While estrogens such as E2 stimulate breast cell proliferation, androgens – notably testosterone (T) – act as inhibitors . Likewise, negative associations between circulating biologically active T and markers of breast cell proliferation and mammographic density have been reported , , . It is also shown that women with polycystic ovary syndrome having elevated circulating concentrations of biologically active T may carry a lower breast cancer risk. One mode of action of T in this respect may include inhibition of the synthesis of the progesterone receptor  and references cited therein. Another mode of action may include inhibition of matrix metalloproteinase (MMP) activity . This family of proteolytic enzymes is essential for breast cell proliferation and normal breast function as well as for breast tumor invasion and metastasis. Several studies have reported inhibitory effects of androgens on MMPs in animal experiments and in cell cultures in vitro , .
Being a well-known estrogen receptor agonist, estrone (E1) has also been shown to act as a partial estradiol-17β (E2) antagonist in different in vitro systems and in human urogenital tissue collagen turnover and breast tissue cell proliferation in vivo , , . Dehydroepiandrosterone sulfate (DHEAS) is the most abundant circulating steroid in humans. While DHEAS has no affinity to the androgen receptor (AR), the unconjugated steroid dehydroepiandrosterone (DHEA) has a weak androgenic activity with an affinity to AR corresponding to 0.04% of that of T and has also been shown to act as an AR antagonist in human MDA-MB-231 mammary tumor and LNCaP prostate cancer cells in vitro .
The aim of the present investigation was to elucidate if circulating DHEA could counteract the inhibitory action of T on mammographic breast density in healthy postmenopausal women.
Materials and methods
The clinical material comprised 84 healthy postmenopausal women aged 50–70 years (mean age 57.8 + 5.7 years), being a part of the clinical material in our previous studies on hormonal replacement therapy (HRT) , , .
We assessed mammographic breast density on pretreatment mammograms from the three studies in this clinical material. Visual mammographic breast density of the left breast was judged according to a five category percentage scale; 0–20%, 21–40%, 41–60%, 61–80% and 81–100% of dense breast area . Furthermore all films were digitized and the dense area of the left CC-view was measured using a computer-assisted program (Cumulus, Sierra plus, Vidar Systems Corporations, Medical Imaging, Herndon, VA, USA) that has been shown to give highly reproducible results , , , . The operator (EL) was unaware of any patient data and in what order the mammograms were taken.
Venous blood samples were collected before hormonal treatment at approximately 10 am on the day of mammography. Pretreatment serum concentrations of T were determined by radioimmunoassay (RIA) using a commercial kit obtained from Siemens Medical Solutions, Los Angeles, CA (Coat-a-Count® Testosterone®). At the time when the T assays were performed, the method used (“Coat-a-Count® Testosterone®”) turned out to be the best out of ten clinical routine methods when compared with gas chromatography–mass spectrometry , . As far as we know from the literature there is no corresponding comparison published between immunoassay and methods including mass spectrometry for the DHEA and E2 assays. However, using the DHEA assay used in the present communication, we found a median serum DHEA level of 17.1 nmol/L in 46 healthy women aged 20–40 years  to be compared with 19.0 nmol/L in 117 healthy controls of the same age, found in a recent study using liquid chromatography – tandem mass spectrometry (LC-MS/MS) by Eklund and co-workers . Sex hormone-binding globulin (SHBG) was determined by chemiluminescence enzyme immunoassay using a commercial kit obtained from the same manufacturer/Immulite®). Apparent concentrations of free testosterone (fT) were calculated from values for total T, SHBG and a fixed albumin concentration of 40 g/L by successive approximation using a computer program based upon an equation system derived from the law of mass action . Serum concentrations of DHEA were determined after extraction with diethyl ether by RIA using an in house method . Serum E2 was determined by RIA using a commercial kit (“Spectria®”, Orion Diagnostica Ab, Esbo, Finland). Concerning E2, our finding of a median pretreatment E2 level of 21 pmol/L in the present study shall be compared with the median E2 level of 19 pmol/L in 72 healthy untreated postmenopausal women found by Naessén and co-workers using LC-MS/MS . This indicates a good specificity also of the DHEA and E2 immunoassays used.
Detection limits and within and between assay coefficients of variation were for T 0.1 nmol/L, 6% and 10%; for SHBG 0.2 nmol/L, 6.5% and 8.7%; 6% and 10%, for DHEA 1.6 nmol/L, 5% and 7% and for E2 5 pmol/L, 5% and 6%. Serum concentrations are presented as median and 25–75th percentile. Correlations were carried out by Spearman’s rank correlation test. The significance level was set at p < 0.05.
Basal anthropometric and endocrine data for the women are given in Table 1. Spearman correlations between DHEAS vs. DHEA, A4 vs. DHEA and A4 vs. DHEAS were 0.68, 0.85 and 0.51, respectively (p < 0.001) for all.
