Estrogen signaling pathway and hormonal therapy
Hormonal therapy, such as estrogen-targeting therapy, has undergone remarkable development in recent several years, using drugs such as LH–RH agonists, new SERMs and third-generation aromatase inhibitors. Several ongoing large-scale international clinical trials for hormonal therapy are establishing the standard protocol for treatments with these drugs. On the other hand, there have been attempts to predict the individual efficacy of hormonal therapy using classical molecular biomarkers such as ER and PgR. However, approximately one-third of ERα-positive patients do not respond to endocrine therapy, while some ERα-negative patients are responsive. These discrepancies may be due to the different estrogen-related intracellular signaling pathways in breast cancer cells. Furthermore, the ineffectiveness of hormonal therapy in some individuals (due to, for example, aromatase inhibitor resistance) may be caused by these mechanisms. In this paper, we discuss the molecular mechanisms of these different responses to hormonal therapies and their implications for the estrogen signaling pathway in breast cancer cells. Furthermore, we touch upon basic studies into predicting the efficacy of hormonal therapy and new strategies in this field.
KeywordsEstrogen Hormonal therapy SERM Aromatase Phosphorylation
Intracellular estrogen signaling pathways
Estrogen signals via ligand-dependent genomic pathways
Estrogen exerts its biological effects by binding to ER, which mainly exists in the nucleus as a member of the nuclear receptor superfamily of transcription factors. ER acts through the formation of homo- or heterodimers of ERα and ERβ, and ERα has been widely used as a predictive marker for hormonal therapy. In the classical pathway, estrogen-bound ERs dimerize and function as a transcription factor which binds to a specific DNA sequence named the estrogen response element (ERE) present in the promoter or enhancer regions of target genes. ER binds to ERE through its DNA-binding domain (DBD) and recruits coactivators such as SRC-1, AIB1 and p300/CBP to form a functional ER complex [8, 9]. It is now known that ER target genes which have full or half ERE sites include pS2, cathepsin D, PgR, Efp, EGR3, etc. [10, 11, 12, 13].
In the genomic pathway, ER can also regulate transcription without binding directly to DNA. ER acts as a coactivator—ER interacts with other transcription factors such as AP-1, SP-1 and NF-κB via protein–protein interactions, and it could regulate the transcription of genes that lack ERE but has a binding element for its partner’s protein. It has been suggested that genes activated in this way include ovalbumin, IGF-1, collagenase, VEGF, c-Myc, cyclin D1, c-fos, NF-κB, and LDL receptor [14, 15, 16].
Activation of ER by protein phosphorylation
Estrogen signals via nongenomic pathways
Estrogen also exerts rapid effects that are not accounted for by transcriptional mechanisms. As some signals from several ligands for the steroid receptor superfamily have been shown to be mediated through plasma membrane, there is accumulating evidence to support the idea that estrogen receptors are also located at the plasma membrane and are responsible for extranuclear, rapid and nongenomic actions. The existence of membrane ER has been demonstrated using cell-membrane impermeable BSA-conjugated estradiol [24, 25], although some concerns have been pointed out regarding these agents. Membrane ER has been reported to associate with many growth factor receptors, such as IGF-1R, EGFR, HER2. In the activation of IGF-1R, E2 induces the formation of a ternary complex among ERα, IGF1-R and Shc, the adaptor protein, in the plasma membrane, which induces phosphorylation of IGF-1R . The estrogen-bounded membrane ER rapidly activates several signals in a cell type-specific manner, including calcium currents, cAMP, inositol phosphate, G proteins, Src, and Shc, which leads to the activation of downstream kinases, such as mitogen-activated protein kianse (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt .
Although the origin of membrane ER is not completely resolved, most of the evidence supports the idea that the membrane ER is the same protein as the nuclear ER, based on evidence such as the detection of membrane ER by antibodies to nuclear ER and the codetection of membrane and nuclear ERs after nuclear ER cDNA transfection in ER null cells. In either case, the membrane ER was only a small fraction of the total ER. Recently, several reports have suggested that GPR30, a G protein-coupled receptor, is another candidate for the rapid estrogen signals [27, 28]. However, one report indicated that estrogen signals could not be induced in human breast cancer SKBR-3 (ER negative, GPR30 positive) cell signals , and further studies are needed to clarify the role of GPR30 in rapid signaling by estrogen at the plasma membrane in breast cancer cells.
