Natural history of estrogen receptor-negative,progesterone receptor-positive breast cancer

The biological significance of estrogen receptor-negative but progesterone receptor-positive breast carcinomas is not clear. In the present study the aggressiveness of breast carcinomas in relation to ER and PgR status has been investigated. The probability of disease-free survival in 297 node-negative breast carcinoma patients was monitored during a follow-up ranging from six to 96 months (median 45 months). Steroid hormone receptor content was assayed with the biochemical method recommended by the EORTC. The probability of disease-free survival was significantly worse for patients with ER-negative, PgR-positive carcinomas compared to the other three steroid hormone receptor phenotypes. Our results suggest that ER-negative, PgR-positive breast carcinomas are biologically different in terms of aggressiveness from the other steroid hormone receptor phenotypes.     

Immunocytochemical detection of estrogen receptors in mammary Paget cells

Immunocytochemistry was used to analyze the estrogen receptor (ER) content in mammary Paget cells obtained by scraping the nipples of six patients. The Paget cells in the smears were ER positive in four cases and ER negative in two cases. Five of the patients underwent a modified radical mastectomy; histologic study of the excision specimens showed three invasive ductal carcinomas and two intraductal carcinomas. Analysis of the ER status of the three invasive tumors, analyzed both by immunohistochemistry and by the radioligand technique, showed that the ER content in the Paget cells reflected that in the tumor in the breast parenchyma. This finding lends support to the hypothesis that Paget cells originate from an epidermotropic cancer in the parenchyma of the breast.     

Estrogen and progestin receptors in intracranial tumors

Tissue samples from 17 intracranial tumors were analysed for the cytosolic estrogen and progestin receptor concentration. Three of the four meningiomas were progestin receptor-positive, but all were estrogen receptor-negative. All the four meningioma patients were postmenopausal women. The intracranial metastasis of a mammary carcinoma contained both estrogen and progestin receptors. Low progestin receptor concentration was found in an astrocytoma of a postmenopausal women. In addition, the presence of estrogen and progestin receptors was studied in four prolactinomas, two glioblastomas, two oligodendrogliomas, two mixed-cell carcinomas and one astrocytoma. All these tumors were estrogen and progestin receptor-negative.     

Metabolism of dehydroepiandrosterone by hormone dependent and hormone independent human breast carcinoma.

The metabolism of 7alpha-3H-dehydroepiandrosterone was studied in six human breast carcinomas in vitro. All mammary tumors transformed DHA to testosterone, dihydrotestosterone, 5alpha-androstane-3alpha, 17beta-diol and 5alpha-androstane-3,17-dione. Of the tumors investigated, three estrogen receptor-negative tumors converted DHA to estradiol and only one estrogen receptor-positive tumor produced estradiol from DHA. Observations on the relationship of androgen metabolism and hormone dependency are discussed.     

Estrogen receptor alpha is required for mammary development and the induction of mammary hyperplasia and epigenetic alterations in the aromatase transgenic mice

Aromatase transgenic mice exhibit hyperplastic and dysplastic changes, attesting to the importance of local estrogen in breast carcinogenesis. These mice also show increased levels of the estrogen receptor and β (ER, ERβ) suggesting that this receptor may play an important role in the initiation of estrogen-mediated mammary hyperplasia observed in these mice. To address the specific role of ER in the mammary development and in the induction of estrogen-mediated hyperplasia in aromatase transgenic mice, we have generated MMTV-aromatase × ER knockout cross (referred as aromatase/ERKO). Even though ERβ is expressed in aromatase/ERKO mice, lack of ER leads to impaired mammary growth in these mice. The data suggest that ER plays an important role in the mammary gland development as well as in the induction of mammary hyperplasia in aromatase transgenic mice. Lack of ER expression in the aromatase/ERKO mice resulted in a decrease in the expression of Cyclin D1, PCNA and TGFβ relative to the aromatase parental strain. The studies involving aromatase/ERKO mice show that lack of ER results in impaired mammary development even in the presence of continuous tissue estrogen, suggesting estrogen/ER-mediated actions are critical for mammary development and carcinogenesis.     

