How does the estrogen receptor work?

In breast cancer, interruption of estrogen receptor (ER)-α function is an effective therapeutic strategy. Despite the clinical benefit of interruption of ER-α function, the precise biological action of ER-α in breast tumors is not completely understood. Results of a recent study show that ER-α promotes growth of breast cancer cells by targeting expression of signaling components of the insulin-like growth factor system. Intriguingly, the authors of this study raise the possibility that unliganded ER-α itself may affect gene expression and breast cancer biology, and they suggest a potential mechanism for ER-α to stimulate proliferation in breast cancer.

gators have attempted to express ER-α in breast cancer cell lines to test the hypothesis that maintenance of ER-α expression in breast tumors would restore estrogen responsiveness and tumor sensitivity to endocrine therapy. In 1992, Jiang and Jordan expressed ER-α in the ER-negative breast cancer cell line MDA-MB-231 [6]. Paradoxically, they discovered that estradiol inhibited, rather than stimulated, cell proliferation. In spite of these findings, both the action of an estrogen-responsive reporter construct and the expression of an endogenous estrogen-responsive gene could be stimulated by estrogen. In the same year, Garcia et al. demonstrated that MDA-MB-231 cells transfected with ER-α had decreased metastatic potential in the presence of estradiol when injected into the tail vein of athymic, ovariectomized nude mice [7]. Subsequent studies that introduced ER-α into breast epithelial cell lines that do not express the receptor have shown similar inhibitory effects of estradiol on cell growth [8][9][10]. However, expression of ER-α in ER-α-negative breast cancer cell lines does not appear to simulate ER-α function. Thus, the mechanism of ER-α function in breast cancer cells has not been elucidated by these studies.
Oesterreich and her colleagues report the creation of a MCF7 cell line that is unresponsive to estrogen [11]. The MCF7 parent cells are ER-α-positive and proliferate in response to estradiol. After starving the cells of estrogen for nine months, the MCF7 cells no longer expressed ERα or proliferated in response to estradiol. Compared to other studies, Oesterreich et al. are the first authors to demonstrate that stable expression of ER-α in a hormonally unresponsive breast cancer cell line re-established estrogen control over proliferation. Why did the introduction of ER-α into ER-negative breast cancer cells in Oesterreich's study reconstitute estrogen-dependent growth, while previous studies have shown growth inhibition by estrogen? These conflicting results show that celldependent effects of estradiol do not depend solely on ER-α expression. The cellular pool of regulatory factors probably influence transcriptional activity of ER-α, and determine the pattern of gene expression. The cells used in Oesterreich's study were initially derived from cells possessing an ER-α-positive, hormonally responsive phenotype. In contrast, MDA-MB-231 cells are not ER-positive or hormonally responsive to estrogen. Even though the MCF7 cells used in the study did not express ER-α, the cells must express essential endogenous coregulatory factors that are required for induction of genes involved in estradiol-induced proliferation. What are the genes regulated by estradiol in this system?
The insulin-like growth factor signaling system is a target of ER-α α action Intriguingly, Oesterreich et al. found that expression of ER-α was directly correlated with expression of the key components of the insulin-like growth factor (IGF) signal-ing system, including the type 1 IGF-receptor and insulinreceptor substrate-1 [11]. Considerable crosstalk has already been documented between the signaling pathways of estrogen and the IGFs [12]. A potent mitogen, IGF-I, in breast cancer cells can act synergistically with estrogen to stimulate ER-α-positive breast cancer cell proliferation [13]. It has also been suggested that signaling components of the IGF-I system are regulated by estrogen [14]. Here, loss of ER-α was associated with decreased expression of proteins in the IGF-I signaling system, diminished IGF signal transduction, and no growth response to estrogen or IGF-I. Re-expression of ER-α in these cells restored expression of molecules critical to IGF signaling as well as the proliferative response to estrogen. These results show that ER-α is a critical regulator of IGF-Iinduced proliferation. In the absence of ER-α, key components of the IGF signaling pathway are lost. Thus, one way that ER-α can stimulate breast cancer growth is by maintaining and regulating responsiveness to the IGFs.
Even though re-introduction of ER-α into the cells restored the hormone-responsive proliferative phenotype, the cells did not completely reconstitute the ER-α phenotype. For example, estrogen treatment failed to induce expression of a key endogenous estrogen-responsive gene, the progesterone receptor gene. Since the cells were derived from MCF7 cells that have been shown to induce progesterone receptor expression following estrogen treatment, one would hypothesize that the cells would express the proper combination of coregulatory factors required for induction of the progesterone receptor gene. Thus, expression of ER-α in the ER-α-negative breast cancer cell line used by Oesterreich et al. [11] was still not able to restore all hormone-dependent features of estrogen.
This study also highlights a paradox concerning the biological role of ER-α in breast tissue. Cells expressing ER-α require the receptor for growth and can be growthretarded by inhibition of the receptor. However, cells that originally expressed ER-α in Oesterreich's study [11], and were subsequently selected to lose its expression, appeared to have a growth advantage. It is possible that other growth regulatory pathways were upregulated while cells were selected for the loss of ER-α. For example, in human breast cancer specimens, ER-α expression correlates inversely with epidermal growth factor receptor (EGFR). A study using MCF7 cells overexpressing EGFR demonstrated that cell growth rate could be augmented by omitting estrogen from the growth media, indicating that ER-α can be a growth inhibitor under some circumstances [15]. Recently, hyperactivation of mitogenactivated protein kinase was shown to downregulate ER-α expression in breast cancer cells that were overexpressing EGFR [16]. Considering this, perhaps other growth stimulatory pathways, such as the epidermal growth factor system, became upregulated in the ER-α-negative cells used in Oesterreich's study [11] and provided an alternative growth stimulus for estrogen and the IGF-I system.

