Tumor-stromal interaction through the estrogen-signaling pathway in human breast cancer

In postmenopausal breast cancers, locally produced estrogen by adipose stromal cells causes the progression of tumor growth. Although aromatase, a key enzyme of estrogen synthesis, is highly expressed in the adipose stromal cells, and aromatase inhibitors show greater efficacy in postmenopausal breast cancers, the mechanism of increasing aromatase activity in the stromal cells remains unclear. To analyze the estrogen signals and to detect the estrogen receptor (ER)-activating ability of adipose stromal cells for individual human breast cancers, we developed a new reporter cell system. To visualize the activation of ER, we first established a stable transformant, named E10, of human breast cancer MCF-7 cells by transfection with the estrogen-responsive element-green fluorescent protein (GFP) gene. E10 cells specifically express GFP when ER is activated by estrogen or by coculture with adipose stromal cells isolated from breast tumor tissues in the presence of testosterone, a substrate for aromatase. Treatment of adipose stromal cells with dexamethasone, a stimulator of aromatase gene expression, resulted in an increase in the expression of GFP in E10 cells in the coculture. Using this system, we characterized the adipose stromal cells of 67 human breast cancers and found that GFP expression levels vary among the cases, suggesting that the ability of adipose stromal cells to activate ERs is unique for individual breast cancers. High induction levels of GFP were observed more frequently in postmenopausal cases than in premenopausal cases, whereas they did not significantly correlate with the ER expression status. Aromatase inhibitors inhibited the induction of GFP expression in the coculture, but the sensitivities to the drugs varied among the individual cases. Aromatase gene expression levels in adipose stromal cells did not always correlate with their ability to induce GFP. These results suggest that this system to detect total ER activation based on the interaction with adipose stromal cells is a useful tool for analyzing local estrogen signals and for tumor-stromal interactions.     

Characterisation of aromatase expression in the human adipocyte cell line SGBS

Aromatase is a member of the cytochrome P450 superfamily of enzymes which catalyses the rate-limiting step in the biosynthesis of estrogens. A number of clinical studies have highlighted the importance of local estrogen production in adipose tissue. In particular, in the postmenopausal woman, the degree of her estrogenization is mainly determined by the extent of her adiposity and it is this extragonadal source of estrogen that likely contributes to breast cancer development and progression. The mechanisms regulating aromatase expression in adipose tissue however, have not been fully elucidated. In this study, we have characterised the expression of aromatase and its activity in a human preadipocyte cell strain, SGBS. Aromatase is expressed in SGBS cells and its expression and activity are strongly stimulated by forskolin (FSK) and phorbol 12-myristate-13-acetate (PMA) treatment. Consistent with this, FSK and PMA treatment also increased activation of the proximal aromatase promoter, promoter II. These findings mimic those that have previously been shown in isolated primary human preadipocytes. These data suggest that SGBS cells are a valuable model with which to further elucidate the mechanisms regulating aromatase expression, and therefore local estrogen synthesis in human adipose tissue.     

Aromatase and prostaglandin inter-relationships in breast adipose tissue: significance for breast cancer development

The role of prostaglandins in the development of breast cancer is a topic of growing interest. Stimulation of aromatase expression within the breast has been proposed as a mechanism whereby prostaglandins could influence breast cancer growth. In the present study, we show that PGE₂ is a powerful stimulator of aromatase expression in human breast adipose stromal cells. Moreover, TNF, which also stimulates aromatase expression in breast adipose stromal cells, acts to increase the secretion of PGE₂ by these cells, as well as the expression of COX 2 and PGE synthase, but not that of COX 1. On the other hand, class I cytokines had no effect, either by themselves or in the presence of estradiol. These factors had little influence on secretion of 15-deoxy-^12,14-PGJ₂, which is inhibitory of aromatase expression by breast adipose stromal cells. These results further substantiate an important role for PGE₂ to stimulate estrogen biosynthesis within the local environment of the breast.     

