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Expression of Wild-Type Estrogen Receptor and Variant Isoforms in Human Breast Cancer¹

Department of Medicine, Baylor College of Medicine, Houston, Texas 77030 [S. A. W. F., R. S., I. P., W. E. F.], and Departments of Molecular Sciences, Molecular Endocrinology and Medicinal Chemistry, Glaxo Wellcome Research and Development, Research Triangle Park, North Carolina 27709-3398 [J-L. S., D. D. M., K. S-K., L. B. M., T. M. W., J. T. M.]

	ABSTRACT

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It has been shown in previous studies that a variety of estrogen receptor (ER) mRNA transcripts are expressed in human breast cancer cell lines and tumors. To complement the RNA expression studies, we have developed ER--specific antibodies to characterize ER- protein expression in breast cancer cell lines and tumors. Monoclonal antibodies were made against a peptide representing the first 18 amino acids of the longest ER- open reading frame reported to date, and polyclonal antibodies were made against a peptide within the ER- B domain. By Western blot analysis, we show that ER- protein is expressed in all cancer cell lines tested and in three of five breast tumor samples. The breast cancer cell lines showed variation in the size of the expressed ER- protein. The longest form detected was consistent with the 530-amino acid, full-length ER- sequence. Shorter ER- isoforms were detected in the ER--negative MDA-MB-231 and MDA-MB-435 breast cancer cell lines, likely corresponding to previously described COOH-terminal RNA variant isoforms.

	Introduction

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Estrogens play an integral role in the growth of most ER³ -positive mammary carcinomas (1 , 2) . Considerable data indicate that many of the growth-promoting characteristics of estrogens are mediated through their mitogenic effects on breast cancer cells. The ability of the antiestrogen tamoxifen to inhibit the growth of many ER-positive breast tumors is consistent with this basic model, but the detailed molecular events involved in this process are not understood. An important goal in understanding estrogen and antiestrogen action on breast tumor cells is to define the molecular forms of ER expressed in breast tumors and how they might relate to tumor progression and drug-resistant phenotypes. The effects of estrogen and antiestrogens in breast tumor cells have been known for years to be mediated, at least in part, by ER- (3) . More recently, ER- has been shown to be expressed in breast tumors at the RNA level, and it has been suggested that changes in the relative levels of ER- and ER- might be associated with tumorigenesis (4 , 5) . Thus, the accurate determination of ER- expression is of clinical importance.

To make an accurate clinical assessment of ER status in breast cancer cells, it is critical to determine which forms of ER are expressed and stable at the protein level. Discrepancies exist in the size of the full-length human ER- receptor. The first human cDNA sequence published reported a length of 487 amino acids (6) . Ogawa et al. (7) and Moore et al. (8) subsequently published a human cDNA sequence with an additional 53 amino acids in the NH₂-terminal region, predicting a primary translation product of 530 amino acid residues. Similarly, various sizes and isoforms of rat ER- have been reported (9, 10, 11) . Finally, the existence of numerous COOH-terminal-truncated ER- isoforms (8) , along with their potential to influence ER function (12) , emphasizes the need to profile ER- protein isoform expression in breast cancer samples as well. In this report, we provide the first characterization of ER- expression at the protein level in breast cancer cells.

	Materials and Methods

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Generation of ER- Antiserum.
Synthesis of peptide DIKNSPSSLNSPSSYNC representing ER- residues 2–18 (amino acid numbering was based on the longest ER- ORFs described to date; see Ref. 7 and 8 ) and conjugation of peptide to KLH using m-maleimido-benzoic acid n-hydroxysuccinimide ester via the COOH-terminal cysteine were performed by ZENECA Inc. (Wilmington, DE). Hybridomas to the peptide were produced from a SJL mouse (Jackson Laboratories, Bar Harbor, ME) after 3 months of immunization with ER peptide-KLH conjugates using a previously published protocol (13) . Hybridoma supernatants were screened for their immunoreactivity by ELISA, Western blotting, BIAcore analysis, and immunocytochemistry.⁴ Hybridoma ER15.64, secreting IgG1 immunologlobulins, was isolated after limiting dilution cloning and used for Western blot analysis.

