Aromatase expression and its localization in human breast cancer

Aromatization or in situ estrogen production by aromatase has been considered to play an important role in the development of human breast carcinoma. In the human breast, aromatase overexpression is observed in the stromal or interstitial cells of the carcinoma, especially at the sites of frank invasion and/or adipose tissue. Transplantation experiments in the nude mouse employing MCF-7 and/or SF-TY human fibroblast cell lines revealed that aromatase activity and expression were much higher in the tumour with MCF-7 and SF-TY than that with MCF-7 alone. Aromatase overexpression in human breast carcinoma tissue is considered to occur as a result of carcinomastromal cell interactions, i.e. paracrine communication between stromal and carcinoma cells. Aromatase overexpression is correlated with the malignant phenotype in the human breast, but not with stage, age, clinical stages, clinical course, or proliferative activity of breast carcinoma. Aromatase overexpression may be correlated with development, rather than the biological behaviour of breast malignancy. Aromatase overexpression is not necessarily correlated with expression of 17β-hydroxysteroid dehydrogenase type 1, which converts estrone to estradiol and estrogen receptor. Different mechanisms may be involved in the regulation of expression of these two important estrogen-metabolizing enzymes and estrogen receptor in human breast cancer. Aromatase overexpression in intratumoral stromal cells was much more frequently detected in male breast cancer than in female counterparts, which confers a growth advantage on cancer cells in a male hormonal environment with low serum estrogen levels.     

Aromatase and in situ estrogen production in DCIS (ductal carcinoma in situ) of human breast

Ductal carcinoma in situ or DCIS belongs to intraductal proliferative lesions, which are a group of cytologically and architecturally diverse ductal proliferations, typically originating from the terminal duct–lobular units. In these intraductal proliferative diseases, estrogens are considered to be involved in the progression of the disease especially from ductal non-neoplastic hyperplasia to DCIS and possibly development of invasive carcinoma from DCIS. Estrogen receptor (ER) alpha is abundantly expressed in atypical ductal hyperplasia and low grade DCIS. Suppression of estrogenic actions using tamoxifen resulted in inhibition of recurrence of DCIS and/or of progression into invasive carcinoma. Intratumoral estrogen concentration in DCIS determined by liquid chromatography/electrospray tandem mass spectrometry is significantly higher than that in non-neoplastic breast tissues with statistically not lower than that in invasive carcinoma. Aromatase mRNA expression in both stromal and parenchymal cells of DCIS determined by quantitative RT-PCR following laser capture microdissection was also much higher than that in non-neoplastic breast, although lower than that in invasive carcinoma. Immunohistochemistry of aromatase also revealed the similar patterns of immunolocalization as in invasive carcinoma. Aromatase is overexpressed in noninvasive breast malignancies including DCIS and results in elevated concentrations of intratumoral estradiol. These findings could provide the scientific rationale as to employing aromatase inhibitors in the management of ER positive DCIS patients.     

Endogenous aromatization of testosterone results in growth stimulation of the human MCF-7 breast cancer cell line

Estrogens produced within breast tumors may play a pivotal role in growth stimulation of the breast cancer cells. However, it is elusive whether the epithelial breast cancer cells themselves synthesize estrogens, or whether the surrounding tumor stromal cells synthesize and supply the cancer cells with estrogen. The aromatase enzyme catalyzes the estrogen production, aromatizing circulating androgens into estrogens. The aim of this study was to investigate aromatase expression and function in a model system of human breast cancer, using the estrogen responsive human MCF-7 breast cancer cell line. Cells were cultured in a low estrogen milieu and treated with estrogens, aromatizable androgens or non-aromatizable androgens. Cell proliferation, expression of estrogen-regulated proteins and aromatase activity were investigated. The MCF-7 cell line was observed to express sufficient aromatase enzyme activity in order to aromatize the androgen testosterone, resulting in a significant cell growth stimulation. The testosterone-mediated growth effect was completely inhibited by the aromatase inhibitors letrozole and 4-hydroxy-androstenedione. Expression studies of estrogen-regulated proteins confirmed that testosterone was aromatized to estrogen in the MCF-7 cells. Thus, the results indicate that epithelial breast cancer cells possess the ability to aromatize circulating androgens to estrogens.     