Correlations between MD and fT at decreasing serum DHEA levels are given in Table 2. The negative correlations were strengthened from −0.29** and −0.32** at a median DHEA level of 10.2 nmol/L to −0.43*** and −0.43* at 7.6 nmol/L, for visual and digitized MD classifications, respectively, becoming weakened at even lower DHEA levels. There were no changes in mammographic density or in serum E2 with the decreasing DHEA levels (data not shown). There was a highly significant positive correlation between visual vs. digitized assessment of mammographic breast density at baseline, Rs = 0.85, p < 0.0001 as shown in Figure 1.
Circulating DHEA/DHEAS is the main substrate in women for the formation of many circulating C19 steroids, most of them with varying androgenic potency, but also of one, 5-androstene-3β, 17β-diol, with considerable direct estrogenic activity , , , , .
The effect of steroid hormones such as androgens and estrogens on target organs are complex. Previous studies have indicated that naturally occurring weaker steroids can have a modifying impact on their more potent counterparts. We investigated if lowering the level of the putative AR antagonist, DHEA could have such an effect, i.e. hamper the negative action of free testosterone on mammographic breast density. In Table 2, the changes in DHEA-levels were done in such a way that a real change in the number of subjects took place, analogously with the method from our previous studies on the influence of serum E1 levels on the association between serum E2 and MD and urogenital tissue collagen markers , . The correlations between fT and MD were strengthened when the upper DHEA limit was lowered down to a threshold value where the fT concentration became too low and/or the sample too small.
Despite negative correlations between fT and proliferation and/or mammographic density in normal breast tissue in several studies by our group and others, the effects of different androgens on breast cancer are much more complex. In general, studies indicate a protective effect of androgens in breast cancers positive for ER α, PR B, Erb B2 and AR. With increasing receptor negativity androgens instead seem to be more and more detrimental being able to instead stimulate breast cancer tissue and cell line growth . Different results also exist for different concentrations of DHEA where in one recent study, only supra-physiologic concentrations were hampering proliferation of T47 D cells in vitro . Furthermore, some epidemiologic studies indicate an increased risk for developing breast cancer by androgen addition in HRT . This adds to the complexity of androgen effects on the breast also in normal women receiving such therapy.
Our results indicate that DHEA and/or its metabolites may act as partial AR antagonists in vivo, in accordance with previous findings in vitro . The results of the present investigation as well as of our previous studies on the of interaction between serum E1 and E2 (vide supra) again points at the necessity to take into consideration the presence of naturally occurring receptor antagonistic steroids when studying relations between circulating bioactive steroids, e.g. E2 and fT, and their biological effects in vivo. The findings also might contribute to the explanation of diverging results concerning the effects of testosterone addition in HRT as well as in breast cancer therapy , , .
Skilful technical assistance was provided by Catharina Karlsson, Birgitta Byström and Berit Legerstam.
Birrell SN, Bentel JM, Hickey D, Riciardelli C, Weger MA, Horsfall DI, et al. Androgens induce divergent proliferative response in human breast cell lines. J Steroid Biochem Mol Biol. 1995;52:459–67. CrossrefGoogle Scholar
Isakson E, von Schoultz E, Odlind V, Söderqvist, G, Csemiczky G, Carlström K, et al. Effects of oral contraceptives on breast epithelial proliferation. Breast Cancer Res Treat. 2001;65:163–9. CrossrefPubMedGoogle Scholar
Hofling M, Lundström E, Azavedo E, Svane G, Lindén-Hirschberg A, von Schoultz B. Testosterone addition during postmenopausal therapy: effects on mammographic breast density. Climacteric. 2007;10:155–63. PubMedCrossrefGoogle Scholar
Hofling M, Löfgren L, von Schoultz E, Carlström K, Söderqvist G. Associations between serum testosterone levels, cell proliferation and progesterone receptor content in normal and malignant breast tissue. Gynecol Endocrinol. 2008;24:405–10. Web of SciencePubMedCrossrefGoogle Scholar
McCulloch DR, Akl P, Samaratunga H, Herington AC, Odorico DM. Expression of the disintegrin metallo-protease, ADAM-10, in prostate cancer and its regulation by dihydrotestosterone, insulin-like growth factor I and epidermal growth factor in the prostate cancer cell model LNCaP. Clin Cancer Res. 2004;10:314–23. CrossrefGoogle Scholar
