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.     

Aromatase expression in the human breast

The plasma levels of free estradiol are very low in postmenopausal women. However, concentrations of estrogens within breast tissue have been reported to be higher than in plasma and similar to plasma concentrations in premenopausal women. One mechanism by which this may occur is for breast cells to synthesize estrogens themselves and produce high concentrations locally. Thus, tumor aromatase may be a significant source of estrogen which stimulates tumor growth. To address the question of the importance of this pathway, we have investigated the expression of aromatase within the normal breast and breast cancers. Because conventional biochemical assays for measuring aromatase activity require relatively large amounts of tissue, we developed an immunocytochemical method using a monoclonal antibody to determine the expression of aromatase. The method can be applied to sections of tumors embedded in paraffin blocks as routinely prepared for pathology. Since we have previously shown that mRNA for aromatase (P450 arom) and the protein are expressed in the same cells of the human placenta, we used in situ hybridization of sequence specific probes to P450 arom mRNA in breast tissue as one method to verify the specificity of the immunocytochemical detection of the enzyme. Both immunocytochemistry and in situ hybridization identified aromatase enzyme and mRNA expression in the epithelial cells of the terminal ductal lobula units (TDLU) and surrounding stromal cells of the normal human breast, and in the tumor epithelial cells and stromal cells of breast cancers. In addition, evidence for the functional significance of tumor aromatase was indicated by a correlation between aromatase activity and expression of proliferating cell nuclear antigen (PCNA) in the tumor, and by increased thymidine incorporation into DNA in response to testosterone in tumors in histoculture which had high aromatase activity but not in those with low activity. The findings suggests that estrogen produced locally is important in enhancing proliferation of the tumor.     

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.     

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.     

The liver receptor homolog-1 regulates estrogen receptor expression in breast cancer cells

Estrogen receptor-?? (ER) is expressed in the great majority of breast cancers, and the inhibition of ER action is a key part of breast cancer treatment. The inhibition of ER action is achieved using anti-estrogens, primarily tamoxifen, and with aromatase inhibitors that inhibit estrogen biosynthesis, thereby preventing ER activation. However, resistance to these therapies is common. With the aim of identifying new molecular targets for breast cancer therapy, we have identified the liver receptor homolog-1 (LRH-1) as an estrogen-regulated gene. RNA interference and over-expression studies were used to investigate the role of the LRH-1 in regulating breast cancer growth and to identify the targets of an LRH-1 action. Promoter recruitment was determined using reporter gene and chromatin immunoprecipitation (ChIP) assays. We show that LRH-1 regulates breast cancer cell growth by regulating the ER expression. Reporter gene and in vitro DNA-binding assays identified an LRH-1-binding site in the ER gene promoter, and ChIP assays have demonstrated in vivo binding at this site. We also provide evidence for new LRH-1 variants in breast cancer cells arising from the use of alternative promoters. Previous studies have shown that LRH-1 functions in estrogen biosynthesis by regulating aromatase expression. Our findings extend this by highlighting LRH-1 as a key regulator of the estrogen response in breast cancer cells through the regulation of ER expression. Hence, inhibition of LRH-1 could provide a powerful new approach for the treatment of endocrine-resistant breast cancer.     

Semi-quantitative immunohistochemical analysis of aromatase expression in ductal carcinoma in situ of the breast

Although estrogens whose production is catalyzed by aromatase are considered to play a role in human breast carcinogenesis, it remains unclear whether aromatase expression occurs in ductal carcinoma in situ (DCIS) of the breast. Aromatase expression in 61 cases of pure DCIS and 101 cases of invasive ductal carcinoma (IDC) was investigated by immunohistochemical analysis using a polyclonal anti-aromatase antibody. The level of aromatase expression was semiquantified by the H-score which was estimated by the percentage of positive-staining cells and the intensity of staining. The levels of aromatase expression were compared between the DCIS and IDC samples, and were also compared among the tumor cells and stromal cells in the DCIS and IDC samples. Positive cytoplasmic staining for aromatase expression was found not only in stromal cells but also in tumor cells. The levels of aromatase expression in the tumor cells and stromal cells from the DCIS samples were significantly higher than those in the respective cells from the IDC samples. Among the DCIS samples, those specimens from patients of ages 50 years or over showed higher levels of aromatase expression in stromal cells, than those from patients below 50 years. The finding that significantly higher aromatase expression levels were found in DCIS than in IDC indicates that it may be possible to treat DCIS patients with aromatase inhibitors, especially as an adjuvant hormonal therapy for postmenopausal patients.     