For synthesis of polyclonal antiserum, another synthetic peptide, GNRCASPVTGPGSK (representing ER- residues 130–143), was synthesized. After linking to KLH via an introduced NH₂-terminal cysteine residue, the conjugate was used to immunize rabbits. The polyclonal antiserum obtained was also used for Western blot analysis. Neither of these two antibodies recognized in vitro translated ER- protein in Western blot experiments (data not shown).

Western Blot Analysis.
All breast cancer cell lines and the SaOS2 osteosarcoma cells were obtained from the American Type Culture Collection. In addition, five ER-positive primary breast tumors from the San Antonio Breast Tumor Bank were examined. These five tumors were defined as ER positive (<3 fmol/mg receptor) using a ligand binding assay. Total cell or tumor lysates were prepared as described previously (14) . Briefly, cell pellets were homogenized in a high-salt buffer [20 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.4 M KCl, and 20% glycerol] containing a mixture of protease inhibitors (2.5 µg/ml aprotinin, antipain, leupeptin, and pepstatin plus 0.3 mM phenylmethylsulfonyl fluoride; Sigma). Homogenates were then centrifuged at 100,000 x g for 1 h, and the supernatants were stored at -20°C until use. Extract (50–100 µg) was separated by electrophoresis on 10% SDS-PAGE and transferred onto nylon membranes (Schleicher and Schuel, Keene, NH). The blots were first stained with StainAll Dye (Alpha Diagnostic International, Inc., San Antonio, TX) to confirm uniform transfer of all samples and then incubated in blocking solution (5% nonfat dry milk in TBST). After brief washes with TBST, the filters were then reacted with ER- antibody at a dilution of 1:20 or 1:200 (monoclonal antibody ER 15-64) or 1:2,000 (polyclonal antiserum) for 1 h at room temperature, followed by extensive washes with TBST. Blots were then incubated with horseradish peroxidase-conjugated secondary antibody (Amersham) for 1 h, washed with TBST, and developed using the enhanced chemiluminescence procedure (Amersham). In some experiments, primary antibody was omitted, and secondary antibody was incubated alone to control for background immunostaining with the secondary antibody. As positive controls, mammalian expression vectors (pSG5) containing the complete ORFs of ER-1 and ER-2 (8) were translated in vitro in a 50-µl rabbit reticulocyte translation reaction (Promega, Madison, WI), and 5 µl were analyzed by Western blotting.

	Results

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Expression of the 530-Amino Acid Form of ER- Is Confirmed in MCF-7 Breast Carcinoma.
Total cellular extracts from MCF-7 breast carcinoma cells were prepared using a high-salt extraction procedure that has previously been used to efficiently extract ER- (14) . Three in vitro translated controls were also included along with the MCF-7 extract in Western blot analysis (Fig. 1) . Control ER-1 corresponds to the predicted 530-amino acid product (7 , 8) , control ER-2 is a COOH-terminal-truncated isoform (8) , and control 5'-truncated corresponds to the originally reported human ER- cDNA (6) that contains an ORF that begins at amino acid 54 relative to ER-1. Using a polyclonal antibody that was prepared using a peptide (residues 130–143) within the B domain of ER-, we found that in vitro translated ER-1 migrated at approximately 58–60 kDa under our Western blotting gel conditions, whereas both the ER-2 and 5'-truncated controls migrated faster. The 58–60 kDa size is consistent with the size predicted (62 kDa) from the sequence of the 530-amino acid form of the receptor. For each control reaction, the polyclonal antibody also recognized a smaller minor band. It is likely that the shorter translation product (in each case, approximately 4–5 kDa smaller than the primary band) represents initiation from an internal methionine. This is supported by the fact that the monoclonal ER 15-64, which was directed at the extreme NH₂ terminus (the first 18 amino acids) of the predicted 530-residue ER- (7 , 8) does not recognize the shorter in vitro translation products (see below). When the polyclonal antibody was used to analyze MCF-7 cell extracts in Western blots, we detected one band migrating at the same position as the longest product in the ER-1 in vitro translated control (Fig. 1A) , demonstrating that the major ER- product in MCF-7 cells is the predicted 530-amino acid form. To further confirm the identity of this ER- isoform in MCF-7 cells, we analyzed the MCF-7 extracts using monoclonal ER 15-64, an antibody specific to a stretch of amino acids present in ER-1 and ER-2, but not in the 5'-truncated sequence. As would be predicted, based on its specificity, ER 15-64 recognized specific products from in vitro translated ER-1 and ER-2, but not the 5'-truncated ER- product (Fig. 1B) . As described above, the monoclonal antibody only recognized a single band in each lane, consistent in size with translation of the longest ORF in each clone. When MCF-7 cell extracts were analyzed with ER 15-64, we detected a single band that comigrated with the band detected from the in vitro translation of ER-1, the same size as the band recognized by the polyclonal antibody. Based on the antigen specificity of the antibodies and the size of the detected product, we conclude that MCF-7 cells express detectable levels of the 530-residue ER- product.

