The potential role of estrogen in aromatase regulation in the breast

Aromatase is expressed in both normal and malignant breast tissues. Aromatase activity in the breast varies over a wide range. Our previous studies have demonstrated that in situ aromatization contributes to the estrogen content of breast tumors to a major extent. Consequently, alterations of aromatase activity could serve as a major determinant of tissue estradiol content. However, the mechanisms and extent of aromatase regulation in breast tissues have not been fully established. We have observed an inverse correlation between tumor aromatase activity and estrogen content in nude mice bearing xenografts of MCF-7 cells transfected with the aromatase gene. To investigate the potential role of estrogen in aromatase regulation in the breast, studies were carried out in an in vitro model. In this model, MCF-7 cells were cultured long term in estrogen-deprived medium and called by the acronym, LTED cells. We found that long-term estrogen deprivation enhanced aromatase activity by 3–4-fold when compared to the wild-type MCF-7 cells. Re-exposure of LTED cells to estrogen reduced aromatase activity to the levels of the wild-type MCF-7 cells. We also measured aromatase activity in 101 frozen breast carcinoma specimens and compared tumor aromatase activities in pre-menopausal patients versus post-menopausal patients and in post-menopausal patients with or without hormone replacement therapy (HRT). Although statistically not significant, there was a trend paralleling that observed in the in vitro studies. Aromatase activity was higher in breast cancer tissues from the patients with lower circulating estrogen levels. Our data suggest that estrogen may be involved in the regulation of aromatase activity in breast tissues.     

Menopausal estrogen deprivation activates steroid sensitive stem cells (3SC) and local estrogen biosynthesis: A model for breast cancer development

We propose a hypothesis for breast cancer (BC) development and its implications for BC prevention. We describe a model in which some breast cells function as both stem cells and steroid sensors (steroid sensitive stem cells). Estrogen receptors on those cells could be upregulated in women who had increased cumulative exposure to estrogen, leading to their progressive sensitization. At menopause, such women experience considerable decline of estrogen concentration in their blood. Consequently, the sensitized stem cells activate mechanisms of local estrogen synthesis including the activation of aromatase. The intracrine build-up of estrogen and its metabolites induces proliferation and genetic dysfunction. Eventually, a normal stem cell transforms into an estrogen-sensitive cancer stem cell that is capable of tumor initiation and delineation into other phenotypes of cancer cells. This hypothesis is supported by significant in-vitro and clinical research evidence. According to this model, we suggest that estrogen therapy could be protective against BC. Alternatively, aromatase inhibitors are expected to be effective in BC prevention. A combination of AIs and estrogen might augment the preventative merits of both drugs and maintain a good tolerability profile for long-term prevention protocols.     

Recent data on intratumor estrogens in breast cancer

The contribution of local synthesis versus circulatory delivery of normal breast as well as breast cancer tissue estrogens has remained a controversial area for decades. Novel data on tissue estrogen levels confirm a positive normal breast tissue to plasma concentration gradient for estrone, and to a smaller extent estradiol. Remarkably, this gradient is similar for pre- and post-menopausal women. Together with pharmacokinetic data on estrogen disposition, these findings suggest plasma and breast tissue estrogens to rapidly equilibrate, with circulating estrogens being a major contributor to breast tissue estrone levels. A likely explanation to the concentration gradient could be the fact that non-polar estrogens easily dissolve into tissue fat compartments as compared to plasma. While intratumor estrone levels are low as compared to benign tissue concentrations, intratumor estradiol is elevated in ER+ tumors. The correlation between intratumor estradiol levels and expression levels of dehydrogenases reducing estrone into estradiol but also intratumor ER concentrations are consistent with intratumor estrogen activation but also a scavenger effect of the ER.     

Development and evolution of therapies targeted to the estrogen receptor for the treatment and prevention of breast cancer

