Analysis of estrogen receptor isoforms and variants in breast cancer cell lines
- Authors:
- Published online on: March 10, 2011 https://doi.org/10.3892/etm.2011.226
- Pages: 537-544
Abstract
Introduction
Estrogen receptor (ER)α was first cloned in rats by Koike et al (1); almost 10 years later, a gene encoding a second type of ER, ERβ, was cloned in rats (2), humans (3) and mice (4), prompting the re-evaluation of estrogen signaling systems. ERα and ERβ are homologous, particularly in the DNA binding domain (95%) and in the C-terminal ligand binding domain (55%) (2–4). The genes for both ERα and ERβ are encoded by eight exons, located on different chromosomes, with ERα found on the long arm of chromosome 6q25.1 and ERβ on chromosome 14q22-24 (5). This confirms that each receptor is the product of independent genes. ERs have six functional domains: domain A/B, containing the N-terminal activation function-1 (AF-1); domain C, the DNA binding domain; domains D/E, bearing both the activation function-2 (AF-2) and the ligand binding domains; and finally, domain F, the C-terminal domain (6,7).
The actions of estrogens are mediated by binding to ERs (ERα and/or ERβ). These receptors, which are co-expressed in a number of tissues, form functional homodimers or heterodimers. When bound to estrogens as homodimers, the transcription of target genes is activated (8,9), while as heterodimers, ERβ exhibits an inhibitory action on ERα-mediated gene expression and, in many instances, opposes the actions of ERα (7,9). Estrogen binding to ERβ also inhibits gene transcription via AP1 sites, while binding to ERα leads to their activation (8,10,11). Thus, as several ER-negative breast cancer cell lines respond to estrogens and anti-estrogens, this suggests that these compounds may act through an alternative mechanism, not the classical ERα pathway (12), or that ER-negative cell lines are not truly ER-negative.
Much of our knowledge on breast carcinomas is based on in vitro studies performed with various breast cancer cell lines. These cell lines provide a source of homogenous self replicating material, free of contaminating stromal cells, that can be grown in culture in standard media. Cell lines that have retained the luminal epithelial phenotype of breast cells include MCF7, T-47D and ZR-75-1; those with a weak luminal epithelial-like phenotype include MDA-MB-453 and SK-BR-3; finally, those that do not express epitheloid markers, but exhibit a high level of vimentin (a marker found in mesenchymal cells), include MDA-MB-231 (13). Although rare, there have been reports of ER-positive cell lines converting to an ER-negative phenotype (13). However, certain breast cancer cell lines reported as being negative for ERα have since been shown to express ERβ at least at the mRNA level. In addition to the aforementioned ER isoforms, several ER variants have been identified for both receptors. A summary of the reported ERα and ERβ isoforms and their variants to date is shown in Tables I and II, respectively.
There have been some discrepancies between the results of researchers studying the mitogenic effects of estradiol and various estrogen agonists and/or antagonists using a number of breast cancer cell lines (both ER-positive and ER-negative). Although this can be attributed to many factors, in this study we aimed to determine the true ER status of breast cancer cells by studying ERβ isoform expression in breast cancer cell lines that have been reported, in the literature, to be ER-positive (MCF7, T-47D and ZR-75-1) or ER-negative (MDA-MB-231, SK-BR-3, MDA-MB-453 and HCC1954). Additionally, we aimed to determine the expression of ERα and ERβ variants in these cell lines using reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Our results revealed that ER-positive and ER-negative cell lines used extensively in breast cancer research have variable degrees of expression of ERα and/or ERβ isoforms and variants at the mRNA and/ or protein level.
Materials and methods
Materials
All media and supplements for cell culture were obtained from Invitrogen (Paisley, UK). The ERβ polyclonal antibody used corresponds to amino acids 1-150 (H-150; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Mouse monoclonal anti-ERα was raised against the steroid binding domain of ERα [amino acid residues 582–595 (referred to in this article as ERα-S)] (SRA-1010, clone C-542; Stressgen, Ann Arbor, MI, USA). For the detection of actin, a mouse monoclonal IgG1 anti-human actin antibody was used (Santa Cruz Biotechnology, Inc.). PVDF membranes were obtained from Amersham Pharmacia Biotech Ltd. (RPN303F; Buckinghamshire, UK). General laboratory chemicals were purchased from Merck (Dagenham, Essex, UK) and all fine chemicals were obtained from Sigma Chemical Co. Ltd. (Poole, Dorset, UK). All buffers, enzymes and reagents used in the RT-PCR experiments were purchased from Invitrogen, and reagents for real-time PCR (ReT-PCR) were purchased from Applied Biosystems (Foster City, CA, USA).
