John B. Adams
Former visiting Professor, School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, NSW, Australia.
...they are ill discoverers that think there is no land when they can see nothing but sea.
Sir Francis Bacon
The Advancement of Learning
Address for offprints and correspondence: J.B. Adams, do School of Biochemistry and Molecular Genetics, University of N ew South Wales, Sydney, NSW 2052, Australia; Tel: 61 2 9948 1833; Fax: 61. 2 9385 1483.
Key words: adrenal androgens, breast cancer, 5-androstene-3β,17β-diol, hermaphrodiol, estrogen receptor.
Summary
A clearer picture of the role of adrenal androgens in the etiology of breast cancer is beginning to emerge. Women who develop breast cancer in premenopausal years tend to have subnormal scrum levels of adrenal androgens, while subjects who develop the disease in postmenopausal years have supranormal levels of these hormones. Androgens, by acting via the androgen receptor, oppose estrogen-stim ulated cell growth in premenopausal years. In postmenopausal women, elevated adrenal androgen levels stimulate cell growth by the action of the unique adrenal androgen 5-androstene-3β,17β-diol, also termed hermaphrodiol, via its combination with the estrogen receptor in a hormone milieu lacking, or having low concentrations of, the classical estrogen 17β-estradiol.
Introduction
Within the broad field of hormones and breast cancer, perhaps no other area has had such a chequered and stormy history as that of the role of the so-called adrenal androgens in the etiology of the disease. The adrenal androgens are comprised of a group of C19-steroids: dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), 5andr ostene-3β,17β-diol (ANDROSTENED1GL), and 4-androstenedionc (ADIONE). Despite the finding that DHEAS was secreted by the human adrenal by Beaulieu et al. in 1965 [1], and quantitatively is the most important steroid in human blood, the exact role of this hormone remains an enigma.
Studies aimed at establishing a possible link between adrenal androgens and human breast cancer first appeared in 1957 [2] when measurements of 11deoxy-17-ketosteroids in urine were made; these being chiefly derived from metabolism of adrenal androgens. Many studies were reported over the subsequent decade. Some were aimed at determining a discriminant, based on the ratio of 11-deoxy-17-ketosteroids to 17-hydroxycorticosteroids in the urine of breast cancer subjects, as a guide to predicting results of hormone ablative procedures such as adrenalectomy and hypophysectomy. The results of these studies were discussed as part of an excellent review on hormone profiles and the epidemiology of breast cancer by Zumoff [3]. Suffice it to say the data on urinary steroid hormone metabolites were very controversial and could probably be explained by distortions due to the non-specific effect of illness, or operative stress. As an example, operative stress, such as mastectomy prior to urine collection, was shown to alter the pattern of adrenal androgen metabolites. I6-hydroxylated-5-androstenes were formed in relatively large amounts, and, as these do not react with the Zimmerman reagent normally used in the assay of 17-ketosteroids at the time, incorrect interpretations of adrenal androgen production were made [4].
Problems such as those described above do not apply to pi ospcclive studies based on liuimune assessment of urine collected from healthy subjects. Three such prospective studies, each involving 5,000 healthy women on the island of Guernsey, were carried out in a remarkable study by Bulbrook and his colleagues between 1961 and 1986 [5]. Urine, and later blood, samples were collected, stored frozen, and when breast cancer occurred in subsequent years, they were subjected to endocrine analysis and comparison made with a number of controls suitably matched for age, weight, parity, etc. In the first study, the main urinary metabolites of DHEAS, i.e. etiocholanolone and androsterone, were assayed in 110 women who subsequently developed breast cancer and 1335 women who did not.. Results from women who developed Ihe disease in the first 9 years of the study, and were mainly premenopausal, showed they had significantly lower levels of urinary androgen metabolites than agematchcd controls. However, as the follow-up continued over 25 years, the results became less clear cut. Women with androgen metabolites at the lower end of the normal range generally had a diagnosis of their disease in the laic-prcmcnopausal period; those with higher levels developed breast cancer at older ages [5]. In analysing their overall data. Bulbrook and Thomas |6j concluded that low blood levels of androgens, as reflected in subnormal excretion of their metabolites, are markers for rapid tumor growth. Androgens may be acting to inhibit tumor growth and therefore time of onset of the actual appearance of a tumor, the hormone environment influencing the growth rate of a clone of neoplastic cells initiated by previous carcinogenic events.
