Endogenously expressed estrogen receptor and coactivator AIB1 interact in MCF-7 human breast cancer cells

Abstract

Coactivators are believed to mediate estrogen-induced gene responses via interaction with estrogen receptors (ER). Currently, a major challenge is to determine the importance of each coactivator in a specific cell type and promoter context in response to a particular ligand. The potential of ER to interact with a growing list of coactivators has been shown in a variety of in vitro and gene transfer assays, yet very few data have demonstrated the interaction of endogenous coactivators with ER in intact cells. We report here a ligand-specific interaction of endogenous human ER (hER) and the AIB1 coactivator in MCF-7 human breast cancer cells by using immunoprecipitation analyses. Complexes between endogenously expressed hER and AIB1 were detected in estradiol-treated cells and to a much lesser extent in cells treated with the partial agonist, monohydroxytamoxifen. We were unable to detect an hER-SRC-1 complex in our immunoprecipitations from MCF-7 cells. The in vitro-binding affinity for mouse ER interaction with AIB1 was estimated to be 40 -120 nM. We conclude that AIB1 is a major coactivator for hER in MCF-7 human breast cancer cells. It is well established that estrogen action in the cell is mediated by estrogen receptors (ERs) ␣ and ␤ (1). Detailed regulation of gene expression is believed to be mediated by coregulator proteins that bind ER in a ligand-dependent manner (2, 3). Several structurally distinct subclasses of nuclear receptor coregulators have been identified, including the SRC family of coactivators. This family includes SRC-1 (also termed p160͞ NcoA-1͞ERAP-160), TIF-2 (also termed SRC-2͞GRIP-1), and AIB1 (also termed SRC-3͞ACTR͞RAC-3͞TRAM-1) (4). These proteins have been shown to physically bind several agonist-occupied nuclear receptors in vitro and to enhance hormone-dependent transcription in transient transfection assays. It is probable that coactivators preferentially interact with receptors dependent on cell type, ligand, and promotor context, which could contribute to the specificity of the physiological response. However, very few data are available on the existence and importance of endogenous receptor-coactivator complexes actually formed in response to a specific ligand in the whole cell.Several studies suggest that the newest member of the SRC family, AIB1, has a special role in breast tissue. AIB1 is expressed in a wide variety of tissues, but the highest expression is in breast and ovary. Mice that lack the ability to express AIB1 show greatly reduced sensitivity of breast tissue to estrogen and progesterone administration (5). In addition, recent findings demonstrating that SRC-1 does not colocalize with ER␣ in rat mammary epithelial cells suggest that other SRC family members likely play a more important role in ER␣-mediated hormone actions in breast tissue (6). In addition, AIB1 was found to be overexpressed in 64% of primary breast tumors and in four of five ERϩ breast and ovarian cancer cell lines (7). In a study of 1,157 human breast tumors, overexpression of AIB1 was shown to ...

Expression of RAC 3, a steroid hormone receptor co-activator in prostate cancer

“…Bautista et al (1998) also demonstrated that RAC 3 amplification correlated with both tumour size and ER and progesterone receptor (PR) positivity in primary breast cancers. It has further been shown that endogenous RAC 3 interacts with the human ER in a ligand-dependent manner in MCF-7 breast cancer cells (Tikkanen et al, 2000). Interestingly the RAC 3 gene has also been found to be amplified in pancreatic cancers and primary gastric cancers suggesting that RAC 3 is functional in non-primarily steroid-dependent organs (Ghadimi et al, 1999;Sakakura et al, 2000).…”

Identification of a Novel Family of Ankyrin Repeats Containing Cofactors for p160 Nuclear Receptor Coactivators

“…Among the p160 coactivators, RAC3 AIB1 is clinically important because it is amplified in breast cancers (17). Furthermore, RAC3 forms a stable complex with estrogen receptor ␣ in breast cancer cells (27), suggesting that RAC3 may play an important role in the development of breast cancer.…”

Impact of the nuclear receptor coactivator AIB1 isoform AIB1-Δ3 on estrogenic ligands with different intrinsic activity

“…We have determined, using a regulatable AIB1-directed ribozyme, that downregulation of endogenous AIB1 levels results in loss of estrogen induction of MCF-7 breast cancer cell proliferation in vivo and in vitro (List et al, 2001a). AIB1 has been shown to directly interact with the ER in breast cancer cells (Tikkanen et al, 2000). Consistent with this, deletion of the AIB1 gene in mice leads to reduced development of the mammary gland (Xu et al, 2000).…”

“…Whole cell extracts were prepared as described previously (Harris et al, 2000), and equal portions (30 mg of protein) were resolved on denaturing 4-20% polyacrylamide gradient gels containing Tris-glycine. The separated proteins were transferred to a nitrocellulose membrane and then subjected to immunoblot analysis with a 1 : 500 dilution of a mouse monoclonal antibody specific for amino acids 376-389 of human AIB1 (Transduction Laboratories, Lexington, KY, USA), horseradish peroxidase-conjugated goat antibodies to mouse immunoglobulin (1 : 10 000 dilution; Amersham Pharmacia Biotech, Piscataway, NJ, USA), and enhanced chemiluminescence reagents (Amersham Pharmacia Biotech).…”