Woraluk Somboonporn[a]✸, Susan R Davis[b]

[a] Department of Obstetrics and Gynaecology, Khon Kaen University, Khon Kaen, Thailand [b] NHMRC Centre of Clinical Research Excellence, Department of Medicine (CECS) Monash University, Melbourne, Australia

Abstract

Background: Testosterone therapy is being increasingly used in the management of postmenopausal women. However, as clinical trials have demonstrated a significantly increased risk of breast cancer with oral combined estrogen-progestin therapy, there is a need to ascertain the risk of including testosterone in such regimens. Objective: Evaluation of experimental and epidemiological studies pertaining to the role of testosterone in breast cancer. Design: Literature review. Setting: The Jean Hailes Foundation, Research Unit. Main Outcome Measures: Mammary epithelial proliferation, apoptosis and breast cancer. Results: In experimental studies, testosterone action is anti-proliferative and pro-apoptotic, and mediated via the AR, despite the potential for testosterone to be aromatized to estrogen. Animal studies suggest that testosterone may serve as a natural, endogenous protector of the breast and limit mitogenic and cancer promoting effects of estrogen on mammary epithelium. In premenopausal women, elevated testosterone is not associated with greater breast cancer risk. The risk of breast cancer is also not increased in women with polycystic ovary syndrome who have chronic estrogen exposure and androgen excess. However, in postmenopausal women, who are oestrogen deplete and have increased adipose aromatase activity, higher testosterone has been associated with greater breast cancer risk. Conclusion: Available data indicate the inclusion of testosterone in estrogen-progestin regimens has the potential to ameliorate the stimulating effects of hormones on the breast. However, testosterone therapy alone cannot be recommended for estrogen deplete women because of the potential risk of enhanced aromatisation to estrogen in this setting.

Keywords: Testosterone therapy; Menopause; Breast cancer

Introduction

Testosterone exhibits important physiological effects in women, being both a precursor hormone for ovarian and extragonadal estrogen biosynthesis [1], and acting directly via androgen receptors (AR) throughout the body. Some postmenopausal women may develop symptomatic androgen insufficiency and may benefit from testosterone therapy in terms of improved libido, sexual satisfaction, quality of life and bone mineralization [2-9]. However, as the most recent large randomized placebo-controlled clinical trials have demonstrated a significantly increased risk of breast cancer in postmenopausal women taking oral combined hormone therapy [10,11] there is a need to establish whether adding testosterone to hormone therapy regimens will influence this risk.

2.Testosterone aromatization after menopause

Testosterone may exert biological effects by acting directly via the AR or indirectly through conversion to estrogen by the aromatase enzyme and dihydrotestosterone (DHT), which is a non-aromatizable androgen, by 5α-reductase type 1 and 2 [12,13]. Following menopause, the former conversion occurs primarily in adipose and target tissues such as breast, brain, bone, skin, vascular endothelium and vascular smooth muscle [13]. Increased aromatase activity and p450 aromatase gene expression has been demonstrated in adipose tissue with increasing age [14,15]. Plasma cytokines, known to be aromatase stimulating factors, such as interleukin-6, increase with age [16,17] and this may partially account for the increase in peripheral aromatase activity that is detected in older women.

Although this change is not influenced by menopause status [18], it is also possible that estrogen itself may inhibit its own bioformation in both human breast cancer cell lines [19] and animal models [20]. Consistent with these findings, intra-tumoral aro-matase activity has been shown to be higher in woman with lower circulating estrogen [21,22] and relative low activity of aromatase was found in women taking hormone therapy [19,23]. These data indirectly suggest that after menopause, when circulating estrogen levels are low, an increase in aromatase levels in the breast may maintain tissue concentrations of estrogen. Thus, aromatase may control the local production of estrogen through an autocrine loop. During the process of tranformation to malignancy, locally produced estrogen may stimulate the proliferation of tumor cells and vascular endothelial growth factor production. These effects are also likely to enhance tumor progression, development of angiogenesis, and, ultimately, metas-tasis of cancer. This has negative implications for the use of testosterone therapy in women who are post-menopausal and not receiving estrogen therapy.

With regard to 5α-reductase activity, a number of studies have substantiated 5α-reductase modulation by sex steroids [24-29]. Evidence for the relationship between 5α-reductase activity and menopausal status is lacking.

3.Effects of testosterone and DHT on breast: experimental studies

In experimental studies, estradiol has been consis-tently been shown to be a major mitogen in the breast [30-34] while testosterone and DHT have been shown to inhibit such estrogenic effects as follows.

