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Pathogenesis and growth of three common women's cancers (breast, endometrium and ovary) are linked to estrogen. A single gene encodes the key enzyme for estrogen biosynthesis named aromatase, inhibition of which effectively eliminates estrogen production in the entire body. Aromatase inhibitors successfully treat breast cancer, whereas their roles in endometrial and ovarian cancers are less clear. Ovary, testis, adipose tissue, skin, hypothalamus and placenta express aromatase normally, whereas breast, endometrial and ovarian cancers overexpress aromatase and produce local estrogen exerting paracrine and intracrine effects. Tissue-specific promoters distributed over a 93-kb regulatory region upstream of a common coding region alternatively control aromatase expression. A distinct set of transcription factors regulates each promoter in a signaling pathway- and tissue-specific manner. In cancers of breast, endometrium and ovary, aromatase expression is primarly regulated by increased activity of the proximally located promoter I.3/II region. Promoters I.3 and II lie 215 bp from each other and are coordinately stimulated by PGE(2) via a cAMP-PKA-dependent pathway. In breast adipose fibroblasts exposed to PGE(2) secreted by malignant epithelial cells, PKC is also activated, and this potentiates cAMP-PKA-dependent induction of aromatase. Thus, inflammatory substances such as PGE(2) may play important roles in inducing local production of estrogen that promotes tumor growth.

A single gene encodes the key enzyme for estrogen biosynthesis termed aromatase, inhibition of which effectively eliminates estrogen production. Aromatase inhibitors successfully treat breast cancer and endometriosis, whereas their roles in endometrial cancer, uterine fibroids, and aromatase excess syndrome are less clear. Ovary, testis, adipose tissue, skin, hypothalamus, and placenta express aromatase normally, whereas breast and endometrial cancers, endometriosis, and uterine fibroids overexpress aromatase and produce local estrogen that exerts paracrine and intracrine effects. Tissue-specific promoters distributed over a 93-kilobase regulatory region upstream of a common coding region alternatively control aromatase expression. A distinct set of transcription factors regulates each promoter in a signaling pathway- and tissue-specific manner. Three mechanisms are responsible for aromatase overexpression in a pathologic tissue versus its normal counterpart. First, cellular composition is altered to increase aromatase-expressing cell types that use distinct promoters (breast cancer). Second, molecular alterations in stromal cells favor binding of transcriptional enhancers versus inhibitors to a normally quiescent aromatase promoter and initiate transcription (breast/endometrial cancer, endometriosis, and uterine fibroids). Third, heterozygous mutations, which cause the aromatase coding region to lie adjacent to constitutively active cryptic promoters that normally transcribe other genes, result in excessive estrogen formation owing to the overexpression of aromatase in many tissues.

Endometrial tissue from uterine disease-free women does not exhibit aromatase activity. In contrast, aromatase enzyme activity and mRNA levels are readily detectable in endometriosis. PGE2 stimulates both aromatase expression and activity in endometriotic stromal cells via promoter II region of the aromatase gene. This results in local production of estradiol, which induces PGE2 formation and establishes a positive feedback cycle. This mechanism seems to contribute to continuous production of estradiol and PGE2. Aromatase mRNA levels and enzyme activity are also present in uterine leiomyomata that are estrogen-dependent benign tumors of the myometrium. Successful treatment of endometriosis and uterine leiomyomata using aromatase inhibitors by recent pilot trials underscores the clinical significance of these molecular studies.

Aromatase p450 (p450arom) is the key enzyme for biosynthesis of estrogen, which is an essential hormone for the establishment and growth of endometriosis. There is no detectable aromatase enzyme activity in normal endometrium; therefore, estrogen is not locally produced in endometrium. Endometriosis tissue, however, contains very high levels of aromatase enzyme, which leads to production of significant quantities of estrogen. Moreover, one of the best-known mediators of inflammation and pain, prostaglandin E (2), strikingly induces aromatase enzyme activity and formation of local estrogen in this tissue. Additionally, estrogen itself stimulates cyclo-oxygenase-2 and therefore increases the formation of prostaglandin E (2) in endometriosis. We were able to target this positive feedback cycle in endometriosis using aromatase inhibitors. In fact, pilot trials showed that aromatase inhibitors could decrease pelvic pain associated with endometriosis.

