Occurrence, regulation, and significance of progesterone receptors in human meningioma

The abundant expression of progesterone receptors (PR) in human meningiomas is well established. It is unknown, however, how PR expression is regulated, especially since estrogen receptors (ER) are virtually absent in these tumors. At the mRNA level, ER splice variants occur in meningioma but these appear not to be involved in the apparently autonomous PR expression. In an earlier study, because other ER-inducible proteins were either not expressed at all (pS2) or were expressed at a very low level compared to their expression in breast cancer (Cathepsin-D), the authors have postulated that the autonomous PR expression in meningioma is PR promoter-related rather than ER-related and have studied PR expression in cultured meningioma cells. PR levels appeared to decrease rapidly in vitro in monolayer as well as in three dimensional spheroid cultures. Culture conditions thus are not yet sufficient for the quantitative evaluation of PR expression. To evaluate whether PR deterioration is associated with cell turnover (meningiomas grow much faster in vitro than in vivo), the relationship between expression of the apoptotic proteins Bcl-2 and Bax and PR expression was investigated. Bcl-2 expression was found to be highest in meningioma with low PR levels, and in breast cancer tissue with high PR levels. Bax expression was not related to PR expression in any of the two tissues. Given the potential benefit of antiprogestin treatment and the occurrence in meningiomas of a protein capable of binding to the estrogen-responsive element, the expression of PR in meningioma remains a fascinating phenomenon which requires further investigation.     

Correlation between sex hormone receptors in normal and cancerous target tissues

According to the classic model of regulation of sex hormone receptors biosynthesis in target tissues, oestrogen stimulates and progesterone inhibits biosynthesis in both receptors. One of the consequences of this model is a direct correlation between oestrogen (ER) and progesterone receptors (PR) in target tissues. Here we investigate a correlation between ER and PR in calf endometrium and breast cancer (BC) tissues of women. A direct correlation was found between receptors in the calf endometrium (r = +0.70; p < 0.01). There were three variants of BC tissues: without correlation, with positive correlation (r = +0.49; p < 0.01), and with non-linear negative correlation between ER and PR. The last variant of samples were detected exclusively in patients operated in spring and fall. The non-linear negative correlation between ER and PR in BC tissues in spring and fall may indicate disregulation of sex hormone receptors biosynthesis under the influence of external factors.     

Cooperation of proto-signals for nuclear accumulation of estrogen and progesterone receptors.

Multiple proto-signals (p-NLSs) for nuclear targeting, none of which suffices on its own, cooperate in the estrogen (ER) and progesterone (PR) receptors. In the ER, an estrogen-inducible p-NLS was found in the hormone binding domain (HBD), in addition to three lysine/arginine-rich motifs resembling prototype constitutive nuclear localization signals (NLSs). The inducible and the constitutive ER p-NLSs cooperate in the presence of estrogen and hydroxy-tamoxifen, but not in the presence of ICI 164,384. In the PR, three p-NLSs, two of which are located within and directly adjacent to the second zinc finger, cooperate with each other and a weak hormone-inducible p-NLS in the PR HBD. No 'masking' of p-NLSs by the HBD was observed for ER and PR, while the ligand-free glucocorticoid receptor HBD inhibited the activity of both homologous and heterologous NLSs. Nuclear co-translocation experiments indicated that in vivo the stability of ER and PR dimers is hormonally controlled, but that, in the absence of the cognate ligand, ER dimers are more stable than PR dimers. This is likely to account for the differential hormone requirement of ER and PR DNA binding in vitro.     