With the advent of radioimmunoassay techniques, scrum concentrations of adrenal androgens were able to be determined in breast cancer subjects and controls. Although no general conclusions were reached, the trend was towards a lower concentration of these hormones in breast cancer cases, but again stress of operation was not considered, nor was menopausal status recorded, in many cases [3J. In one carefully controlled study, DHEA and DHEAS were measured in pooled 24 hr scrum samples, collection being made every 20 min. Subjects were 11 women with primary operable breast cancel aged 31 to 78 years and controls wer e 37 normal women aged 21 to 75 years. In contrast to the marked decline in adrenal androgens with age in the blood of normal women, the concentrations of both steroids were age invariant in the cancer patients; the premenopausal patients had subnormal while the postmenopausal patients had supranortnal levels of each steroid hormone [7].
More recently, the results of two prospective studies have been published wherein analyses were determined on serum samples stored in blood banks. In the first of these, scrum was obtained from the Washington County, Maryland, serum bank, which holds specimens from 25,620 volunteers collected in 1974. Such banks are unique and the policy is to allow analyses of 30 cases and appropriate controls; if statistically significant results arc achieved, the study stops. Gordon et al. [8] measured scrum levels of DHEA and DHEAS in 30 postmenopausal women who subsequently, at least 9 years later, developed breast cancer, and in 59 matched controls. Significantly elevated serum DHEA levels were found among cases prior to diagnosis compared to controls. DHEAS levels were slightly increased among cases. In a parallel investigation involving 15 women who developed breast cancer while still in their premenopausal years, the risk ratio Tor women in the highest tertile, compared to the lowest tertile of serum DHEA, was 0.4 with a suggestion of a dose-response trend with increasing levels. No consistent association between serum DHEAS and risk of premenopausal breast cancer was evident [9].
In a very recent study. Dorgan ct al. [10] measured DHEAS, DHEA, and ANDROSTENEDLOL in serum samples from the Columbia Missouri Breast Cancer Serum Bank. Seventy one healthy postmenopausal volunteers, not taking replacement estrogen when they donated blood, subsequently developed breast cancer up to 10 years later. Two randomly selected controls, who were also postmenopausal and not taking estrogen, were matched to each case on exact age (date ± 1 year) and time (± 2 hours) of blood collection. Significant gradients of increased risk of breast cancer were observed for increasing concentrations of DHE-A and ANDROSTENEDIOL. Women whose scrum levels of these hormones were in the highest quartiles were at significantly elevated risk compared to those in the lowest. The relationship ofDHEAS to breast cancer was less consistent, but women with levels in the highest quartile also exhibited a significantly elevated risk ratio.
All of the above prospective studies arc then in general agreement; lowered levels of adrenal androgens being associated with risk of breast cancer in premenopausal years and elevated levels with risk of developing breast cancer in postmenopausal years (Figure 1).
Figure 1. The decrease in scrum levels of adrenal androgens with advancing age in normal women is contrasted with the age invariant behaviour seen in women with breast cancer, or at higher risk of developing the disease.