This article describes the origins and evolution of "antiestrogenic" medicines for the treatment and prevention of breast cancer. Developing drugs that target the estrogen receptor (ER) either directly (tamoxifen) or indirectly (aromatase inhibitors) has improved the prognosis of breast cancer and significantly advanced healthcare. The development of the principles for treatment and the success of the concept, in practice, has become a model for molecular medicine and presaged the current testing of numerous targeted therapies for all forms of cancer. The translational research with tamoxifen to target the ER with the appropriate duration (5 years) of adjuvant therapy has contributed to the falling national death rates from breast cancer. Additionally, exploration of the endocrine pharmacology of tamoxifen and related nonsteroidal antiestrogen (e.g. keoxifene now known as raloxifene) resulted in the laboratory recognition of selective ER modulation and the translation of the concept to use raloxifene for the prevention of osteoporosis and breast cancer. However, the extensive evaluation of tamoxifen treatment revealed small but significant side effects such as endometrial cancer, blood clots and the development of acquired resistance. The solution was to develop drugs that targeted the aromatase enzyme specifically to prevent the conversion of androstenedione to estrone and subsequently estradiol. The successful translational research with the suicide inhibitor 4-hydroxyandrostenedione (known as formestane) pioneered the development of a range of oral aromatase inhibitors that are either suicide inhibitors (exemestane) or competitive inhibitors (letrozole and anastrozole) of the aromatase enzyme. Treatment with aromatase inhibitors is proving effective and is associated with reduction in the incidence of endometrial cancer and blood clots when compared with tamoxifen and there is also limited cross resistance so treatment can be sequential. Current clinical trials are addressing the value of aromatase inhibitors as chemopreventive agents for postmenopausal women.     

A novel gene STYK1/NOK is upregulated in estrogen receptor-alpha negative estrogen receptor-beta positive breast cancer cells following estrogen treatment

The human STYK1/NOK protein is approximately 30–35% similar to mouse fibroblast growth factor receptor 3 and a kinase homologue in D. melanogaster in the tyrosine protein kinase region. STYK1/NOK was identified as being up regulated in MDA-MB-231, an estrogen receptor-alpha negative breast cancer cell line, following 12 h of estrogen treatment at 1 × 10⁻⁹ M. On further investigation of STYK1/NOK in estrogen treated cell line MDA-MB-231, STYK1/NOK was up regulated at 6 h post treatment when compared to untreated cells. We also investigated the expression levels of STYK1/NOK in other breast cancer cell lines MCF-7, MDA-MB-231, BT-549, and MDA-MB-435S using QRT-PCR. In addition, the analysis of message accumulation was increased with other synthetic estrogen response modifiers. We propose that the regulation of STYK1/NOK is achieved independent of ERα and suggests further investigation to the relevance of this kinase in breast cancer progression.     

Update on estrogen signaling

Our understanding of estrogen signaling has undergone a true paradigm shift over recent years, following the discovery in 1995 of a second estrogen receptor, estrogen receptor beta (ERbeta). In many contexts ERbeta appears to antagonize the actions of ERalpha (yin/yang relationship) although there also exist genes that are specifically regulated by one of the two receptors. Studies of ERbeta knockout mice have shown that ERbeta exerts important functions in the ovary, central nervous system, mammary gland, prostate gland, hematopoiesis, immune system, vessels and bone. The use of ERbeta-specific ligands against certain forms of cancer represents one of the many pharmaceutical possibilities that have been created thanks to the discovery of ERbeta.     

17beta-Estradiol inhibits osteoprotegerin production by the estrogen receptor-alpha-positive human breast cancer cell line MCF-7

Estrogen regulates various cytokines and growth factors in estrogen receptor (ER)-positive human breast cancer. Receptor activator of NF-κB ligand (RANKL) is an essential cytokine for osteoclasts, whereas osteoprotegerin (OPG) is a soluble inhibitor for RANKL. We analyzed the regulation of the RANKL/OPG system by estrogens and androgens in the ER-positive breast cancer cell line MCF-7 and the ER-negative breast cancer cell line MDA-MB-231. In MCF-7 cells, which predominantly express ER-α, 17β-estradiol and testosterone dose-dependently decreased OPG mRNA levels and protein secretion by 70 and 65%, respectively (p < 0.0001 by ANOVA). The inhibition of OPG production by 17β-estradiol and testosterone was specifically prevented by the pure anti-estrogen ICI 182,780, and the testosterone effect was prevented by an aromatase inhibitor. In conclusion, 17β-estradiol suppressed OPG production by human breast cancer cell lines in a dose-dependent and specific manner, indicating that the RANKL/OPG cytokine system is an estrogen-responsive target in breast cancer.     

Comparison between aromatase inhibitors and sequential use

The biochemical efficacy of aromatase inhibitors and inactivators in vivo may be determined by two types of methods; by measuring plasma or tissue estrogen levels, or assessment of the conversion of the androgen substrate (in practice, androstenedione) into estrogens (estrone) by the use of tracer methods. While methods to determine plasma and tissue estrogens are limited through lack of sensitivity required to measure the very low concentrations recorded in postmenopausal women on treatment with these compounds, measurement of in vivo aromatization is an extensive procedure, applicable to a limited number of patients only. While we may correlate the mean level of aromatase inhibition achieved with different compounds to clinical efficacy, data correlating individual estrogen suppression to clinical outcome among patients treated with a specific compound is limited. The now well-characterized phenomenon of lack of cross-resistance between non-steroidal aromatase inhibitors and steroidal aromatase inactivators are likely due to biochemical effects not related to differences in total body aromatase inhibition.     

