Estradiol and progesterone receptor levels in human breast cancer in relation to cytosol and plasma estrogen level

Estradiol receptor (ER) and progesterone receptor (PR) content along with the cytosol and plasma estrone and estradiol levels in 15 premenopausal and 26 postmenopausal women with breast cancer in different clinical stages (T₁₂₃, N₀₁, M₀) were measured. ER-positive tumor frequency and the ER content tended to be higher in postmenopausal than in premenopausal patients. There was no evidence for a relationship between high cytosol estrogen levels and low receptor measurements. The estrogen concentration was higher in ER-positive tumor cytosols than in those of ER-negative tumors; the differences were significant in postmenopausal women, only, with P < 0.05 for estrone and P < 0.01 for estradiol values. Twelve pairs of tumor and normal tissue from the same breast, were also studied: seven of which contained ER-positive and five ER-negative tumors. The ER-positive tumors showed a clear trend to higher estradiol content as compared to the corresponding normal tissues. The circulating level of estradiol in postmenopausal women, was higher (P < 0.05) in ER-positive tumors than that in ER-negative tumors. Our results indicate that: (a) false negative ER assays are not likely to be due to the presence of endogenous estrogens, (b) higher amounts of estrone and estradiol are contained in ER-positive tumors than in negative ones.     

Vitronectin in human breast carcinomas

We have analysed the occurrence of the extracellular glycoprotein vitronectin in carcinomas and normal tissue of human breast. Immunohistochemical analysis of carcinomas revealed a strong vitronectin accumulation in extracellular matrix (ECM) around some cancer cell clusters and in the subendothelial area of some blood vessels. In normal tissue, vitronectin had a homogeneous periductal occurrence, with local accumulation much lower than that in the carcinomas. Using a new solid phase radioligand assay, the vitronectin concentrations of extracts of carcinomas and normal breast tissue were determined and found to be indistinguishable. Comparison of the vitronectin and the hemoglobin concentrations of the extracts showed that their vitronectin content was not derived from blood contamination. Vitronectin mRNA was undetectable in both carcinomas and normal tissue. We conclude that vitronectin is not synthesised locally in breast tissue but derived by leakage from vessels, followed by extracellular accumulation in patterns distinctly different in carcinomas and normal tissue. The observation of a high vitronectin content in the carcinomas and its localisation in the tissue contributes to the clarification of the role of vitronectin in tumour biology in interaction with the plasminogen activation system and integrins.     

In vitro estrogen-binding by human breast carcinomas.

Patients whose breast carcinomas possess only low concentrations of a receptor molecule that binds estrogens with high affinity are unlikely to respond to hormonal manipulative therapy when the disease recurs. The estrogen-binding capacity of 106 breast carcinomas was measured by an in vitro method and was expressed per milligram wet weight and in some cases related to the concentration of deoxyribonucleic acid (DNA) of the tumours. The ability of tumors to bind 3H-estradiol ranged from 0 to 1.3 fm/mg in pre- and perenopausal women, and from 0 to 16.8 fm/mg in postmenopausal women. Menopausal status or serum concentrations of endogenous estrogen, or both, should therefore be considered when tumours are classified into low and high estrogen-binding capacity. It is not necessary to carry out Scatchard analysis for every tumour, and expressing estradiol binding on the basis of DNA concentration may be preferable to expressing in on a wet-weight basis.     

Estradiol inhibits glucocorticoid receptor expression and induces glucocorticoid resistance in MCF-7 human breast cancer cells

