In 1896, physician George Beatson observed that removal of the ovaries caused regression of mammary tumors in women. His experiments were the first to establish a link between ovarian secretions and breast cancer. Later in the early 1930s, the “female” sex hormones, estrone and estriol, could be isolated from human pregnancy urine, followed by isolation of a third estrogen, estradiol (E2), from pig follicular fluid. Shortly thereafter, a number of estrogenic compounds were synthesized and used therapeutically, but the mechanisms of estrogen action remained obscure. However, in 1958 Elwood Jensen produced a major breakthrough when he used a radioactive marker to demonstrate that only estrogen-responsive tissues were able to concentrate injected estrogen from the blood, suggesting the existence of estrogen-binding components, which were called “estrogen receptors (ERs).” Further on, Noteboom and Gorski reported that the estrogen receptor is stereospecific and probably a protein. In 1966, the cytosolic estrogen-binding component was isolated from rat uterus. By treating the estrogen receptor with proteolytic enzymes, binding of estrogen was abolished, indicating that this component was a protein. Later in 1968, O’Malley observed changes in ovalbumin mRNA levels upon estrogen stimulation of the chick oviduct, indicating that ER functions as a transcription factor. In 1986, an ER was cloned from the uterus and its role as a ligand-dependent transcription factor was established (Welboren et al. 2007). This receptor, now known as ERα (NR3A1), was long believed to be the only existing ER that mediates estrogenic effects, until a second ER, now denoted ERβ (NR3A2), was cloned from rat prostate in 1996 (Kuiper et al. 1996). The first ERα knockout mouse was created in 1993, while the ERβ knockout mice became available in 1998. It is known today that ERs are members of the superfamily of nuclear receptors and specifically the family of steroid receptors that act as ligand-regulated transcription factors.
ER Gene and Protein Structure
ERα and ERβ are transcribed from different genes located on distinct chromosomes. The human ERα gene is located on chromosome 6q25.1 spanning a total of 140 kb, while the human ERβ gene is located on chromosome 14q23.2 spanning 60 kb. Both ERs consist of eight coding exons. In humans, ERα is expressed in the reproductive tissues (e.g., uterus, testis, breast), kidney, bone, white adipose tissue, and liver. ERβ has been found to be expressed in the ovary, prostate, lung, gastrointestinal tract, bladder, and hematopoietic and central nervous systems (Matthews and Gustafsson 2003).
Mechanisms of ER Signaling
Estrogen action is exerted in target tissues via binding to one or both of the two ERs, ERα and ERβ. Like other steroid hormone receptors, ERs act as dimers to regulate transcriptional activation. Full transcriptional activity of the ERs is thought to proceed through a synergism between two activation domains, AF-1 at the N terminus and AF-2 in the LBD. Both ERα and ERβ contain a potent AF-2 function, but unlike ERα, ERβ seems to have a weaker corresponding AF-1 function and thus depends more on the ligand-dependent AF-2 for its transcriptional activation function (Dahlman-Wright et al. 2006). These AFs have been shown to exhibit distinct transactivation properties that depend on both cell and promoter contexts.
In addition to these ligand-induced transcriptional activities of ERs, ligand-independent pathways to activate ERs have been described. Growth factor signaling or stimulation of other signaling pathways leads to activation of kinases that can phosphorylate and thereby activate ERs or associated coregulators in the absence of ligand. For example, it has been shown that the HER2 downstream signaling molecules ERK1 and ERK2 can phosphorylate ER, leading to ligand-independent receptor activation (Martin et al. 2005).