The role of estrogen in the initiation of breast cancer

Estrogens are considered to play a major role in promoting the proliferation of both the normal and the neoplastic breast epithelium. Their role as breast carcinogens has long been suspected and recently confirmed by epidemiological studies. Three major mechanisms are postulated to be involved in their carcinogenic effects: stimulation of cellular proliferation through their receptor-mediated hormonal activity, direct genotoxic effects by increasing mutation rates through a cytochrome P450-mediated metabolic activation, and induction of aneuploidy. Recently it has been fully demonstrated that estrogens are carcinogenic in the human breast by testing in an experimental system the natural estrogen 17β-estradiol (E₂) by itself or its metabolites 2-hydroxy, 4-hydroxy, and 16-a-hydroxy-estradiol (2-OH-E₂, 4-OH-E₂, and 16--OH E₂), respectively, by inducing neoplastic transformation of human breast epithelial cells (HBEC) MCF-10F in vitro to a degree at least similar to that induced by the chemical carcinogen benz(a)pyrene (BP). Neither Tamoxyfen (TAM) nor ICI-182,780 abrogated the transforming efficiency of estrogen or its metabolites. The E₂ induced expression of anchorage independent growth, loss of ductulogenesis in collagen, invasiveness in Matrigel, is associated with the loss of 9p11-13 and only invasive cells that exhibited a 4p15.3-16 deletion were tumorigenic. Tumors were poorly differentiated ER- and progesterone receptor negative adenocarcinomas that expressed keratins, EMA and E-cadherin. The E₂ induced tumors and tumor-derived cell lines exhibited loss of chromosome 4, deletions in chromosomes 3p12.3-13, 8p11.1-21, 9p21-qter, and 18q, and gains in 1p, and 5q15-qter. The induction of complete transformation of the human breast epithelial cell MCF-10F in vitro confirms the carcinogenicity of E₂, supporting the concept that this hormone could act as an initiator of breast cancer in women. This model provides a unique system for understanding the genomic changes that intervene for leading normal cells to tumorigenesis and for testing the functional role of specific genomic events taking place during neoplastic transformation.     

Reversal of tamoxifen resistant breast cancer by low dose estrogen therapy

Currently, the standard of care for estrogen receptor (ER)-positive breast cancer is 5 years of tamoxifen (TAM) or an aromatase inhibitor (AI) such as anastrozole. New studies indicate that extending antiestrogen therapy beyond 5 years with sequential regimens will improve disease-free survival. Based on the emerging concept that longer therapies are better, we have developed sequential models of tamoxifen-resistant breast cancer in vivo to mimic the clinical scenario of long-term antiestrogen therapy. The goal of the current study was to investigate the consequences of long-term treatment with tamoxifen on the growth of breast tumors in athymic mice. The results demonstrate that there are distinct phases of resistance to tamoxifen that correlate with time of treatment and expression of HER2/neu mRNA. In the treatment phase, 17β-estradiol (E₂) stimulated growth, while TAM inhibited growth of MCF-7 tumors (MCF-7E₂). The withdrawal of treatment, mimicking the use of an AI, completely prevented growth. In Phase I resistance, the tumors (MCF-7TAMST) were growth-stimulated by either E₂ or TAM, but inhibited by no treatment, fulvestrant, or E₂ + fulvestrant. Phase II-resistant tumors (MCF-7TAMLT) were treated for more than 5 years and growth-stimulated by TAM. However, no treatment, fulvestrant, or E₂ completely inhibited growth. Interestingly, the few tumors (MCF-7TAMLT) that survived in response to E₂ were robustly re-stimulated by E₂ after transplantation into new generations of athymic mice. These E₂-stimulated tumors (MCF-7TAME) were inhibited by TAM in a dose-dependent similar to their parental tumors (MCF-7E₂). In addition, the MCF-7TAME tumors were inhibited by either no treatment or fulvestrant. HER2/neu and HER3 mRNAs were over-expressed in TAM-stimulated MCF-7TAMLT tumors and remained high in E₂-stimulated MCF-7TAME tumors. The data indicate that complete reversal of resistance to TAM can be achieved with the use of low dose E₂ therapy. Also, these data suggest that over-expression of HER2/neu alone is insufficient to predict resistance to TAM. Based on the results, we suggest using an alternating treatment regimen, cycling antiestrogen with estrogen therapy to avoid drug-resistance.     