Our study has shown that treatment of MCF-7 human breast cancer cells with 17-beta estradiol (E(2)) produced significant decreases in glucocorticoid receptor (GR) concentrations and GR mRNA levels. E(2) pre-treatment of MCF-7 cells stably transfected with the GR responsive pMTV-CAT reporter (MCF-7-MTV cells), caused significant attenuation of dexamethasone (DEX)-induced chloramphenicol acetyl transferase (CAT). In MCF-7 cells transiently transfected with [(GRE)(3)-Luc] reporter plasmid, E(2) pre-treatment significantly suppressed DEX-induced luciferase, which was abolished by the estrogen receptor antagonist ICI 182,780. We examined the effect of chronic E(2) treatment as well as E(2) withdrawal on GR function and abundance. MCF-7-MTV cells were treated with vehicle (control) or E(2) for up to 16 days. A third group received E(2) for 5 days followed by E(2) withdrawal from day 6 to 16. Chronic E(2) treatment almost totally abrogated DEX-induced CAT and reduced GR to very low levels. Interestingly, in the group subjected to E(2) withdrawal, neither the DEX response nor GR abundance recovered and reached control values suggesting that the estrogen mediated suppression is long lasting and could not be easily reversed. The E(2) induced resistance to glucocorticoid action may be of potential clinical significance in a number of settings including breast cancer, neuroendocrine response to stress and osteoporosis and could possibly contribute to the differences in glucocorticoid responsiveness among patients.     

Secretion of immunoreactive calcitonin by human breast carcinomas.

Twenty-three out of 28 patients with metastatic breast carcinoma and one out of 13 patients with localised disease had raised levels of plasma immunoreactive calcitonin. Monolayer cultures of breast carcinomas maintained for up to 10 weeks released immunoreactive calcitonin, and a primary breast carcinoma passaged in "nude" mice for over a year contained material immunologically and chromatographically resembling the monomeric form of human calcitonin. These studies indicate that breast carcinomas can produce calcitonin and that plasma calcitonin measurements may be useful in staging patients with breast carcinomas.     

Estradiol increases VEGF in human breast studied by whole-tissue culture

Sex steroid exposure constitutes a risk factor for breast cancer, but little is known about the effects of sex steroids on the normal breast, largely because of the lack of convenient models. We have developed a method of culturing normal breast tissue ex vivo. We have applied this method to investigate the effects of estradiol and progesterone on the key angiogenic mediator, vascular endothelial growth factor (VEGF), in the breast. Whole breast tissue was obtained from routine reduction mammoplasty. Tissue biopsies were cultured in vitro for 1-3 weeks, and the expression of luminal cytokeratin 18 was determined by immunohistochemistry. As an application, tissue biopsies were treated in vitro for 1 week with or without estradiol or estradiol and progesterone. Estrogen receptor, progesterone receptor, and Ki-67 were analyzed, and VEGF levels were examined by quantitative immunoassay and immunohistochemistry. Whole breast tissue was cultured ex vivo for 1 week with preserved morphology. Increased detachment of the luminal epithelium was observed after 2 weeks. Estradiol increased extracellular levels of VEGF in normal breast tissue biopsy medium. The addition of progesterone had neither stimulatory nor inhibitory effects on secreted VEGF. The method of whole breast tissue culturing thus provide a means by which to explore the biology of normal breast tissue. Our results suggest that estradiol exerts pro-angiogenic effects in normal breast by increasing levels of biologically active VEGF.     