Aromatase and cyclooxygenases: enzymes in breast cancer

Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C₁₉ androgens to C₁₈ estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin PGE₂ increases intracellular cAMP levels and stimulates estrogen biosynthesis, and previous studies in our laboratories have shown a strong linear association between aromatase (CYP19) expression and expression of the cyclooxygenases (COX-1 and COX-2) in breast cancer specimens. To further investigate the pathways regulating COX and CYP19 gene expression, studies were performed in normal breast stromal cells, in breast cancer cells from patients, and in breast cancer cell lines using selective pharmacological agents. Enhanced COX enzyme levels results in increased production of prostaglandins, such as PGE₂. This prostaglandin increased aromatase activity in breast stromal cells, and studies with selective agonists and antagonists showed that this regulation of signaling pathways occurs through the EP₁ and EP₂ receptor subtypes. COX-2 gene expression was enhanced in breast cancer cell lines by ligands for the various peroxisome proliferator-activated receptors (PPARs), and differential regulation was observed between hormone-dependent and -independent breast cancer cells. Thus, the regulation of both enzymes in breast cancer involves complex paracrine interactions, resulting in significant consequences on the pathogenesis of breast cancer.     

Role of steroid sulfatase in local formation of estrogen in post-menopausal breast cancer patients

More than two-thirds of breast cancers occur in post-menopausal women, and depend on the estrogens for their proliferation and survival. For the treatment of estrogen-dependent breast cancers, two major treatment options are now available. One is selective estrogen receptor modulator (SERM) such as Tamoxifen and another is aromatase inhibitor such as Anastrozole, Letrozole and Exemestane, which reduce local in situ formation of estrogens. Although these therapies are clinically active for advanced and early breast cancers, de novo and/or acquired resistance to SERM and/or aromatase inhibitors are also clinical problem. Recent studies suggest that local formation of estrogens in the breast tumors is more important than circulating estrogen in plasma for the growth and survival of estrogen-dependent breast cancer in post-menopausal women. The rationale for the importance of local formation of estrogens is based on the following evidences. Estradiol (E2) levels in breast tumors are equivalent to those of pre-menopausal patients, although plasma E2 levels are 50-fold lower after menopause. E2 concentrations in breast tumors of post-menopausal women are 10–40 times higher than serum level. Biosynthesis of estrogens in breast tumors tissues occurs via two major different routes, one is aromatase pathway and another is steroid-sulfatase (STS) pathway. Whereas many studies has been reported about aromatase inhibitor and its clinical trial results in breast cancer patients, limited information are available regarding to other estrogen regulating enzymes including STS, its role in breast tumors and STS inhibitors. STS is the enzyme that hydrolyses estrone 3-sulfate (E₁S) and dehydroepiandrosterone-sulfate (DHEA-S) to their active un-sulfoconjugated forms, thereby stimulating the growth and survival of estrogen-dependent breast tumors. It has been well known that E₁S level are much higher than E2 level both in plasma and tumor of post-menopausal patients. Recent reports show that more than 80% of breast tumors are stained with anti-STS antibody and the expression of STS is an independent prognostic factor in breast cancer. Taking these findings into consideration, local formation of estrogens could be partially synthesized from large amount of E₁S by STS, which exist in breast cancer. On the other hand, aromatase localizes in stroma and adipocyte surrounding breast cancer. Furthermore, since estrogen formation from E₁S and DHEA-S (STS pathway) cannot be blocked by aromatase inhibitors, STS is thought to be a new molecular target for the treatment of estrogen-dependent tumor post-SERM and/or aromatase inhibitors. In this symposium, these recent rationale for the importance of STS in post-menopausal breast cancer patients is reviewed as well as STS inhibitor.     