Expression of oestrogen receptor-beta in invasive breast tumours

Steroid hormone receptors are used routinely to predict endocrine responsiveness in patients with breast cancer. Two oestrogen receptors (ERs): ER alpha and ER beta have been identified. Although ER alpha and ER beta genes share a large degree of homology, it is generally thought that their distribution and function are substantially different in many tissues. Both of them may be expressed in normal and neoplastic tissues of the breast. While much is known about ER alpha, the role of ER beta is still undefined, especially at the protein level. Recent development of reliable antibodies to ER beta has provided opportunity to test immunohistochemical reactions detecting ER beta in archival breast tumours. The aim of our study was to learn more about the cellular mechanisms underlying the relationship of ER beta and progesterone receptor (PR) in breast cancer tissues, discriminating between hormone-dependent and hormone-independent tumours. ER alpha and PR content of tumour tissues of 154 patients with breast cancer were tested by in situ indirect immunohistochemical method parallel with ligand binding biochemical assay. ER beta was detected in 8 ER alpha-/PR+ breast carcinomas by immunohistochemical method too. Steroid hormone receptor content was analysed comparing to the histologic type and grading of the tumours. CONCLUSIONS: A considerable part of breast carcinomas belongs to the ER+/PR+ and ER-/PR- groups. About 1-2% of the tumours is expected to be ER alpha-/ER beta+/PR+ type. In such cases ER alpha negative reaction together with PR positivity can signal the necessity of the immunohistochemical detection of ER beta in routine histopathological practice, presenting the precise steroid hormone receptor status for the most effective endocrine therapy of the patients.     

Immunohistochemical localisation of progesterone and oestrogen receptors at the placental interface in mares during early pregnancy

Previous reports documenting progesterone receptors (PR) and oestrogen receptors (ER) in the endometrium of early pregnant mares included specimens only up to Day 20 post ovulation. This study aimed to localise PR and ERα on equine feto-maternal tissues between Days 20 and 68 to encompass the period around fixation of the conceptus, development of the endometrial cups and attachment and initial interdigitation of the allantochorion. During early pregnancy mares had the same pattern of PR in the endometrium as that reported for other mammals; namely, a loss of PR from the endometrial epithelia but continued localisation in stromal cells. The spatial arrangement of ERα over the same time period showed cytoplasmic staining of endometrial epithelia and in the nuclei of occasional stromal cells. In the fetal tissues, no cells had PR although ERα was evident in some tissue compartments. No major change in localisation of either receptor was noted throughout the time period examined despite important changes occurring at the placental interface. Nevertheless, these steroid receptor molecules probably play important roles in the production of histotroph and growth factors by the endometrium which go on to stimulate differentiation and growth of the feto-maternal tissues.     

WW domain binding protein-2, an E6-associated protein interacting protein, acts as a coactivator of estrogen and progesterone receptors

WW domain binding protein-2 (WBP-2) was cloned as an E6-associated protein interacting protein, and its role in steroid hormone receptors functions was investigated. We show that WBP-2 specifically enhanced the transactivation functions of progesterone receptor (PR) and estrogen receptor (ER), whereas it did not have any significant effect on the androgen receptor, glucocorticoid receptor, or the activation functions of p53 and VP-16. Depletion of endogenous WBP-2 with small interfering RNAs indicated that WBP-2 was required for the proper functioning of PR and ER. We also demonstrated that WBP-2 contains an intrinsic activation domain. Moreover, chromatin immunoprecipitation assays demonstrate the hormone-dependent recruitment of WBP-2 onto an estrogen-responsive promoter. Mutational analysis suggests that one of three polyproline (PY) motifs of WBP-2 is essential for its coactivation and intrinsic activation functions. We show that WBP-2 and E6-associated protein each enhance PR function, and their effect on PR action are additive when coexpressed, suggesting a common signaling pathway. In this study, we also demonstrate that the WBP-2 binding protein, Yes kinase-associated protein (YAP) enhances PR transactivation, but YAP's coactivation function is absolutely dependent on WBP-2. Taken together, our data establish the role of WBP-2 and YAP as coactivators for ER and PR transactivation pathways.     

Characterization and biological relevance of a 29-kDa, oestrogen receptor-related protein

The properties of a monoclonal antibody (D5) that can immunoprecipitate human oestradiol receptor (ER) under some but not all conditions are described. The antibody recognises a 29-kDa serine phosphoprotein that is qualitatively and quantitatively related to ER but not other steroid receptors or binding proteins. p29 will not complex with untreated cytosol ER but, after ammonium sulphate, KCl, heat or phosphatase treatments, interaction occurs that can be detected by immunoprecipitation with D5; molybdate and GTP inhibit complex formation. In human endometrium, p29 is increased by oestrogen and decreased by progestins. IRMA and histochemical assays for p29 have been developed and applied to a large series of human breast tumours. Most, but not all ER+ tumours are p29+, whilst ER-tumours are rarely p29+ unless they are also PR+. p29 predicts for clinical response to hormone therapy. ER+ p29+ tumours have a higher response rate than the ER+ p29-tumours. We do not know if p29 is a previously undetected component of the oestradiol receptor machinery or whether it is a product of oestrogen action.     