Microenvironmental regulation of estrogen signals in breast cancer

In breast cancers, estrogen activates estrogen receptor (ER) through genomic and nongenomic pathways, which leads to nuclear and extranuclear processes that promote the proliferation of breast cancer cells. Growth factor receptor signaling pathways also activate ER via phosphorylation through the signal crosstalks between estrogen and growth factors. The intratumoral levels of estrogen and growth factors, therefore, profoundly influence ER activity, which are regulated by the tumor-stromal interactions in the microenvironment. In postmenopausal breast cancers, tumor cells activate stromal fibroblasts to express aromatase, a key enzyme in estrogen biosynthesis, resulting in intratumoral estrogen production. At present, aromatase inhibitors are used as a first-line endocrine therapy for breast cancers. We developed a comprehensive system to evaluate the ER-activating ability of stromal fibroblasts for individual patients, and found that it varied among individual cases. This system might be useful for predicting the individual response to endocrine therapy and analyzing the tumor microenvironment. In addition to estrogen production, tumor-associated fibroblasts lead to the progression of breast cancer via different pathways. A study to differentiate the microenvironmental regulation of estrogen-dependent and -independent breast cancer growth would also be useful to improve hormone therapy for breast cancer.     

Interactions between prostaglandin E(2), liver receptor homologue-1, and aromatase in breast cancer

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 E(2) (PGE(2)) 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 PGE(2) induced expression of both LRH-1 and aromatase. We suggest that PGE(2) 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.     

Influence of J series prostaglandins on apoptosis and tumorigenesis of breast cancer cells.

This study was undertaken to investigate the influence of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonists on the proliferation, apoptosis and tumorigenesis of breast cancer cells. PPARgamma investigation has been largely restricted to adipose tissue, where it plays a key role in differentiation, but recent data reveal that PPARgamma is expressed in several transformed cells. However, the function of PPARgamma activation in neoplastic cells is unclear. Activation of PPARgamma with the known prostanoid agonist 15-deoxy-Delta12,14-prostaglandin J(2) (15dPGJ(2)) or the thiazolidinedione (TZD) agonist troglitazone (TGZ) attenuated cellular proliferation of the estrogen receptor-negative breast cancer cell line MDA-MB-231, as well as the estrogen receptor-positive breast cancer cell line MCF-7. This was marked by a decrease in total cell number and by an inhibition of cell cycle progression. Addition of 15dPGJ(2) was not associated with an increase in cellular differentiation, as has been seen in other neoplastic cells, but rather induction of cellular events associated with programmed cell death, apoptosis. Video time-lapse microscopy revealed that 15dPGJ(2) induced morphological changes associated with apoptosis, including cellular rounding, blebbing, the production of echinoid spikes, blistering and cell lysis. In contrast, TGZ caused only a modest induction of apoptosis. These results were verified by histochemistry using the specific DNA stain DAPI to observe nuclear condensation, a marker of apoptosis. Finally, a brief exposure of MDA-MB-231 cells to 15dPGJ(2) initiated an irreversible apoptotic pathway that inhibited the growth of tumors in a nude mouse model. These findings illustrate that induction of apoptosis may be the primary biological response resulting from PPARgamma activation in some breast cancer cells and further suggests a potential role for PPARgamma ligands for the treatment of breast cancer.     

Correlation of aromatase and cyclooxygenase gene expression in human breast cancer specimens

Aromatase, the enzyme system catalyzing estrogen biosynthesis, is found in stromal tissue in the breast. The increased expression of the aromatase CYP19 gene in breast cancer tissues was recently associated with a promoter region regulated through cAMP-mediated pathways. PGE₂, derived from cyclooxygenase, increases intracellular cAMP levels and stimulates estrogen biosynthesis. This association suggest that local production of PGE₂ via cyclooxgenase isozymes may influence estrogen biosynthesis. The present study represents the first to examine the levels of mRNA expression of CYP19, COX-1, and COX-2 genes in human breast cancer specimens and normal breast tissue samples using semi-quantitative RT-PCR methods. Positive correlations were observed between CYP19 and COX-2 and the greater extent of breast cancer cellularity. Linear regression analysis using a bivariate model shows a strong linear association between CYP19 expression and the sum of COX-1 and COX-2 expression. This significant relationship between the aromatase and cyclooxygenase enzyme systems suggests that autocrine and paracrine mechanisms may be involved in hormone-dependent breast cancer development via growth stimulation from local estrogen biosynthesis.     

