Regulation of aromatase in estrogen-producing cells

Human adipose stromal cells in monolayer culture aromatize androstenedione to estrone. The rate of aromatization is stimulated 20- to 30-fold by glucocorticoids when fetal calf serum is present in the culture medium and by dibutyryl cyclic AMP in the absence of serum. The action of dibutyryl cyclic AMP to stimulate aromatase activity is potentiated markedly by phorbol esters and inhibited by growth factors, such as EGF. In order to investigate the mechanisms underlying this multifactorial regulation, we have prepared polyclonal and monoclonal antibodies specific for aromatase cytochrome P-450. By use of these antibodies it was demonstrated that the action of these various factors to regulate aromatase activity was caused by alterations in the rate of synthesis of aromatase cytochrome P-450, whereas the synthesis of the reductase component of the aromatase enzyme complex was relatively unaffected. The changes in the rate of synthesis of aromatase cytochrome P-450 were, in turn, reflective of changes in the levels of translatable mRNA specific for this protein. In order to analyze the levels of aromatase cytochrome P-450 mRNA directly, we have isolated a cloned cDNA insert complementary to the mRNA encoding aromatase cytochrome P-450, by screening a lambda gt 11 human placental cDNA library utilizing the polyclonal anti-aromatase P-450 IgG. Use of this cDNA probe in Northern analysis of RNA extracted from human adipose stromal cells revealed that the changes in translatable mRNA resulting from incubation of the cells with the various regulatory factors were due to changes in the absolute levels of mRNA encoding this protein.     

Regulation of aromatase induction by nuclear receptor coregulator PELP1

Estradiol (E2), estrogen receptor (ER), ER-coregulators have been implicated in the development and progression of breast cancer. In situ E2 synthesis is implicated in tumor cell proliferation through autocrine or paracrine mechanisms, especially in post-menopausal women. Several recent studies demonstrated activity of aromatase P450 (Cyp19), a key enzyme that plays critical role in E2 synthesis in breast tumors. The mechanism by which tumors enhance aromatase expression is not completely understood. Recent studies from our laboratory suggested that PELP1 (Proline, Glutamic acid, Leucine rich Protein 1), a novel ER-coregulator, functions as a potential proto-oncogene and promotes tumor growth in nude mice models without exogenous E2 supplementation. In this study, we found that PELP1 deregulation contributes to increased expression of aromatase, local E2 synthesis and PELP1 cooperates with growth factor signaling components in the activation of aromatase. PELP1 deregulation uniquely up-regulated aromatase expression via activation of aromatase promoter I.3/II. Analysis of PELP1 driven mammary tumors in xenograft as well as in transgenic mouse models revealed increased aromatase expression. PELP1-mediated induction of aromatase requires functional Src and PI3K pathways. Chromatin immuno precipitation (ChIP) assays revealed that PELP1 is recruited to the Aro 1.3/II aromatase promoter. HER2 signaling enhances PELP1 recruitment to the aromatase promoter and PELP1 plays a critical role in HER2-mediated induction of aromatase expression. Mechanistic studies revealed that PELP1 interactions with orphan receptor ERRα, and histone demethylases play a role in the activation of aromatase promoter. Accordingly, ChIP analysis showed alterations in histone modifications at the aromatase promoter in the model cells that exhibit local E2 synthesis. Immunohistochemical analysis of breast tumor progression tissue arrays suggested that deregulation of aromatase expression occurs in advanced-stage and node-positive tumors, and that cooverexpression of PELP1 and aromatase occur in a sub set of tumors. Collectively, our results suggest that PELP1 regulation of aromatase represent a novel mechanism for in situ estrogen synthesis leading to tumor proliferation by autocrine loop and open a new avenue for ablating local aromatase activity in breast tumors.     

Future uses for aromatase inhibitors in breast cancer

Over recent years highly potent, well-tolerated aromatase inhibitors have been developed, which essentially obliterate peripheral aromatase activity in postmenopausal women. Their role as the optimal second-line agents (post-tamoxifen) for the treatment of advanced breast cancer has recently been established in large comparative clinical trials. Their testing as adjuvant therapy is warranted, but their eventual application in this (or the prophylactic) setting will be dependent on the currently unknown effects of profound oestrogen deprivation on the physiology of postmenopausal women as well as on its efficacy. It is also possible that these new compounds could suppress oestrogen synthesis in premenopausal women, but the consequences on ovarian folliculogenesis might prevent their widespread use in this group of patients.     

