Current medical treatment of estrogen receptor-positive breast cancer

Approximately 80% of breast cancers(BC) are estrogen receptor(ER)-positive and thus endocrine therapy(ET) should be considered complementary to surgery in the majority of patients. The advantages of oophorectomy, adrenalectomy and hypophysectomy in women with advanced BC have been demonstrated many years ago, and currently ET consist of(1) ovarian function suppression(OFS), usually obtained using gonadotropinreleasing hormone agonists(Gn RHa);(2) selective estrogen receptor modulators or down-regulators(SERMs or SERDs); and(3) aromatase inhibitors(AIs), or a combination of two or more drugs. For patients aged less than 50 years and ER+ BC, there is no conclusive evidence that the combination of OFS and SERMs(i.e., tamoxifen) or chemotherapy is superior to OFS alone. Tamoxifen users exhibit a reduced risk of BC, both invasive and in situ, especially during the first 5 years of therapy, and extending the treatment to 10 years further reduced the risk of recurrences. SERDs(i.e., fulvestrant) are especially useful in the neoadjuvant treatment of advanced BC, alone or in combination with either cytotoxic agents or AIs. There are two types of AIs: type Ⅰ are permanent steroidal inhibitors of aromatase, while type Ⅱ are reversible nonsteroidal inhibitors. Several studies demonstrated the superiority of the third-generation AIs(i.e., anastrozole and letrozole) compared with tamoxifen, and adjuvant therapy with AIs reduces the recurrence risk especially in patients with advanced BC. Unfortunately, some cancers are or became ET-resistant, and thus other drugs have been suggested in combination with SERMs or AIs, including cyclin-dependent kinase 4/6 inhibitors(palbociclib) and mammalian target of rapamycin(m TOR) inhibitors, such as everolimus. Further studies are required to confirm their real usefulness.     

Protein kinase C subspecies in estrogen receptor-positive and -negative human breast cancer cell lines

Estrogen receptor-positive (MCF7) and -negative (BT20) human breast cancer cell lines, which are frequently used for studies on cancer chemotherapy with triphenylethylene (TPE) anti-estrogens, express at least three protein kinase C subspecies. Two of them are identified as type II PKC having the beta-sequence and type III PKC having the alpha-sequence. The other one shows typical characteristics of PKC which responds to Ca2+, phosphatidylserine and diacylglycerol, but shows kinetic properties subtly different from the previously known PKC subspecies. Immunoblot analysis has shown that this enzyme does not correspond to any of the well defined subspecies with known sequence structures. All of these PKC subspecies are similarly susceptible to the TPE antiestrogens.     

Estrogen receptor beta in the breast: role in estrogen responsiveness and development of breast cancer

Breast cancer is one of the most common forms of cancer observed in women. Endogenous estrogen is thought to play a major role in its development and estrogen receptor blockers are the most important drugs in its treatment. It has long been thought that any conditions or exposures, which enhance estrogenic responses, would result in an increased risk for breast cancer. The discovery of the second estrogen receptor, ERbeta, which can have effects opposite to those of the well-known 'original' estrogen receptor (now called ERalpha) challenges this simplistic view. In order to understand breast cancer one must first understand how the normal breast is maintained. The functions of ERbeta in the breast remain to be defined but from what we have learnt about its activities in in vitro systems, this estrogen receptor may have a protective role in the breast. Studies in human and rodent breasts as well as in human breast cancer biopsies reveal that ERbeta is by far the more abundant of the two ERs. Despite the role of estrogen in proliferation of the breast, neither of the two ERs appears to located in epithelial cells which divide in response to estrogen. In order to define the functions of ERbeta in the normal and malignant breast, we have created mice in which the ERbeta gene has been inactivated. Studies of the breasts of ERbeta knock out mice (BERKO) revealed abnormal epithelial growth, overexpression of Ki67 and severe cystic breast disease as mice age.     