Cell lines
All of the cell lines used in this study were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). Seven breast cancer cell lines were used, three of which are known to be ER-positive in the literature (MCF7, T-47D and ZR-75-1) and four of which are reported to be ER-negative (MDA-MB-231, MDA-MB-453, SK-BR-3 and HCC1954). Cell lines were grown as monolayers in the following media: RPMI-1640 (T-47D, ZR-75-1 and HCC1954), Eagle’s MEM (MDA-MB-231 and MCF7), McCoy’s 5A (SK-BR-3) and Leibovitz’s (MDA-MB-453) containing 10% fetal bovine serum (FBS), penicillin (100 IU/ml) and streptomycin (100 μg/ml). Other supplements were added to the medium for some of the cell lines, as per the ATCC data sheet supplied with the cell lines. When required for assays, 5 ml of a 1:10 dilution of trypsin-EDTA in phosphate buffered saline (PBS) was added to PBS-washed monolayers, followed by incubation at 37˚C for 5–10 min. Cells were centrifuged for 7 min at 130 × g, reconstituted in the medium, and counted.
Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was isolated from a minimum number of 5×106 cells using the method of Chomczynski and Sacchi (54). Following isolation, the RNA samples were DNase-treated, then reverse transcribed using random hexamer primers (55). PCR reactions were carried out in a programmable thermal cycler (Perkin Elmer, model 9700) in a reaction mixture consisting of 1X PCR buffer (20 mM Tris/50 mM KCl), 3 mM MgCl2, 0.5 mM dNTPs and 0.3 μM each of forward and reverse primers (primer sets are shown in Table III), 0.5 μl template and 1.25 units recombinant Taq DNA polymerase in a final volume of 25 μl. The PCR reactions were then cycled as follows: 5 min at 94˚C (1 cycle); 30 sec at 94˚C (denaturation step), 30 sec (annealing step) and 1 min (extension step) at 72˚C for the required number of cycles (Table III). Tubes were then incubated for a further 7 min at 72˚C (1 cycle).
 | Table III.Primers used for RT-PCR, expected PCR product sizes, annealing temperatures and cycle numbers. |
Protein analysis using Western blotting and immunodetection
Trypsinized cells (∼2.65×106) were centrifuged at 1,000 x g at 4˚C for 10 min to remove the medium and then washed twice with PBS buffer. The pellet was resuspended in homogenization buffer (20–50 μl) and then vortexed, sonicated for 30 min at 4˚C, and frozen for 15 min. This step was repeated twice. Finally, the samples were centrifuged at 4˚C for 30 min at 20,000 × g, the supernatant was collected, and the total protein concentration was measured (20 μg of protein was loaded per lane). Proteins were separated using SDS-PAGE. A monoclonal antibody for ERα raised against the steroid binding domain (ERα-S) and a polyclonal antibody against ERβ were used. Western blot analysis and immunodetection of total ER proteins together with analysis of protein sizes were performed as previously described (55). In preliminary experiments, the primary antibody was omitted and filters were incubated with the secondary antibody only. No bands were detected with this antibody. Once the membranes were probed with the anti-ER antibodies, they were stripped and re-probed with actin, which was present equally in all the samples (data not shown). The results obtained from this experiment were compiled for each cell line and are shown as the percentage of expression for each band per group of study.
Results
ER mRNA expression using RT-PCR
Representative images for all cell lines using the various primers are shown in Fig. 1. Overall results for all cell lines studied are shown in Table IV. The housekeeping gene β-actin was used as a control and expression was verified in all the cell lines studied. Wild-type (wt) ERα was expressed in all the ER-positive cell lines (MCF7, T-47D and ZR-75-1), as well as in the ER-negative cell lines (MDA-MB-231 and HCC1954). The ERα Δ3, Δ5 and Δ7 spliced variants were present in both the MCF7 and T-47D ER-positive cell lines. Regarding the ZR-75-1 cell line, only the ERα Δ5 and Δ7 spliced variants were detected. Concerning the ER-negative cell lines, both MDA-MB-231 and HCC1954 showed mainly weak expression of the ERα Δ5 and Δ7 spliced variants. The ERα Δ6 and Δ6+7 variants were not expressed in any of the cell lines. The ERβ1 variant was expressed in the ER-positive and ER-negative cell lines; however, ZR-75-1 and SK-BR-3 cells exhibited weak expression. The ERβ2 variant was expressed in all of the ER-positive and two of the ER-negative cell lines (MDA-MB-231 and MDA-MB-453), with very weak expression noted in SK-BR-3. MCF7, ZR-75-1, MDA-MB-453 and HCC1954 clearly expressed ERβ5, with weak expression noted only in the T-47D cell line.