ER does not have a transmembrane domain, and is localized at the inner face of the plasma membrane through binding to other proteins such as caveolin-1 , the major structural protein of caveolae. Shc and growth factor receptors also facilitate the membrane localization of ER as described above, and the cytoplasmic proteins MTA1-S or MNAR (modulator of the nongenomic activity of ER)/PELP1 can sequester ER outside of the nucleus [31, 32, 33]. Overexpression of HER2 in ER-positive breast cancer cells, which is associated with the development of hormone-resistant breast cancer, promotes the cytoplasmic sequestration of ER, and the interaction of ER with HER2 activates mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (ERK1/2) .
As described above, the molecular mechanism of nongenomic estrogen action has not yet been established. Moreover, the clinical significance of this action has not been assessed in breast cancer, because of methodological issues.
Extracellular factors for estrogen signal stimulation
Recent data have demonstrated that the intercellular communication between tumor and stromal cells profoundly affects the proliferation of breast cancer cells, which is mediated by the production of estrogen, growth factors, chemokines, and cytokines [35, 36, 37]. Among the stromal cells, fibroblasts are the most abundant cell type, and tumor-associated fibroblasts are known to express aromatase, a key enzyme of estrogen synthesis, resulting in the intratumoral estrogen production frequently observed in postmenopausal breast cancers [38, 39]. Aromatase expression levels in breast cancer tissues are significantly higher than those in benign breast lesions. Several reports suggest that the tumor–stromal interaction regulates aromatase gene expression via the production of various factors such as prostaglandin E2, COX2, tumor necrosis factor-α, interleukin-6 and interleukin-11 . Therefore, aromatase is a target of endocrine therapy for breast cancers, and several aromatase inhibitors have been developed to attenuate estrogen biosynthesis, such as anastrozole, letrozole, and exemestane. The recent reports on the efficacies of these drugs again strongly indicate that the estrogen is a crucial factor in the survival of breast cancer .
ERE-GFP reporter cells (E10) and estrogen-depletion resistance
To investigate estrogen signaling in breast cancer and in order to improve predictions of the efficacy of aromatase inhibitors for individual breast cancers, we developed a comprehensive system to visualize the ER-activating ability (not only of estrogen, but also of other crosstalk signals, as described above) stimulated by the adjacent microenvironment. We first established a stable transformant, named E10, of human breast cancer MCF-7 cells which had an integrated ERE-GFP reporter gene. These E10 cells express GFP when endogenous ERα is activated. We characterized the stromal fibroblasts of individual breast cancers by establishing a co-culture system with E10 cells, and found that the induced GFP expression varied among the cases, indicating that the stromal fibroblasts in each case had their own properties with respect to the activation of estrogen signals .
Prediction of hormonal therapy
Recent advances in molecular diagnostic tools such as Oncotype DX (Genomic Health Inc.) and MammaPrint (Agendia Inc.) have opened up a new vista for personalized medicine in the future. On the other hand, a more accurate predictive method or a novel predictive biomarker from among ER and PgR is also desired for predicting the individual efficacy of hormonal therapy. To address this issue, we studied the gene expression profiles of estrogen-responsive genes in breast cancer using a DNA microarray technique , and further study revealed several candidate genes for assessing response to hormonal therapy . Recently, a molecular signature of neoadjuvant hormonal therapy for ER-positive patients has been reported . The identified 50-gene subset could be useful for monitoring the short-term effects of hormonal therapy on ER-positive cancers. A new predictive biomarker for hormonal therapy may be found in these genes. Further studies using comprehensive techniques such as microarray analysis to search for new biomarkers will be common in the future.
However, these expression analyses are not able to reveal the specific pathway of estrogen signaling in the cells. Other novel methods, such as phosphorylation-specific proteomics or the in vitro bioassay system using GFP that we reported previously , will be needed to analyze the signaling pathway at work in individual cancer cells. In order to be able to predict the individual response to aromatase inhibitor or SERMs, the ability to distinguish between a so-called ligand (estrogen)-independent/ERα-dependent signaling pathway and a ligand-dependent/ERα-dependent signaling pathway is crucial.
The estrogen signaling pathway must be closely associated to the efficacy of hormonal therapy. The discrepancy between ERα expression status and response to hormonal therapy probably derives from the different estrogen signaling pathway conditions in the cells. The resistance to aromatase inhibitor or antiestrogen may be also caused by changes in this pathway. The last two decades have revealed the structure and function of the estrogen receptor to be a nuclear transcription factor which controls its target genes and plays an important role in various physiologic functions. However, as described in this paper, there are still many questions about and ambiguities regarding the estrogen signaling pathway. In particular, the elucidation of the mechanism of ligand-independent ER-activation and nongenomic estrogen action is extremely important for translational research in breast cancer. Further studies of the estrogen signaling pathway may provide new clues about the estrogen-dependent mechanisms of breast cancer development, and may dramatically advance the accuracy with which the efficacy of molecular therapeutics can be predicted, as well as new strategies in this field.
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