Alternative splicing of the estrogen receptor primary transcript normally occurs in estrogen receptor positive tissues and cell lines

Several laboratories have described estrogen receptor mRNA variants created by skipping internal exons. Some of the putative proteins encoded for by these variants have been functionally characterized by transfection analyses. The variant lacking exon 5 would lead, if translated, to a truncated receptor which shows dominant positive transactivation activity in the absence of hormone. It has been postulated that the variant could account for anti-estrogen resistant tumor growth and for expression of the progesterone receptor in estrogen receptor negative tumors. In order to understand the possible role this and other variants may have in the tumorigenesis of mammary tissue we have carried out a thorough analysis of variants expressed in a tumor cell line (MCF-7), in a tumor sample and in a sample of normal breast tissue derived from mammary reduction surgery. We performed rt-PCR analyses followed by hybridization with exon specific oligonucleotide probes. By these means we have detected nine different variants co-expressed in MCF-7 cells and at least the major variants were equally expressed in normal and neoplastic breast tissue. The same is true for the variant lacking exon 5 which, however, resulted to be a variant of low expression in the three samples analyzed. Variant formation appeared to be restricted to the estrogen receptor messenger since several other members of the superfamily of nuclear receptors did not show variant formation. We also have analyzed the effect of the most abundantly expressed variant, the exon 4 lacking variant, on normal estrogen receptor function, on the growth and on the response to estradiol and to tamoxifen of MCF-7 cells. Although over-expressed at high levels this variant has, if any, only marginal effects on the expression of endogenous estrogen regulated genes and on growth and response to the hormone and its antagonist. Although the lack of function of this variant cannot be extrapolated to other variants, their involvement in tumor formation appears rather unlikely since they are also expressed in normal tissue and the single variant is expressed in addition to many others, some of which might have opposing effects. Variant formation is, however, specific for the estrogen receptor and apparently regulated with tissue specificity as our expression analysis in normal mouse tissues shows. Therefore the variants probably have a physiological significance yet to be discovered.     

Estrogen receptor variants and mutations

There is a large and increasing body of experimental and clinical data supporting the existence of variant estrogen receptor (ER) proteins in both normal and neoplastic estrogen target tissues, including human breast. Therefore, future examination of ER signal transduction and/or measurement of ER protein must take into account variant ER expression. The functions of variant ER proteins, either physiological or pathological, remain unclear, although a role(s) for some ER variants in breast tumorigenesis and breast cancer progression would be consistent with the accumulated data. Possible tissue specific expression leads to the speculation that ER variants may have a role in tissue specific estrogen action. The following review focuses on the current knowledge available in the scientific literature with respect to the type and characteristics of estrogen receptor variants and mutations that have been identified to occur naturally in tissues and cell lines.     

A yeast-based bioassay for the determination of functional and non-functional estrogen receptors

The response to endocrine therapy of breast cancer is not entirely predictable from hormone receptor status alone since some point mutated or splicing variants of the estrogen receptor (ER) show altered biological activities. In order to characterize the activities of all forms of ER in a heterogeneous breast tumor, a functional assay in Saccharomyces cerevisiae was developed. Total RNA isolated from breast cancer cells and one breast cancer specimen was reverse transcribed and the ER cDNA was amplified by PCR. The products were then cloned into an expression vector by in vivo homologous recombination in yeast. The yeast strain carries a reporter gene ( ADE2 ) coupled to an estrogen response element. Activation of the reporter by ER yielded white colonies whereas lack of ER activity produced red colonies. This permitted the testing for functionality of individual ER molecules and subsequent analysis by rescuing of the ER expression plasmids and complete DNA sequencing. This simple visual test allows discrimination between wild-type ER, constitutively active ER and inactive ER.     

The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer

Extracts containing wild-type or mutant human estrogen receptor (ER) have been used to study the binding of ER to its responsive element (ERE). Estradiol (E2) or the antiestrogen hydroxytamoxifen is required for ER binding as assayed by gel retardation. The DNA binding domain (DBD) encompasses the highly conserved region C. Both intact ER-E2 complexes and ER mutants truncated for the hormone binding domain (HBD) bind as dimers to an ERE. However, an HBD-truncated ER binds less tightly to an ERE than an intact ER-E2 complex. The DBD and HBD contain a constitutive and a stronger ER-induced dimerization function, respectively. Thus, in addition to inducing the activation function associated with the HBD, estrogen plays a crucial role in the formation of stable ER dimers that bind tightly to ERE.     

Estrogen receptor-alpha 36 mediates the anti-apoptotic effect of estradiol in triple negative breast cancer cells via a membrane-associated mechanism

17β-Estradiol can promote the growth and development of several estrogen receptor (ER)-negative breast cancers. The effects are rapid and non-genomic, suggesting that a membrane-associated ER is involved. ERα36 has been shown to mediate rapid, non-genomic, membrane-associated effects of 17β-estradiol in several cancer cell lines, including triple negative HCC38 breast cancer cells. Moreover, the effect is anti-apoptotic. The aim of this study was to determine if ERα36 mediates this anti-apoptotic effect, and to elucidate the mechanism involved. Taxol was used to induce apoptosis in HCC38 cells, and the effect of 17β-estradiol pre-treatment was determined. Antibodies to ERα36, signal pathway inhibitors, ERα36 deletion mutants, and ERα36-silencing were used prior to these treatments to determine the role of ERα36 in these effects and to determine which signaling molecules were involved. We found that the anti-apoptotic effect of 17β-estradiol in HCC38 breast cancer cells is in fact mediated by membrane-associated ERα36. We also showed that this signaling occurs through a pathway that requires PLD, LPA, and PI3K; Gαs and calcium signaling may also be involved. In addition, dynamic palmitoylation is required for the membrane-associated effect of 17β-estradiol. Exon 9 of ERα36, a unique exon to ERα36 not found in other identified splice variants of ERα with previously unknown function, is necessary for these effects. This study provides a working model for a mechanism by which estradiol promotes anti-apoptosis through membrane-associated ERα36, suggesting that ERα36 may be a potential membrane target for drug design against breast cancer, particularly triple negative breast cancer.     

Cooperation of proto-signals for nuclear accumulation of estrogen and progesterone receptors.

Multiple proto-signals (p-NLSs) for nuclear targeting, none of which suffices on its own, cooperate in the estrogen (ER) and progesterone (PR) receptors. In the ER, an estrogen-inducible p-NLS was found in the hormone binding domain (HBD), in addition to three lysine/arginine-rich motifs resembling prototype constitutive nuclear localization signals (NLSs). The inducible and the constitutive ER p-NLSs cooperate in the presence of estrogen and hydroxy-tamoxifen, but not in the presence of ICI 164,384. In the PR, three p-NLSs, two of which are located within and directly adjacent to the second zinc finger, cooperate with each other and a weak hormone-inducible p-NLS in the PR HBD. No 'masking' of p-NLSs by the HBD was observed for ER and PR, while the ligand-free glucocorticoid receptor HBD inhibited the activity of both homologous and heterologous NLSs. Nuclear co-translocation experiments indicated that in vivo the stability of ER and PR dimers is hormonally controlled, but that, in the absence of the cognate ligand, ER dimers are more stable than PR dimers. This is likely to account for the differential hormone requirement of ER and PR DNA binding in vitro.