As members of the nuclear receptor superfamily, ER-alpha and -beta have a similar architecture. The N-terminal A/B domain is involved in transactivation of gene expression. The C-domain contains a two-zinc finger structure, which plays an important role in receptor specific DNA-binding and receptor dimerization. The C-terminal ligand-binding domain (E/F domain) is crucial for binding of receptor specific ligands, nuclear translocation, receptor dimerization, and modulation of target gene expression in association with corepressors and coactivators (Barkhem T. et al., 1998).
ER-alpha and -beta have the potential to function as heterodimers (Cowley S. et al., 1997), but the two receptors are more likely to function as homodimers in the majority of cells.
ER-alpha stimulates transcription of target genes by means of two distinct activation functions, AF1 in the N-terminal domain and AF2 in the ligand binding domain, whose activities vary depending upon the target promoter and cell type. The activity of AF1 is ligand independent and constitutive but can be modulated by phosphorylation by the mitogen activated protein kinase (MAPK) pathway in response to growth factors (Kato S. et al., 1998). The activity of AF2, which depends on the binding of estradiol, is reduced or abolished by mutations in a C-terminal helix which is conserved in most nuclear receptors. ER-alpha and ER-beta exhibit similar but not identical ligand binding properties (Kuiper G.G.J.M. et al., 1997 (reference in
ER-beta). Both receptors appear to contain a functionally conserved AF2 which is stimulated by binding the coactivator
SRC-1. Although the two receptors are poorly conserved in the N-terminal domain, ER-beta, like ER-alpha, appears to contain a MAPK phosphorylation site that results in enhanced transcriptional activity. However, the activity of AF1 in ER-beta is negligible compared with that of ER-alpha on estrogen responsive element (ERE) based promoters. As a consequence, when transcription from a gene depends on both AF1 and AF2 (such as observed with pS2), the activity of ER-alpha greatly exceeds that of ER-beta, but when AF1 is not required, ER-alpha and Er)beta have similar transcriptional activities (Cowley S.M. and Parker M.G., 1999).
It has been shown that tamoxifen, 4-OH-tamoxifen, raloxifene, and ICI 164,384 have an ER-alpha-selective partial agonist/antagonist function but a pure antagonist effect through ER-beta. In addition, raloxifene was found to display an ER-alpha-selective antagonist potency, in agreement with its ER-alpha selective affinity. However, although ICI 164,384 showed an ER-beta -selective affinity, it had a similar potency to antagonize the effect of 17beta-estradiol in the ER-alpha- and ER-beta-specific reporter cell lines used. Thus, the ligand binding cavity of ER-beta is probably more different from that of ER-alpha than can be anticipated from the primary sequences of the two ER subtypes (Barkhem T. et al., 1998).
It is accepted that the presence of ER-alpha identifies those breast cancer patients with a lower risk of relapse and better overall survival, and the measurement of ER-alpha has become a standard assay in the clinical management of breast cancer.
Cell lines:
- The presence of ER-alpha has been investigated in a number of widely used breast cancer cell (BCC) lines. ER was detected in MCF-7 (high amount), T-47D, ZR-75, CAMA-1, BT483, BT474, MDA-MB-361, MDA-MB-134,...In these cell lines, ER and
E-cadherin were frequently coexpressed, while
vimentin was absent. ER was not found in MDA-MB-231, -435, -436, -453, 468, Hs578T, SK-BR-3, Evsa-T, BT549, MCF-7/Adr (doxorubicin-resistant MCF-7)...Most of these BCC lines were also found to express
vimentin, but not
E-cadherin and rarely
zonula occludens-1 (ZO-1)(C.L. Sommers et al., 1994).
- Upon treatment with the demethylating agents 5-azacytidine or 5-aza-2'-deoxycytidine, MDA-MB-231 were found to reexpress ER-alpha mRNA and protein. This was associated with partial demethylation of the cytosine- and guanine-rich area (CpG island) of the ER-alpha gene (Ottaviano Y.L. et al., 1994; Ferguson A.T. et al., 1995).