Intracrinology of estrogens and androgens in breast carcinoma

Intratumoral metabolism and synthesis of biologically active steroids such as estradiol and 5-dihydrotestosterone as a result of interactions of various enzymes are considered to play very important roles in the pathogenesis and development of hormone-dependent breast carcinoma. Among these enzymes involved in estrogen metabolism, intratumoral aromatase play an important role in converting androgens to estrogens in situ from serum and serving as the source of estrogens, especially in postmenopausal patients with breast carcinoma. However, other enzymes such as 17β-hydroxysteroid dehydrogenase (17β-HSD) isozymes, estrogen sulfatase (STS), and estrogen sulfotransferase, which contribute to in situ availability of biologically active estrogens, also play pivotal roles in this intratumoral estrogen production above. Androgen action on human breast carcinoma has not been well-studied but are considered important not only in hormonal regulation but also other biological features of carcinoma cells. Intracrine mechanisms also play important roles in androgen actions on human breast carcinoma cells. Among the enzymes involved in biologically active androgen metabolism and/or synthesis, both 17β-hydroxysteroid dehydrogenase type 5 (17βHSD5; conversion from circulating androstenedione to testosterone) and 5-reductase (5Red; reduction of testosterone to DHT (5-dihydrotestosterone) were expressed in breast carcinoma tissues, and in situ production of DHT has been proposed in human breast cancer tissues. However, intracrine mechanisms of androgens as well as their biological or clinical significance in the patients with breast cancer have not been fully elucidated in contrast to those in estrogens.     

New steroidal aromatase inhibitors: Suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death

Background  

Aromatase, the cytochrome P-450 enzyme (CYP19) responsible for estrogen biosynthesis, is an important target for the treatment of estrogen-dependent breast cancer. In fact, the use of synthetic aromatase inhibitors (AI), which induce suppression of estrogen synthesis, has shown to be an effective alternative to the classical tamoxifen for the treatment of postmenopausal patients with ER-positive breast cancer. New AIs obtained, in our laboratory, by modification of the A and D-rings of the natural substrate of aromatase, compounds 3a and 4a, showed previously to efficiently suppress aromatase activity in placental microsomes. In the present study we have investigated the effects of these compounds on cell proliferation, cell cycle progression and induction of cell death using the estrogen-dependent human breast cancer cell line stably transfected with the aromatase gene, MCF-7 aro cells.     

Aromatase and its inhibitors

Inhibitors of aromatase (estrogen synthetase) have been developed as treatment for postmenopausal breast cancer. Both steroidal substrate analogs, type I inhibitors, which inactivate the enzyme and non-steroidal competitive reversible, type II inhibitors, are now available. 4-hydroxyandrostenedione (4-OHA), the first selective aromatase inhibitor, has been shown to reduce serum estrogen concentrations and cause complete and partial responses in approximately 25% of patients with hormone responsive disease who have relapsed from previous endocrine treatment. Letrozole (CGS 20, 269) and anastrozole (ZN 1033) have been recently approved for treatment. Both suppress serum estrogen levels to the limit of assay detection. Letrozole has been shown to be significantly superior to megace in overall response rates and time to treatment failure, whereas anastrozole was found to improve survival in comparison to megace. Both were better tolerated than the latter. The potential of aromatase within the breast as a significant source of estrogen mediating tumor proliferation and which might determine the outcome of inhibitor treatment was explored. Using immunocytochemistry and in situ hybridization, aromatase and mRNA_arom was detected mainly in the epithelial cells of the terminal ductal lobular units (TDLU) of the normal breast and also in breast tumor epithelial cells as well as some stromal cells. Increase in proliferation, measured by increased thymidine incorporation into DNA and by PCNA immunostaining in response to testosterone was observed in histocultures of breast cancer samples. This effect could be inhibited by 4-OHA and implies that intratumoral aromatase has functional significance. An intratumoral aromatase model in the ovariectomized nude mouse was developed which simulated the hormone responsive postmenopausal breast cancer patient. This model also allows evaluation of the efficacy of aromatase inhibitors and antiestrogens in tumors of estrogen receptor positive, human breast carcinoma cells transfected with the human aromatase gene. Thus, the cells synthesized estrogen which stimulated tumor formation. Both aromatase inhibitors and antiestrogens were effective in suppressing tumor growth in this model. However, letrozole was more effective than tamoxifen. When the aromatase inhibitors were combined with tamoxifen, tumor growth was suppressed to about the same extent as with the aromatase inhibitors alone. Thus, there was no additive or synergistic effects of combining tamoxifen with aromatase inhibitors. This suggests that sequential treatment with these agents is likely to be more beneficial to the patient in terms of longer response to treatment.     