Analysis of estrogen receptor (ER) and estrogen-dependent pS2 protein expression in cells of mammary ductal carcinoma

Not every case of mammary carcinoma with expression of estrogen receptors (ER) responds by remission to anti-estrogen treatment. Possible causes of the phenomenon may involve abnormalities of the receptor or defects in mechanisms of signal transmission. One of the ways in which function of the receptor may be detected involves examination of expression of the antigens which appear as a consequence of estrogen stimulation, e.g., expression of pS2 protein. The present study was aimed at comparing ER and pS2 expression in cells of mammary carcinoma, and hence, at finding out whether the presence of ER is equivalent to the sensitivity to estrogen action. In paraffin sections of ductal mammary carcinoma samples obtained from 56 patients, immunocytochemical reactions were performed using monoclonal antibodies against ER and pS2. The results documented positive correlation between the presence of ER and the presence of pS2 in cells of mammary carcinoma (Spearman's rank correlation: r=0.43, p <0.001). Thus, the intensity of pS2 expression was directly related to the expression of ER and the latter was found to be functional.     

Cell-specific mechanisms of estrogen receptor in the pituitary gland

Analysis of the mechanism of action of estrogen receptor shows protein and mRNA polymorphism within distinct pituitary receptor-positive cells. The lactotropes exhibit unique properties in these mechanisms that distinguish them from gonadotropes. Therefore, this cell type constitutes an especially interesting model in the male as well as in the female for estrogen receptor studies.Abbreviations PRL prolactin - E2 estradiol - ER estrogen receptor - GnRH gonadotropin releasing hormone - PMSG pugnant mare serum gonadotropin     

The ontogeny and cellular distribution of estrogen receptors in normal mouse mammary gland.

The appearance, epithelial and stromal cell distribution of estrogen receptors (ER) in normal mouse mammary gland were determined between 1 and 10 weeks of age using immunohistochemistry. The effect of ovariectomy and estrogen (E)-treatment on the distribution and concentration of ER-positive cells at various ages was also analyzed. These studies demonstrate that ER are present in both mammary epithelial and stromal cells before the mammary gland exhibits a proliferative response or increase in progesterone receptor concentration as a result of E-treatment. Furthermore, an analysis of E-treatment suggests that although ER are present at an early age, there may be additional factors that determine the nature and extent of E-responsiveness.     

Binding of glucocorticoid receptors to mammary chromatin acceptor sites

We have recently characterized the interaction of mouse mammary estrogen receptors (ER) with mammary chromatin acceptor sites and demonstrated that ER from estrogen resistant lactating mammary glands do not bind to chromatin. In this study we have characterized the chromatin binding of the glucocorticoid receptor from mouse mammary glands isolated from nulliparous and lactating mice in order to better understand the relationship between receptor binding to chromatin and steroidogenic sensitivity of the tissue. Mammary chromatin was linked covalently to cellulose and deproteinized sequentially by 0-8 M Gdn-HCl. Binding to intact chromatin as well as to chromatin deproteinized by Gdn-HCl was determined using partially purified [3H]dexamethasone labelled glucocorticoid-receptor complexes (GR) obtained by fractionation on DEAE-cellulose columns. The binding of [3H]GR from mammary glands of nulliparous mice to chromatin fractions from the same tissue revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 5-6 M Gdn-HCl. Binding of [3H]GR was of high affinity (Kd = 0.2 nM) and saturable. A simultaneous comparison of the chromatin binding patterns for [3H]ER and [3H]GR isolated from mammary glands of nulliparous mice revealed that the chromatin subfractions obtained with 4-6 M Gdn-HCl extraction contained acceptor sites for both [3H]ER and [3H]GR; however, while the [3H]ER bound to a 4.5 M and a 5.5 M site, the [3]GR bound a 5 M and a 6 M site. Competition experiments supported the steroid receptor specificity of the chromatin acceptor sites. Thus, the 4-6 M chromatin fractions contain distinct acceptor sites for the glucocorticoid receptor and for the estrogen receptor. In addition our studies reveal that the binding patterns of [3H]GR isolated from mammary glands of nulliparous and lactating mice to their homologous chromatin is essentially similar. Thus, in contrast to estrogen receptors, glucocorticoid receptors from lactating mammary glands are able to effectively bind to chromatin acceptor sites which supports our previous suggestion that the estrogenic insensitivity of lactating mouse mammary glands may at least be in part due to the impeded interaction of ER with chromatin acceptor sites.     