Estrogen response in the hFOB 1.19 human fetal osteoblastic cell line stably transfected with the human estrogen receptor gene

The gene coding for the human wild-type estrogen receptor (ER) was stably transfected into the human fetal osteoblastic cell line hFOB 1.19, a clonal cell line which is conditionally immortilized with a temperature sensitive mutant of SV40 large T antigen (tsA58). Five subclones were obtained which express various levels of ER mRNA and protein. The subclone with the highest level of functional (nuclear bound) ER, hFOB/ER9, contained 3,931 (±1,341) 17β-estradiol molecules bound/nucleus as determined by the nuclear binding (NB) assay. Using the dextran coated charcoal (DCC) method, the level of total cytosolic ER measured was 204 (±2) fmol/mg protein. This subclone was examined further for estradiol (E₂) responsiveness. The ER expressed in hFOB/ER9 cells was shown to be functional using a transiently transfected ERE-TK-luciferase construct. Expression of luciferase from this construct increased ～25-fold in hFOB/ER9 cells following 10^?9M E₂ treatment. This effect on ERE-TK-luciferase expression was both dose and steroid dependant. Further, treatment of hFOB/ER9 cells with 10^?9M E₂ resulted in a 2.5–4.0-fold increase in endogenous progesterone receptor (PR) levels detected by steroid binding assays, and a noticeable increase in both the A and B forms of PR by western blot assay. The establishment of this estrogen responsive human osteoblastic cell line should provide an excellent model system for the study of estrogen action on osteoblast function. © 1995 Wiley-Liss, Inc.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Estrogen receptor alpha negative breast cancer patients: estrogen receptor beta as a therapeutic target

Clinical management of breast cancer is increasingly guided by assessment of tumor phenotypic parameters. One of these is estrogen receptor (ER) status, currently defined by ERalpha expression. However with the discovery of a second ER, ERbeta and its variant isoforms, the definition of ER status is potentially more complex. In breast tumors there are two ERbeta expression cohorts. One where ERbeta is co-expressed with ERalpha and the other expressing ERbeta alone. In the latter subgroup of currently defined ER negative patients ERbeta has the potential to be a therapeutic target. Characterization of the nature and role of ERbeta in ERalpha negative tumors is essentially unexplored but available data suggest that the role of ERbeta may be different when co-expressed with ERalpha and when expressed alone. This review summarizes available data and explores the possibility that ERbeta signaling may be a therapeutic target in these tumors. Evidence so far supports the idea that the role of ERbeta in breast cancer is different in ERalpha negative compared to ERalpha positive tumors. However, cohort size and numbers of independent studies are small to date, and more studies are needed with better standardization of antibodies and protocols. Also, the ability to determine the role of ERbeta in ERalpha negative breast cancer and therefore assess ERbeta signaling pathways as therapeutic targets would be greatly facilitated by identification of specific downstream markers of ERbeta activity in breast cancer.     

A therapeutic target for hormone-independent estrogen receptor-positive breast cancers

BACKGROUND: The action of the steroid hormone estradiol (E2) is mediated via interaction with a specific receptor (ER) that initiates a series of events downstream, leading to the modulation of hormone-responsive genes and cell proliferation. Antihormones also bind, but do not confer the active configuration to ER, thereby, blocking the transmission of E2-ER-initiated signals for cell proliferation. Although these compounds qualify for successful therapy of ER-positive [ER (+)] breast cancer patients, only a fraction of patients responds to antihormone treatment. In this study, the functional status of ER is determined to identify alternative targets for therapy of antihormone-resistant ER (+) breast cancers. METHOD: The interaction of ER with a specific DNA sequence, designated as E2 response element (ERE), was targeted to assess the functional state of ER. ER-ERE complex formation was measured by electrophoretic mobility shift assay (EMSA) and by a newly developed technique, based on the preferential binding of DNA-protein complex to a nitrocellulose membrane (NMBA) that measures both total and functional fraction of ER. RESULTS: The NMBA assay identified functional variants of ER among ER (+) breast cancer cell lines and breast tumor biopsy specimens. ER of (21PT) cells did not bind E2 and these cells were tamoxifen (TAM) resistant. However 21PT cells were sensitive to a calmodulin (CaM) antagonist, W7, that blocked ERE-ER complex formation. CONCLUSIONS: ER variants of the 21PT type were detected among breast cancer biopsy specimens, emphasizing the significance of an alternative therapeutic target for TAM-resistant ER (+) human breast cancers with compounds such as W7.     