Combined inhibitory effect of formestane and herceptin on a subpopulation of CD44+/CD24low breast cancer cells

In breast cancer, stromal cells surrounding cancer epithelial cells can influence phenotype by producing paracrine factors. Among many mediators of epithelial–stromal interactions, aromatase activity is perhaps one of the best studied. Clinical data suggest that estrogen‐independent signaling leads to increased proliferation even during therapy with aromatase inhibitors (AIs). Molecular mechanism of crosstalk between the estrogen receptor (ER) and the epidermal growth factor receptor (HER) family have been implicated in resistance to endocrine therapy, but this interaction is unclear. The ability of aromatase to induce estradiol biosynthesis provides a molecular rationale to combine agents that target aromatase activity and the HER pathway. We targeted stromal–epithelial interactions using formestane, which exerts antiaromatase activity, combined with the monoclonal anti‐HER2 antibody herceptin, in a subpopulation of CD44+/CD24low cells sorted from epithelial‐mesenchymal co‐cultures of breast cancer tissues. The growth inhibition was respectively 16% (P < 0.01) in the response to herceptin, 25% to formestane (P < 0.01), and 50% (P < 0.001) with the combination of the two drugs, suggesting that herceptin cooperates with formestane‐induced inhibition of aromatase and this effect could be mediated through HER family receptors. In cells which expressed ERα, formestane/herceptin combination suppressed the mRNA expression of aromatase and HER2 and decreased cyclin D1 expression. These results show that combination therapies involving AIs and anti‐HER2 can be efficacious for the treatment of cancer in experimental models and suggest that subtyping breast tumors gives useful information about response to treatment. (Cancer Sci 2010)     

芳香化酶mRNA在肺癌组织中表达的初步研究

目的 探讨芳香化酶Ｐ４５０ ｍＲＮＡ在肺癌组织中的表达及其定位分布情况。方法 以芳香化酶ｃＲＮＡ作探针,采用原位杂交检测２３例肺癌及其相对应的癌旁组织和７例肺良性疾病及其相应正常肺组织中芳香化酶ｍＲＮＡ的表达。结果 １１例肺鳞癌、６例腺癌和２例腺鳞癌芳香化酶ｍＲＮＡ表达均为阳性,而３例小细胞肺癌和１例肺泡细胞癌均为弱阳性。阳性信号主要位于癌细胞的胞浆和胸核内,肿瘤的间质细胞和血管内皮细胞也有少量表     

Vascular stroma formation in carcinoma in situ, invasive carcinoma, and metastatic carcinoma of the breast.

The generation of vascular stroma is essential for solid tumor growth and involves stimulatory and inhibiting factors as well as stromal components that regulate functions such as cellular adhesion, migration, and gene expression. In an effort to obtain a more integrated understanding of vascular stroma formation in breast carcinoma, we examined expression of the angiogenic factor vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF); the VPF/VEGF receptors flt-1 and KDR; thrombospondin-1, which has been reported to inhibit angiogenesis; and the stromal components collagen type I, total fibronectin, ED-A+ fibronectin, versican, and decorin by mRNA in situ hybridization on frozen sections of 113 blocks of breast tissue from 68 patients including 28 sections of breast tissue without malignancy, 18 with in situ carcinomas, 56 with invasive carcinomas, and 8 with metastatic carcinomas. A characteristic expression profile emerged that was remarkably similar in invasive carcinoma, carcinoma in situ, and metastatic carcinoma, with the following characteristics: strong tumor cell expression of VPF/VEGF; strong endothelial cell expression of VPF/VEGF receptors; strong expression of thrombospondin-1 by stromal cells and occasionally by tumor cells; and strong stromal cell expression of collagen type I, total fibronectin, ED-A+ fibronectin, versican, and decorin. The formation of vascular stroma preceded invasion, raising the possibility that tumor cells invade not into normal breast stroma but rather into a richly vascular stroma that they have induced. Similarly, tumor cells at sites of metastasis appear to induce the vascular stroma in which they grow. We conclude that a distinct pattern of mRNA expression characterizes the generation of vascular stroma in breast cancer and that the formation of vascular stroma may play a role not only in growth of the primary tumor but also in invasion and metastasis.     

Expression of a novel factor, com1, in early tumor progression of breast cancer.

Tumor cells and their surrounding microenvironment produce a variety of growth factors and proteolytic enzymes to promote tumor growth and metastasis. We have recently identified a novel factor, termed com1, which is up-regulated in human breast carcinoma cells upon formation of experimental metastatic tumors and assumed to act as a growth-promoting factor in breast cancer. In attempts to explore the biological role of com1 in clinical tumor growth and metastasis, expression of com1 mRNA in primary carcinomas from 81 breast cancer patients and 27 samples of uninvolved adjacent breast tissue from these patients was compared and related to known prognostic parameters and outcome. The levels of com1 mRNA were significantly up-regulated (P < 0.0001) in the tumors compared to the normal breast tissues. Tumor expression of com1 mRNA, however, did not correlate with the mRNA levels for urokinase-type plasminogen activator (uPA), its receptor, or the type 1 inhibitor, which are factors that define a phenotype of tumor aggressiveness when elevated. And whereas the mRNA levels of uPA and the uPA receptor were elevated in tumors from the patients who subsequently had poor outcome, no correlations were observed between tumor com1 mRNA expression and prognosis or histological and biochemical characteristics of the tumors. We therefore assume that com1 may mediate some growth-promoting function early in development of the primary breast carcinoma, but not in later stages of tumorigenesis or metastasis.