Both DHEA and ANDROSTENEDIOL arc mainly derived by peripheral conversion from DHEAS [11] secreted by cells of the zona reticularis of the human adrenal [4]. Although ANDROSTENEDIOI, is a member of the adrenal androgen
family, it was shown to possess unique estrogenic properties by Huggins et al. in 1954 [12]. At physiological concentrations, it is estrogenic in a wide variety of experimental systems, including the stimulation of growth of human mammary cancer cells in culture. This action occurs by combination with the estrogen receptor (ER) and without prior con version to estradiol [13-16]. There arc some other remarkable similarities between ANDROSTENEDIOL and estradiol which need to be mentioned. These involve Ihe transformations which the two hormones undergo when exposed to human mammary cancer cells in culture. Firstly, both steroids are converted to biologically inactive esters by combination with a variety of long-chained fatty acids. These esters, which are retained within the cell, then undergo a slow transformation to release the biologically active free hormone. In this manner, occupancy of the ER can be maintained a process thought to be necessary for hormone-stimulated DNA synthesis and cell replication. Secondly, elimination of hormone from the cell, necessary for signal termination, occurs mainly by formation of water-soluble sulfate esters catalysed by two separate sulfolransferase enzymes. One is specific for phenolic estrogens such as estradiol, and the other for hydroxysteroids such as ANDROSTENEDIOL. Both enzymes arc under estrogen control, have a very high affinity for their respective steroid substrates, and show coopcrativity in their binding. These enzymes may serve to eliminate the hormone from the cell after processing of the ligand-charged receptor [17].
Rochefort and Garcia [18] demonstrated that high concentrations of potent androgens such as 5a-dihydrotcstostcrone (UHf), could act as estrogens via combination with ER. The concentrations required to induce estrogenic effects were far higher than those required to saturate the androgen receptor (AR). Such results were of paramount importance in acceptance of the proposal that the specificity of response to steroid hormone, in a defined largel tissue, is determined by interaction of charged receptor with n uclear components, and not by the nature of the hormone complcxed to this receptor. Estrogenic and antiestrogenic activities of androgens in female target tisscs were the subject of DLOL in serum samples from the Columbia Missouri Breast Cancer Serum Bank. Seventy one healthy postmenopausal volunteers, not taking replacement estrogen when they donated blood, subsequently developed breast cancer up to 10 years later. Two randomly selected controls, who were also postmenopausal and not taking estrogen, were matched to each case on exact age (date ± 1 year) and time (± 2 hours) of blood collection. Significant gradients of increased risk of breast cancer were observed for increasing concentrations of DHE-A and ANDROSTENEDIOL. Women whose scrum levels of these hormones were in the highest quartiles were at significantly elevated risk compared to those in the lowest. The relationship ofDHEAS to breast cancer was less consistent, but women with levels in the highest quartile also exhibited a significantly elevated risk ratio.
All of the above prospective studies arc then in general agreement; lowered levels of adrenal androgens being associated with risk of breast cancer in premenopausal years and elevated levels with risk of developing breast cancer in postmenopausal years (Figure 1).
Both DHEA and ANDROSTENEDIOL arc mainly derived by peripheral conversion from DHEAS [11] secreted by cells of the zona reticularis of the human adrenal [4]. Although ANDROSTENEDIOI, is a member of the adrenal androgen family, it was shown to possess unique estrogenic properties by Huggins et al. in 1954 [12]. At physiological concentrations, it is estrogenic in a wide variety of experimental systems, including the stimulation of growth of human mammary cancer cells in culture. This action occurs by combination with the estrogen receptor (ER) and without prior con version to estradiol [13-16]. There arc some other remarkable similarities between ANDROSTENEDIOL and estradiol which need to be mentioned. These involve Ihe transformations which the two hormones undergo when exposed to human mammary cancer cells in culture. Firstly, both steroids are converted to biologically inactive esters by combination with a variety of long-chained fatty acids. These esters, which are retained within the cell, then undergo a slow transformation to release the biologically active free hormone. In this manner, occupancy of the ER can be maintained a process thought to be necessary for hormone-stimulated DNA synthesis and cell replication. Secondly, elimination of hormone from the cell, necessary for signal termination, occurs mainly by formation of water-soluble sulfate esters catalysed by two separate sulfolransferase enzymes. One is specific for phenolic estrogens such as estradiol, and the other for hydroxysteroids such as ANDROSTENEDIOL. Both enzymes arc under estrogen control, have a very high affinity for their respective steroid substrates, and show coopcrativity in their binding. These enzymes may serve to eliminate the hormone from the cell after processing of the ligand-charged receptor [17].