Signaling pathways regulating aromatase and cyclooxygenases in normal and malignant breast cells

Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C(19) androgens to C(18) estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin PGE(2) increases intracellular cAMP levels and stimulates estrogen biosynthesis, and our recent studies have shown a strong linear association between CYP19 expression and the sum of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) expression in breast cancer specimens. Knowledge of the signaling pathways that regulate the expression and enzyme activity of aromatase and cyclooxygenases (COXs) in stromal and epithelial breast cells will aid in understanding the interrelationships of these two enzyme systems and potentially identify novel targets for regulation. The effects of epidermal growth factor (EGF), transforming growth factor-beta (TGFbeta), and tetradecanoyl phorbol acetate (TPA) on aromatase and COXs were studied in primary cultures of normal human adipose stromal cells and in cell cultures of normal immortalized human breast epithelial cells MCF-10F, estrogen-responsive human breast cancer cells MCF-7, and estrogen-unresponsive human breast cancer cells MDA-MB-231. Levels of the constitutive COX isozyme, COX-1, were not altered by the various treatments in the cell systems studied. In breast adenocarcinoma cells, EGF and TGFbeta did not alter COX-2 levels at 24h, while TPA induced COX-2 levels by 75% in MDA-MB-231 cells. EGF and TPA in MCF-7 cells significantly increased aromatase activity while TGFbeta did not. In contrast to MCF-7 cells, TGFbeta and TPA significantly increased activity in MDA-MB-231 cells, while only a modest increase with EGF was observed. Untreated normal adipose stromal cells exhibited high basal levels of COX-1 but low to undetectable levels of COX-2. A dramatic induction of COX-2 was observed in the adipose stromal cells by EGF, TGFbeta, and TPA. Aromatase enzyme activity in normal adipose stromal cells was significantly increased by EGF, TGFbeta and TPA after 24h of treatment. In summary, the results of this investigation on the effects of several paracrine and/or autocrine signaling pathways in the regulation of expression of aromatase, COX-1, and COX-2 in breast cells has identified more complex relationships. Overall, elevated levels of these factors in the breast cancer tissue microenvironment can result in increased aromatase activity (and subsequent increased estrogen biosynthesis) via autocrine mechanisms in breast epithelial cells and via paracrine mechanisms in breast stromal cells. Furthermore, increased secretion of prostaglandins such as PGE(2) from constitutive COX-1 and inducible COX-2 isozymes present in epithelial and stromal cell compartments will result in both autocrine and paracrine actions to increase aromatase expression in the tissues.     

Effect of antiestrogens and aromatase inhibitor on basal growth of the human breast cancer cell line MCF-7 in serum-free medium

Antiestrogens are efficient inhibitors of estrogen-mediated growth of human breast cancer. Besides inhibiting estradiol-stimulated growth, antiestrogens may have a direct growth-inhibitory effect on estrogen receptor (ER) positive cells and thus be more efficient than aromatase inhibitors, which will only abrogate estrogen-dependent tumor growth. To address this issue, we have used the human breast cancer cell line MCF-7/S9 as a model system which is maintained in a chemically defined medium without serum and estrogen. The addition of estradiol results in an increase in cell growth rate. Thus, the MCF-7/S9 cell line is estrogen-responsive but not estrogen-dependent. Three different types of antiestrogens, namely tamoxifen, ICI 182,780 and EM-652 were found to exert a significant and dose-dependent inhibition of basal growth of MCF-7/S9 cells. The growth-inhibitory effect of the three antiestrogens was prevented by simultaneous estradiol treatment. Antiestrogen treatment also reduced the basal pS2 mRNA expression level, thus indicating spontaneous estrogenic activity in the cells. However, treatment with the aromatase inhibitor had no effect on basal cell growth, excluding that endogenous estrogen synthesis is involved in basal growth. These data demonstrate that in addition to their estrogen antagonistic effect, antiestrogens have a direct growth-inhibitory effect which is ER-mediated. Consequently, in the subset of ER positive breast cancer patients with estrogen-independent tumor growth, antiestrogen therapy may be superior to treatment with aromatase inhibitors which only inhibit estrogen formation but do not affect cancer cell growth in the absence of estrogens.     