- Methylation has been shown to play an important role in the down-regulation of oestrogen receptors (ER) in breast cancer cells (see above). One critical question that remains unclear is whether methylation can account for the loss of ER expression in cells derived from an ER-positive cell line. It has been shown that, in a ER-negative clonal cell line obtained by culturing ER-positive T-47D BCC in oestrogen-free medium, the ER CpG island remained unmethylated. The loss of ER in this cell line must be due to mechanisms other than methylation (Chen Z. et al., 1998).
- In transient transfection assays in T-47D, MCF-7, and MDA-MB-231 BCC,
BRCA1 was found to inhibit signaling by the ligand-activated estrogen receptor-alpha through the estrogen-responsive enhancer element and to block the transcriptional activation function AF2 of ER-alpha. This raises the possibility that wild-type
BRCA1 suppresses estrogen-dependent transcriptional pathways related to mammary epithelial cell proliferation and that loss of this ability contributes to tumorigenesis (Fan S. et al., 1999).
- Recombinant adenoviral vectors expressing a dominant negative ER or, for comparison, antisense ER, or the sense wild-type ER were constructed. Expression of the dominant negative ER or antisense ER, but not wild-type ER, blocked estradiol stimulation of the estrogen-responsive genes
pS2 and c-myc. The dominant negative ER also fully abolished the estradiol-induced increase in proliferation of MCF-7 BCC, as did the antisense ER. The antiproliferative effects of the dominant negative and antisense ERs are explained by an increase in the number of cells in the G
0/G
1 stage of the cell cycle and decrease in the number of cells in G
2/M as determined by flow cytometry, and also by a significant increase in the percentage of cells undergoing apoptosis (Lazennec G. et al., 1999).
- 23 BCC lines were studied for their
in vitro invasiveness and for their ER, progesterone receptor (PgR), estrogen inducible pS2, and plasminogen activator inhibitor-1 (PAI-1) mRNA levels. In general, cells with ER, PR, pS2 but no PAI-1 expression showed a non-invasive phenotype, while cells expressing PAI-1 mRNA but not ER mRNA were invasive (Tong D. et al., 1999b).
Tumors:
- In a series of 27 breast tumors, ER mRNA (measured by dot-blot) and protein (measured by the dextran-coated charcoal assay) levels were found to be in close agreement. ER mRNA level also significantly correlated to ER protein level estimated by immunocytochemical assay. ER-negative tumors produced no detectable mRNA (Barrett-Lee P.J. et al., 1988).
- Southern analysis of the ER gene in a series of ER-negative and -positive breast tumour biopsies failed to provide evidence of gross rearrangements and in only 1 of 37 tumour DNA samples was significant gene amplification observed. Methylation of the ER gene as assessed by Hpa II and Msp I restriction enzyme digests varied between tumours but the degree of methylation was not correlated with levels of expression of the receptor protein. Similar finding applied in a series of ER-negative and -positive breast cancer cell lines and clonal lines of MCF-7 BCC, which were developed as an in vitro model for the acquisition of oestrogen and antioestrogen resistance (Watts C.K.W. et al., 1992).
- ER-alpha mutations were searched in 188 breast cancer patients using single-strand conformation polymorphism (SSCP) analysis, denaturing gradient electrophoresis, and DNA sequencing. In the majority of primary breast cancers, the ER-negative phenotype was due to deficient ER expression at the transcriptional or post-transcriptional level, and was not the result of mutations in the coding region of the ER-alpha gene (Roodi N. et al., 1995). In another study, there were neither germline nor somatic mutations in the ER gene in 14 patients with ER-negative and
progeterone receptor (PgR)-positive breast tumors as assessed by SSCP analysis and DNA sequencing (Iwase H. et al., 1996). A unique mutated ER (Tyr 537 Asn, T 1609 A) has been identified in 1 of 30 metastatic breast cancers. This substitution confers constitutive transcriptional activity to ER and its activity cannot be antagonized with antiestrogens such as tamoxifen and pure antiestrogen ICI164384 (Zhang Q.X. et al., 1997).
