Controversies of aromatase localization in human breast cancer--stromal versus parenchymal cells

Aromatase is a key enzyme of estrogen production through conversion from serum androgens in estrogen-dependent postmenopausal breast cancer. Aromatase has been reported to be predominantly located in intratumoral stromal cells and adipocytes but not in parenchymal or carcinoma cells in breast cancer tissue. It is, however, true that there have been controversies regarding intratumoral localization of aromatase in human breast carcinoma, especially whether intratumoral production of estrogens through aromatase occurs in parenchymal or stromal cells. Results of several studies suggested that aromatase present in parenchymal carcinoma cells plays more important roles in the growth and invasion of breast carcinomas than that in stromal cells through providing higher levels of estrogens to carcinoma cells. Aromatase inhibitors are increasingly being used in place of tamoxifen after results of various clinical trials demonstrated that aromatase inhibitors are more effective in increasing survival and recurrence of estrogen-dependent breast cancer patients. Therefore, it is important to clarify the estrogen supplying pathway by aromatase inside of breast carcinoma tissues in order to evaluate the possible efficacy of aromatase inhibitor treatment. In this review, the controversies regarding these intratumoral localization patterns in human breast carcinoma will be briefly summarized.     

Aromatase and COX-2 expression in human breast cancers

We have investigated aromatase and the inducible cyclooxygenase COX-2 expression using immunocytochemistry in tumors of a series of patients with advanced breast cancer treated with aromatase inhibitors. Aromatase was expressed in 58/102 breast cancers. This is similar to the percentage previously reported for aromatase activity. Interestingly, aromatase was expressed in a variety of cell types, including tumor, stromal, adipose, and endothelial cells. Since prostaglandin E₂ is known to regulate aromatase gene expression and is the product of COX-2, an enzyme frequently overexpressed in tumors, immunocytochemistry was performed on the tissue sections using a polyclonal antibody to COX-2. Aromatase was strongly correlated (P<0.001) with COX-2 expression. These results suggest that PGE₂ produced by COX-2 in the tumor may be important in stimulating estrogen synthesis in the tumor and surrounding tissue. No correlation was observed between aromatase or COX-2 expression and the response of the patients to aromatase inhibitor treatment. However, only 13 patients responded. Nine of these patients were aromatase positive. Although similar to responses in other studies, this low response rate to second line treatment suggests that tumors of most patients were no longer sensitive to the effects of estrogen. Recent clinical studies suggest that greater responses occur when aromatase inhibitors are used as first line treatment. In the intratumoral aromatase mouse model, expression of aromatase in tumors is highly correlated with increased tumor growth. First line treatment with letrozole was effective in all animals treated and was more effective than tamoxifen in suppressing tumor growth. Letrozole was also effective in tumors failing to respond to tamoxifen, consistent with clinical findings. In addition, the duration of response was significantly longer with the aromatase inhibitor than with tamoxifen, suggesting that aromatase inhibitors may offer better control of tumor growth than this antiestrogen.     

Aromatase in endometriosis and uterine leiomyomata

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.     

The potential role of estrogen in aromatase regulation in the breast

Aromatase is expressed in both normal and malignant breast tissues. Aromatase activity in the breast varies over a wide range. Our previous studies have demonstrated that in situ aromatization contributes to the estrogen content of breast tumors to a major extent. Consequently, alterations of aromatase activity could serve as a major determinant of tissue estradiol content. However, the mechanisms and extent of aromatase regulation in breast tissues have not been fully established. We have observed an inverse correlation between tumor aromatase activity and estrogen content in nude mice bearing xenografts of MCF-7 cells transfected with the aromatase gene. To investigate the potential role of estrogen in aromatase regulation in the breast, studies were carried out in an in vitro model. In this model, MCF-7 cells were cultured long term in estrogen-deprived medium and called by the acronym, LTED cells. We found that long-term estrogen deprivation enhanced aromatase activity by 3–4-fold when compared to the wild-type MCF-7 cells. Re-exposure of LTED cells to estrogen reduced aromatase activity to the levels of the wild-type MCF-7 cells. We also measured aromatase activity in 101 frozen breast carcinoma specimens and compared tumor aromatase activities in pre-menopausal patients versus post-menopausal patients and in post-menopausal patients with or without hormone replacement therapy (HRT). Although statistically not significant, there was a trend paralleling that observed in the in vitro studies. Aromatase activity was higher in breast cancer tissues from the patients with lower circulating estrogen levels. Our data suggest that estrogen may be involved in the regulation of aromatase activity in breast tissues.     

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.     

An integrated view of aromatase and its inhibition

Aromatase inhibition has become a major treatment strategy for postmenopausal women with oestrogen-dependent breast cancer. Its optimal application is, however, dependent upon (i) the accurate identification of cancers which are ultimately dependent upon the activity of the aromatase enzyme, (ii) the use of the best method/inhibitor by which to blockade aromatase activity.

The single best predictor of response to aromatase inhibitors is the presence of tumour oestrogen receptors; receptor-negative cancers rarely respond whereas those with high levels seem particularly likely to benefit. However, there is a need for additional discriminatory markers. The use of microarray technology coupled with neoadjuvant therapy is likely to yield promising candidate genes. The finding that, amongst peripheral tissues, the tumour itself may have high activity has led to the suggestion that the tumour aromatase measurements may be predictive; however, in situ studies and the lack of robust assays for tumour aromatase suggest that tumour aromatase may not be an influential marker.

