IntroductionIn the last decade or so, our knowledge of the roles of the steroids referred to as 'sex hormones' namely testosterone and estradiol, has undergone a quiet revolution. In the first place, models of androgen and estrogen insufficiency, whether they be natural or engineered, have revealed new and unexpected roles for sex steroids, some of which have nothing to do with reproduction.Furthermore both categories of steroid have roles in both sexes which blunts the definition of the terms 'androgen' and 'estrogen'. Secondly, the gradual acceptance of the role of local steroid hormone action, particularly as it applies in postmenopausal women and in men, provides new insights into the significance of paracrine and intracrine actions, and requires a re-evaluation of the importance of circulating steroid hormone levels. This article will attempt to review both of these developments, particularly in the context of the work of this laboratory on the aromatase knock-out (ArKO) mouse and the role of local aromatase expression within the breast and breast cancer. The concept of local estrogen biosynthesisModels of estrogen insufficiency have revealed new and unexpected roles for estrogens in both males and females. These models include natural mutations in the aromatase gene, as well as mouse knockouts of aromatase and the estrogen receptors (1)(2)(3)(4)(5)(6) . In addition there is one man described with a natural mutation in the ERα (7) . Some of the roles of estrogens apply equally to males and females and do not relate to reproduction, for example the bone, vascular and Metabolic Syndrome phenotypes.In postmenopausal women and in men, estradiol does not appear to function as a circulating hormone, instead it is synthesised in a number of extragonadal sites such as breast, brain and bone where its actions are mainly at the local 2 level as a paracrine or intracrine factor. Thus in postmenopausal women and in men, circulating estrogens are not the drivers of estrogen action, rather they reflect the metabolism of estrogens formed in these extragonadal sites, they are reactive rather than proactive (8) . Importantly, estrogen biosynthesis in these sites depends on a circulating source of androgenic precursors such as testosterone. Table 1 shows the plasma steroid levels in postmenopausal women and in men. As can be seen, the levels of estrone and estradiol in the plasma of postmenopausal women are extremely low, lower in fact than those in the circulation of men; and moreover the levels of circulating testosterone are an order of magnitude greater than those of estrogens in postmenopausal women.This in itself would suggest that circulating testosterone is better placed to serve as a precursor of estradiol in target tissues than is circulating estradiol.On the other hand, the levels of testosterone in the blood of men are an order of magnitude higher than those of women. Significantly, levels of DHEA and DHEAS in the blood of both men and women are orders of magnitude higher than those of the circulating active ste...

Aromatase inhibitors target the production of estrogen in breast adipose tissue, but in doing so, also decrease estrogen formation in bone and other sites, giving rise to deleterious side effects, such as bone loss and arthralgia. Thus, it would be clinically useful to selectively inhibit aromatase production in breast. In this regard, we have determined that the orphan nuclear receptor liver receptor homologue-1 (LRH-1) is a specific transcriptional activator of aromatase gene expression in human breast preadipocytes but not in other tissues of postmenopausal women. In this study, we show that the coactivator peroxisome proliferator-activated receptor ; coactivator-1A (PGC-1A) is a physiologically relevant modulator of LRH-1, and that its transcriptional activity can be inhibited effectively using receptor-interacting peptide antagonists that prevent PGC-1A recruitment. Interestingly, we note that all of these peptides also interact in an agonistdependent manner with retinoid X receptor A (RXRA), suggesting that these two receptors may compete for limiting cofactors within target cells. In support of this hypothesis, we show that 9-cis-retinoic acid, acting through RXR, inhibits both the basal and PGC-1A-induced transcriptional activity of LRH-1. The importance of this finding was confirmed by showing that LRH-1-dependent, PGC-1A-stimulated regulation of aromatase gene expression in primary human breast preadipocytes was effectively suppressed by RXR agonists. We infer from these data that LRH-1 is a bona fide target whose inhibition would selectively block aromatase expression in breast, while sparing other sites of expression. (Cancer Res 2005; 65(24): 11762-70)

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Estrogen synthesis from C19 precursors is catalyzed by aromatase cytochrome P450. Overexpression of aromatase through atypical promoter usage (use of promoter II) in adipose tissue contributes to breast cancer development and progression. One tumor-derived factor that appears to contribute to this process is prostaglandin E2 (PGE2). A factor that regulates aromatase expression downstream of PGE2 is liver receptor homolog-1 (LRH-1). In a study of factors that inhibit LRH-1, we have examined the ability of short heterodimer partner (SHP) to inhibit aromatase transcription mediated by LRH-1 in preadipocytes. RT-PCR analysis indicated that both LRH-1 and SHP are expressed in human preadipocytes. To assess the effect of SHP on aromatase transcription, a transient transfection system was established using 3T3-L1 preadipocytes. Expression of SHP completely inhibited activity of an aromatase promoter II reporter gene induced by LRH-1. The combined treatment of forskolin and phorbol ester (which mimic PGE2) as well as LRH-1, which maximally induced reporter gene expression (140-fold), was also completely inhibited by SHP. This effect of SHP was mediated by inhibition of LRH-1 transcriptional activity, as measured by activity of GAL4-LRH-1 fusion constructs, and by inhibition of LRH-1 binding to promoter II. We conclude that SHP is a potent inhibitor of aromatase transcription in preadipocytes. Modulation of SHP expression and/or activity in adipose tissue may therefore have significant effects on aromatase expression and estrogen production in breast adipose tissue.

Local synthesis of estrogens within breast adipose tissue by cytochrome P450 aromatase contributes to the growth of postmenopausal breast cancers. One of the major stimulators of aromatase expression in breast is prostaglandin E2 (PGE2) derived from tumorous epithelium and/or infiltrating macrophages. Recently, the orphan nuclear receptor, liver receptor homologue-1 (LRH-1), has also been shown to regulate aromatase expression in breast adipose tissue. We therefore examined the expression of, and correlations between, aromatase and LRH-1 mRNA in a panel of breast carcinoma tissues and adjacent adipose tissue. LRH-1 mRNA expression was low in normal breast tissue but markedly elevated in both breast carcinoma tissue and adipose tissue surrounding the tumor invasion (thereby paralleling aromatase expression). Laser capture microdissection localized the site of LRH-1 expression to tumor epithelial cells but not to intratumoral stromal cells. A strong correlation between LRH-1 and aromatase mRNA levels was observed in tumor-containing adipose tissue but not in tumor tissue. Ectopic expression of LRH-1 in primary human adipose stromal cells strongly activated endogenous aromatase mRNA expression and enzyme activity. Finally, treatment of adipose stromal cells with PGE2 induced expression of both LRH-1 and aromatase. We suggest that PGE2 derived from breast tumor tissue may increase aromatase expression in the surrounding adipose stroma in part by inducing LRH-1 in these cells. The roles of LRH-1 in breast cancer proliferation merit further study.