Genome-Wide Profiling of ER Gene Expression Programs
There have been a number of studies in the past few years aimed at comprehensively unraveling the complete estrogen-regulated gene expression programs in cancer cells. These reports can be attributed to the introduction of microarrays for global gene expression profiling. DNA microarray technology allows quantitative monitoring of changes in the expression of thousands of genes simultaneously and has been described in several configurations including oligonucleotide arrays and microarrays of cDNAs spotted on glass slides. During the past few years, the development of high throughput DNA sequencing (HTS) methods for global gene expression profiling, also known as “RNA-Seq,” has challenged microarray technology because of its superior capability for detection of low-expressed genes, alternative splice variants, and novel transcripts (Cloonan et al. 2008). However, to our knowledge, no studies that explore HTS to assay genome-wide transcriptional regulation by estrogen have been reported.
Several reports have described the gene expression profiles in ERα-expressing breast cancer cell lines in response to E2 treatment. The available studies have reported different numbers of E2/ERα-regulated genes in MCF7 breast cancer cells, ranging from ∼200 to ∼1500. These discrepancies can be attributed to differences in the length of the E2 treatment, application of different microarray platforms and different analysis strategies (Kininis and Kraus 2008). Two studies that aimed to identify E2/ERα-direct targets by short-term E2 treatment (3 h) in MCF7 cells identified similar numbers of E2 target genes. In one of the studies, 122 genes were identified as stimulated by E2 and 95 genes were identified as inhibited by E2 (Kininis et al. 2007). In the other study, 134 genes were up-regulated and 141 genes were down-regulated after E2 treatment (Carroll et al. 2006). However, a comparison of the E2-regulated genes between the studies has not been reported. Overall, gene expression profiling and candidate gene analysis have identified several well-known estrogen-regulated genes in breast cancer cells such as TFF1, CCND1, IGFBP4, C3, ADORA1, GREB1, and MYC. Furthermore, gene expression profiling has identified categories of genes regulated by estrogen, including those that modulate the cell cycle, transcriptional regulation, morphogenesis, and apoptosis, compatible with a role of estrogen in inducing breast cancer cell proliferation and survival (Carroll and Brown 2006).
Due to the lack of high levels of endogenous ERβ in breast cancer cell lines, gene expression profiling studies aimed at revealing the genes regulated by ERβ have been performed in breast cancer cell lines stably expressing ERβ. Of the categories of genes down-regulated by ERβ, the “regulation of cell proliferation” category was the most overrepresented one, consistent with the observations that ERβ expression was associated with suppression of breast cancer cell proliferation (Williams et al. 2008). Gene expression profiles for ER-subtypes showed that ERα and ERβ share some common target genes, although each receptor also appears to have distinct sets of downstream target genes.
ER and Breast Cancer
Estrogen is essential for growth and development of the mammary glands and has been associated with promotion and growth of breast cancer. ERβ is found in both ductal and lobular epithelial and stromal cells of the rodent, whereas ERα is only found in the ductal and lobular epithelial cells and not in stroma. The presence of significant amounts of ERα in breast cancer at the time of diagnosis is taken as an indication of hormone dependence. On this basis, treatment with ERα antagonistic compounds, such as tamoxifen, is first line for adjuvant therapy. ERα is also an important prognostic factor in breast cancer. ERα-negative breast cancers are associated with poor prognosis and a more aggressive phenotype (Spears and Bartlett 2009).
To date, ERβ expression in normal human breast and breast cancer specimens and the relationship between ERβ and clinicopathological features and response to endocrine treatment has been extensively investigated at both mRNA and protein levels. Overall, these studies suggest a protective role of ERβ in breast cancer development. ERβ is lost in a majority of breast tumors, which has been shown to be correlated with ERβ promoter methylation in breast cancer cells (Zhao et al. 2003). Promoter methylation is frequently observed for cancer suppressor genes. Several studies have demonstrated that ERβ is an important modulator of proliferation and invasion of breast cancer cells, thus supporting the hypothesis that loss of ERβ expression could be one of the events leading to breast cancer development. Currently, only the ERα form is measured for clinical decision-making and treatment of breast cancer patients.