3.1.Breast cancer cell lines

3.1.1.Anti-proliferative effects of androgens

Normal circulating levels in women are approxi-mately 0.2-0.8nmol/L for DHT and 0.5-2.3 nmol/L for testosterone. At physiological doses (1nM/L), testosterone and DHT inhibit proliferation of MCF- 7 cells in both the absence of estradiol [35-37] or presence [35,38], although not all studies have found this association [39]. These inhibitory effects have also been reported in other breast cancer cell lines, ZR-75- 1 [39] andT47-D [37,39]. Birrelletal. [39] reported no effect on proliferation of BT-20 cells, whereas Ortmann et al. [37] demonstrated a significant dose-dependent inhibition of cell growth by testosterone and DHT in this cell line. The contradictory result may be explained by the difference in cell proliferation assays used. Birrel et al. used a modification of a methylene blue whole cell binding assay while Ortman et al. usedMTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- 2H-tetrazolium bromide) method.

3.1.2.Apoptotic effects

Bcl-2 is considered an anti-apoptotic gene [40], and Bax is a pro-apoptotic gene [41]. DHT down-regulates Bcl-2 protooncogene levels via an AR-mediated mechanism in the ZR75-1 breast cancer cell line in either the presence or absence of 17β-estradiol [42]. In addition, in the AR positive human breast cancer cell lines, T47-D and ZR-75-1, DHT has been reported to be pro- apoptotic with maximal effects at 10 nmol/L concentration [43]. These findings are in-line with the growth inhibitory effects reported by Birrell et al. [44] in these two cell lines. This leads us to conclude that DHT may oppose the mitogenic action of estrogen in the breast by promoting apoptosis. Table 1. summarizes the results of breast cancer cell line studies.

Table 1. Effects of testosterone and DHT on breast cancer cell lines.

3.2. Animal studies

3.2.1.Normal model

In the Noble rat model, treatment with testosterone, either alone or in combination with estrogen, gave rise to high levels of Bax expression in ‘pre-malignant’ mammary glands [41]. This supports the hypothesis that testosterone may oppose the mitogenic action of estrogen in the breast by promoting apoptosis [45].

The addition of methyltestosterone to oral contra-ceptive therapy in rats causes a significant suppression of epithelial proliferation and a reduction in the number of progesterone receptor-labelled cells [46]. In rhesus monkeys either supra- or physiologic testosterone treatment reduced estradiol-induced epithelial proliferation [47,48], suggesting that testosterone can reduce breast cancer risk associated with estrogen treatment [48].

3.2.2.Tumor model

DHT therapy reduced the number of progressing tumors and increased the number of tumors that completely regressed in the DMBA-induced mammary carcinoma model in ovariectomized rats [49] and on ZR-75-1 breast cancer cells implanted into ovariec-tomized athymic mice in both the presence or absence of exogenous estradiol [50]. Table 2. summarizes the results from all animal studies.

Table 2. Effects of testosterone and DHT in animal studies.

4.Effects of testosterone on breast tissue: epidemiological studies

4.1. Endogenous testosterone and breast cancer

In premenopausal women, there are inconsistent results from two cross-sectional studies [51,52] and two prospective case-control studies have found no association between breast cancer risk and total testosterone levels [53,54].

Polycystic ovarian syndrome (PCOS) is a useful model of the effects of long-term exposure to hormone imbalance. Concentrations of testosterone, androstene- dione, and DHEAS, and the calculated free androgen index (total testosterone nmol/L divided by SHBG nmol/L x 100) are significantly higher in women with PCOS regardless of hirsutism [55]. An increased risk of endometrial cancer has been well documented in women with this condition [56] whereas the risk of breast cancer is not increased [56,57] despite hyper- androgenism and long term exposure to unopposed estrogen. Infact, Gammon and Thompson [58] have reported an aged-adjusted odds ratio for breast cancer in women with this syndrome of0.52 (95%CI 0.32-0.87). Although this risk reduction might be related to the hy- perandrogemia of this condition, cause and effect cannot be established.

In postmenopausal women, there are inconsistent results in both cross-sectional studies and prospective studies [53,59-68]. Table 3. shows the results from the prospective case-control studies. A reanalysis of prospective studies [69] reported that the relative risks associated with a doubling of total testosterone levels within the normal range were 1.37 (95%CI 1.15-1.65) for the three studies incorporating a purification step in their testosterone assay [66,67,70] and 1.44 (95%CI 1.21-1.72) for four studies that used a direct testosterone assay [62-65]. The considerable inaccuracy of direct total testosterone assays limits the interpretation of any data obtained by this methodology [71]. Furthermore, a subgroup analysis was not undertaken to determine the association between breast cancer and free testosterone. This is important as free testosterone, not total testosterone, is considered the best indicator of tissue exposure to testosterone.