| Table IV.RT-PCR results for ERα and ERβ isoforms (and/or variants) and actin gene expression for various ER-positive and ER-negative cell lines. |
Western blotting and immunodetection
Representative images for ERα and ERβ protein expression are shown in Fig. 2. The percentage of positivity for ERα and ERβ in all the samples studied is shown in Table V. All cell lines (ER-positive and ER-negative) expressed a ∼66 kDa protein corresponding to ERα (reported size for ERα). Smaller molecular weight bands (<66 kDa) were noted in some of the ER-positive and ER-negative cell lines. These may be spliced variants of ERα, as spliced variants have been reported for this gene (27,29,56). All of the cell lines were found to express a 52–54 kDa protein (the reported size for ERβ1). Certain cell lines also expressed a smaller molecular weight band that may be an ERβ spliced variant (46,57–59).
| Table V.Percentage of positive expression of ERα and ERβ isoform/variant protein determined by Western blotting. |
Discussion
It has been reported that breast cancer cell lines from different laboratories may differ in their sensitivity to estradiol (13). This discrepancy may be attributed to lack of proper investigation of the ER status. We demonstrated that all of the cell lines used in this study express cytoplasmic and/or membrane ER when analyzed by flow cytometry (unpublished data). In this study, we demonstrated that cell lines that have been known to be positive or negative for classical ER (ERα) show various degrees of positivity for the ERβ isoform and for the ERα and ERβ variants at both the protein and mRNA levels. This is important, as the presence of the ERβ isoforms together with the ERα isoforms in a tissue may have functional implications for binding and response to a particular ligand.
As several variants have been shown to exist for ERα (Table I) differences in estrogen responsiveness of cell lines may be due to varying ratios of wild-type to variant ER mRNA. The Δ1 variant lacking exon 1 (N terminal AF1 region) results in a 46-kDa protein that heterodimerizes with the wild-type ERα, suppressing its activity (33), and the Δ3 (60), Δ4 (61) and Δ7 variants (16) also inhibit gene transcription by interfering with the ability of the wild-type ER to initiate transcription (15,16,18). Conversely, the Δ5 variant acts in a dominant-positive manner to activate the gene transcription of an ER-regulated gene (15,16,18). Certain cell lines, misclassified as ER-negative, exhibit the Δ5 variant, which activates gene transcription in the absence of the hormone and inhibits wild-type activity by competing for steroid receptor co-activator-1e (SRC-1e) (62). Thus, the presence of this variant may explain hormone independence and tamoxifen-resistance, and may contribute to the hormone-independent proliferation of ER-negative cell lines (16).
Although the presence of variants in cell lines has been reported by several investigators, a complete analysis of variant expression has not been attempted. Two of the ER-positive cell lines, MCF7 and ZR-75-1, have been shown to exhibit the ERα wild-type Δ5 and Δ7 variants (63). However, the present results also show that in addition to these variants, MCF7 cells express Δ3, in agreement with previous reports (34,37). Strom et al (64) reported that the predominant ER in T-47D cell lines is ERα (9:1 with ERβ) and that estradiol stimulates growth of T-47D cells, while anti-estrogens do not induce proliferation. As we demonstrated, T-47D expresses ERα Δ5, Δ7 and Δ3 (albeit little of this variant) and ERβ (explained below), and these variants may act to inhibit or enhance wild-type ERα action. As Δ3 and Δ7 act in a dominant-negative fashion to suppress ERα wild-type activity, and Δ5 acts to enhance gene transcription, investigation of the relative expression of these variants in comparison to the wild-type gene is of critical importance. The presence of these variants may be the cause of reported discrepancies in results between different laboratories.