SERMs in Breast Cancer Treatment
Selective estrogen receptor modulators, referred to as SERMs, are a class of compounds with mixed ER-agonist/antagonist activities. Tamoxifen is the most commonly used SERM for treating all stages of ERα-positive breast cancer. In primary breast cancer, adjuvant tamoxifen significantly decreases relapse rates and mortality in pre- and postmenopausal patients, and the therapy benefit from 5 years of adjuvant tamoxifen is maintained, even >10 years after the primary diagnosis. Tamoxifen has also been used as a chemopreventive agent in women who have high risk for breast cancer. The mechanism behind the anti-tumorigenic function of tamoxifen is through competing with estrogens for the LBD of ERα, thereby inhibiting ERα-mediated mitogenic estrogen signaling in the breast. However, tamoxifen has been shown to have an agonistic effect on endometrium and thus may increase the risk for endometrial cancer.
Raloxifen is a newly developed SERM and has been shown to be as effective as tamoxifen in reducing the incidence of breast cancer in postmenopausal women who are at increased risk of the disease. Because of its nonproliferative effects in the uterus and estrogenic effects on bone, this drug has also been approved for prevention of osteoporosis in high-risk women. Aromatase inhibitors, which potently suppress estrogen synthesis in postmenopausal women, are now considered to be more effective in treating metastatic breast cancer in postmenopausal women than tamoxifen. Fulvestrant (ICI 182,780) is a new class of compounds for endocrine treatment, functioning as a pure ER-antagonist with no agonist effects.
Despite substantial improvements in the treatment of breast cancer, resistance to therapy remains a major clinical problem. Approximately one third of the patients with ERα-positive tumors fail to respond to tamoxifen treatment due to intrinsic or de novo resistance of the tumor. Furthermore, even patients who initially respond eventually acquire tamoxifen resistance, leading to tumor progression and death. Several mechanisms have been proposed to account for the observed resistance, including changes in the expression of ERα or ERβ, altered levels of co-regulatory proteins, and the influences of cellular kinase signal transduction pathways (Ring and Dowsett 2004). Today, circumvention of endocrine resistance represents a major challenge for clinicians and cancer researchers.
Estrogens are thought to exert their biological effects predominantly via ERs through their interactions with DNA. Interestingly, more evidence has indicated that estrogens, like androgen and progesterone, can exert nongenomic effects. Nongenomic steroid activity typically involves the rapid induction of secondary messengers of signal transduction cascades via membrane-localized ERs, including activation of protein kinase A (PKA), protein kinase C (PKC), and MAP kinase. Interestingly, recent studies have suggested that GPR30 functions as a novel type of extranuclear ER that mediates nongenomic estrogen signaling. It is still possible that additional membrane receptors for estrogen are involved in mediation of the nongenomic estrogen action. The mechanistic details of activation through these nongenomic pathways, such as target genes, remain to be characterized.
Gene expression profiling has furthered our understanding of the role of ERs and provided important glimpses into the molecular basis of ER-mediated estrogen action in target cells. Future studies will need to explore time courses of estrogen-regulated genes thus facilitating the identification of primary, secondary, and higher order estrogen-regulated genes, finally revealing the gene regulatory networks affected by estrogen signaling.
Currently only ERα status is an important marker for routine prognostic and predictive evaluation in breast cancer. Over the last decade, the role of ERβ in estrogen action in breast cancer involving cooperation, as well as competition with ERα, has been revealed. Future studies should include validation of ERβ as a target for breast cancer and exploration of ERβ as a marker for clinical decision-making and treatment. The discovery of clinically useful ERβ-agonists might hold great promise. Furthermore a better understanding of the biological pathways of tamoxifen resistance should allow the circumvention of tamoxifen resistance using rational therapeutic approaches.
- Kininis M, Chen BS, Diehl AG, Isaacs GD, Zhang T, Siepel AC, et al. Genomic analyses of transcription factor binding, histone acetylation, and gene expression reveal mechanistically distinct classes of estrogen-regulated promoters. Mol Cell Biol. 2007;27:5090–104.PubMedPubMedCentralCrossRefGoogle Scholar