曲格列酮增强乳腺癌细胞对表阿霉素敏感性的研究

背景与目的:过氧化物酶增殖体激活受体γ(peroxisomeproliferator-activatedreceptorγ,PPARγ)在乳腺癌组织中呈高表达,其特异性配体噻唑烷二酮(thiazolidinediones,TZD)类药物作用于肿瘤细胞后可抑制细胞增殖、诱导细胞凋亡。本研究探讨TZD类药物曲格列酮的使用与雌激素受体(estrogenreceptor,ER)阴性乳腺癌细胞对表阿霉素化疗敏感性的关系。方法:通过MTT法和流式细胞仪检测曲格列酮、表阿霉素单独和联合使用时,对ER阴性人乳腺癌细胞系MDA-MB-435S和MDA-MB-231增殖、凋亡的影响。Westernblot和划痕实验分别检测不同药物处理下,乳腺癌细胞中Bcl-2蛋白表达水平和细胞迁移能力的变化。结果:在4～24μmol/L浓度范围内,曲格列酮与表阿霉素协同抑制乳腺癌细胞的增殖,IC50是表阿霉素单独使用时的60%。曲格列酮和表阿霉素单独作用于乳腺癌细胞时并无明显的凋亡发生,而两种药物联合作用时,MDA-MB-435S和MDA-MB-231细胞的凋亡率分别为(5.48±0.45)%、(10.08±1.89)%。联合使用曲格列酮下调了Bcl-2蛋白的表达,降低了乳腺癌细胞的迁移能力。结论:曲格列酮不仅促进了表阿霉素对乳腺癌细胞的增殖抑制和凋亡诱导作用,而且明显抑制了乳腺癌细胞的迁移能力,从而增强乳腺癌细胞对表阿霉素的敏感性。     

Obesity is associated with inflammation and elevated aromatase expression in the mouse mammary gland

Elevated circulating estrogen levels are associated with increased risk of breast cancer in obese postmenopausal women. Following menopause, the biosynthesis of estrogens through CYP19 (aromatase)-mediated metabolism of androgen precursors occurs primarily in adipose tissue, and the resulting estrogens are then secreted into the systemic circulation. The potential links between obesity, inflammation, and aromatase expression are unknown. In both dietary and genetic models of obesity, we observed necrotic adipocytes surrounded by macrophages forming crown-like structures (CLS) in the mammary glands and visceral fat. The presence of CLS was associated with activation of NF-κB and increased levels of proinflammatory mediators (TNF-α, IL-1β, Cox-2), which were paralleled by elevated levels of aromatase expression and activity in the mammary gland and visceral fat of obese mice. Analyses of the stromal-vascular and adipocyte fractions of the mammary gland suggested that macrophage-derived proinflammatory mediators induced aromatase and estrogen-dependent gene expression (PR, pS2) in adipocytes. Saturated fatty acids, which have been linked to obesity-related inflammation, stimulated NF-κB activity in macrophages leading to increased levels of TNF-α, IL-1β, and Cox-2, each of which contributed to the induction of aromatase in preadipocytes. The discovery of the obesity → inflammation → aromatase axis in the mammary gland and visceral fat and its association with CLS may provide insight into mechanisms underlying the increased risk of hormone receptor-positive breast cancer in obese postmenopausal women, the reduced efficacy of aromatase inhibitors in the treatment of breast cancer in these women, and their generally worse outcomes. The presence of CLS may be a biomarker of increased breast cancer risk or poor prognosis.     

Aromatase activity in breast adipose tissue from women with benign and malignant breast diseases

In order to determine the significance of local oestrogen biosynthesis within the breast, aromatase activity has been measured in adipose tissue from the breasts of women with either benign (n = 36) or malignant breast disease (n = 51). Particulate fractions from all samples possessed aromatase activity, but levels in adipose tissue adjacent to malignant tumours were significantly higher than those in tissue close to benign breast lesions (P less than 0.0001). Elevated aromatase activity in adipose tissue from breast cancer patients may be of importance in view of the central role played by oestrogen in the natural history of breast cancer.     