- On the basis of a downstream functional assay of ER interaction with estrogen response element (ERE), it has been suggested that ER+ breast tumor cells can be subclassified into two categories. The first is E2-dependent (ERd+) and these cells should respond to anti-hormone therapy. The second type of ER interacts with ERE independent of E2 (ERi+) and constitutively transactivates responsive genes. It is predicted that the latter type of breast cancers will not respond to antihormone therapy (Biswas D.K. et al., 1998).
- It has been suggested that in ER-positive human breast tumors with increased polyamine (especially spermine) content, ER structure and function might be directly altered by tight-ion polyamine complexing that results in loss of ER-mediated gene regulation (Lu B. et al., 1998).
- Tumor samples of 240 patients with primary breast cancer were biochemically and immunohistochemically investigated for estrogen receptors (ER) and, in 130 of the samples, for
progesterone-receptors (PgR). The biochemical (DCCA) and immunohistochemical assays (ICA) yielded positivity in 71% for ER, and in 44% for
PgR. Concordant ER-DCCA and ER-ICA results were obtained in 84%; two thirds of the discordant ER-findings manifested as DCCA-neg/ICA-pos. Concordance in the case of
PgR amounted to 72%, and of the discordances 60% were DCCA-neg/ICA-pos. Significant association with postmenopausal status existed only for ER positivity in ICA (p = 0.01), whereas ER-DCCA,
PgR-DCCA and
PgR-ICA were all more or less independent of the menopausal status. The frequency of discordances was independent of menopausal status. Discordance for ER-assays increased significantly near the respective cut-off point; this was not unequivocally true for
PgR-assays (Stierer M. et al., 1998).
- The expression of estrogen (ER) and
progesterone (PgR) receptors was analyzed in a retrospective series of 3000 patients who had operable primary breast cancer. Patients were stratified according to ER and
PgR status and the study was focused on the two groups (ER-
PgR+ and ER-
PgR-) of patients whose tumors contained low levels of ER (< 15 fmol/mg protein), regarding potential response to endocrine therapy. The comparison of clinical or histological characteristics between ER-
PgR+ and ER-
PgR- patients was analyzed as well as the disease-related death and survival. The mean follow-up was 86.3 months. Among the 529 ER-patients, 62 were
PgR+ (12%), whereas 467 were
PgR- (88%). The ER-
PgR+ and ER-
PgR- populations represented 2% and 15.6% of the overall population, respectively. In ER- tumors, the
PgR status was significantly related to: age, menopausal status, tumor size, SBR grade, and histological type, but not to the type of surgical treatment or to lymph node involvement. ER-
PgR+ tumors had smaller size (64% T1 vs 43%) (p=0.004) and were more frequently grade I (28% vs 12%) than ER-
PgR- tumors (p < 0.001). In addition, the patients with ER-
PgR+ tumors were significantly younger (49.4 years vs 58.4 years; p < 0.0001), and were more frequently premenopausal (76% vs 36%, p < 0.001). The disease-free interval and the metastasis-free survival tended to be worse for ER-
PgR- than for ER-
PgR+ patients, but the difference was not statistically significant at 10 years. However, a small but significant difference in overall survival, in favor of the
PgR+ group, was observed between the two groups during the first 5 years (p=0.03) (Bernoux A. et al., 1998).
- ER protein and mRNA (determined by RT-PCR) levels were found to be significantly correlated in a series of 51 breast tumors. The mRNA levels for ER,
PgR, and
pS2 in breast tumors (n=100) were significantly correlated to each other, but none of them was associated with the mRNA expression of
PAI-1. ER protein and mRNA were significantly correlated to the age of patients. With respect to clinical data, ER and
PgR mRNA were found to be inversely correlated to tumor size and histological grade, but not to the lymph node status (Tong D. et al., 1999a).
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