Estradiol as an anti-aromatase agent in human breast cancer cells

Estradiol (E(2)) is an important risk factor in the development and progression of breast cancer. However, a "direct effect" of E(2) in breast cancerization has not yet been demonstrated. The estrogen receptor complex can mediate the activation of oncogens, proto-oncogens, nuclear proteins and other target genes that can be involved in the transformation of normal to cancerous cells. Breast cancer cells possess all the enzymes (sulfatase, aromatase, 17beta-hydroxysteroid dehydrogenase (17beta-HSD)) necessary for the local bioformation of E(2). In the last years, many studies have shown that treatment of breast cancer patients using anti-aromatase agents has beneficial therapeutic effects. The aromatase activity is very low in most breast cancer cells but was significantly increased in a hormone-dependent breast cancer cell line: the MCF-7aro, using the aromatase cDNA transfection and G-418 (neomycin) selection. In the present study, we explore the effect of E(2) on the aromatase activity of this cell line. The MCF-7aro cell line was a gift from Dr. S. Chen (Beckman Research Institute, Duarte, U.S.A.). For experiments the cells were stripped of endogenous steroids and incubated with physiological concentrations of [(3)H]-testosterone (5 x 10(-9)mol/l) alone or in the presence of E(2) (5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol/l) for 24h at 37 degrees C. The cellular radioactivity uptake was determined in the ethanolic supernatant and the DNA content in the remaining pellet. [(3)H]-E(2), [(3)H]-estrone ([(3)H]-E(1)) and [(3)H]-testosterone were characterized by thin layer chromatography and quantified using the corresponding standard. It was observed that [(3)H]-testosterone is converted mainly into [(3)H]-E(2) and not to E(1), which suggests very low or absence of oxidative 17beta-HSD (type 2) activity in these experimental conditions. The aromatase activity, corresponding to the conversion of [(3)H]-testosterone to [(3)H]-E(2) after 24h, is relatively high, since the concentration of E(2) was 2.74+/-0.11pmol/mg DNA in the non-treated cells. E(2) inhibits this conversion by 77, 57 and 21%, respectively, at the concentrations of 5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol. In previous studies, it was demonstrated that E(2) exerts a potent anti-sulfatase activity in the MCF-7 and T-47D breast cancer cells. The present data show that E(2) can also block the aromatase activity. The dual inhibition of the aromatase and sulfatase activities, two crucial enzymes for the biosynthesis of E(2) by E(2) itself in breast cancer add interesting and attractive information for the use of estrogen therapeutic treatments.     

Serum TAG 72 levels in different human carcinomas

Since December 1988, we have measured the TAG 72 serum levels in 326 patients with different carcinomas, especially breast, gastrointestinal and ovarian, using a RIA kit. With a cut-off value of 5 U/mL, a specificity of 100% in the controls and an overall sensitivity of 22% in the neoplastic patients was obtained, with the highest positivities in ovarian (63%) and gastric (58%) carcinomas. Therefore, TAG 72 can be associated with other tumor markers for these latter neoplasms.     

Estradiol induced proteins in the MCF7 human breast cancer cell line

MCF₇ cells were cultured with steroids, labelled with (³⁵S)-methionine and the secreted and intracellular proteins were examined by one and two dimensional gel electrophoresis. Estradiol (0.1 nM) increased the synthesis of some of the secreted proteins; the induction of a protein of molecular weight 46,000 daltons being the most dramatic. The 46,000 daltons secreted protein was heterogeneous with respect to molecular weight and isoelectric point. The antiestrogen Tamoxifen did not stimulate the synthesis of any of the estrogen induced proteins, but completely inhibited the induction by estradiol. The effect of estradiol on internal proteins was much more subtle; only 3 proteins out of about 250 were stimulated. The functions of these Proteins are unknown, however they appear to be good markers for studying the mechanism of action of estrogens and antiestrogens in breast cancer and might be related to the control of cell proliferation by estrogen.     

Estradiol esterification in the human preovulatory follicle.

In the preovulatory follicle, the LH surge stimulates progesterone production, reduces estradiol synthesis, and scales up the permeability of the blood-follicle barrier. The purpose of this study was to investigate whether the extent of these changes is correlated with the levels of estradiol, estradiol esters, and cholesteryl esters in the follicular fluid. The follicular levels of progesterone, estradiol, estradiol linoleate, cholesterol, and cholesteryl linoleate were measured by HPLC. The estradiol linoleate/estradiol ratio, which reflects the efficiency of in vivo estradiol esterification, and the cholesteryl linoleate/cholesterol ratio were calculated and found negatively correlated. The estradiol level was positively correlated with the cholesteryl linoleate/cholesterol ratio while negatively correlated with the estradiol linoleate/estradiol ratio. The in vitro activity of lecithin-cholesterol acyltransferase, the enzyme esterifying both cholesterol and estradiol, was assayed by incubating the fluid with labeled substrates. This activity was not correlated with either the estradiol linoleate/estradiol or the cholesteryl linoleate/cholesterol ratio. The enzyme K(m) and V(max) values were lower with estradiol than with cholesterol. Higher estradiol linoleate/estradiol ratios and lower cholesteryl linoleate/cholesterol ratios were associated with higher level of Haptoglobin penetration into the follicle. This level, which was determined by ELISA, was found increased with increased progesterone concentration and, therefore, used as a marker of the LH-stimulated permeability of the blood-follicle barrier. Our data suggest that early preovulatory follicles contain more cholesteryl esters and less estradiol esters than follicles closer to ovulation.     

