Expression Levels of Estrogen Receptor-a, Estrogen Receptor-b, Coactivators, and Corepressors in Breast Cancer

Recent studies have indicated that a complex machinery of transactivation of target genes by estrogen or antiestrogen through estrogen receptor (ER) exists. However, the substantial roles of ER-b, coactivators, and corepressors in the development and progression of breast cancer remain to be elucidated. To obtain some clue to these roles, we screened the expression levels of ER-a, ER-b, coactivators (SRC-1, TIF2, AIB1, CBP, and P/CAF) and corepressors (N-CoR and SMRT) in 6 normal mammary glands, 6 intraductal carcinomas, 22 invasive ductal carcinomas, and 7 breast cancer cell lines using a multiplex reverse transcription-PCR. ER-a mRNA expression levels significantly correlated with ER-a protein levels measured by enzyme immunoassay in the breast cancer tissues and cell lines. A significant correlation of expression levels was observed between ER-a and TIF2, AIB1, P/CAF, and N-CoR, and between ER-b and AIB1 and CBP in the tissue samples. A significant correlation was also observed between ER-a and ER-b and between ER-b and CBP in the cell lines. The expression levels of ER-a, TIF2, and CBP were significantly higher in the intraductal carcinomas than those in the normal mammary glands. In addition, the expression levels of ER-a and N-CoR were significantly higher in the intraductal carcinomas than those in the invasive ductal carcinomas. These findings suggest a positive correlation of expression levels among ER-a and cofactors and among ER-b and cofactors, an up-regulation of expression levels of ER-a and cofactors during the development of intraductal carcinomas from normal mammary glands, and a decrease in their expression levels during the progression of breast cancer. INTRODUCTION ER belongs to the steroid/thyroid nuclear receptor family and is an estrogen-dependent transcriptional factor that regulates growth, development, differentiation, and homeostasis by binding to estrogen response elements in DNA to modulate transcription of target genes, including progesterone receptors and transforming growth factors, in target organs, such as the breast and uterus. Recent studies have disclosed a complex machinery of transactivation mediated by ER. There are several cofactors, termed coactivators and corepressors, which interact with ER and basal transcriptional machinery and activate or repress ER-mediated transcription (1–3). In addition, a new subtype of ER, ER-b, also participates in the transcriptional machinery as homodimers or heterodimers with ER-a (4–6). The definitive roles of ER in the development and progression of breast cancer have been elucidated (7–9), but the substantial roles of ER-b and cofactors in breast cancer have not been clarified. Recently, it has been suggested that the relative expression levels of ER-b versus those of ER-a decrease during human breast tumorigenesis (10). There have been some reports suggesting a possible relationship between the expression levels of a coactivator, SRC-1, and the clinical responses to an antiestrogen, tamoxifen (11), between the expression levels of another coactivator, AIB1 and ER-a expression levels (12, 13), and between the development of tamoxifen resistance and the expression levels of a corepressor, N-CoR, in breast cancer (14). Taken together with the fundamental knowledge of ER-mediated transcriptional machinery, these recent findings suggest ER-b and cofactors may play certain roles in the development and progression of breast cancer. To obtain some clue to the roles of ER-b and cofactors in breast cancer, we decided to use a semiquantitative multiplex RT-PCR method to measure the relative expression levels of ER-a, ER-b, and cofactors. We screened their expression levels in 7 human breast cancer cell lines, and then 6 normal mammary glands, 6 intraductal carcinomas, and 22 invasive ductal carcinomas were studied in the same manner. MATERIALS AND METHODS Breast Cancer Cell Lines and Tissue Samples. The KPL-1, KPL-3C, and KPL-4 cell lines were established in our laboratory, and their characterization has been published elsewhere (15–17). All these cell lines were derived, respecReceived 8/16/99; revised 11/1/99; accepted 11/9/99. 