The effects of sequential treatment with ethynyloestradiol and medroxyprogesterone acetate on oestrogen and progestogen receptors in breast cancer

As part of a clinical study designed to modulate oestrogen and progestogen receptor (ER and PR) binding site concentrations prior to chemotherapy ER and PR levels have been estimated immediately before treatment, after ethynyloestradiol (EE, 10 micrograms/day, 1 week) and after medroxyprogesterone acetate (MPA, 500 mg/day i.m., 2 weeks) in tumour tissue from 14 women with advanced breast cancer. There was no consistent change after EE treatment. MPA tended to decrease ER and PR levels, though some increases were also seen. Responses (10 complete and 3 partial remissions) to sequential cyclical hormono-chemotherapy were not related to ER or PR levels prior to chemotherapy.     

Discrepancies between antibody (EIA) and saturation analysis of oestrogen receptor content in breast tumour samples

The use of different techniques for assay of oestrogen receptors (ER) in breast cancer raises the question of their relative effectiveness in measuring concentrations of functional receptors. Data were obtained on soluble receptors from supernatants from 58 primary breast tumour homogenates, using the ligand ([3H]oestradiol) binding assay with dextran-coated charcoal (DCC) separation, either at a single saturating ligand dose, or by Scatchard analysis, and by using the Abbott enzyme immunoassay (EIA) kit. As previous reports have shown, the two methods gave reasonably good correlation (r = 0.8), but EIA values were systematically higher than DCC (slope = 3.0). Similar values were obtained when the ER + ve/progesterone receptor (PR) + ve subgroup were examined separately (n = 34, r = 0.86, slope = 3.0). However the two sets of data were in much better agreement in the ER + ve/PR - ve subgroup (n = 10, r = 0.98, slope = 1.24). When analysed by isoelectric focusing on polyacrylamide gels (IEF), two major specific binding components were identified, at pI 6.1 and at pI 6.6. Both isoforms were present in 50/66 ER + ve PR + ve breast tumour samples, but only the pI 6.6 (4S) was present in most ER + ve/PR - ve samples (13/20). It appears that, compared with DCC, the EIA method gives much higher values for the 8S isoform, whereas the two methods detect the 4S isoform with similar sensitivity. In assays on the tumour cell lines, T47D and MCF-7, still greater discrepancies, at least 10-fold, were found between EIA and DCC data.     

Hormonal regulation of vitellogenin genes: an estrogen-responsive element in the Xenopus A2 gene and a multihormonal regulatory region in the chicken II gene

Expression of the vitellogenin genes in avian and amphibian liver is regulated by estrogens. The DNA elements mediating estrogen induction of the various vitellogenin genes of chicken and Xenopus encompass one or more copies of a 13-mer palindromic sequence called the estrogen-responsive element (ERE). Here we show that upon incubation with the purified estrogen receptor (ER) from calf uterus the Xenopus vitellogenin A2 gene yields a DNase-I footprint over the ERE between -331 and -319. This element does not mediate the response to glucocorticoids or progestins in T47D cells. The three guanine residues in each half of the palindrome are protected against methylation by dimethylsulfate after incubation with ER, but not with glucocorticoid (GR) or progesterone (PR) receptors. In contrast, the chicken vitellogenin II gene exhibits multihormonal regulation by estrogens, progestins, and glucocorticoids in T47D and MCF7 cells. Regulation is mediated by the DNA region between -721 and -591 that contains four binding sites for hormone receptors, as demonstrated by DNase-I footprints and methylation protection experiments. The two distal and most proximal binding sites are recognized by ER, GR, and PR, whereas the central binding site is only bound by ER and GR. At suboptimal concentrations, estrogens and progestins or glucocorticoids act synergistically. In experiments using a DNA fragment containing an ERE adjacent to a glucocorticoid-responsive element/progesterone-responsive element, ER and PR bind synergistically to their corresponding sites, perhaps explaining the functional synergism of both hormones. Thus, two very different regulatory elements are used to mediate estrogen induction of related genes in chickens and amphibians.     