View larger version (56K): [in this window] [in a new window]   Fig. 1. Western blot comparison of ER- polyclonal and monoclonal (ER- 15–61) antibodies. Lysate (5 µl) from in vitro translation reactions primed with pSG5 expression plasmid containing either ER-1 amino-acids 1–530 (1), ER-2 (2), or ER-1 amino acids 54–530 (5' truncated) and cell lysate (50–100 µg) prepared from MCF-7 breast carcinoma cell line were electrophoresed on SDS-PAGE gels and transferred to nylon filters. The blots were incubated with (A) rabbit polyclonal ER- antibody serum diluted 1:2000 or (B) ER- 15-64 ER- monoclonal antibody serum diluted 1:200. Binding of primary antibody was detected with horseradish peroxidase-conjugated secondary antibody.

Breast Cancer Cell Lines Express ER- Variants.

View larger version (46K): [in this window] [in a new window]   Fig. 2. Western blot analysis of ER- expression in breast carcinoma cell lines. Cell lysates (50–100 µg) from three human breast carcinoma cell lines (MDA-231, MCF-7, and MDA-435) and a human osteosarcoma cell line (SaOS2) were analyzed by Western blot analysis. For size comparison, the lysates were electrophoresed adjacent to 5 µl of in vitro translation product primed with pSG5 expression vector containing ER-1 amino acids 1–530. The blot was probed with monoclonal ER- antibody serum (ER- 15–64) at a 1:200 dilution and visualized using horseradish peroxidase-conjugated secondary antibody.

Human Breast Tumor Specimens Express ER- Protein.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Western blot analysis of ER- expression in breast tumor biopsy samples. Extracts from five ER-positive breast tumors were analyzed by Western blot analysis, and the blots were probed with monoclonal ER- antibody serum (ER- 15–64) at a 1:200 dilution and visualized using horseradish peroxidase-conjugated secondary antibody. For size comparison, the lysates were electrophoresed adjacent to 5 µl of in vitro translation product primed with pSG5 expression vector containing ER-1 and ER-2 as well as the SaOS2 extract. Contaminating tumor IgG is indicated.

	Discussion

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Most ER- functional studies reported in the literature to date have used clones that express the short form of the receptor, corresponding to amino acids 54–530, because this was the first human form of the receptor reported (6) . For example, the recent report describing differences in AP-1 activity between ER- and ER- used the truncated form of ER- in the studies (15) . In such assays, in which the complete molecular details involved in determining the assay read-out are unknown, differences might be noted if the full-length receptor is not used in the study. For example, the contributions of the NH₂ terminus, a region in ER- known to contain the autonomous transactivation function AF-1 (16) , may be critical. This is especially hard to assess at the present time because of the plethora of genomic (such as interactions with the estrogen response element in various promoter contexts in vivo) and nongenomic interactions involving the ERs. For example, Bhat et al. (17) have found that only the 530-amino acid form of ER- is functional in a nuclear factor B promoter inhibition assay. A large effort is ongoing in many laboratories to determine the differences in functional profiles between ER- and ER-; hence, it is critical that the correct in vivo expressed form of the receptor be used in these studies. Defining the correct translational start site of ER- is an important first step toward defining the exact molecular mechanisms involving ER- function.