Aromatase and breast cancer

Aromatase activity may be detected using in vitro and in vivo techniques in most breast cancers and mammary adipose tissue. This activity makes a variable contribution to endogenous estrogens within the breast and in many cases represents the major source of these hormones. Such local biosynthesis may maintain the growth of some hormone-dependent tumors. The factors which regulate aromatase activity within the breast are not defined but are likely to include growth factors and cytokines which may be produced by breast tissues so that autocrine and paracrine loops may exist. Estrogen biosynthesis within the breast, like other peripheral systems, appears sensitive to classical aromatase inhibitors and the new generation of drugs are capable of profoundly blocking the activity and markedly reducing endogenous estrogen levels; in turn these endocrine effects are translated in dramatic anti-tumor influences in hormone-dependent breast cancer.     

The evolving role of aromatase inhibitors in breast cancer

 Anti-aromatase agents inhibit the cytochrome P-450 component of the aromatase enzyme complex responsible for the final step of estrogen biosynthesis in peripheral tissues. These drugs can be classified into first-generation (e.g., aminoglutethimide), second-generation (e.g., formestane and fadrazole), and third-generation (e.g., anastrozole, letrozole, and exemestane) agents. Anti-aromatase agents can also be divided into type I and type II inhibitors. Type I inhibitors have a steroidal structure similar to androgens and inactivate the enzyme irreversibly by blocking the substrate-binding site, and are therefore known as aromatase inactivators. Type II inhibitors are nonsteroidal and their action is reversible. This article reviews the recent evidence regarding the role of third-generation aromatase inhibitors in the management of breast cancer. Relevant PubMed listed articles and presentations at recent international symposia were reviewed. There is a growing body of evidence supporting the role of third-generation aromatase inhibitors (anastrozole, letrozole, and exemestane) as first-line and second-line therapy for estrogen receptor (ER)- and/or progesterone receptor (PgR)-positive advanced breast cancer in postmenopausal women, and as a neoadjuvant therapy in postmenopausal women with hormone receptor-positive invasive breast cancer unsuitable for breast-conserving surgery. Furthermore, the preliminary results of the ATAC (Arimidex, Tamoxifen, Alone and in Combination) study have shown that adjuvant anastrozole is superior to tamoxifen in terms of disease-free survival (DFS), adverse effects, and prevention of contralateral breast cancer in postmenopausal women with early, ER-positive breast cancer. However, longer follow-up is required to assess the long-term effects of these agents on bone mineral density, cognitive function, and overall survival prior to considering their routine use in the adjuvant setting instead of tamoxifen. The potential role of these drugs in the management of ductal carcinoma in situ (DCIS), premenopausal breast cancer, and breast cancer prevention is worth investigating. Received: January 21, 2002 / Accepted: May 7, 2002 Correspondence to:K. Mokbel     

Aromatase inhibitors in breast cancer therapy

Estrogens are involved in numerous physiological processes and have crucial roles in certain disease states, such as mammary carcinomas. Estradiol, the most potent endogenous estrogen, is biosynthesized from androgens by the cytochrome P450 enzyme complex called aromatase. Aromatase is found in breast tissue and the importance of intratumoral aromatase and local estrogen production is being unraveled. Inhibition of aromatase is an important approach for reducing growth stimulatory effects of estrogens in estrogen-dependent breast cancer. Steroidal and nonsteroidal aromatase inhibitors have shown clinical efficacy for the treatment of breast cancer. The initial nonselective nature of nonsteroidal inhibitors, such as aminoglutethimide, has been greatly reduced in the later generations of inhibitors, anastrozole and letrozole. Mechanism-based steroidal inhibitors, such as 4-hydroxyandrostenedione and exemestane produce potent aromatase inhibition in patients. The potent and selective third-generation aromatase inhibitors, anastrozole, letrozole and exemestane, are approved for clinical use as first-line endocrine therapy in postmenopausal women with metastatic hormone-dependent breast cancer and as second-line endocrine therapy in postmenopausal patients failing antiestrogen therapy alone or multiple hormonal therapies.     

