The use of AI as first line endocrine therapy for post-menopausal, estrogen receptor positive breast cancer has increased dramatically over the last few years with the publication of clinical trials suggesting that these compounds may be more effective than the anti-estrogen tamoxifen [4
]. Although the AIs are effective, well tolerated drugs, a significant percentage of patients experience disease relapse during AI therapy, suggesting that resistance to this class of compound is a significant problem. Anecdotal evidence suggests that some women that fail to respond to AIs may still respond to other modes of endocrine therapy, even though their serum estrogen concentrations have been successfully suppressed by AI therapy. This suggests that at least a proportion of these resistant tumors in these women are still estrogen dependent, and implies that non-classical estrogens may be playing a role in resistance to AI therapy.
Women on AI therapy have circulating concentrations of estrogen that are below the limit of detection (low pg/ml), however, the concentrations of androgens remain unchanged (low nM range) [5
]. We hypothesized that under conditions of profound estrogen deprivation, the weak estrogenic activity of other steroids might be sufficient to drive the proliferation of estrogen dependent breast cancer, thereby providing a mechanism for AI resistance. Specifically, we hypothesized that androgens and their metabolites, generated independent of aromatase activity, may contribute to breast cancer growth in the absence of estrogens.
A considerable amount of work has been done over the years studying the effects of androgens on the proliferation of breast cancer cells [1
]. The literature is, however, somewhat confusing, with conflicting data coming from relatively similar experimental systems. For example, 30 years ago we demonstrated that under serum-free conditions, androgens stimulate thymidine incorporation in breast cancer cell lines apparently through the androgen receptor [8
]. Similarly, the testosterone metabolite DHT was shown by Birrell et al. to inhibit the growth of some breast cancer cell lines, but induce the growth of others [7
]. Anti-androgens demonstrated mixed ability to inhibit the effects of DHT on growth, and this was attributed to the potential activity of un-indentified DHT metabolites [7
]. Macedo et al. later showed that DHT is growth-inhibitory in MCF-7 cells under low-estrogen conditions, and that this effect was mediated by the androgen receptor [1
]. One common thread of much of this work is that many of the studies use culture systems in which it is possible that low, but significant, amounts of residual estrogen remain, and so may not adequately model the conditions present in a woman on AI therapy. We have previously made use of a culture system in which residual estrogen concentrations are extremely low (sub pM)[14
], and decided to make use of this system to revisit the effects of androgens and their metabolites on the proliferation of estrogen dependent breast cancer cells.
In this study we have demonstrated that profound estrogen depravation results in the up-regulated expression of two important steroid metabolizing enzymes, CYP19 aromatase and 3β-HSD. MCF-7 and T47D cells are generally considered to express very low levels of aromatase and the finding that estrogen withdrawal can substantially increase expression levels has important implications. The induced expression of aromatase may not be important in the context of AI therapy, since the newly expressed enzyme should be efficiently inhibited by the drug. However, the induction of 3β-HSD and potentially other enzymes raises the possibility of significant local metabolism of androgens and other steroids, and generation of estrogens, by the breast cancer cells.
The downstream metabolite of DHT, 5α-androstane-3β,17β-diol (3βAdiol), is generated by the action of 3β-HSD. It has been known for some time that 3βAdiol can bind to both ERα and ERβ with approximately 30-fold and 14-fold lower affinity relative to that of E2, respectively, suggesting slight specificity for ERβ [13
]. 3βAdiol has been extensively characterized as an ERβ ligand in in vitro
ERβ-promoter driven luciferase assays [18
], gene expression assays [20
], and in vivo
prostate and prostate cancer models [22
]. 3βAdiol has been shown to play a well defined role in prostate cancer etiology as an ERβ ligand. Weihua et al. demonstrated that 3βAdiol is anti-proliferative in prostate cancer via activation of ERβ [22
]. The cytochrome P450 CYP7B1 has been shown to be the primary enzyme responsible for the inactivation and elimination of 3βAdiol [24
]. Activation of ERβ by 3βAdiol and elimination of 3βAdiol by CYP7B1 have been shown to be critical regulators of prostate cancer growth as an anti-proliferative pathway [22
]. Recently, increased CYP7B1 levels were correlated with increased prostate cancer grade, suggesting that increased elimination of 3βAdiol removes tumor growth inhibition by ERβ [26
]. Surprisingly, in spite of this work elucidating a role for 3βAdiol in prostate cancer, little is known about the importance of this steroid in breast cancer. Reporter studies have suggested that androgen metabolites (largely undefined in these studies) can induce the expression of an estrogen-responsive luciferase construct, but little further analysis of the function of these metabolites has been reported [11
]. Interestingly, female knockout mice generated by Omoto et al that lack expression of CYP7B1 (the enzyme responsible for the elimination of 3βAdiol ) showed increased proliferation of both mammary and other reproductive tissues, as well as early onset of puberty and early ovarian failure, suggesting that 3βAdiol is indeed estrogenic in the breast and reproductive tissues [28
In this study, we report for the first time that 3βAdiol can induce the proliferation of breast cancer cells through direct activation of ERα. This growth-stimulation is antagonized by the anti-estrogens 4-hydroxytamoxifen and ICI 182,780. In addition to inducing growth, 3βAdiol also induces the expression of the ERα-specific downstream gene GREB1 which we have previously shown is a critical mediator of estrogen stimulated proliferation. These findings raise the possibility that in the absence of conventional estrogens, 3βAdiol may be an important mediator of estrogen dependent breast cancer growth. We hypothesize that the generation of 3βAdiol from testosterone via aromatase-independent pathways, represents a potential mechanism for resistance to AIs. The enzymes required for generation of 3βAdiol, 5α-reductase and 3β-HSD, are both expressed in a wide variety of tissues, primarily the adrenal glands and liver [29
]. In addition, we have demonstrated that 3β- HSD is expressed in estrogen-deprived breast cancer cells. Thus, in the context of AI therapy, while circulating testosterone cannot be converted to 17β-estradiol due to inhibition of aromatase activity, it may readily be converted to 3βAdiol both systemically and, potentially, locally in the mammary tumor. In one study, plasma concentrations of 3βAdiol in humans were reported to be approximately 1.5nM [30
]. These relatively low concentrations of circulating 3βAdiol may be sufficient to drive tumor growth in the absence of estrogen, particularly in women undergoing treatment with AIs. In addition, prior reports have demonstrated that breast cancer cells can become hypersensitive to extremely low concentrations of estrogens after long-term estrogen deprivation [31
]. These data suggest that tumors may be sensitive to very low concentrations of a weak ERα agonist such as 3βAdiol. Further, the reported 3βAdiol plasma concentrations are greater than the calculated EC50
values for growth induction of breast cancer cells in culture (as shown in ).
In summary, these data demonstrate the important concept that the metabolism of testosterone by aromatase does not represent the only mechanism by which estrogen-like steroids may be generated in post-menopausal women. While inhibition of aromatase may be sufficient to block the productions of the more well know estrogens in the majority of patients treated with AIs, conditions causing an increase in activity of the enzymes responsible for 3βAdiol production, particularly locally within the tumor, may lead to production of estrogen-like steroids independent of aromatase. These pathways may represent an important mechanism for resistance to AI therapy and a more thorough understanding of the diversity of hormone metabolism may be extremely valuable in the refinement of optimal endocrine therapy for breast cancer.