Regulation of sex-specific formation of oestrogen in brain development: Endogenous inhibitors of aromatase

Brain sexual differentiation occurs during steroid-sensitive phases in early development, and is affected particularly by exposure to oestrogens formed in the brain by aromatisation of androgen. The organisational effects of oestrogen result in male-specific neuronal morphology, control of reproductive behaviour, and patterns of gonadotrophin secretion. A question which still has to be resolved is what determines changes in aromatase activity effective for the differentiation of sexually dimorphic brain development during sensitive periods of growth. In the mouse, a sex difference exists at early stages of embryonic development in aromatase-containing neurones of the hypothalamus. The embryonic aromatase system is regulated later in foetal development by androgens. Testosterone treatment increases the numbers of aromatase-immunoreactive hypothalamic neuronal cell bodies. Kinetic evidence from studies on the avian brain suggest that endogenous steroid inhibitors of aromatase, probably formed within neuroglia, also have a role in the control of oestrogen production. Inhibitory kinetic constant determination of endogenous androgenic metabolites formed in the brain showed that preoptic aromatase is potently inhibited by 5-androstanedione (K_i = 6 nM) and less strongly by 5β-dihydrotestosterone (K_i = 350 nM). Regulation by steroidal and possibly non-steroidal inhibitors may contribute to the special characteristics and plasticity in aromatase activity which develops at certain stages in ontogeny.     

Regulation of pig Leydig cell aromatase activity by gonadotropins and Sertoli cells

The present work was done to investigate the cell localization of testicular aromatase activity and its regulation in immature pig testis using an in vitro model. Leydig cells and Sertoli cells were isolated from immature pig testes and cultured alone or together in the absence or presence of human chorionic gonadotropin (hCG) or porcine follicle-stimulating hormone (pFSH) for 2 days. At the end of incubation, the amounts of testosterone (T), estrone sulfate (E1S) and estradiol (E2) were measured. Then the cells were incubated for 4 h in the presence of saturating concentrations of delta 4-androstenedione (3 microM) and the amounts of E1S and E2 were measured again (aromatase activity). The ability of Sertoli cells to produce estrogens was very low and neither hCG nor pFSH had any significant effect. hCG stimulated, in a dose-dependent manner, the secretion of T and E1S by Leydig cells cultured alone as well as the aromatase activity of these cells. The main estrogen produced by Leydig cells was E1S. pFSH also stimulated the above parameters of Leydig cell function; this may have been due to the contamination of this hormone with luteinizing hormone (LH). Coculture of Leydig cells with Sertoli cells without gonadotropins had very small effects on T and E1S production and on aromatase activity. However, treatment of coculture with increasing concentrations of hCG had a dramatic effect on Leydig cell functions. For each hCG concentration, the amounts of T and E1S secreted, as well as the aromatase activity of the coculture, were 2- to 3-fold higher than those of Leydig cells cultured alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Overview of adjuvant trials of aromatase inhibitors in early breast cancer

The third-generation aromatase inhibitors are an important class of drugs for use in adjuvant therapy for postmenopausal women with resected estrogen receptor positive breast cancer. Multiple large prospective randomized trials have established their value in this setting and provided guidance for their use in clinical management. This review will outline the trials that have provided evidence on the value of the aromatase inhibitors in the adjuvant setting as well as the ongoing trials that will expand our knowledge of how to use them most effectively.     

Breast cancer prevention--clinical trials strategies involving aromatase inhibitors

Estrogens and their metabolites have been implicated in both the initiation and the prevention of breast cancer. The reduction in breast cancer incidence seen in the tamoxifen arms of the four prospective trials to date has established the proof of principle that antagonizing estrogen is a potential means of reducing breast cancer risk. However, the areas to improve on these results include: (a) enhanced efficacy, (b) reduction in the incidence of receptor-negative tumors, (c) improved overall and endocrinological side effects, and (d) improved function on end-organs other than the breast. The aromatase inhibitors offer the potential to achieve these goals in part in the following ways: (a) greater reduction in risk of disease as evidenced by superior efficacy in advanced breast cancer and by inhibition of both initiation and promotion of breast cancer, (b) reduction in receptor-negative tumors by synergy with COX-2 inhibitors resulting in growth factor inhibition, anti-angiogenesis and inhibition of tumor-associated aromatase expression, (c) fewer vasomotor and urogenital abnormalities, and (d) reduced thromboembolism and cardiovascular complications and satisfactory effects on bone metabolism. Important differences may exist between non-steroidal reversible inhibitors and steroidal irreversible inactivators in particular related to the androgenic/anabolic effects of the steroidal inactivators. Pilot studies of aromatase inhibitors described elsewhere in this session have begun in healthy women with dense mammography, or a high-risk genetic and/or histocytopathologic profile, to determine potential efficacy, as well as effects on end-organ function. A number of phase three trials with aromatase inhibitors are also underway or in planning. Among these are the BRCA 1 and 2 study of exemestane versus placebo in unaffected postmenopausal carriers, the International Breast Intervention Study 2 (IBIS 2) of anastrozole versus placebo in women with a high-risk profile, and the National Cancer Institute of Canada’s Clinical Trial Group (NCIC CTG) study of exemestane with or without celecoxib versus placebo in women at risk of the disease. For premenopausal women, combination strategies of gonadotrophin agonists and aromatase inhibitors are being investigated. The potential of using low doses of aromatase inhibitors to lower “in breast” estrogen levels without unduly perturbing plasma concentrations is also being explored. The potential of the aromatase gene functioning as an oncogene within the breast may be tied to breast density which in turn may represent both a selection tool for elevated risk and an intermediate marker of prevention. The strong link between postmenopausal estrogen levels and breast cancer risk suggests the possibility that plasma estrogen levels may be a useful intermediate marker of prevention. The aromatase inhibitors offer us the first ever tool to render women virtually free of estrogen and are potentially an exciting tool for the prevention of breast cancer.     