Oestradiol-BSA conjugates for receptor histochemistry: problems of stability and interactions with cytosol

Summary The validity of histochemical methods for the localization of hormone receptors based on the binding of fluorescent bovine serum albumin conjugates of oestradiol was examined with respect to their stability and their interactions with the oestrogen receptor type I. Stability was assessed by measuring free oestrogen in conjugates by radioimmunoassay and/or receptor protein binding assay. Sufficient free oestrogen-in order to saturate type I and type II binding sites (ER I, ER II)-was detected in freshly prepared conjugates. This free oestrogen originates in inadequate removal of adsorptively bound original ligand after synthesis. Apart from this fact, conjugates appeared to be unstable in aqueous solutions, especially under the conditions used for histochemical methods. Free oestrogen extracted from the conjugates was subjected to high performance liquid chromatography. Amongst the eluted peaks, oestradiol and/or the original ligand used for synthesis were identified. Thein vitro interaction of conjugates with oestrogen receptors was studied by competitive binding analysis and by incubation of cytosol with a Sepharose-bound conjugate. The results, especially those concerning the amount of free oestrogen, suggest that neither ER I nor ER II is involved in the staining mechanism of conjugates.This paper is dedicated to Professor Dr E. Schauenstein on the occasion of his 65th birthday.     

Immunocytochemical versus biochemical receptor determination in normal and tumorous tissues of the female reproductive tract and the breast

The development of highly specific and sensitive monoclonal antibodies directed against human estrogen (ER) and progesterone receptors (PR) provides a new approach in precise histochemical receptor location independent of hormone binding. Over the years receptor determination was the domain of the radioligand-binding assay, in which receptors are measured by tritiated ligand and unbound ligand is removed by the dextran-coated charcoal (DCC) procedure. Presented here are the results and experiences obtained by the classic DCC and the immunocytochemical method in the different normal and tumorous tissues of the female reproductive tract and the breast. The results of both methods were compared, and overall concordance of the results was found to vary considerably among the different types of tissue analyzed. Best agreement (86%) was found for PR determination in breast cancer, and the lowest rate of concordance for ER determination in fibrocystic disease of the breast. Special attention was directed toward the heterogeneity of receptor distribution in the specimens examined. In all tissues investigated, ER and PR were located in the nuclei of cells in both paraffin and frozen sections. Staining intensity varied among different cell types and from cell to cell for a single cell type, as well as in tumorous and normal tissues. In breast cancer, randomly scattered single cell receptor positivity was distinguished from focal/clonal positivity. Paraffin-embedded lymph node metastases showed significantly weaker staining as compared with their respective primary tumors. In the normal ovary, the corpus luteum and the stromal layer of the outer cortex were revealed as highly receptive elements for progestins, whereas ER was barely demonstrable in the normal ovary. Benign serous and mucinous ovarian tumors showed opposite ER and PR distribution among the stromal and epithelial components. Of special interest were the highly significant changes in ER and PR content in the stromal and glandular cells of the different layers of the normal endometrium throughout the menstrual cycle.     

Structural plasticity in the oestrogen receptor ligand-binding domain

The steroid hormone receptors are characterized by binding to relatively rigid, inflexible endogenous steroid ligands. Other members of the nuclear receptor superfamily bind to conformationally flexible lipids and show a corresponding degree of elasticity in the ligand-binding pocket. Here, we report the X-ray crystal structure of the oestrogen receptor alpha (ERalpha) bound to an oestradiol derivative with a prosthetic group, ortho- trifluoromethlyphenylvinyl, which binds in a novel extended pocket in the ligand-binding domain. Unlike ER antagonists with bulky side groups, this derivative is enclosed in the ligand-binding pocket, and acts as a potent agonist. This work shows that steroid hormone receptors can interact with a wider array of pharmacophores than previously thought through structural plasticity in the ligand-binding pocket.     