Estrogen receptor-beta agonist diarylpropionitrile counteracts the estrogenic activity of estrogen receptor-alpha agonist propylpyrazole-triol in the mammary gland of ovariectomized Sprague Dawley rats

Although estrogen can bind both types of estrogen receptors, estrogen receptor-alpha (ERα) is dominant in mediating estrogenic activity in the mammary gland and uterus. Excessive estrogenic activity such as estrogen-based postmenopausal hormone replacement therapy increases the risk for breast and endometrial cancers. The adverse effect of estrogen on uterine endometrium can be opposed by progestins; however, estrogen-plus-progestin regimen imposes substantially greater risk for breast cancer than estrogen alone. In this study, we used ERα-selective agonist propylpyrazole-triol (PPT) and ERβ-selective agonist diarylpropionitrile (DPN) to activate ERα and estrogen receptor-beta (ERβ) separately in an ovariectomized rat model and determined whether PPT-activated ERα function in the mammary gland can be suppressed by DPN activated ERβ. Ovariectomized rats were randomly divided into six groups and treated with DMSO (control), DPN, PPT, PPT/DPN, PPT/Progesterone, and PPT/Progesterone/DPN, respectively. In the mammary gland, PPT but not DPN increased cell proliferation and amphiregulin gene expression; importantly, the stimulatory effect of PPT on mammary cell proliferation and amphiregulin gene expression can be suppressed by DPN. In the uterus, the effect of PPT on uterine weight and endometrial cell proliferation was not inhibited by DPN but can be inhibited by progesterone. These data provide in vivo evidence that PPT activated ERα activity in the mammary gland can be opposed by ERβ-selective agonist DPN, which may be explored for the development of better hormone replacement therapy regimen with less risk for breast cancer.     

Prognostic value of immunocytochemical estimation of estrogen receptor (ER) and of pS2 estrogen-dependent protein in cells of mammary ductal carcinoma. Analysis of five-year course of the disease

The pS2 protein belongs to the so called estrogen-dependent proteins, which appear in cells as a result of estrogen stimulation. The present study was aimed at determining prognostic value of estrogen receptor (ER) and pS2 protein expression in mammary cancer cells. The immunocytochemical reactions were performed in paraffin sections of tissue samples from 62 patients with ductal mammary carcinoma using monoclonal antibodies against ER and pS2. The analysis included also survival time of the patients, determined during the five-year observations. The studies documented a correlation between survival time and the level of ER expression (p=0.014) and absence of correlations between survival time and pS2 expression (p=0.55). The results indicate that evaluation of ER expression in mammary cancer cells may assist in defining prognosis of the patients while parallel estimation of pS2 expression in the cells is useless in this respect.     

Immunocytochemical detection of estrogen receptors in a hormone-unresponsive mammary tumor

Summary A combination of two monoclonal antibodies and high resolution immunocytochemical technique was applied to label estrogen receptors in spontaneous mouse mammary tumors. Protein A-colloidal gold complex was used as an electron opaque marker. With this procedure estrogen receptors were labelled in the nuclei of cancer cells, predominantly over heterochromatin. In the cytoplasm a slight tagging of the rough endoplasmic reticulum was detected, apparently related with the sites of receptor biosynthesis. Other organelles and the mammary tumor viruses (MuMTV) were not stained immunocytochemically.The immunocytochemical procedure applied in this investigation allowed the detection of low levels of estrogen receptors in an estrogen-unresponsive mammary carcinoma. The presence of estrogen receptors with a specific distribution in estrogen-independent tumors suggests the need of a reevaluation of their capacity as indicators of hormone-dependence in mammary carcinomas.     

Immunocytochemical detection of estrogen receptors in a hormone-unresponsive mammary tumor

A combination of two monoclonal antibodies and high resolution immunocytochemical technique was applied to label estrogen receptors in spontaneous mouse mammary tumors. Protein A-colloidal gold complex was used as an electron opaque marker. With this procedure estrogen receptors were labelled in the nuclei of cancer cells, predominantly over heterochromatin. In the cytoplasm a slight tagging of the rough endoplasmic reticulum was detected, apparently related with the sites of receptor biosynthesis. Other organelles and the mammary tumor viruses (MuMTV) were not stained immunocytochemically. The immunocytochemical procedure applied in this investigation allowed the detection of low levels of estrogen receptors in an estrogen-unresponsive mammary carcinoma. The presence of estrogen receptors with a specific distribution in estrogen-independent tumors suggests the need of a reevaluation of their capacity as indicators of hormone-dependence in mammary carcinomas.     