Aromatase, aromatase inhibitors, and breast cancer

Estrogens are known to be important in the growth of breast cancers in both pre and postmenopausal women. As the number of breast cancer patients increases with age, the majority of breast cancer patients are postmenopausal women. Although estrogens are no longer made in the ovaries after menopause, peripheral tissues produce sufficient concentrations to stimulate tumor growth. As aromatase catalyzes the final and rate-limiting step in the biosynthesis of estrogen, inhibitors of this enzyme are effective targeted therapy for breast cancer. Three aromatase inhibitors (AIs) are now FDA approved and have been shown to be more effective than the antiestrogen tamoxifen and are well tolerated. AIs are now a standard treatment for postmenopausal patients. AIs are effective in adjuvant and first-line metastatic setting. This review describes the development of AIs and their current use in breast cancer. Recent research focuses on elucidating mechanisms of acquired resistance that may develop in some patients with long term AI treatment and also in innate resistance. Preclinical data in resistance models demonstrated that the crosstalk between ER and other signaling pathways particularly MAPK and PI3K/Akt is an important resistant mechanism. Blockade of these other signaling pathways is an attractive strategy to circumvent the resistance to AI therapy in breast cancer. Several clinical trials are ongoing to evaluate the role of these novel targeted therapies to reverse resistance to AIs. Article from the special issue on 'Targeted Inhibitors'.     

Impact of aromatase inhibitors on bone health in breast cancer patients

Following the implementation of the third generation aromatase inhibitors in the treatment algorithms for early breast cancer, special attention has been given to the influence of these drugs on bone health. Due to their potent estrogen suppression, the aromatase inhibitors anastrozole and letrozole, as well as the aromatase inactivator exemestane, enhance bone loss in postmenopausal women reflected in decreasing levels of bone mineral density. Moreover, all major phase III trials involving aromatase inhibitors in the adjuvant setting have reported increased fracture rates. All in all, there is no hard evidence to suggest major differences between the individual compounds concerning their side-effects on bone. The consequences of AI therapy on bone are in addition modified by a variety of factors like the BMD level prior to therapy, time since menopause, and vitamin D status. Strategies to avoid bone loss during AI therapy have shown promising results. Thus, bisphosphonates have been shown to prohibit bone loss during AI therapy if used upfront. Novel treatment strategies, like antibodies against RANKL have been developed and promising preliminary results have been published from early trials. Standardized guidelines to avoid or minimize bone loss during AI therapy have been developed, in most countries involving calcium and vitamin D supplementation, as well as BMD measurements to identify patient subgroups demanding bisphosphonate therapy.     

The control and biological importance of intratumoural aromatase in breast cancer

The existence of aromatase activity in human breast carcinomas has been established for about 20 years but the clinical and biological importance of this remains unclear. A number of studies in clinical material suggest that aromatase activity may be a prerequisite of response to aromatase inhibitors and that aromatase activity may be enhanced in those tumours relapsing during treatment with one such inhibitor, aminoglutethimide. These results would carry more significance, however, if it was demonstrable that the growth of breast carcinomas is affected by the conversion of androgens to oestrogens by intratumoural aromatase. We have tried to address this by establishing model systems with aromatase-transfected MCF7 breast cancer cells. We have demonstrated that these cells can be stimulated mitogenically with androgen and that this proliferation is suppressible with aromatase inhibitors. Similarly the growth of aromatase transfected cells but not wild type cells as xenografts is supported by androstenedione and inhibitable by both the steroidal aromatase inhibitor, 4-hydroxyandrostenedione and the non-steroidal inhibitor, CGS 20267. Work with the former of these, which is a suicide inhibitor allowed us to demonstrate that growth can proceed with aromatase activity approximating to the highest level seen in breast carcinomas indicating that at least at this extreme level the intratumoural conversion of androgens to oestrogens may indeed be able to support tumour growth. Further work with this model system should allow us to define the minimal amount of intratumoural activity which can support tumour growth.