Reports that the MDA-MB-453 cell line is negative for ERα wild-type mRNA are in agreement with our results; however, MDA-MB-231 was found to exhibit positivity for wild-type ERα. This was confirmed by using three different sets of primers that detect the wild-type ERα and different variants. Although ERα was not expressed at the transcript level, it was detected in both cell lines at the protein level both by Western blotting, as indicated above, and by using flow cytometry (unpublished data). This may be due to a high turnover of mRNA and protein accumulation. HCC1954 was also positive for wild-type ERα and for the Δ5 and Δ7 variants. ERα Δ7 is able to form heterodimers with ERα and ERβ in a ligand-independent manner resulting in a dominant-negative effect on both ER isoforms (65,66), and the presence of ERα Δ5, which has AF-1 activity and DNA binding ability, leads to a constitutively active receptor (65). This may explain resistance to tamoxifen and hormone-independent proliferation in ER-negative cell lines (67).
Five spliced isoforms of the human ERβ, designated ERβ1-5, were cloned by Moore et al (10). The amino acid sequences diverge at amino acid 469 within the ligand binding domain and extend to the C-terminus (42). Longer forms of the ERβ – 485, 530 and 548 aa – have also been reported (5,10,11,68,69). In addition to the expression of wild-type ERβ of various lengths due to the use of alternative transcription start sites, a number of ERβ variants have been identified (Table II) arising from alternative splicing (10,41,42,70). As with ERα, these spliced variants, when expressed with the wild-type ERβ, alter the response of the wild-type to estradiol; thus, the relative expression levels of the wild-type vs. variant ERβ is of significance in predicting cellular responsiveness to various estrogen and anti-estrogen therapies (59,71,72).
Tumors that express ERβ2 (or ERβcx), a splice variant of ERβ that utilizes an alternative exon 8, show a poor response to tamoxifen (48,72). The ERβ2 variant does not bind ligands and heterodimerizes with ERα, having an overall dominant-negative effect on ERα reporter gene activity (10,73). In the present study, ERβ1, ERβ2 and ERβ5 mRNA expression in the cell lines was investigated; however, we did not study ERβ3 and ERβ4, as it has been indicated that they are barely detectable in breast tumor samples. However, the expression of the ERβ4 variant cannot be ruled out in breast cancer cell lines; Tong et al (47) used a different primer set and were able to amplify ERβ4 in MCF7, T-47D, ZR-75-1, MDA-MB-231 and SK-BR-3 cells (MDA-MB-453 and HCC1954 were not studied), although its expression was very low in comparison to the other variants, and thus it may have limited physiological significance.
The ER-positive cell lines MCF7, T-47D and ZR-75-1 were positive for ERβ1, ERβ2 and ERβ5. Other investigators have shown that MCF7 contains high levels of the ERβ2 and ERβ5 isoforms (51), and that the T-47D cell line is positive for ERβ1 and ERβ2 and negative for ERβ5 (47). Conversely, our results showed very weak ERβ5 expression in this cell line. Moreover, Tong et al showed that SK-BR-3 was negative for ERβ1 and positive for ERβ2 and ERβ5, while we detected some ERβ1 and ERβ2 expression. The MDA-MB-231 cell line has been reported to express ERβ1, ERβ2 and ERβ5 (47), while our results confirm expression of ERβ1 and ERβ2 only, in agreement with other reports (42,46,51). Others have been unable to detect ERβ in SK-BR-3 (74), but in the present study SK-BR-3 cells were found to express ERβ by flow cytometry (unpublished data), as well as to express the ERβ 268 and 214-bp products at the mRNA level, and the 52–54 and 38–44 kDa products at the protein level.
Many of the ERα and ERβ variants have been shown to be translated into proteins (Tables I and II). In the present study, all of the cell lines showed wild-type ERα and ERβ1 expression, albeit to varying degrees. In addition, some cell lines clearly exhibited a 42-kDa variant that could be the translated protein product of the exon 5-deleted ERα variant. The expression of a smaller (38-44 kDa) ERβ variant by all cell lines, the significance of which is not clear at this stage, demonstrates that our level of understanding of the expression of ER variants at the functional level requires further investigation.
Acknowledgements
The authors would like to acknowledge the skillful technical assistance of Dr Beryl G. Rego and Mrs. Ani Mathew for handling the cell culture aspect of the project, and Dr Sureikah S. Mohan, Mrs. Lizamma Jacob and Ms. Jocelin Jacob for the processing of samples for protein and gene analysis. Financial support for this study was provided by the Kuwait University Grant no MY01/02. The authors would also like to acknowledge the support of the Department of Physiology, Faculty of Medicine.
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