Table 3. Endogenous testosterone and risk of breast cancer in postmenopausal women.

There are only two prospective studies available that have utilized measures of free testosterone [62,67]. Cauley et al. [67] measured free testosterone by equilibrium dialysis, considered to be a reasonably accurate methodology, in 97 cases and 244 controls and reported that breast cancer risk was three times greater in women with the highest concentration(≥13.17pmol/L), but that this was no longer significant after adjustment for estradiol. In contrast Berrino et al. [62] measured free testosterone by radioimmunoassay in 25 cases and 100 controls and reported a significant association between higher free testosterone levels(≥0.86pg/ml) and breast cancer after adjustment for estradiol. Again, as direct free testosterone assays are considered unreliable, the value of this data is questionable [71].

4.2. Exogenous testosterone and breast cancer

The specific evidence pertaining to the use of testosterone therapy and breast cancer risk has significant methodological limitations and provides inconclusive results. Breast cancer risk was not a primary endpoint for two of these studies [72,73]. Consequently, each had only a small sample size for this subgroup analysis [72,73]. A recent retrospective study had no control group [74]. Table 4 summarized study characteristics and their results.

Table 4. Exogenous testosterone and risk of breast cancer in postmenopausal women.

5.Clinical consideration

The term female androgen insufficiency (FAI) was proposed as a pattern of clinical symptoms in the presence of decreased bioavailable testosterone and normal estrogen status [75]. The addition of testosterone to hormone therapy has shown benefits for sexual dysfunction, mood, energy, psychological wellbeing, bone mineral density, muscle mass and strength and adipose tissue distribution [2-7,76]. However, there are some potential risks have been reported, for example acne, excess facial and body hair, permanent deepening of voice, weight gain, emotional changes (e.g. increased anger) and adverse effects on the lipid profile [77]. As androgens are converted to estrogens in vivo, estrogenic side-effects are also potential consequences of androgen therapy, including increased risk of breast and ovarian cancer [77].

Based on experimental studies covered in this re-view it is unlikely that physiological testosterone ther-apy in addition to estrogen therapy increases the risk of breast cancer. Alternatively, it appears that testosterone serves as a natural safe guard of the breast and limits mitogenic and cancer promoting effects of estrogen within the breast [35,37-39,42,43,45,49,50]. However, the existing evidence in women specific to exogenous testosterone therapy and the risk of breast cancer [72-74] has a significant limitations of research methodology to address this issue.

In case-control studies, high endogenous total testosterone levels have been associated with increased breast cancer risk in postmenopausal women [69], but not in premenopausal women [53,54]. It is almost impossible to be sure of a direct causal relationship between high levels of free testosterone and breast cancer although the positive association was found. Increased aromatase activity in the setting of estrogen depletion after menopause, and thus increased capacity to convert testosterone to estradiol in adipose tissue with age may be the major factors. That there is no longer an association between high levels of free testosterone and breast cancer risk after adjustment for estradiol levels would support this [67]. Furthermore, there has been no report of an increase in breast cancer risk in women with PCOS in which hyperandrogenism coexists with elevated estrogen.

As the epidemiological studies specific to exoge-nous testosterone therapy and breast cancer risk have major limitations, with the available data pertaining to the effects of testosterone on the breast, the inclusion of testosterone in hormonal regimens should be limited to women symptomatic of androgen insufficiency despite adequate estrogen replacement and involve regular measurements of circulating levels of free testosterone to avoid androgen excess.

6.Conclusion

Breast cancer is the most common carcinoma in women [78,79]. Evidence from the most recent large randomized, placebo-controlled clinical trials shows the risk significantly increases in postmenopausal women taking estrogen plus medroxyprogesterone ac-etate [10,11,80] but does not increase in those taking estrogen only [81] while the clinical studies specific to exogenous testosterone therapy and breast cancer risk yield inconclusive result due to significant limitations [72-74]. However, the preclinical studies suggest that testosterone may serve as a natural, endogenous protector of the breast and limit mitogenic and cancer promoting effects of estrogen on mammary epithelium [35,37-39,42,43,46,48-50]. In contrast, in the setting of estrogen depletion, ie postmenopause, aromatase activity is increased and higher free testosterone has been associated in one study with a small increase in breast cancer risk. Thus, we propose that whereas benefits may be achieved by adding testosterone to estrogen-progestin therapy, greater caution is required for the use of testosterone alone at this time. There is no doubt that further studies are required so that firm clinical advice can be given pertaining to the use of testosterone with or without estrogen therapy.

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