Edwall L, Carlström K, Fianu-Jonasson A. Endocrine status and markers of collagen synthesis and degradation in serum and urogenital tissue from women with and without stress urinary incontinence. Neurourol Urodynam. 2007;26:410–5. CrossrefWeb of ScienceGoogle Scholar
Davies ME, Gumucio JP, Sugg KB, Bedi A, Mendias CL. MMP inhibition as a potential method to augment the healing of skeletal muscle and tendon extracellular matrix. J Appl Physiol. 2013;115:884–91. Web of ScienceCrossrefPubMedGoogle Scholar
Lundström E, Conner P, Naessén S, Löfgren L, Carlström K, Söderqvist G. Estrone a partial estradiol antagonist in the normal breast. Gynecol Endocrinol. 2015;31:747–9. Web of ScienceCrossrefPubMedGoogle Scholar
Lundström E, Christow A, Kersemaekers W, Svane G, Azavedo E, Söderqvist G, et al. Effects of tibolone and continuous combined hormone replacement therapy on mammographic breast density. Am J Obstet Gynecol. 2002;186:717–22. CrossrefPubMedGoogle Scholar
Lundström E, Lindén Hirschberg A, Söderqvist G. Digitized assessment of mammographic breast density – effect of continuous combined therapy, tibolone and black cohosh compared to placebo. Maturitas. 2011;70:361–4. CrossrefGoogle Scholar
Lundström E, Söderqvist G, Svane G, Azawedo E, Olovsson M, von Schoultz E, et al. Digitized assessment of mammographic breast density in patients who received low dose intrauterine levo-norgestrel in continuous combination with oral estradiol valerate: a pilot study. Fertil Steril. 2006;85:989–95. CrossrefGoogle Scholar
Hofling M, Carlström K, Svane G, Azavedo E, Klosterboer H, von Schoultz B. Different effects of tibolone and continuous combined estrogen plus progestogen hormone therapy on sex hormone-binding globulin and free testosterone levels – an association with mammographic density. Gynecol Endocrinol. 2005;20:110–5. PubMedCrossrefGoogle Scholar
Taieb J, Mathian B, Millot F, Patricot MC, Mathieu E, Queyrel N, et al. Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography–mass spectrometry in sera from 116 men, women and children. Clin Chem. 2003;49:1381–95. PubMedCrossrefGoogle Scholar
Carlström K, Brody S, Lunell NO, Lagrelius A, Möllerström G, Pousette A, et al. Dehydroepiandrosterone sulfate and dehydroepiandrosterone in serum: age and sex-related differences. Maturitas. 1988;10:297–306. CrossrefGoogle Scholar
Eklund E, Berglund B, Labrie F, Carlström K, Ekström L, Hirschberg AL. Serum androgen profile and physical performance in women Olympic athletes. Br J Sports Med. 2017;51:1301–8. Web of SciencePubMedCrossrefGoogle Scholar
Södergård R, Bäckström T, Shanbhag V, Carstensen H. Calculation of free and bound fractions of testosterone and estradiol-17β to plasma proteins at body temperature. J Steroid Biochem. 1982;16:801–10. PubMedCrossrefGoogle Scholar
Naessen T, Sjogren U, Bergquist J, Larsson M, Lind L, Kushnir MM. Endogenous steroids measured by high-specificity liquid chromatography-tandem mass spectrometry and prevalent cardiovascular disease in 70-year-old men and women. J Clin Endocrinol Metab. 2010;95:1889–97. Web of SciencePubMedCrossrefGoogle Scholar
Zumoff BV, Bradlow HL. Sex difference in the metabolism of dehydroisoandrosterone sulfate. JCEM. 1980;51:334–6. Google Scholar
Matsuoka LY, Wortsman J, Lifrak ET, Parker LN, Mehta RG, Parker LN. Effect of isotretinoin in acne is not mediated by adrenal androgens. J Am Acad Dermatol. 1989;20:128–9. PubMedCrossrefGoogle Scholar
Longcope C. Dehydroepiandrosterone metabolism. J Endocrinol. 1996;150:125–7. Google Scholar
Montt-Guevara MM, Shortrede JE, Giretti MS, Giannini A, Mannella P, Russo E, et al. Androgens regulate T47D cells motility and invasion through actin cytoskeleton remodeling. Front Endocrinol. 2016;7:136. Web of ScienceGoogle Scholar
Tamimi RM, Hankinson SE, Chen WY, Rosner B, Colditz GA. Combined estrogen and testosterone use and risk of breast cancer in postmenopausal women. Arch Intern Med. 2006;166:1483–9. PubMedCrossrefGoogle Scholar
Glaser RL, Dimitrakakis C. Reduced breast cancer incidence in women treated with subcutaneous testosterone, or testosterone with anastrozole: a prospective, observational study. Maturitas. 2013;76:342–9. PubMedCrossrefWeb of ScienceGoogle Scholar
About the article
Published Online: 2018-08-25
Research funding: This study was supported by grants from the Swedish Cancer Society, the Swedish Research Council (project No. 5982), the Karolinska Institutet Research Funds and NV ORGANON, Oss, The Netherlands.
Conflict of interest: All authors declare no conflict of interest.
Informed consent: Informed consent has been obtained from all individuals included into the study.
Ethical approval: The research results related to human use complied with all the relevant national regulations and institutional policies, was performed in accordance to the tenets of the Helsinki Declaration and has been approved by the local institutional review board.