Aromatase in the normal breast and breast cancer

Adipose tissue and muscle constitute the larger proportion of body mass, and therefore aromatization in these tissues is the major source of circulating estrogens in postmenopausal women. Although plasma estrogen concentrations are very low, levels in breast cancers from postmenopausal patients are reported to be 10-fold higher than in plasma and normal tissue. Whereas studies on aromatase activity in the tumor suggest that estrogen may be produced locally, the significance of this contribution has been questioned. Using immunocytochemistry (ICC) to an anti-aromatase antibody, a relatively strong immunoreaction was detected in tumor epithelial cells as well as in the terminal ductal lobular units (TDLUs) of the normal breast. Aromatase expression was detected in the cytoplasm of tumor epithelial cells and the surrounding stromal cells of over 50% of tumors in a series of 19 breast cancers. In situ hybridization (ISH) to aromatase mRNA confirmed the immunocytochemical result that the epithelial cells are the primary site of estrogen synthesis in the breast and breast cancers. In the 10 tumors which showed immunoreaction to aromatase, the average aromatase activity measured in cryosections was 286.5 ± 18.6 fmol estrogen/mg protein/h (SE), whereas in nine tumors with weak aromatase immunoreaction, the enzyme activity was 154.7 ± 19.3 fmol estrogen/mg protein/h (P < 0.05) (SE). The functional significance of tumor aromatase and locally produced estrogens on the growth of tumors was suggested by the correlation between aromatase activity and proliferating cell nuclear antigen (PCNA), a marker of cell proliferation (P < 0.005). Although intratumoral aromatase activity did not correlate with steroid receptors significantly, there was a trend for estrogen receptor (ER)-positive tumors to express aromatase. In addition, proliferation ([³H]-thymidine incorporation into DNA) during histoculture, was increased by both estradiol and testosterone in tumors with high aromatase activity. Our results suggest that some tumors synthesize sufficient estrogen to stimulate their proliferation. It may thus be important to inhibit tumor aromatase as well as to reduce circulating levels of estrogen for effective breast cancer treatment.     

Inhibition of aromatase: insights from recent studies

Aromatase is the rate limiting enzyme that catalyzes the conversion of androgens to estrogens. Blockade of this step allows treatment of diseases that are dependent upon estrogen. Over the past two decades, highly potent and specific aromatase inhibitors have been developed which block total body aromatization by over 99%. An important recent question is whether aromatase inhibitors are superior to the antiestrogens for treatment of hormone-dependent breast cancer. The third generation aromatase inhibitors have been compared to tamoxifen for the treatment of breast cancer in the advanced, adjuvant, and neoadjuvant settings. All of these studies suggest the superiority of aromatase inhibitors over tamoxifen. The mechanism responsible for the superiority of the aromatase inhibitors relates to the estrogen agonistic effects of tamoxifen. During exposure to estrogen deprived conditions and to tamoxifen, breast cancer cells adapt and upregulate the MAP kinase and PI-3 kinase pathways. These growth factor signaling pathways potentiate the estrogen agonistic properties of tamoxifen. Data from a large adjuvant therapy trial (ATAC trial) provide evidence that the aromatase inhibitors may also be superior for breast cancer prevention. The mechanism for superiority in this setting probably relates to the genotoxic effects of estradiol metabolites. The aromatase inhibitors may be also useful for the treatment of endometriosis and for ovulation induction as evidenced by preliminary data. The recent advances in development of the aromatase inhibitors clearly demonstrate the utility of these agents for treatment of breast cancer and potentially for other indications.     