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 a grant from the Ministry of Education, Science, Sports and Culture of Japan and by Research Project Grants 10-113 and 10-307 from Kawasaki Medical School. 2 To whom requests for reprints should be addressed, at the Department of Breast and Thyroid Surgery, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan. Phone: 81-86-462-1111; Fax: 81-86-462-1199; E-mail: [email protected]. 3 The abbreviations used are: ER, estrogen receptor; RT-PCR, reverse transcription-PCR; HAT, histone acetyltransferase; TIF2, transcription intermediary factor 2; AIB1, amplified in breast cancer 1; CBP, CREBbinding protein; N-CoR, nuclear receptor corepressor; SMRT, silencing mediator of retinoid and thyroid receptors; P/CAF, p300/CBP-associated factor. 512 Vol. 6, 512–518, February 2000 Clinical Cancer Research Research. on October 24, 2017. © 2000 American Association for Cancer clincancerres.aacrjournals.org Downloaded from tively, from the malignant pleural effusion of three different Japanese patients with recurrent breast cancer. The KPL-1 and KPL-3C cell lines are ER positive, but the KPL-4 cell line is ER negative. Four other human breast cancer cell lines, the MCF-7 (ER-positive), T-47D (ER-positive), MDAMB-231 (ER-negative), and SkBr-3 (ER-negative) cell lines were kindly provided by Dr. Robert B. Dickson (at the Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC). A total of 28 breast cancer specimens and 6 normal mammary glands, which were located more than 5 cm away from the breast cancer in mastectomy specimens, were selected from the tumor bank of our department. Each breast cancer specimen was confirmed to include tumor tissues by pathologists of our institute. Six tumors were derived from patients with intraductal carcinomas, 11 tumors from patients with node-negative invasive ductal carcinomas, and the other 11 tumors were from patients with node-positive invasive ductal carcinomas. In breast cancer specimens, ER in 26 of 28 samples were measured by an enzyme immunoassay (Dinabot, Tokyo, Japan). Tumor samples with .10 fmol/mg protein of ER were defined as ER positive. Multiplex RT-PCR Method. Total cellular RNA from the human breast cancer cell lines and tissue samples was extracted with a TRIzol RNA extraction kit (Life Technologies, Inc., Gaithersburg, MD), according to the manufacturer’s recommendations. One mg of total RNA and 1mM Oligo(dT)-18 primer in 12.5 ml of diethyl pyrocarbonate-treated water were heated to 70°C for 2 min, followed by cooling on ice for 1 min. cDNA synthesis was initiated with 200 units of recombinant Moloney murine leukemia virus reverse transcriptase (Clontech Laboratories, Inc., Palo Alto, CA) under conditions recommended by the manufacturer, and the reaction was allowed to proceed at 42°C for 1 h. The reaction was terminated by heating at 94°C for 5 min. cDNA was dissolved to a final volume of 100 ml by adding 80 ml of diethyl pyrocarbonate-treated water and then was frozen at 220°C until use. Oligonucleotide primers for the RT-PCR were designed using a published sequence of each target gene and synthesized by the solid-phase triester method. The primers and conditions used in this study and the expected sizes from the reported cDNA sequence are shown in Table 1. To amplify both b-actin, the housekeeping control gene, and the target gene in a single reaction, the multiplex RT-PCR was carried out. The ratios of primer sets between the target gene and b-actin are also shown in Table 1. These ratios and PCR cycles were determined to amplify both products logarithmically and in relatively similar amounts. Each RT-PCR reaction contained 1/100 of cDNA, the indicated concentrations of primers of each target gene and b-actin, 200 mM deoxynucleotide triphosphates, 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl2, 50 mM KCl, 0.08% NP40, and 1 unit of recombinant Thermus aquaticus DNA polymerase (MBI Fermentas, Vilnius, Lithuania) in a final volume of 20 ml. After an initial denaturation at 94°C for 4 min, various cycles of denaturation (at 94°C for 15 s), annealing (at various temperatures, as shown in Table 1 for 15 s), and extension (at 72°C for 30 s) for the respective target genes were performed on a DNA Thermal Cycler 2400 (PC-960G Microplate Gradient TherT ab le 1 Pr im er se qu en ce s, co nd iti on s, an d pr od uc t si ze s fo r th e m ul tip le x R T -P C R