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.     

Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance

Estrogen receptors (ERs) mediate most of the biological effects of estrogen in mammary and uterine epithelial cells by binding to estrogen response elements in the promoter region of target genes or through protein-protein interactions. Anti-estrogens such as tamoxifen inhibit the growth of ER-positive breast cancers by reducing the expression of estrogen-regulated genes. However, anti-estrogen-resistant growth of ER-positive tumors remains a significant clinical problem. Here we show that phosphatidylinositol (PI) 3-kinase and AKT activate ERalpha in the absence of estrogen. Although PI 3-kinase increased the activity of both estrogen-independent activation function 1 (AF-1) and estrogen-dependent activation function 2 (AF-2) of ERalpha, AKT increased the activity of only AF-1. PTEN and a catalytically inactive AKT decreased PI 3-kinase-induced AF-1 activity, suggesting that PI 3-kinase utilizes AKT-dependent and AKT-independent pathways in activating ERalpha. The consensus AKT phosphorylation site Ser-167 of ERalpha is required for phosphorylation and activation by AKT. In addition, LY294002, a specific inhibitor of the PI 3-kinase/AKT pathway, reduced phosphorylation of ERalpha in vivo. Moreover, AKT overexpression led to up-regulation of estrogen-regulated pS2 gene, Bcl-2, and macrophage inhibitory cytokine 1. We demonstrate that AKT protects breast cancer cells from tamoxifen-induced apoptosis. Taken together, these results define a molecular link between activation of the PI 3-kinase/AKT survival pathways, hormone-independent activation of ERalpha, and inhibition of tamoxifen-induced apoptotic regression.     

Estrogen-related receptors-stimulated monoamine oxidase B promoter activity is down-regulated by estrogen receptors

Although there are studies published about the neuroprotective effect of estrogen, little is known about the mechanisms and cellular targets of the hormone. Recent reports demonstrate that estrogen down-regulates the expression of monoamine oxidase A and B (MAO-A and MAO-B) in the hypothalamus of the Macaques monkey, both of which are key isoenzymes in the neurotransmitter degradation pathway. Additionally, estrogen-related receptor alpha (ERRalpha) up-regulates MAO-B gene expression in breast cancer cells. ERRalpha recognizes a variety of estrogen response elements and shares many target genes and coactivators with estrogen receptor alpha (ERalpha). In this study, we investigate the interplay of ERs and ERRs in the regulation of MAO-B promoter activity. We demonstrate that ERRalpha and ERRgamma up-regulate MAO-B gene activity, whereas ERalpha and ERbeta decrease stimulation in both a ligand-dependent and -independent manner. Ectopically expressed ERRalpha and ERRgamma stimulate the expression of MAO-B mRNA and protein as well as increase the MAO-B enzymatic activity in ER-negative HeLa cells. The ability of ERRs to stimulate MAO-B promoter activity was reduced in ER-positive MCF-7 and T47D cells. Several AGGTCA motifs of the MAO-B promoter are responsible for up-regulation by ERRs. Interestingly, ERalpha or ERbeta alone have no effect on MAO-B promoter activity but can down-regulate the activation function of ERRs, whereas glucocorticoid receptor does not. By using chromatin immunoprecipitation assay, we demonstrate that ERs compete with ERRs for binding to the MAO-B promoter at selective AGGTCA motifs, thereby changing the chromatin status and cofactor recruitment to a repressed state. These studies provide new insight into the relationship between ERalpha, ERbeta, ERRalpha, and ERRgamma in modulation of MAO-B gene activity.     

Activation function-1 domain of estrogen receptor regulates the agonistic and antagonistic actions of tamoxifen

The antiestrogen tamoxifen has been widely used for decades as selective estrogen receptor (ER) modulator for ERalpha-positive breast tumors. Tamoxifen significantly reduces tumor recurrence by binding to the activation function-2 (AF-2) domain of the ER. Acquired resistance to tamoxifen in breast cancer patients is a serious therapeutic problem. Antiestrogen-resistant breast cancer often shows increased expression of the epidermal growth factor receptor (EGFR) family members, EGFR and ErbB2. In this report we now show that overexpression of EGFR or activated AKT-2 in MCF-7 cells leads to phosphorylation of Ser167 in the AF-1 domain of ERalpha, enhanced ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of tamoxifen, and resistance to tamoxifen. In contrast, transfection of activated MAPK kinase, an immediate upstream activator of MAPK (ERK 1 and 2) into MCF-7 cells leads to phosphorylation of Ser118 in the AF-1 domain of ERalpha, inhibition of ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of Tam, and maintenance of sensitivity to tamoxifen. Inhibition of AKT by short inhibitory RNA blocked Ser167 phosphorylation in ER and restored tamoxifen sensitivity. However, maximum sensitivity to tamoxifen was observed when both AKT and MAPK were inhibited. Taken together, these data demonstrate that different phosphorylation sites in the AF-1 domain of ERalpha regulate the agonistic and antagonistic actions of tamoxifen in human breast cancer cells.     

Estrogen receptor alpha/beta isoforms, but not betacx, modulate unique patterns of gene expression and cell proliferation in Hs578T cells

The actions of 17beta-estradiol (E2) and selective estrogen receptor modulators (SERMs) have been extensively investigated regarding their ability to act through estrogen receptor-alpha (ERalpha) to perturb estrogen receptor positive (ER+) breast cancer (BC) growth. However, many BCs also express ERbeta, along with multiple estrogen receptor (ER) splice variants such as ERbetacx, an ERbeta splice variant incapable of binding ligand. To gain a more comprehensive understanding of ER action in BC cells, we stably expressed ERalpha, ERbeta, or ERbetacx under doxycycline (Dox) control in Hs578T cells. Microarrays performed on E2 or 4OH-tamoxifen (4HT) treated Hs578T ERalpha and ERbeta cells revealed distinct ligand and receptor-dependent patterns of gene regulation, while the induction of ERbetacx did not alter gene expression patterns. E2 stimulation of Hs578T ERbeta cells resulted in a 27% decrease in cellular proliferation, however, no significant change in proliferation was observed following the exposure of Hs578T ERalpha or ERbeta cells to 4HT. Expression of ERbetacx in Hs578T cells did not effect cellular proliferation. Flow cytometry assays revealed a 50% decrease in E2-stimulated Hs578T ERbeta cells entering S-phase, along with a 17% increase in G0/G1 cell-cycle arrest. We demonstrate here that ERalpha and ERbeta regulate unique gene expression patterns in Hs578T cells, and such regulation likely is responsible for the observed isoform-specific changes in cell proliferation. Hs578T ER expressing cell-lines provide a unique BC model system, permitting the comparison of ERalpha, ERbeta, and ERbetacx actions in the same cell-line.