Intracellular aromatase and its relevance to the pharmacological efficacy of aromatase inhibitors

An important feature of the pharmacological profile of aromatase inhibitors is the ability of the various inhibitors to inhibit intracellular aromatase. It is now well documented that a large proportion of breast tumors express their own aromatase. This intratumoral aromatase produces estrogen in situ and therefore may contribute significantly to the amount of estrogen to which the cell is exposed. Thus it is not only important that aromatase inhibitors potently inhibit the peripheral production of estrogen and eliminate the external supply of estrogen to the tumor cell, but that they in addition potently inhibit intratumoral aromatase and prevent the tumor cell from making its own estrogen within the cell. To study the inhibition of intracellular aromatase we have compared the aromatase-inhibiting potency of the non-steroidal aromatase inhibitors, letrozole, anastrozole and fadrozole in a variety of model cellular endocrine and tumor systems which contain aromatase. We have used hamsters ovarian tissue fragments, adipose tissue fibroblasts from normal human breast, the MCF-7Ca human breast cancer cell line transfected with the human aromatase gene and the JEG-3 human choriocarcinoma cell line. Although letrozole and anastrozole are approximately equipotent in a cell-free aromatase system (human placental microsomes), letrozole is consistently 10–30 times more potent than anastrozole in inhibiting intracellular aromatase in intact rodent cells, normal human adipose fibroblasts and human cancer cell lines. Whether these differences between letrozole and anastrozole are seen in the clinical setting will have to await the results of clinical trials which are currently in progress.     

X-ray structure of human aromatase reveals an androgen-specific active site

Aromatase is a unique cytochrome P450 that catalyzes the removal of the 19-methyl group and aromatization of the A-ring of androgens for the synthesis of estrogens. All human estrogens are synthesized via this enzymatic aromatization pathway. Aromatase inhibitors thus constitute a frontline therapy for estrogen-dependent breast cancer. Despite decades of intense investigation, this enzyme of the endoplasmic reticulum membrane has eluded all structure determination efforts. We have determined the crystal structure of the highly active aromatase purified from human placenta, in complex with its natural substrate androstenedione. The structure shows the binding mode of androstenedione in the catalytically active oxidized high-spin ferric state of the enzyme. Hydrogen bond-forming interactions and tight packing hydrophobic side chains that complement the puckering of the steroid backbone provide the molecular basis for the exclusive androgenic specificity of aromatase. Locations of catalytic residues and water molecules shed new light on the mechanism of the aromatization step. The structure also suggests a membrane integration model indicative of the passage of steroids through the lipid bilayer.     

Insight into the enzyme-inhibitor interactions of the first experimentally determined human aromatase

Aromatase is an important pharmacological target in the anti-cancer therapy as the intratumoral aromatase is the source of local estrogen production in breast cancer tissues. Suppression of estrogen biosynthesis by aromatase inhibition represents an effective approach for the treatment of hormone-sensitive breast cancer. Because of the membrane-bound character and heme-binding instability, no crystal structure of aromatase was reported for a long time, until recently when crystal structure of human placental aromatase cytochrome P450 in complex with androstenedione was deposited in PDB. The present study is towards understanding the structural and functional characteristics of aromatase to address unsolved mysteries about this enzyme and elucidate the exact mode of binding of aromatase inhibitors. We have performed molecular docking simulation with twelve different inhibitors (ligands), which includes four FDA approved drugs; two flavonoids; three herbal compounds and three compounds having biphenyl motif with known IC(50) values into the active site of the human aromatase enzyme. All ligands showed favorable interactions and most of them seemed to interact to hydrophobic amino acids Ile133, Phe134, Phe221, Trp224, Ala306, Val370, Val373, Met374 and Leu477 and hydrophilic Arg115 and neutral Thr310 residues. The elucidation of the actual structure-function relationship of aromatase and the exact binding mode described in this study will be of significant interest as its inhibitors have shown great promise in fighting breast cancer.     

