[CANCER RESEARCH 38. 4036-4040. November 1978] 0008-5472/78/0038-OOOOS02.00 Adrenal Dehydroepiandrosterone and Human Mammary Cancer1 John B. Adams,2 Lesley Archibald, and Christine Clarke School ot Biochemistry. University of New South Wales, Sydney. Australia Abstract tion to weight (13, 15). Calculations suggested that over one-half of the differences in breast cancer incidence be A hypothesis implicating adrenal dehydroepiandroster- tween regions in Holland and Japan can be attributed to one (DHEA) (sulfate) in the etiology of human breast cancer of the "adrenal" or "Western" type has been differences in body weight and height (15). As pointed out by de Waard, such a combination can be interpreted as a presented (Adams, J. B. Steroid Hormones and Human measure of body surface area (15). A recent hypothesis has Breast Cancer. An Hypothesis. Cancer, 40: 325-333, implicated adrenal DHEA3-DHEAS in the etiology of human 1977). High concentrations of DHEA sulfate in the blood breast cancer, particularly of the "Western" or "adrenal" provide a potentially high flux of the free steroid to type (2). It was demonstrated that the metabolism of these mammary tumors, due to the presence therein of a sulfa steroids by human breast carcinoma tissue was very similar tase. The free steroid, in turn, is metabolized by human to that by skin; one possibility for this similarity is the mammary tumors in vitro to 5-androstene-3/i,17/J-diol reputed derivation of the mammary gland from primitive (ADIOL) and 7-hydroxydehydroepiandrosterone. It has sweat glands. It is possible that DHEA-DHEAS, the exact now been found that ADIOL when administered s.c. to physiological function of which remains unknown, may immature female rats will deplete the estrogen receptor in utilize skin as a major target organ and hence may also the uterine cytosol. Similarly, dimethylbenzanthracene- influence the mammary gland. induced rat mammary tumors, when incubated in vitro Recently, it has been shown that ADIOL, which is pro with 1 /IM ADIOL, show translocation of the estrogen duced from DHEA-DHEAS in both skin (20) and mammary receptor from cytosol to the nucleus, as measured by cancer tissue (7, 22), can compete very effectively for the exchange assays. The magnitude of depletion of cytosol estrogen receptor (28). Thus ADIOL could conceivably be estrogen receptor by ADIOL was less than that obtained one of the factors linking the observations of de Waard on with 17/i-estradiol, studied at 0.03 /.IM concentrations, but body weight and height (surface area) with the proposed greater than that with 1 fiM dihydrotestosterone. Among a involvement of adrenal DHEA-DHEAS in postmenopausal wide group of C steroids examined as possible inhibitors breast cancer (2). In this paper the ability of ADIOL to cause of estrogen sulfotransferase, both DHEA and ADIOL translocation of the estrogen receptor and to act as a potent showed marked inhibitory properties. By contrast, both inhibitor of estrogen sulfotransferase is demonstrated. 7a- and 7/i-hydroxydehydroepiandrosterone showed neg ligible inhibitory effects. This paralleled previous findings on the influence of a 7-hydroxyl group in modifying the Materials and Methods ability of ADIOL to compete effectively for the estrogen Chemicals. DHEA, ADIOL, 17/3-estradiol, and DHT used receptor. Thus the high levels of 7-hydroxylase found in in animal experiments were obtained from Sigma Chemical human mammary tumors, and acting on both DHEA and Co., St. Louis, Mo. Steroids used in the enzyme inhibition ADIOL, may function in controlling the intracellular con studies were obtained from the following sources: testos centrations of these steroids. terone, androsterone, and epiandrosterone from Koch- Light Laboratories, Colnbrook, England; 1-androstene- Introduction 3,17-dione, DHT, and 5a-androstane-3,17-dione from Ster- aloids, Wilton, N. H. 4-Androstene-3,17-dione, 50-andros- De Waard (14, 16) first suggested that 2 types of human tan-3a-ol-11,17-dione, DHEA, ADIOL, and Norit A from breast cancer may exist, one associated with ovarian hor Sigma; etiocholanolone, 5-androstene-3ß,16«-diol-17-one, mones in the premenopausal period and the other with a and 5-androstene-30,16/3-diol-17-one from Ikapharm, Ra- postmenopausal hormonal environment. He termed the mat-Gan, Israel; 7«-hydroxytestosterone, 5«-androstane- latter the "Western" environmental type, due to the low 3a,16/3,17/3-triol, 5a-androstane-3«,16o,17/3-triol, 7«- and incidence of this type of breast cancer in Asian countries, 7/3-hydroxydehydroepiandrosterone, and 5-androstene- particularly Japan. Variation in international breast cancer 3/3,17a-diol kindly supplied by the Medical Research Coun incidence was suggested to be related to body weight and cil, Steroid Reference Collection, London, England; 17/3- nutritional status (14, 16). More recently, the emphasis on [2,4,6,7-3H]estradiol (specific activity, 96 Ci/mmol) from the weight alone has been modified to include height in addi Radiochemical Centre, Amersham, England; Eagle's basal medium (dry powder) through the Commonwealth Serum ' Presented at the John E. Fogarty International Center Conference on Hormones and Cancer, March 29 to 31, 1978, Bethesda, Md. Supported by grants from the Australian National Health and Medical Research Council. 