Binding activity of steroidogenic factor-1 (SF-1) to promoters of the majority of steroidogenic genes in response to gonadotropins is a critical mechanism that regulates steroidogenesis in gonads. Thus, the modulation of SF-1 action may be essential for the differential regulation of formation of sex steroids in the ovary. Aromatase P450 (P450arom) is the rate-limiting enzyme for estrogen formation. In this study, we characterize another nuclear receptor half site in the gonadal aromatase promoter which we show to be important for aromatase regulation. We also show herein that the stimulation of P450arom promoter activity by SF-1 in ovarian granulosa, testicular Sertoli and JEG-3 choriocarcinoma cells is inhibited by two transcription factors, Wilms' tumor suppressor gene (WT1) and dosage sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1). Given the characterized roles of these transcription factors in gonadal development and function, modulation of SF-1 action by WT1 and DAX-1 may represent an important key mechanism in steroidogenesis.

The orphan nuclear receptor steroidogenic factor-1 (SF-1) induces the expression of Müllerian inhibiting substance (MIS) and many steroidogenic genes, including aromatase P450 (P450arom). Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1) inhibits SF-1-mediated induction of MIS and other steroidogenic genes, whereas Wilms' tumor suppressor gene (WT1) augments SF-1-mediated MIS expression. The effects of WT1 on steroidogenesis or P450arom expression have not been explored to date. In human endometriotic stromal cells, extremely high levels of P450arom mRNA and enzyme activity are present. Prostaglandin E(2) stimulates cAMP formation, SF-1 binding activity, P450arom mRNA levels, and estrogen synthesis in endometriotic stromal cells. Stromal cells of eutopic endometrium from disease-free women, on other hand, do not contain readily detectable levels of P450arom mRNA. Thus, we evaluated herein the possible roles of WT1 and DAX-1 in cAMP/SF-1-mediated regulation of P450arom expression in endometriotic and endometrial stromal cells. We also determined the cellular distribution and levels of these transcription factors in pathological endometriotic vs. normal eutopic endometrial tissues by immunohistochemistry to understand their in vivo roles. In vitro transcriptional regulation studies showed that both WT1 and DAX-1 inhibited cAMP and/or SF-1-induced P450arom promoter activity in a dose-dependent fashion in cultured human endometriotic and endometrial stromal cells. Site-directed disruption of the SF-1 binding site (-136/-124 bp) in the P450arom promoter abolished basal or cAMP/SF-1-induced promoter activity in the presence or absence of WT1 or DAX-1. Immunohistochemistry and H-scoring showed that DAX-1 was ubiquitously present in epithelial and stromal cells of both tissues. WT1, on the other hand, was preferentially expressed in stromal (vs. epithelial) cells. Moreover, WT1 levels in endometriotic stromal cells are significantly down-regulated compared with normal endometrial stromal cells. In summary, WT1 or DAX-1 inhibits cAMP-SF-1 pathway-dependent P450arom expression in cultured human endometriotic and endometrial stromal cells. In vivo down-regulation of WT1 in endometriotic stromal cells (vs. normal endometrial stromal cells) may in part be responsible for aberrantly increased P450arom expression and estrogen formation in this pathological tissue.

Aromatase P450 (P450arom) is the key enzyme for the biosynthesis of estrogen that is essential for the growth of human endometriosis, a pathology characterized by endometrium-like tissue on the peritoneal surfaces of abdominal organs manifest by pelvic pain and infertility. Surgically transplanted autologous uterine tissue to ectopic sites on the peritoneum in mice has been used as an animal model to study endometriosis. Using this mouse model, we evaluated the roles of the P450arom gene and aromatase enzyme activity in the growth of endometriosis represented by ectopic uterine tissues in mice. Endometriosis was induced surgically in the following groups of mice: 1) untreated transgenic mice with disrupted P450arom gene (ArKO); 2) ArKO mice treated with systemic estrogen; 3) untreated wild-type (WT) mice; 4) WT mice treated with estrogen; 5) WT mice treated with the aromatase inhibitor, letrozole; and 6) WT mice treated with letrozole and estrogen. Each group contained eight mice; +/+ littermates of ArKO mice were used as WT controls. Treatment with estrogen increased the size of ectopic uterine tissues in ArKO and WT mice significantly. The ectopic uterine lesions in untreated and estrogen-treated ArKO mice were strikingly smaller than those in untreated and estrogen-treated WT controls, respectively. Systemic treatment of WT mice with letrozole significantly decreased the lesion size in a dose-dependent manner. The addition of estrogen to letrozole treatment increased the ectopic lesion size, although these lesions were significantly smaller than those in mice treated with estrogen only. As tissue controls, the effects of these conditions on normally located (eutopic) uterine tissue were evaluated. The effects of disruption of the P450arom gene and treatments with letrozole and estrogen seemed to be more profound on ectopic tissues, suggesting that ectopic tissues might be more sensitive to estrogen for growth. We conclude that both an intact P450arom gene and the presence of aromatase enzyme activity are essential for the growth of ectopic uterine tissue in a mouse model of endometriosis.