Rochefort and Garcia [18] demonstrated that high concentrations of potent androgens such as 5a-dihydrotcstostcrone (UHf), could act as estrogens via combination with ER. The concentrations required to induce estrogenic effects were far higher than those required to saturate the androgen receptor (AR). Such results were of paramount importance in acceptance of the proposal that the specificity of response to steroid hormone, in a defined largel tissue, is determined by interaction of charged receptor with n uclear components, and not by the nature of the hormone complcxed to this receptor. Estrogenic and antiestrogenic activities of androgens in female target tisscs were the subject of act as an cslrngen at the concentration found in the blood of Western women. In postmenopausal women, when production of estradiol from the ovaries ceases, elevated scrum ANDROSTENEDIOL lev els could stimulate growth of a clone of neoplastic, or preneoplastic, cells transformed by carcinogenic events occurring sonic years earlier. High seiuni concentrations of ANDROSTENEDIOL derived from DHEAS, and other adrenal androgens derived from ADIONE, would oppose estradiol-stimulated cell growth in premenopausal subjects; subnormal levels of these androgens being manifested in the appearance of tumors at an early age. ANDROSTENEDIOL is then a novel hormone possessing both estrogenic and androgenic action at physiological concentrations. The name ‘Hermaphrodiol’ conferred on it is appropriate to describe its unique features (Figure 2).
Figure 2. Diagrammatic presentation of binding of sex hormones to ER and AR in transformed mammary epithelial cells, In premenopausal women, high concentrations of estradiol (E) bind with high affinity to ER resulting in a stimulation of cell growth. ANDROSTENEDIOL, also known as hermaphrodiol (H), has a lower affinity for binding to ER compared to E, and is shown binding to AR. In postmenopausal women, when secretion of E from the ovaries ceases, II is able to bind to ER for which it has higher affinity compared to AR. More potent androgens such as DHT, though present at lower concentration, can bind with high affinity to AR (inset) but with negligible affinity Lo ER at these physiological concentrations.
The lower incidence of breast cancer in postmenopausal Japanese women in Japan compared to Western women, and the rise of incidence in migratory Japanese populations, run parallel to serum DHEAS levels measured in these groups of women [23]. These data are then in harmony with the above interpretation ol the role of adrenal androgens in the etiology of the disease.
Acknowledgement
I am indebted to Dr. Frank Stanczyk for sending details of results obtained from the Missouri Breast Cancer Serum Bank prior to publication.
References
Beaulieu EE, Corpechot C, Dray E Emilozzi R, Lebean M, Mauvais-Jarvis F, Robel P: An adrenal-secreted ‘androgen’: dchydroepiandrosteronc sulfate, its metabolism and a tentative generalisation on the metabolism of other steroid conjugates in man. Recent Prog Hormone Res 21: 411-500, 1965
Allen BJ, Hayward .1L, Merivale WMH: The excretion of 17-kestostcroids in the urine of patients with carcinoma of the breast. Lancet 1: 496-497, 1957
Zumolf B: Hormone profiles and the epidemiology of breast cancer. In: Stoll BA (ed) Endocrine Relationships in Breast Cancer. Hcinman, London. 19S2, pp 3—47
Adams JB: Control of secretion and the function of C19-Δ5-steroids of the human adrenal gland. Mol Cell Hndiermol 41: 1-17.1985