Whilst drugs such as anastrozole, exemestane, formestane and letrozole are all effective and specific inhibitors of aromatase, they differ in structure, potency and mechanism of action. Thus, differential sensitivity of tissues/tumours and non-cross resistance mean inhibitors are not equivalent and individual agents may have differing roles according to the setting in which they will be used. Aromatase inhibitors have evolved as key endocrine agents in the treatment of breast cancer. They offer the promise of rational treatment management based on the accurate identification of individual cohorts of tumours responsive to specific drugs.     

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.     

Binding features of steroidal and nonsteroidal inhibitors

Aromatase is the rate-limiting enzyme in estrogen biosynthesis. As a cytochrome P450, it utilizes electrons from NADPH-cytochrome P450 reductase (CPR) to produce estrogen from androgen. Estrogen is a key factor in the promotion of hormone-dependent breast cancer growth. Aromatase inhibitors (AIs) are drugs that block estrogen synthesis, and are widely used to treat estrogen-dependent breast cancer. Structure-function experiments have been performed to study how CPR and AIs interact with aromatase to further the understanding of how these drugs elicit their effects. Our studies have revealed a strong interaction between aromatase and CPR, and that the residue K108 is situated in a region important to the interaction of aromatase with CPR. The published X-ray structure of aromatase indicates that the F221, W224 and M374 residues are located in the active site. Our site-directed mutagenesis experiments confirm their importance in the binding of the androgen substrate as well as AIs, but these residues interact differently with steroidal inhibitors (exemestane) and non-steroidal inhibitors (letrozole and anastrozole). Furthermore, our results predict that the residue W224 also participates in the mechanism-based inhibition of exemestane, as time-dependent inhibition is eliminated with mutation on this residue. Together with previous research from our laboratory, this study confirms that W224, E302, D309 and S478 are important active site residues involved in the suicide mechanism of exemestane against aromatase.     

Breast cancer tissue estrogens and their manipulation with aromatase inhibitors and inactivators

Despite the dramatic fall in plasma estrogen levels at menopause, only minor differences in breast tissue estrogen levels have been reported comparing pre- and postmenopausal women. Thus, postmenopausal breast tissue has the ability to maintain concentrations of estrone (E₁) and estradiol (E₂) that are 2–10- and 10–20-fold higher than the corresponding plasma estrogen levels. This finding may be explained by uptake of estrogens from the circulation and/or local estrogen production. Local aromatase activity in breast tissue seems to be of crucial importance for the local estrogen production in some patients while uptake from the circulation may be more important in other patients. Beside aromatase, breast tissue expresses estrogen sulfotransferase and sulfatase as well as dehydrogenase activity, allowing estrogen storage and release in the cells as well as conversions between estrone and estradiol. The activity of the enzyme network in breast cancer tissue is modified by a variety of factors like growth factors and cytokines. Aromatase inhibitors have been used for more than two decades in the treatment of postmenopausal metastatic breast cancer and are currently investigated in the adjuvant treatment and even prevention of breast cancer. Novel aromatase inhibitors and inactivators have been shown to suppress plasma estrogen levels effectively in postmenopausal breast cancer patients. However, knowledge about the influence of these drugs on estrogen levels in breast cancer tissue is limited. Using a novel HPLC-RIA method developed for the determination of breast tissue estrogen concentrations, we measured tissue E₁, E₂ and estrone sulfate (E₁S) levels in postmenopausal breast cancer patients before and during treatment with anastrozole. Our findings revealed high breast tumor tissue estrogen concentrations that were effectively decreased by anastrozole. While E₁S was the dominating estrogen fraction in the plasma, estradiol was the estrogen fraction with the highest concentration in tumor tissue. Moreover, plasma estrogen levels did not correlate with tissue estrogen concentrations. The overall experience with aromatase inhibitors and inactivators concerning their influences on breast tissue estrogen concentrations is summarized.     

Molecular pharmacology of aromatase and its regulation by endogenous and exogenous agents

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 E₂ (PGE₂) 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 COX-1 and COX-2 expression in breast cancer specimens. PGE₂ can bind to four receptor subtypes, EP₁–EP₄, which are coupled to different intracellular signaling pathways. In primary human breast stromal cell cultures, aromatase activity was significantly induced by PGE₂, dexamethasone, and agonists for the EP₁ and EP₂ receptor subtypes. An EP₁ antagonist, SC-19220, inhibited the induction of enzyme activity by PGE₂ or 17-phenyltrinor-PGE₂, an EP₁ agonist. Sulprostone, an EP₃ agonist, did not alter aromatase activity levels. Investigations are also underway on the regulation of aromatase by exogenous medicinal agents. Selective steroidal and nonsteroidal agents are effective in inhibiting breast tissue aromatase. The benzopyranone ring system is a molecular scaffold of considerable interest, and this scaffold is found in certain flavonoid natural products that have weak aromatase inhibitory activity. Our novel synthetic route for benzopyranones utilizes readily available salicylic acids and terminal alkynes as starting materials. The synthesis of flavones with diversity on the benzopyranone moiety and at the C-2 position occurs with good to excellent yields using these reaction conditions, resulting in an initial benzopyranone library of thirty compounds exhibiting enhanced and differential aromatase inhibition. Current medicinal chemistry efforts focus on diversifying the benzopyranone scaffold and utilizing combinatorial chemistry approaches to construct small benzopyranone libraries as potential aromatase inhibitors.