A new Suzuki synthesis of triphenylethylenes that inhibit aromatase and bind to estrogen receptors α and β

The design and synthesis of dual aromatase inhibitors/selective estrogen receptor modulators (AI/SERMs) is an attractive strategy for the discovery of new breast cancer therapeutic agents. Previous efforts led to the preparation of norendoxifen (4) derivatives with dual aromatase inhibitory activity and estrogen receptor binding activity. In the present study, some of the structural features of the potent AI letrozole were incorporated into the lead compound (norendoxifen) to afford a series of new dual AI/SERM agents based on a symmetrical diphenylmethylene substructure that eliminates the problem of E,Z isomerization encountered with norendoxifen-based AI/SERMs. Compound 12d had good aromatase inhibitory activity (IC₅₀ = 62.2 nM) while also exhibiting good binding activity to both ER-α (EC₅₀ = 72.1 nM) and ER-β (EC₅₀ = 70.8 nM). In addition, a new synthesis was devised for the preparation of norendoxifen and its analogues through a bis-Suzuki coupling strategy.     

Estrogen receptor agonists/antagonists in breast cancer therapy: A critical review

Estrogens display intriguing tissue selective action that is of great biomedical importance in the development of optimal therapeutics for the prevention and treatment of breast cancer. There are also strong evidences to show that both endogenous and exogenous estrogens are involved in the pathogenesis of breast cancer. Tamoxifen has been the only drug of choice for more than 30 years to treat patients with estrogen related (ER) positive breast tumors. There is a need therefore, for identifying newer, potential and novel candidates for breast cancer. Keeping this in view, the present review focuses on selective estrogen receptor modulators and estrogen antagonists such as sulfatase and aromatase inhibitors involved in breast cancer therapy. A succinct and critical overview of the structure of estrogen receptors, their signaling and involvement in breast carcinogenesis are herein described.     

Estrogen receptor β 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, ERβ, which can have effects opposite to those of the well-known ‘original’ estrogen receptor (now called ER) challenges this simplistic view. In order to understand breast cancer one must first understand how the normal breast is maintained. The functions of ERβ 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 ERβ 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 ERβ in the normal and malignant breast, we have created mice in which the ERβ gene has been inactivated. Studies of the breasts of ERβ knock out mice (BERKO) revealed abnormal epithelial growth, overexpression of Ki67 and severe cystic breast disease as mice age.     

Association between estradiol, estrogen receptors, total lipids, triglycerides, and cholesterol in patients with benign and malignant breast tumors

This study addresses the correlation between the levels of estradiol (E₂), total lipids, triglycerides, and cholesterol in serum and tissue samples of age-matched patients with benign (40 cases; 16 were premenopausal and 24 were postmenopausal) and malignant (50 cases; 17 were premenopausal and 33 were postmenopausal) breast tumors. Estradiol levels were determined in serum and cytosol, estrogen receptors (ER) were assayed in cytosol, and total lipids, triglycerides and cholesterol were determined in serum and membrane fractions of all benign and malignant breast disease patients. Serum E₂ was significantly higher in malignant cases than benign ones (P<0.05) with a significant reduction (40%) in postmenopausal than premenopausal women. ER-positive tumors were significantly higher in postmenopausal women with malignant breast tumors than benign cases (P<0.05). Tissue levels of total lipids, triglycerides, and cholesterol were highly significantly increased in breast cancer women than women with benign breast diseases (P<0.05, P<0.005 and P<0.05 respectively) and they were also significantly correlated with estradiol levels. It could be concluded that the uptake of lipids from plasma by the tumor tissue is greatly correlated to estradiol and it may confirm the possible role of lipids as risk factor in breast cancer.     