In human endometriotic stromal cells, markedly high levels of aromatase P450 (P450arom) mRNA and promoter II activity are present and can be vigorously stimulated by PGE(2) via a cAMP-dependent pathway to give rise to physiologically significant estrogen biosynthesis. Stromal cells of eutopic endometrium, on the other hand, do not express sufficient levels of P450arom for detectable enzyme activity. Because P450arom is up-regulated in the ovaries of CCAAT/enhancer binding protein (C/EBP) beta knockout mice and activation of the ovarian-type P450arom promoter (II) is responsible for aberrant P450arom expression in endometriosis, we sought here to evaluate the possible roles of C/EBP isoforms in the regulation of P450arom expression in endometriotic vs. eutopic endometrial stromal cells. We previously found that the -517-bp flanking region of promoter II contained the critical cis-acting elements for baseline and cAMP (analog)-induced activity. In this study, we disrupted several potential sequences and found that mutations of a -211/-197-bp cAMP-response element (CRE) and a -317/-304-bp C/EBP binding site abolished both baseline and cAMP-induced promoter II activity. Ectopic expression of C/EBPalpha increased both baseline and cAMP-dependent promoter II activity significantly in endometriotic cells, whereas ectopic expression of C/EBPbeta or C/EBPdelta abolished promoter II activity in both untreated and cAMP-treated endometriotic stromal cells. Comparable changes in promoter II activity were observed using endometrial stromal cells, which showed, however, seemingly diminished levels of baseline and cAMP-induced promoter II activity in comparison with endometriotic cells. EMSA using a probe containing the critical -317/-304-bp C/EBP site upstream of promoter II demonstrated a distinct DNA-protein complex in endometriotic, but not in endometrial stromal cells. This specific complex, however, could not be altered using antibodies against C/EBPalpha, -beta, or -delta. Because CRE is another potential DNA motif that can bind C/EBP isoforms, we next used EMSA using a probe containing the -211/-197-bp CRE and demonstrated that specific DNA-protein complexes contained C/EBPalpha but not C/EBPbeta or C/EBPdelta in endometriotic stromal cells. In contrast, C/EBPbeta and C/EBPdelta but not C/EBPalpha were detected in DNA-protein complexes using nuclear extracts from endometrial stromal cells. Western blotting and immunohistochemistry demonstrated expression of C/EBPalpha, -beta, and -delta in human endometriotic and endometrial stroma and epithelium. Intriguingly, C/EBPbeta was expressed at increased levels in stromal cells of human eutopic endometrium compared with simultaneously biopsied endometriotic tissues. We conclude that both -317/-304 and -211/-197-bp elements in promoter II are critical for the robust cAMP-dependent induction in endometriosis. C/EBPalpha up-regulates, whereas C/EBPbeta and C/EBPdelta inhibit P450arom promoter activity via binding primarily to the -211/-197-bp CRE under in vitro conditions. In vivo down-regulation of C/EBPbeta in endometriotic stromal cells and its up-regulation in endometrial stromal cells may in part account for the induction of P450arom expression in endometriosis and its inhibition in endometrium.