Aromatase inhibitors: assessment of biochemical efficacy measured by total body aromatase inhibition and tissue estrogen suppression

The implementation of aromatase inhibitors for treatment of early and metastatic breast cancer has been one of the major improvements in endocrine therapy of breast cancer. Measurement of endocrine effects of aromatase inhibition in vivo has been a major tool in the process of evaluating novel compounds. Biochemical efficacy of aromatase inhibitors in vivo may be determined from their effects on “total body aromatization” as well changes in plasma and tissue estrogen levels. Due to high sensitivity, tracer methods allowing calculation of whole body aromatase inhibition are still considered the gold standard. The method developed by our group in collaboration with the Royal Marsden Hospital and the results of this joint program are summarized and discussed. These studies allowed classification of the different aromatase inhibitors and their optimal dosage, selecting the best compounds for clinical evaluation. In vivo total body aromatase assessment is a work-consuming method, allowing such studies to be conducted in a limited number of patients only. In contrast, plasma estrogen measurement is a cruder but simpler method, allowing screening of larger groups of patients. As plasma estrogens arise through passive diffusion of estrogens synthesized in different body compartments, plasma estrogens, as well as total body aromatase assessment, present a rough estimate of total body tissue estrogen production, and changes associated with treatment with aromatase inhibitors reflect the effects on tissue estrogen production in general. However, plasma estrogen levels do not correlate to breast cancer tissue estrogen levels. This is due to the endocrine autonomy of breast cancer tissue with significant local estrogen production in some tumors. Thus, direct measurement of intratumor estrogens is demanded to evaluate the effects of aromatase inhibitors in malignant target tissues. Our group has developed a highly sensitive HPLC-RIA for the simultaneous measurement of estrone, estradiol, and estrone sulfate in malignant breast tissue samples, and we are currently using this method to assess alterations in intratumor estrogen levels during treatment with different aromatase inhibitors.     

Effect of androstenedione on growth of untransfected and aromatase-transfected MCF-7 cells in culture

Aromatase is present in human breast tumors and in breast cancer cell lines suggesting the possibility of in-situ estrogen production via the androstenedione to estrone and estradiol pathway. However, proof of the biologic relevance of aromatase in breast cancer tissue requires the demonstration that this enzyme mediates biologic effects on cell proliferation. Accordingly, we studied the effects of the aromatase substrate, androstenedione, on the rate of proliferation of wild-type and aromatase-transfected MCF-7 breast cancer cells. Androstenedione did not increase cell growth in wild-type MCF-7 cells which contained relatively low aromatase activity and produced 4-fold more estrone than estradiol. In contrast, aromatase-transfected cell contained higher amounts of aromatase, produced predominantly estradiol, and responded to androstenedione with enhanced growth. An aromatase inhibitor fadrozole hydrochloride, blocked the proliferative effects of androstenedione providing evidence for the role of aromatase in this process. As further evidence of the requirement for aromatase, cells transfected with the neomycin resistance expression plasmid but lacking the aromatase cDNA did not respond to androstenedione. These studies provide evidence that aromatase may have a biologic role for in-situ synthesis of estrogens of breast cancer tissue.     

Leptin enhances,via AP-1, expression of aromatase in the MCF-7 cell line

Leptin, a product of adipocytes, is involved in the regulation of body weight and results strongly correlated to body fat content. An excess of fat mass represents a breast cancer risk factor particularly in postmenopausal women, where estrogen production by adipose tissue through its own aromatase activity stimulates tumor progression. Leptin stimulates estrogen production through the increase of aromatase expression and activity in human luteinized granulosa cells and adipose stromal cells. In the present study, we have examined the possible link that exists between leptin and breast cancer, focusing our attention on the direct effect of leptin on aromatase activity, which may enhance estrogen production and induce tumor cell growth stimulation. We have shown that leptin enhances aromatase mRNA expression, aromatase content, and its enzymatic activity in MCF-7. Aromatase expression appears to be regulated by tissue-specific promoter. It has been demonstrated that promoters II and 1.3 are the major promoters that drive aromatase expression in MCF-7. Transient transfection experiments using vector containing human aromatase promoters II and 1.3 sequence fused with luciferase reporter gene demonstrated that leptin is able to activate this promoter. In the presence of either mitogen-activated protein kinase inhibitor PD 98059 or ERK2 dominant negative as well as in the presence of STAT3 dominant negative, the stimulatory effects of leptin on aromatase promoter, enzymatic activity, and aromatase protein content were inhibited. Functional studies of mutagenesis and electrophoretic mobility shift assay revealed that the AP-1 motif is important in determining the up-regulatory effects induced by leptin on aromatase expression in MCF-7.