Postnatal mammary ductal growth: three-dimensional imaging of cell proliferation,effects of estrogen treatment,and expression of steroid receptors in prepubertal calves

Cows may provide insights into mammary development that are not easily obtained using mouse models. Mammary growth in control and estrogen-treated calves was investigated to evaluate general patterns of proliferation and relationship to estrogen receptor (ER) expression. After in vivo labeling with bromodeoxyuridine (BrdU), serial histological sections of mammary tissue were used to generate three-dimensional reconstructions. BrdU-labeled cells were present throughout the highly branched terminal ducts. ER and progesterone receptors (PR) were colocalized in nuclei of ductal epithelial cells. However, basal cells and epithelial cells that were located in the central region of epithelial cords and those that lined the lumen of patent ducts were ER- and PR-negative, as were stromal cells. Cells along the basal portion of the epithelium were not myoepithelial. ER in mammary epithelial cells but not stromal cells is analogous to patterns in human breast but contrasts with localization in murine mammary gland. After estrogen stimulation, 99% of BrdU-labeled (and Ki67-labeled) epithelial cells were ER-negative. Data suggest that proliferation in response to estrogen treatment was initiated within ER-positive epithelial cells of the developing mammary gland and the signal was propagated in paracrine fashion to stromal elements and ER-negative epithelial cells.     

Characterization of spontaneous mammary gland carcinomas in female baboons

Spontaneous mammary gland carcinomas occurred in five baboons during a 13-year period at Southwest Foundation for Biomedical Research. The affected baboons ranged in age from 21 to 33 years. Menopause in the baboon occurs at approximately 26 years of age. All five animals had typical invasive ductal carcinoma. Morphologically, the tumors were characterized by neoplastic cells arranged from pseudopapillary and cribiform to more poorly differentiated solid cellular growth patterns. Additional features included lack of tubule formation (4/5), marked nuclear pleomorphism (5/5), a high mitotic rate (4/5) and tumor necrosis (4/5). Applying a grading system used for breast cancer in women, four tumors were graded as poorly differentiated carcinomas and one was graded as moderately differentiated. Co-existent ductal carcinoma in situ (DCIS) was observed in three of the mammary tumors. Metastases to the regional lymph nodes were confirmed in two animals, both with histological evidence of lymphovascular invasion in the primary tumor. Distant metastases were observed in only one animal. Immunohistochemical staining for human therapeutic markers revealed 2/5 tumors strongly positive for estrogen receptor, 1/5 strongly positive for progesterone receptor and 4/4 negative for HER2 expression. Although the incidence appears to be low, these five cases of mammary carcinoma in female baboons suggest that when present baboon mammary carcinoma is usually of ductal origin and behaves similar to a human breast carcinoma.     

Estradiol-dependent regulation of angiopoietin expression in breast cancer cells

Angiopoietin-1 (Ang-1) is a ligand for Tie-2 receptors and a promoter of angiogenesis. Angiogenesis plays an important role in breast cancer, as it is one of the critical events required for tumors to grow and metastasize. In this study, we investigated the influence of estradiol (E2) on the expression of angiopoietins in breast cancer cell lines. Ang-1 mRNA and protein expressions were significantly higher in estrogen receptor-negative (ERα-) breast cancer cells than in estrogen receptor-positive (ERα+) cells. Exposure of ERα+ cells to E2 resulted in further reductions of Ang-1 levels. In mouse mammary pads inoculated with breast cancer cells, both tumor size and Ang-1 production were significantly lower in ERα+ cell-derived xenografts, as compared to those derived from ERα- cells. Reduction of circulating levels of E2 by ovariectomy eliminated this response. Overall, these results indicate that Ang-1 mRNA and protein expressions: (1) negatively correlate with the level of ERα in breast cancer cell lines; (2) are downregulated by E2 in an ERα dependent manner; and (3) positively correlate with the degree of angiogenesis in vivo. We conclude that Ang-1 is an important modulator of growth and progression of ERα- breast cancers.