In breast tumors, ER- RNA expression has been detected in both ER--positive and -negative tumors (18) . RNA-based ER- studies have raised the possibility that the presence of ER- in breast tumors may be a marker of endocrine therapy responsiveness (14) . RNA studies have also indicated that the dual presence of ER- and ER- RNA may correlate with a poor clinical prognosis (19) . Furthermore, ER- RNA expression has been shown to be inversely correlated to levels of the progesterone receptor (19) . Our data indicate that, in fact, full-length ER- is expressed in breast tumor cell lines and in breast cancer biopsy tissue.

In addition to full-length ER-, we also detected truncated forms of ER- in breast cancer cell lines. Because these ER- translated products were detected with the ER- antibody 15-64 (which recognizes amino acids 2–17 of the predicted long form of ER-), the shorter length of these receptors is not due to downstream translation initiation but is most likely due to COOH-terminal truncations. RNA isoforms representing COOH-terminal-truncated receptors have also been reported, including ER-2, ER-4, and ER-5 (6) as well as an exon 5-deleted product (20) , but it was not previously known whether any of these altered proteins were translated into stable products in cells. The sizes of these receptor forms are consistent with the sizes predicted from primary sequences of ER- isoforms (6) . Future studies using isoform-specific antibodies will be necessary to confirm and extend the details of specific isoform expression.

The presence of ER- isoforms is potentially of clinical significance. Multiple studies have detected the presence of RNA isoforms in cancer cell lines and tumors, and more recently, it has been shown that the ratios of ER-2:ER-1 and ER-5:ER-1 RNAs correlate positively with tumor inflammation and nuclear grade, respectively (19) . However, these RNA changes and ratios have not yet been correlated with alterations in protein isoform profiles. It will be important to determine whether a particular protein isoform overexpression is correlated with specific tumor characteristics, such as the antiestrogen-resistant phenotype.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by NIH Grant CA30195 (to S. A. W. F.).

² To whom requests for reprints should be addressed, at Breast Center, Baylor College of Medicine, 1 Baylor Plaza, Alkek N-550, Houston, TX 77030.

³ The abbreviations used are: ER, estrogen receptor; KLH, keyhole limpet hemocyanin; TBST, Tris buffered saline + Tween [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20]; ORF, open reading frame.

⁴ J-L. Su, D. McKee, S. A. W. Fuqua, G. B. Wisely, S. Kadwell, J. Stimmel, and J. T. Moore. Production and characterization of ER--specific monoclonal antibodies, manuscript in preparation.

Received 8/ 3/99. Accepted 9/20/99.

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				S. L. Gadd, G. Hobbs, and M. R. Miller Acetaminophen-Induced Proliferation of Estrogen-Responsive Breast Cancer Cells Is Associated with Increases in c-myc RNA Expression and NF-{kappa}B Activity Toxicol. Sci., April 1, 2002; 66(2): 233 - 243. [Abstract] [Full Text] [PDF]

				G. J. Pepe, R. B. Billiar, M. G. Leavitt, N. C. Zachos, J. A. Gustafsson, and E. D. Albrecht Expression of Estrogen Receptors {alpha} and {beta} in the Baboon Fetal Ovary Biol Reprod, April 1, 2002; 66(4): 1054 - 1060. [Abstract] [Full Text] [PDF]

				E. V. Jensen, G. Cheng, C. Palmieri, S. Saji, S. Makela, S. Van Noorden, T. Wahlstrom, M. Warner, R. C. Coombes, and J.-A. Gustafsson Estrogen receptors and proliferation markers in primary and recurrent breast cancer PNAS, November 29, 2001; (2001) 211556298. [Abstract] [Full Text] [PDF]

				S. Saji, H. Sakaguchi, S. Andersson, M. Warner, and J.-A. Gustafsson Quantitative Analysis of Estrogen Receptor Proteins in Rat Mammary Gland Endocrinology, July 1, 2001; 142(7): 3177 - 3186. [Abstract] [Full Text] [PDF]

				L. G. Horvath, S. M. Henshall, C-S. Lee, D. R. Head, D. I. Quinn, S. Makela, W. Delprado, D. Golovsky, P. C. Brenner, G. O'Neill, et al. Frequent Loss of Estrogen Receptor-{beta} Expression in Prostate Cancer Cancer Res., July 1, 2001; 61(14): 5331 - 5335. [Abstract] [Full Text] [PDF]