Aromatase inhibitors in breast cancer therapy

Estrogens are involved in numerous physiological processes and have crucial roles in certain disease states, such as mammary carcinomas. Estradiol, the most potent endogenous estrogen, is biosynthesized from androgens by the cytochrome P450 enzyme complex called aromatase. Aromatase is found in breast tissue and the importance of intratumoral aromatase and local estrogen production is being unraveled. Inhibition of aromatase is an important approach for reducing growth stimulatory effects of estrogens in estrogen-dependent breast cancer. Steroidal and nonsteroidal aromatase inhibitors have shown clinical efficacy for the treatment of breast cancer. The initial nonselective nature of nonsteroidal inhibitors, such as aminoglutethimide, has been greatly reduced in the later generations of inhibitors, anastrozole and letrozole. Mechanism-based steroidal inhibitors, such as 4-hydroxyandrostenedione and exemestane produce potent aromatase inhibition in patients. The potent and selective third-generation aromatase inhibitors, anastrozole, letrozole and exemestane, are approved for clinical use as first-line endocrine therapy in postmenopausal women with metastatic hormone-dependent breast cancer and as second-line endocrine therapy in postmenopausal patients failing antiestrogen therapy alone or multiple hormonal therapies.     

Metformin inhibits aromatase expression in human breast adipose stromal cells via stimulation of AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is recognized as a master regulator of energy homeostasis. In concert with the AMPK-kinase LKB1, it has been shown to provide a molecular link between obesity and postmenopausal breast cancer via its actions to inhibit aromatase expression, hence estrogen production, within the breast. The anti-diabetic drug metformin is known to increase the activity of AMPK and was therefore hypothesized to inhibit aromatase expression in primary human breast adipose stromal cells. Results demonstrate that metformin significantly decreases the forskolin/phorbol ester (FSK/PMA)-induced expression of aromatase at concentrations of 10 and 50 μM. Consistent with the hypothesized actions of metformin to increase AMPK activity, treatment with 50 μM metformin results in a significant increase in phosphorylation of AMPK at Thr172. Interestingly, metformin also causes a significant increase in LKB1 protein expression and promoter activity, thereby providing for the first time an additional mechanism by which metformin activates AMPK. Furthermore, metformin inhibits the nuclear translocation of CRTC2, a CREB-coactivator known to increase aromatase expression which is also a direct downstream target of AMPK. Overall, these results suggest that metformin would reduce the local production of estrogens within the breast thereby providing a new key therapeutic tool that could be used in the neoadjuvant and adjuvant settings and conceivably also as a preventative measure in obese women.     

Intratumoral estrogen production in breast carcinoma: significance of aromatase

It is well known that estrogens are closely involved in the growth of human breast carcinoma, and that the great majority of breast carcinomas express estrogen receptor. Recent studies have demonstrated that estrogens are locally produced in breast carcinoma by several enzymes. Among these, aromatase is generally considered the most important enzyme, and aromatase inhibitors are currently used in the treatment of breast carcinoma in postmenopausal women as an estrogen deprivation therapy. Therefore, in this review, we summarize the results of recent studies on aromatase in breast carcinoma, and we discuss its biological and/or clinical significance. Aromatase was expressed in various cell types in breast carcinoma, such as carcinoma cells, intratumoral stromal cells and adipocytes adjacent to the carcinoma, and the aromatase expression was regulated by various factors, including carcinoma cell–stromal cell interactions, cytokines and nuclear receptors, depending on the cell types. Aromatase was involved not only in local estrogen production but also the inhibition of intratumoral androgen synthesis in breast carcinoma. Finally, tissue concentrations of sex steroids were significantly higher in noninvasive breast carcinoma, regarded as a precursor lesion to invasive carcinoma, than in non-neoplastic breast tissue, and various sex steroid-producing enzymes (including aromatase) were abundantly expressed in noninvasive breast carcinoma tissue. Therefore, sex steroids are locally produced in noninvasive breast carcinoma as well as invasive carcinoma, and endocrine therapies may be clinically effective in a select group of noninvasive breast carcinoma patients.