Uterine leiomyomas are the most common benign tumours of the female genital tract, often necessitating hysterectomy. The most common symptoms are dysmenorrhoea, menorrhagia, infertility and abortion. Ovarian hormones seem to play an essential role in pathogenesis, and deprivation of ovarian oestrogen causes leiomyomas to shrink significantly. The purpose of this study was to evaluate the effects of the non-steroidal aromatase inhibitor letrozole on uterine leiomyomas and on bone metabolism. A prospective, open clinical trial was conducted in a university-based hospital. Sixteen premenopausal women with symptomatic uterine leiomyomas were treated with letrozole 5 rag/day orally for 3 months. The main outcome measures of uterine and uterine leiomyoma sizes, serum FSH, LH, oestradiol concentrations, ovarian volumes and myoma-related symptoms were noted at baselines and once a month during treatment. Lumbar spine bone mineral density and biochemical markers of bone metabolism were studied at the beginning and at the end of 3 months. Letrozole significantly decreased uterine leiomyoma sizes (P < 0.01) and promptly benefited women with heavy menstrual bleeding associated with leiomyomas without changing bone mineral density. Aromatase inhibitors may represent a new generation of medications for the treatment of leiomyoma and associated symptoms, Larger clinical trials are needed, however, to fully evaluate their safety and efficacy. [ABSTRACT FROM AUTHOR]

Aim. The present study was designed to investigate the effects of two aromatase inhibitors on steroid hormone levels, bone mineral density (BMD) and bone turnover markers in intact female rats. Methods. Letrozole and anastrazole at two different dose levels were investigated for their effect on serum levels of estradiol, androstenedione, testosterone, dehydroepiandrosterone and dehydroepiandrosterone sulfate, BMD of femur and dorsal spine, and osteocalcin and pyridinoline levels as bone turnover markers. Fifty intact female rats were randomly divided in five groups (group 1 (n = 10): control, 2 ml saline; group 2 (n = 10): letrozole 1 mg/kg; group 3 (n = 10): letrozole 2 mg/kg; group 4 (n = 10): anastrazole 0.1 mg/kg; group 5 (n = 10): anastrazole 0.2 mg/kg), and oral gavages were applied for a period of 16 weeks. Results. Both doses of letrozole and anastrazole did not change femoral and vertebral BMD. Serum estradiol levels were reduced significantly at all dose levels by both agents (p < 0.001); all androgen levels were significantly elevated by letrozole (p < 0.05), although anastrazole increased only androstenedione (p < 0.05). The higher dose of letrozole increased osteocalcin levels (p < 0.05), while pyridinoline levels were increased (p < 0.05) by the higher dose of anastrazole. Conclusion. Our results indicate that short-term use of letrozole and anastrazole had no clear effects on BMD in intact rats. Further investigations are needed to understand their effects on bone metabolism in intact females. [ABSTRACT FROM AUTHOR]

Estrogen is produced in a number of human tissues including the ovary, placenta and extraglandular sites such as adipose tissue, skin and the brain. Aromatase is the key enzyme that regulates estrogen formation in these tissues. Aromatase activity is not detectable in normal endometrium. In contrast, aromatase is expressed aberrantly in endometriosis and is stimulated by PGE2. This results in local production of estrogen, which induces PGE2 formation and establishes a positive feedback cycle. Another abnormality in endometriosis, i.e. deficient 17β-hydroxysteroid dehydrogenase (17β-HSD) type 2 expression, impairs the inactivation of estradiol to estrone. These molecular aberrations collectively favor accumulation of increasing quantities of estradiol and PGE2 in endometriosis. The clinical relevance of these findings was exemplified by the successful treatment of an unusually aggressive case of postmenopausal endometriosis using an aromatase inhibitor. [Copyright &y& Elsevier]

Abstract: Endometriosis is a common and chronic disease characterized by persistent pelvic pain and infertility. Estradiol is essential for growth and inflammation in endometriotic tissue. The complete cascade of steroidogenic proteins/enzymes including aromatase is present in endometriosis leading to de novo estradiol synthesis. PGE2 induces the expression of the genes that encode these enzymes. Upon PGE2 treatment, coordinate recruitment of the nuclear receptor SF-1 to the promoters of these steroidogenic genes is the key event for estradiol synthesis. SF-1 is the key factor determining that an endometriotic cell will respond to PGE2 by increased estradiol formation. The presence of SF-1 in endometriosis and its absence in endometrium is determined primarily by the methylation of its promoter. The key steroidogenic enzyme in endometriosis is aromatase encoded by a single gene because its inhibition blocks all estradiol biosynthesis. Aromatase inhibitors diminish endometriotic implants and associated pain refractory to existing treatments in affected women. [Copyright &y& Elsevier]