3The abbreviations used are: DHEA, dehydroepiandrosterone; DHEAS. 2To whom requests for reprints should be addressed, at School of dehydroepiandrosterone 3-sulfate; ADIOL. 5-androstene-30.170-diol; DHT, Biochemistry. University of N.S.W.. P. O. Box 1. Kensington, N. S. W. 2033 17ß-hydroxy-5u-androstan-3-one; DMBA, 1.12-dimethylbenz(a)anthracene; Australia. i g intragastric. 4036 CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1978 American Association for Cancer Research. DHEA and Mammary Cancer Laboratory, Melbourne, Australia; Bio-Gel HTP (DMAgrade) from stock solutions in propylene glycol. The concentration from Bio-Rad Laboratories, Richmond, Calif. of the latter was held constant at 6% (v/v). Estrogen Receptor in Rat Uteri. The experimental pro Liquid Scintillation Counting. This was carried out with a cedure for studying the influence of ADIOL on rat uteri Triton-toluene phosphor system (5) and computerized cytosol receptor was based on that described by Rochefort Packard 2650 instrument. Quench corrections were carried and Garcia (24) in studies of the effects of DHT on estrogen out by the external standard method. receptor translocation. Immature female Wistar rats (20- to 22-day-old) were used in groups of 5. ADIOL was adminis Results tered s.c. in doses of 8 to 10 mg in 1 ml of peanut oil containing 10% (v/v) ethanol. Controls received the vehicle Although ADIOL will compete effectively for the estrogen alone. Cytosol was prepared by centrifugation at 100,000 x receptor in cytosol preparations of human mammary tu g for 1 hr. Estrogen receptor was assayed, after brief mors and myometrium (28), to our current knowledge no treatment of cytosol with charcoal to remove any endoge experiments have been reported wherein the effects of nous free steroid, by a single saturating dose procedure ADIOL have been examined, either in intact animals or in with 20 nM 17/3-[3H]estradiol in the presence and absence tissue preparations. Rochefort and Garcia (24) have shown of 2 fj.M cold 17/3-estradiol. Incubations were continued for that high doses of DHT provoked the in vivo translocation 20 hr at 2°,and unbound steroid was removed by dextran- of estrogen receptor in the immature rat uterus. Under coated charcoal (24). similar conditions high doses of ADIOL caused a depletion In Vitro Studies on Estrogen Receptor Translocation in of accessible estrogen receptor sites in the rat uterine DMBA-induced Rat Mammary Tumors. Mammary tumors cytosol. This reached a maximum after some 4 hr when the were induced in 50-day-old female Sprague-Dawley rats by estrogen receptor sites were reduced to 30% of that in a single i.g. feeding of 20 mg of DMBA in 1 ml of peanut oil. control animals (Chart 1). A dose of 8 mg was administered Animals were sacrificed, and tumors (weighing about 2 g) since a dose of this order achieved the maximum effect in were removed, washed in 0.9% NaCI solution, and minced the case of DHT (24). finely with scissors. The tissue pieces were then mixed DHT at concentrations of 1 to 50 ¡J.Mwill cause the thoroughly to provide a homogeneous sample. Weighed translocation of estrogen receptor in immature rat uteri in portions (usually 0.5 g) were placed in Eagle's medium (3 vitro (25, 26). In DMBA-induced rat mammary tumors in ml), buffered with bicarbonate to pH 7.2. Steroid was added vitro, 17/3-estradiol, ADIOL, and DHT all individually caused to the empty flasks from stock solutions, solvent was depletion of total (exchangeable) estrogen receptor sites in evaporated with N2, and propylene glycol (5 /nl) was added. the cytosol; their relative effectiveness is in the order listed After being gassed with carbogen, the flasks were stop (Charts 2 and 3). pered and shaken at 37°for1 hr. With the newly described exchange assay for mammary The method of isolation of cytoplasmic and nuclear tumor nuclear estrogen receptor, which overcomes the fractions, and the assay of estrogen receptor in these problem of nuclear receptor destruction by proteases (19), fractions, was that of Carola and McGuire (19), the only evidence has been obtained that depletion of cytosol estro exception being that 1 mM dithiothreitol was used in place gen receptor is accompanied by an increase in receptor in of monothioglycerol in the buffers. Essentially, the assay the nucleus (Chart 3). It would appear that estrogen recep involves an exchange at 25°(cytosol) and 30°(nuclear tor accumulation in the nucleus may be more prolonged extract), the receptor in the nuclear extract being absorbed with ADIOL.