Concentration-dependent mitogenic and antiproliferative actions of 2-methoxyestradiol in estrogen receptor-positive human breast cancer cells

We compared in this study the effects of 2-methoxyestradiol (2-MeO-E(2)) on the growth of two estrogen receptor (ER)-negative human breast cancer cell lines (MDA-MB-231 and MDA-MB-435s) and two ER-positive human breast cancer cell lines (MCF-7 and T-47D). 2-MeO-E(2) exerted a concentration-dependent antiproliferative action in the ER-negative MDA-MB-231 and MDA-MB-435s cells. The presence or absence of exogenous 17beta-estradiol (E(2)) in the culture medium did not affect the potency and efficacy of 2-MeO-E(2)'s antiproliferative action in these ER-negative cells. When the ER-positive MCF-7 and T-47D cells were cultured in a medium supplemented with 10nM of exogenous E(2), 2-MeO-E(2) at 750 nM to 2 microM concentrations exerted a similar antiproliferative effect. However, when the ER-positive cell lines were cultured in the absence of exogenous E(2), 2-MeO-E(2) at relatively low concentrations (10-750 nM) had a moderate mitogenic effect, with its apparent efficacy 75-80% of that of E(2). This mitogenic effect of 2-MeO-E(2) was ER-mediated and largely attributable to 2-MeO-E(2)'s residual estrogenic activity on the basis of our following findings: (i) its effect was only manifested in the ER-positive cells but not in the ER-negative cells; (ii) its effect in the ER-positive cells was partially or fully abolished when exogenous E(2) was concomitantly present in the culture medium; (iii) 2-MeO-E(2) retained 1-2% of E(2)'s binding affinity for the human ERalpha and ERbeta, and its mitogenic effect was inhibited in a concentration-dependent manner by ICI-182,780, a pure ER antagonist; and (iv) its effect was not due to its metabolic conversion to 2-hydroxyestradiol. Our timely findings are of importance to the on-going clinical trials designed to evaluate 2-MeO-E(2)'s effectiveness for the treatment of different types (ER-positive or ER-negative) of human breast cancer. This knowledge will improve the design of clinical trials as well as the interpretation of clinical outcomes when 2-MeO-E(2) is used as a single agent therapy or as part of a combination therapy for human breast cancer.     

A variant estrogen receptor messenger ribonucleic acid is associated with reduced levels of estrogen binding in human mammary tumors

An analysis of the human estrogen receptor (ER) mRNA was performed on 71 human breast tumors using an RNase protection assay. Complementary DNA clones to the human estrogen receptor (lambda R8 and lambda R3) were used to generate small antisense 32P-labeled RNA molecules that were hybridized to the tumor RNA. We determined the relative amounts of ER mRNA in each tumor by measuring the amount of RNases A and T1 resistant hybrids. Moreover, because RNase A has the ability to cleave single-base mismatches within RNA/RNA duplexes, we were able to use the assay to screen for possible mutations or deletions in the ER mRNA. A significant correlation was found between the ER mRNA levels and the estrogen binding concentrations determined by a dextran-coated charcoal assay (r = 0.68; P less than 0.0001; n = 58). We also identified a subpopulation of tumors in which a mismatch in the ER mRNA was detected. This message modification, in the B region of the message, significantly correlated with low levels of estrogen binding. This result suggests that the observed B variant might lead to the production of receptors with altered properties.     

