbonic anhydrase II (CA II), and transit the liver without undergoing first-pass inactivation. 667 COUMATE is also a potent inhibitor of CA II activity with an IC₅₀ of 17 nM. Second generation STS inhibitors, such as 2-methoxyoestradiol bis-sulphamate (2-MeOE2bisMATE), in addition to inhibiting STS activity, also inhibit the growth of oestrogen receptor negative (ER⁻) tumours in mice and are anti-angiogenic. As the active pharmacaphores for the inhibition of aromatase and STS are now known it may be possible to develop third generation inhibitors that are capable of inhibiting the activities of both enzymes. Whilst exploring the potential of such a strategy it was discovered that 667 COUMATE possessed weak aromatase inhibitory properties with an IC₅₀ of 300 nM in JEG-3 cells. The identification of potent STS inhibitors will allow the therapeutic potential of this new class of drug to be explored in post-menopausal women with hormone-dependent breast cancer. Second generation inhibitors, such as 2-MeOE2bisMATE, which also inhibit the growth of ER⁻ tumours should be active against a wide range of cancers.     

Steroid metabolism in human breast cancer cell lines

The metabolism of 1,2-³H-androstenedione was studied in 2 cell lines, MCF-7 (estrogen responsive) and BT-20 (estrogen nonresponsive) over 48 hrs. Water soluble and unconjugated metabolites were separated by solvent partition and the former was submitted to chromatography on Sephadex LH-20 and enzyme hydrolysis. The resulting unconjugated steroids were separated by paper chromatography and identities were established by reverse isotope dilution. The unconjugated steroids initially obtained were separated by chromatography and identified by reverse isotope dilution. About 70% of the androstenedione was metabolized by both cell lines. However, the respective conversions to conjugates by MCF-7 and BT-20 were 31% and 0.32%. In the former, glucosiduronates predominated (94%) and consisted of androsterone (55%), etiocholanolone (9.4%) and androstanediol (5α-androstane-3α,17β-diol) (9.3%). Androsterone comprised most of the unconjugated metabolites in both cell lines. Androstanediol was found in both cell lines, 2% in MCF-7 and 12% in BT-20. Testosterone, 5α-androstane-3,17-dione and 3β-hydroxy-5α-androstan-17-one were isolated only from MCF-7. The metabolism of ³H-estriol was studied in a similar way. Both cell lines produced about equal amounts of estriol-3-sulfate (9%) and a compound with properties of estriol-3-glucosiduronate (0.15 – 0.5%). The results worthy of emphasis are: 1. The far greater conjugation of androgens exhibited by the MCF-7 cell lines as compared to the BT-20 cell lines; 2. In MCF-7, the high conversion of androstenedione to etiocholanolone (glucosiduronate form), a metabolite reported to form only in liver and sebaceous cysts; 3. The possible formation in both cell lines of estriol-3-glucosiduronate, normally a metabolite of the intestine.     

Levels of human chorionic gonadotropin in breast cyst fluid

The results of dosage of human chorionic gonadotropin (HCG) in 75 breast cyst fluids taken from 61 patients suffering from breast gross cystic disease (BGCD), 13 of which had multiple mono and/or bilateral cysts are discussed. The corresponding sera were also examined. Assays were carried out using the ELISA method. For comparison, HCG levels were also determined in 21 breast cyst fluids using RIA method. In 66.7% of the breast cyst fluids examined the hormone levels were higher than normal serum values. Corresponding sera showed HCG to be within the range of normal levels. In 5 patients the multiple and/or bilateral cysts showed widely differing hormone levels. The importance of these observations is discussed.     

Androgens and breast cancer risk

Transdermal testosterone supplementation is a treatment option for postmenopausal women with distressful decreased libido. Side effects are minor, but there is a long-term safety concern with respect to breast cancer, as women with high testosterone serum levels appear to be at a significantly increased risk to have or to develop breast cancer within a few years. Epidemiological studies of sufficient duration to study long-term effects of testosterone supplementation are limited, both in number and in methodological quality and are, therefore, inconclusive. Preclinical studies do not provide evidence for an androgen receptor-mediated stimulating effect of androgens on breast epithelium. However, one biologically plausible possibility, which cannot be ruled out, is that exogenous androgens become mitogenic after aromatization into bioactive oestradiol, either in peripheral fat or within the breast or even within small occult tumours. The evidence available so far makes counselling women interested in testosterone supplementation for distressful low sexual desire, more of an art than science.     

Allopregnanolone, pregnenolone sulfate, and epitestosterone in breast cyst fluid

The risk of breast cancer is 2 to 5 times higher in patients suffering from gross cystic disease. Breast cysts are categorized into two groups (type I and type II) according to the concentration of electrolytes in the cyst fluid. The two types also differ with respect to accumulation of steroids and steroidogenic enzyme activity. In type I cysts a higher risk of breast carcinoma could be expected. Here, we studied a possible relationship between the type of cyst and levels of epitestosterone (an endogenous antiandrogen), allopregnanolone (a product of 5alpha-reductase activity), and pregnenolone-sulfate (an activator of N-methyl-D-asparate receptors). We have found five times higher levels of epitestosterone in BCF in comparison with the circulation. Allopregnanolone levels were similar to those in plasma of women in the luteal phase of the menstrual cycle. Pregnenolone-sulfate levels in BCF were about two orders of magnitude higher when compared with the circulation. No differences were found in concentrations of the steroids studied between the types of cysts.