Oestrogen regulates oestrogen receptor mRNA levels in an oestrogen-responsive human breast cancer cell line

The EFM-19 human breast cancer cell line contains high levels of oestrogen receptor mRNA and is oestrogen responsive for growth. The oestrogen receptor gene appears to be specifically regulated by oestrogens in EFM-19 cells. The induction by oestradiol is half-maximal in the presence of 3 x 10(-11) M oestradiol. LY117018 is a potent antioestrogen with a similar affinity to oestradiol for the oestrogen receptor. LY117018 completely blocks the induction of the oestrogen receptor mRNA by oestradiol when it is present in a 50-fold molar excess. The ability of oestrogens to control the levels of their own receptor in human breast cancer cells has implications for the understanding of oestrogen responsiveness and the amelioration of endocrine therapy.     

Estradiol inhibits the estrone sulfatase activity in normal and cancerous human breast tissues

It is well accepted that estradiol (E₂) plays an important role in the genesis and evolution of breast cancer. Quantitative evaluation indicates that in human breast tumor, estrone sulfate (E₁S) ‘via sulfatase’ is a much more likely precursor for E₂ than is androstenedione ‘via aromatase’. In previous studies, it was demonstrated that in isolated MCF-7 and T-47D breast cancer cell lines, estradiol can block estrone sulfatase activity. In the present study, the effect of E₂ was explored using total normal and cancerous breast tissues. This study was carried out with post-menopausal patients with breast cancer. None of the patients had a history of endocrine, metabolic or hepatic diseases or had received treatment in the previous 2 months. Each patient received local anaesthetic (lidocaine 1%) and two regions of the mammary tissue were selected: (A) the tumoral tissue and (B) the distant zone (glandular tissue) which was considered as normal. Samples were placed in liquid nitrogen and stored at –80 °C until enzyme activity analysis. Breast cancer histotypes were ductal and post-menopausal stages were T2. Homogenates of tumoral or normal breast tissues (45–75 mg) were incubated in 20 mM Tris–HCl, pH 7.2 with physiological concentrations of [³H]-E₁S (5 × 10⁻⁹ M) alone or in the presence of E₂ (5 × 10⁻⁵ to 5 × 10⁻⁷ M) during 30 min or 3 h. E₁S, E₁ and E₂ were characterized by thin layer chromatography and quantified using the corresponding standard. The sulfatase activity is significantly more intense with the breast cancer tissue than normal tissue, since the concentration of E₁ was 3.20 ± 0.15 and 0.42 ± 0.07 pmol/mg protein, respectively after 30 min incubation. The values were 27.8 ± 1.8 and 3.5 ± 0.21 pmol/mg protein, respectively after 3 h incubation. Estradiol at the concentration of 5 × 10⁻⁷ M inhibits this conversion by 33% and 31% in cancerous and normal breast tissues, respectively and by 53% and 88% at the concentration of 5 × 10⁻⁵ M after 30 min incubation. The values were 24% and 18% for 5 × 10⁻⁷ M and 49% and 42% for 5 × 10⁻⁵ M, respectively after 3 h incubation. It was observed that [³H]-E₁S is only converted to [³H]-E₁ and not to [³H]-E₂ in normal or cancerous breast tissues, which suggests a low or no 17β-hydroxysteroid dehydrogenase (17β-HSD) Type 1 reductive activity in these experimental conditions. In conclusion, estradiol is a strong anti-sulfatase agent in cancerous and normal breast tissues. This data can open attractive perspectives in clinical trials using this hormone.     

Effect of time and therapy on the hormone receptor status of breast carcinomas.