Bulbrook RD, Hayward JL, Wang DY, Thomas BS, Clark tiMCi, Allen DS, Moore JW: identification ot women at high risk of breast cancer. Breast Cancer Res Treat 7 (Suppl): 5-10,1986
Rulhrook RD, Thomas RS: Hormones are amhiguous factors for breast, cancer. Acta Oncologica 28: 841-847,1989
Zumoff B, Levin J, Rosenfeld RS, Markham M, Strain GW, Fukushima DK: Abnormal 24-hr mean plasma concentration of déhydroépiandrostérone and dchydrocpiandroslerone sulfate in women with primary operable breast cancer. Cancer Res 41: 3360-3363,1981
Gordon GR, Bush TL, Helzlsouer KJ, Miller SR, Comstock GW: Relationship of serum levels of déhydroépiandrostérone and dehydroepiandrosterone sulfate to the risk of developing postmenopausal breast cancer. Cancer Res 50: 3859-3862.1990
HelzlsouerKJ, Gordon GB, Alberg AJ, Bush TL, Comstock GW: Relationship of prediagnostic serum levels of dehydro-piandrostcronc and dehydi ocpiandi ostcronc sulfate to Lite risk of developing premenopausal breast cancer. Cancer Res 52:1-4, 1992
Dorgan JF, Stanczyk F, Longcope C, Stephenson HF., Chang L, Miller R, Franz C, Falk RT, Kahle L: Relationship of serum DHEA, DHEAS and 5-androstenc-3β,7β-diol to risk of breasl cancer in postmenopausal women. Cancer Epidemiol Biomarkers Prevention 6: 177-181,1997
Bonney R, Scanlon MJ, Jones DL, Beranek PA, Reed MJ, James VHT: The interrelationship between plasma 5-ene adrenal androgens in normal women. J Steriod Biochcm 20: 1353-1355,1984
Huggins C, Jensen EV, Cleveland AS: Chemical structure of steroids in relation to promotion of growth of the vagina and uterus of the hypophysectomized rat. J Exp Med 100: 225-240.1954
Adams f, Garcia M. Rochefort H: Estrogenic effects of physiological concentrations of 5-androstene-3β,7β-diol and its metabolism in MCFhuman breast cancer cells. Cancer Res 41: 4720-4726,1981
Seymour-Munn K, Adams J: Estrogenic effects of 5-androstcnc-3β,7β-cliol and its possible implication in the etiology of breast cancer. Endocrinology 112; 486-491.1983
Poulin R, Labrie F: Stimulation of cell proliferation and estrogenic response by adrenal C19-Δ5-steroids in the ZR-75-1
human breast cancer cell line. Cancer Res 46: 4933-4937, 1986
Spinola PG, Marchetti B, Labrie F: Adrenal steroids stimulate growth and progesterone receptor levels in rat uterus and DMBA-induccd mammary tumors. Breast Cancer Res Treat 81: 241-248.1986
Adams JB, Mailyu P, Lee F-T, Phillips NS, Smith DL, Metabolism of 17β-estradiol and the adrenal-derived estrogen 5-androstenc-3β,7β-diol (Hermaphrodiol) in human mammary cell lines. Ann NY Acad Sci 595: 93-105,1990
Rochefort II. Garda M: Androgen tin the estrogen receptor 1. Rinding and in vivo nuclear translocation. Steroids 28: .849-560, 1976
Rochefort H, Garcia M: The estrogenic and antiestrogenic activities of androgens in female target tissues. Thai m 1 her 23:193-216, 1984
Poulin R, Baker D. Labric T: Androgens inhibit basal and estrogen-induced cell proliferation in the ZK-75-1 human breast cancer cell line. Breast Cancer Res Treat 12: 213-225, 1988
Haekenberg R, Schulz K-D: Androgen receptor mediated growth control of breast cancer and endometrial cancer modulated by antiandrogenand androgen-like steroids. J Steroid Biochem Mol Biol 56: 113-117,1996
Bocuzzi G, Brignardello E, Di Monaco M., Gatto V, Leonardi L, Pizzini A, Gallo M; 5-En-androslene-3β,7β-diol inhibits the growth of MCF-7 breast cancer cells when oestrogen receptors are blocked by oestradiol. Br J Cancer 70:1035-1039.1994
Hayward JL. Greenwood FC, Globcr G, Stemmerman G, Bulbrook RD, Wang DY, Kantaoka S: Endocrine status in normal British, Japanese and Hawaiian-Japanese women. Cur .1 Cancer 14: 1221-1228, 1978