Tissue estradiol is selectively elevated in receptor positive breast cancers while tumour estrone is reduced independent of receptor status

Previous studies have suggested elevated estrogen production in tumour-bearing breast quadrants as well as in breast cancers versus benign tissue. Using highly sensitive assays, we determined breast cancer tissue estrogen concentrations together with plasma and benign tissue estrogen concentrations in each quadrant obtained from mastectomy specimens (34 postmenopausal and 13 premenopausal women). We detected similar concentrations of each of the three major estrogens estradiol (E₂), estrone (E₁) and E₁S in tumour-bearing versus non-tumour-bearing quadrants. Considering malignant tumours, intratumour E₁ levels were reduced in cancer tissue obtained from pre- as well as postmenopausal women independent of tumour ER status (average ratio E₁ cancer: benign tissue of 0.2 and 0.3, respectively; p < 0.001 for both groups), suggesting intratumour aromatization to be of minor importance. The most striking finding was a significant (4.1–8.6-fold) increased E₂ concentration in ER positive tumours versus normal tissue (p < 0.05 and <0.001 for pre- and postmenopausal patients, respectively), contrasting low E₂ concentrations in ER− tumours (p < 0.01 and <0.001 comparing E₂ levels between ER+ and ER− tumours in pre- and postmenopausals, respectively). A possible explanation to our finding is increased ligand receptor binding capacity for E₂ in receptor positive tumours but alternative factors influencing intratumour estrogen disposition cannot be excluded.     

Superiority of gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol for monitoring of aromatase inhibitor therapy

Currently available radioimmunoassay methods for estradiol in serum lack sufficient sensitivity and precision to monitor estradiol levels in patients placed on third generation aromatase inhibitors. We recently validated a gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol and determined estrogen levels in normal post-menopausal women and in women with breast cancer before and during administration of aromatase inhibitors. Validation of the GC/MS/MS assay in human plasma and human serum included determination of assay sensitivity (<0.63 pg/ml), precision (all CVs less than 17.8%), recovery (98-103%), and linearity of recovery (R=0.998). Levels of estradiol were lower when assayed by GC/MS/MS compared to RIA under all conditions (7.26+/-4.82 pg/ml versus 11.9+12.0 pg/ml in normal post-menopausal women; 5.88+/-3.43 pg/ml versus 13.8+/-7.5 pg/ml in breast cancer patients prior to treatment; and<0.63 pg/ml versus 5.8+/-4.1 pg/ml during aromatase inhibitor therapy). Fifty-five women treated either with atamestane/toremiphene or letrozole/placebo were monitored for estradiol levels at 4, 8 and 12 weeks of therapy. The mean levels of estradiol during aromatase inhibitor therapy was 5.8+/-4.1 pg/ml as measured by RIA and <0.63 pg/ml by GC/MS/MS. The degree of suppression with the aromatase inhibitors as detected by RIA was 58% versus >89% by GC/MS. These results suggest that most RIA methods detect cross-reacting estrogen metabolites and yield higher measured levels than GC/MS/MS. Several pharmacological and clinical considerations suggest that GC/MS/MS should become the preferred method for monitoring aromatase inhibitor therapy.     

Design and synthesis of tamoxifen derivatives as a selective estrogen receptor down-regulator

We designed and synthesized an estrogen receptor (ER) down-regulator (5), which is a derivative of tamoxifen with a long alkyl side chain. Compound 5 effectively reduced ER protein levels in MCF-7 cells and had an antagonistic effect.     

Contributions of estrogen to ER-negative breast tumor growth

Breast cancer is a hormone-based disease with numerous factors contributing to the lifetime risk of developing the disease. While breast cancer risk is reduced by nearly 50% after one full term pregnancy, women over the age of 25 have a significantly greater risk of developing breast cancer immediately following parturition compared to their nulliparous counterparts. It is widely presumed that the increased risk of developing breast cancer following pregnancy is due to the ability of pregnancy-associated hormones to promote the further proliferation of an initiated target cell population. It is surprising however, that the majority of breast cancers that develop following pregnancy lack appreciable expression of either the estrogen or progesterone receptors. This important observation suggests that if hormones play a part in promoting breast cancer following pregnancy, they may not be doing so through direct binding to hormone receptor molecules expressed by breast cancer cells.

To reconcile this conceptual conflict we investigated the hypothesis that steroid hormones promote the outgrowth of ER-negative cancers by influencing host cell types distinct from the breast epithelium itself. We demonstrated that increasing the levels of circulating estrogens is sufficient to promote the formation and progression of ER-negative cancers while, pharmacologically inhibiting estrogen synthesis following pregnancy prevents ER-negative tumor formation. Moreover, we demonstrate that the effects of estrogen act via a systemic increase in host angiogenesis, in part through increased mobilization and recruitment of bone marrow stromal derived cells into sites of angiogenesis and to a growing tumor mass. Taken together, these data suggest that estrogen may promote the growth of ER-negative cancers by acting on cells distinct from the cancer cells to stimulate angiogenesis.