To determine whether the hormone receptor status of a breast carcinoma can change during the course of the disease or its treatment, the results of estrogen receptor assays in two or more biopsy specimens from 68 patients were examined; progesterone receptors had been assayed in approximately 40% of the specimens, too few to permit statistical analysis of the results. The patients fell into four groups: A, those with at least two primary breast carcinomas, and B to D, those with at least two breast carcinomas, at least one of which was a secondary tumour (usually of lymph node, skin or soft tissue) excised on the same occasion (B), 1 to 76 months later, after no intervening therapy (C), or 3 to 73 months later, after intervening chemotherapy (usually adjuvant), regional irradiation or hormonal therapy, or a combination of these (D). The small numbers in the subgroups precluded statistical analysis of the results for groups A and D. The degree of concordance of the hormone receptor status of the primary and secondary tumours in groups B and C was significant, at 87% (P less than 0.01) and 80% (P congruent to 0.01) respectively. Chemotherapy and regional irradiation did not appear to reduce the degree of concordance. All primary tumours in the same breast removed on the same occasion had the same hormone receptor status, but bilateral primary tumours appeared to have an independent status, which suggests that local tissue factors, as well as the systemic hormonal environment, play a role in establishing the hormone receptor status of breast carcinomas.     

Epidermal growth factor receptors in human breast and endometrial carcinomas

The incidence and levels of epidermal growth factor receptor (EGFR) were studied in 67 breast tumors and 22 endometrial carcinomas. Estrogen receptors (ER) were also measured in all samples and progesterone receptors (PR) were analyzed in 57 breast samples and 21 endometrial tumors. A high level of EGFR expression is found in both breast and endometrial carcinomas, although the incidence of EGFR content is greater in breast carcinomas. 36% of breast tumors had EGFR at levels 3-49.5 fmol/mg membrane protein, whereas this percentage of positivity was 27% for endometrial tumors. In 51% of breast carcinoma and 73% of endometrial tumors, there was a positive ER content, whereas 53% of breast tumors and 62% of endometrial carcinomas were positive. A clear inverse relationship between EGFR content and ER and PR status has been observed in breast tumors. Our data confirm the previously described inverse correlation between expression of EGFR and estrogen receptors in human breast cancer. We also show here that there is a similar inverse relationship between EGFR content and ER levels in endometrial tumors.     

High lib mRNA expression in breast carcinomas.

Lib, first identified as a novel beta-amyloid responsive gene in rat astrocytes, has an extracellular domain of 15 leucine-rich repeats (LRRs) followed by a transmembrane domain and a short cytoplasmic region. It is a distinctly inducible gene and is thought to play a key role in inflammatory states via the LRR extracellular motif, an ideal structural framework for protein-protein and protein-matrix interactions. To evaluate potential roles of Lib, we screened various tumors for Lib expression. Lib mRNA expression was high and uniquely expressed in breast tumor tissues, compared to paired normal breast tissues. Lib mRNA was localized in the ductal carcinoma cells and Lib protein displayed a homophilic association on the surface of cultured cells. These data suggest that Lib may play a role in the progression of breast carcinomas and may be a diagnostic marker for breast tumors.     

Oestrogen receptor content of normal breast cells and breast carcinomas throughout the menstrual cycle

To examine the presence and distribution of oestrogen receptors in the normal breast during the menstrual cycle cytological samples obtained by fine needle aspiration from 69 premenopausal women with normal breasts were analysed immunocyto-chemically with a monoclonal antibody to oestrogen receptor; samples from 15 postmenopausal women were also analysed. The receptor content of breast cancers from 83 premenopausal women was also determined in relation to when during the menstrual cycle excision was performed. In the normal premenopausal women oestrogen receptors were detected in the nuclei of epithelial cells in 21 out of 68 (31%) assessable samples. All 21 of these samples were obtained from the 35 women who were studied during the first half of their menstrual cycle (days 28 to 14). None of the 33 samples obtained during the second half of the cycle contained oestrogen receptors. Samples were assessable in eight of the postmenopausal women, six giving a positive result for oestrogen receptor. Fifty one of the 83 carcinomas were positive for oestrogen receptor, 24 having been excised during the first half of the cycle and 27 during the second half.Production of oestrogen receptor protein is suppressed at the time of ovulation in the normal breast epithelium of premenopausal women. In contrast, breast carcinoma cells either synthesise this protein continuously throughout the cycle or fail to express it despite fluctuations of serum hormone concentrations.