Fine needle aspirate cell blocks are reliable for detection of hormone receptors and HER‐2 by immunohistochemistry in breast carcinoma

S. P. Bueno Angela, R. M. Viero and C. T. Soares Fine needle aspirate cell blocks are reliable for detection of hormone receptors and HER‐2 by immunohistochemistry in breast carcinoma Aims: To evaluate the reliability of fine needle aspirate cell blocks in the assessment of oestrogen receptor (ER), progesterone receptor (PR) and HER‐2/neu proteins by immunohistochemistry in comparison with surgical specimens. Materials and methods: This is a retrospective study of 62 cases of breast carcinoma diagnosed by fine needle aspiration cytology (FNAC) and confirmed using the surgical specimen. Immunohistochemical tests were performed to assess the presence of oestrogen receptor (ER), progesterone receptor (PR) and HER‐2/neu proteins in cell blocks and the corresponding surgical specimens. The cell block method used alcohol prior to formalin fixation. Cases with 10% or more stained cells were considered positive for ER and PR. Positivity for HER‐2/neu was assessed on a scale of 0–3+. The criterion for positivity was a score of 3+. Results: Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of the cell blocks in the investigation of ER, PR and HER‐2/neu protein (3+) were (%): ER, 92.7, 85.7, 92.7, 85.7 and 90.3; PR, 92.7, 94.7, 97.4, 87.0 and 93.5; HER‐2/neu, 70.0, 100.0, 100.0, 94.5 and 95.2. Discrepancies were seen in cell blocks in the 1+ and 2+ HER‐2/neu staining scores: two of 12 cases scoring 2+ and one case of 26 scoring 1+ on cell blocks scored 3+ on surgical specimens. The correlation index between cell block and corresponding surgical specimen varied from 90% to 94%. Conclusion: Cell blocks provide a useful method of assessing ER, PR and HER‐2/neu, mainly for inoperable and recurrent cases, but consideration should be given to carrying out FISH analysis on 1+ as well as 2+ HER‐2/neu results.     

Immunochemistry for oestrogen receptor,progesterone receptor and HER2 on cell blocks in primary breast carcinoma

K. Kumar S, N. Gupta, A. Rajwanshi, K. Joshi and G. Singh Immunochemistry for oestrogen receptor, progesterone receptor and HER2 on cell blocks in primary breast carcinoma Objective: Steroid receptors and human epidermal growth receptor 2 (HER2) have been used for predicting response to treatment in breast cancers. Fine needle aspiration cytology can provide highly cellular material and can be used for such analysis. The present study was undertaken to assess the reliability of oestrogen and progesterone receptor (ER, PR) status and HER2 as demonstrated by immunochemistry (IHC) on cell blocks from breast carcinoma cases, in comparison with histological sections. Methods: IHC for ER, PR and HER2 was performed on cell blocks and their corresponding tissue sections of 50 primary pre‐chemotherapy breast carcinomas. Positivity for ER and PR was scored according to the Allred scoring system. Strong membranous positivity in more than 30% of tumour cells was considered positive for HER2. The tumours were classified as luminal A, luminal B, HER2‐over‐expressing and triple negative on the basis of ER, PR and HER2 status and results on cell blocks compared with histological sections. Results: Correlation between immunostaining on cell blocks and the corresponding tumour tissues revealed a concordance rate for ER, PR and HER2 of 90% [Correlation coefficient (r) = 0.79], 94% (r = 0.86) and 90% (r = 0.76), respectively. Including five cases in which cell blocks were either ER or PR positive, 43/50 cases (86.0%) could be correctly classified on cell block immunostaining alone. The main reasons for seven discordant cases included technical errors (sampling error and staining error) and interpretational error in HER2 evaluation on cell blocks; the core biopsy was inadequate in one, and apparently false negative for HER2 in another. Conclusion: Cell blocks are useful in the assessment of hormone receptor status and HER2 by IHC, especially in cases of locally advanced breast cancer for planning neoadjuvant chemotherapy. It is highly recommended to have good quality cell blocks and quality control of their interpretation.