Does ER-α α affect gene expression in the absence of estrogen?
Since ER-α-positive tumors generally exhibit a less aggressive phenotype than ER-α-negative tumors, ER-α is considered to be a differentiation marker in breast cancer. While Oesterreich et al. showed that re-expression of the receptor restored estrogen-and IGF-mediated growth, their data also suggested that re-expression of ER-α tended to suppress proliferation compared to cells that were selected to lose ER-α expression [11]. The observation that unliganded ER-α affected gene expression and biological function shows that ER-α itself can influence breast cancer cells, even in the absence of estrogen. Although it is possible that low concentrations of estrogen in the media were responsible for the restored gene expression, it seems unlikely because the cells were maintained in serum stripped of endogenous steroids. A previous study has shown that ligand binding to ER-α is not required for receptor dimerization or binding of the receptor to its response elements [17]. Recent findings have also suggested that ER-β and ER-α have ligand-independent effects on motility and migration when expressed in MDA-MB-231 breast cancer cells [18].
Although the biological effects of estrogens on the breast are well known, the specific targets of ER-α to promote growth are largely unknown. Oesterreich et al. have provided two pieces of evidence suggesting how ER-α stimulates proliferation in breast cancer cells [11]. First, the components of the IGF-I signaling system are targets of ER-α action. Cells that lose ER-α expression also lose the IGF signaling pathway. These results provide the clearest demonstration of the crosstalk between estrogen and the IGF-I pathway. Second, ligand-independent actions of ER-α on cell biology are suggested by the finding that expression of the type 1 IGF-receptor and insulin-receptor substrate-1 were restored when ER-α expression was restored. It also appears that cells which have lost ER-α expression grow faster than their ER-α-positive parent cells. One possible explanation for this finding is that unliganded ER-α may affect expression of genes involved in proliferation or differentiation. Oesterreich et al. have clearly shown that expression of ER-α by itself influences expression of IGF signaling components, and that activation of receptor further stimulates gene expression [11]. Are there an opposing set of growth inhibitory genes that are repressed by activation of ER-α? Indeed, the idea that estrogen functions to cancel growth inhibitory signals has also been suggested by another recent study [19].
The results of Oesterreich et al. [11] challenge the dogma that ER-α only depends on ligand to affect gene expression, and introduce the possibility that unliganded ER-α itself may have important biological effects. Clinically, this suggests that inhibition of ER-α function could have substantially different biological effects than loss of ER-α expression.