Estrogen induces estrogen-related receptor alpha gene expression and chromatin structural changes in estrogen receptor (ER)-positive and ER-negative breast cancer cells

Estrogen-related receptor alpha (ERRalpha), a member of the nuclear receptor superfamily, is closely related to the estrogen receptors (ERalpha and ERbeta). The ERRalpha gene is estrogen-responsive in several mouse tissues and cell lines, and a multiple hormone-response element (MHRE) in the promoter is an important regulatory region for estrogen-induced ERRalpha gene expression. ERRalpha was recently shown to be a negative prognostic factor for breast cancer survival, with its expression being highest in cancer cells lacking functional ERalpha. The contribution of ERRalpha in breast cancer progression remains unknown but may have important clinical implications. In this study, we investigated ERRalpha gene expression and chromatin structural changes under the influence of 17beta-estradiol in both ER-positive MCF-7 and ER-negative SKBR3 breast cancer cells. We mapped the nucleosome positions of the ERRalpha promoter around the MHRE region and found that the MHRE resides within a single nucleosome. Local chromatin structure of the MHRE exhibited increased restriction enzyme hypersensitivity and enhanced histone H3 and H4 acetylation upon estrogen treatment. Interestingly, estrogen-induced chromatin structural changes could be repressed by estrogen antagonist ICI 182 780 in MCF-7 cells yet were enhanced in SKBR3 cells. We demonstrated, using chromatin immunoprecipitation assays, that 17beta-estradiol induces ERRalpha gene expression in MCF-7 cells through active recruitment of co-activators and release of co-repressors when ERRalpha and AP1 bind and ERalpha is tethered to the MHRE. We also found that this estrogen effect requires the MAPK signaling pathway in both cell lines.     

Immunochemical analyses of estrogen receptors in human breast tumors by a novel monoclonal estrogen receptor antibody (EVG F9)

The objective of this study was to assess the potential utility of a new site-directed, monoclonal anti-estrogen receptor antibody (EVG F9) in detection and analyses of human breast tumor estrogen receptor (ERalpha), using immunoblotting and immunohistochemical assays. Using Western Blot analyses, we demonstrated that EVG F9 monoclonal antibody binds specifically to ERalpha and does not cross-react with ERbeta. Furthermore, binding of EVG F9 to ERalpha was effectively displaced with the immunogenic peptide in Western Blots and in immunohistochemical analyses. In Western Blot analyses, EVG F9 detected ERalpha at low concentrations approaching 5 to 10 fmol/sample. Determination of ERalpha status of a series of human breast tumor samples by Western Blot analyses or immunohistochemistry using EVG F9 correlated well with ERalpha values measured by ligand binding assays. These observations suggest that EVG F9 monoclonal anti-ERalpha antibody is a valuable immunochemical tool for detection and analyses of ERalpha in human breast tumors.     

Response of estrogen receptor-positive breast cancer tumorspheres to antiestrogen treatments

Estrogen signaling plays a critical role in the pathogenesis of breast cancer. Because the majority of breast carcinomas express the estrogen receptor ERα, endocrine therapy that impedes estrogen-ER signaling reduces breast cancer mortality and has become a mainstay of breast cancer treatment. However, patients remain at continued risk of relapse for many years after endocrine treatment. It has been proposed that cancer recurrence may be attributed to cancer stem cells (CSCs)/tumor-initiating cells (TICs). Previous studies in breast cancer have shown that such cells can be enriched and propagated in vitro by culturing the cells in suspension as mammospheres/tumorspheres. Here we established tumorspheres from ERα-positive human breast cancer cell line MCF7 and investigated their response to antiestrogens Tamoxifen and Fulvestrant. The tumorsphere cells express lower levels of ERα and are more tumorigenic in xenograft assays than the parental cells. Both 4-hydroxytamoxifen (4-OHT) and Fulvestrant attenuate tumorsphere cell proliferation, but only 4-OHT at high concentrations interferes with sphere formation. However, treated tumorsphere cells retain the self-renewal capacity. Upon withdrawal of antiestrogens, the treated cells resume tumorsphere formation and their tumorigenic potential remains undamaged. Depletion of ERα shows that ERα is dispensable for tumorsphere formation and xenograft tumor growth in mice. Surprisingly, ERα-depleted tumorspheres display heightened sensitivity to 4-OHT and their sphere-forming capacity is diminished after the drug is removed. These results imply that 4-OHT may inhibit cellular targets besides ERα that are essential for tumorsphere growth, and provide a potential strategy to sensitize tumorspheres to endocrine treatment.     

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.