ATBF1 inhibits estrogen receptor (ER) function by selectively competing with AIB1 for binding to the ER in ER-positive breast cancer cells

Abstract

Loss of the q22 band of chromosome 16 is a frequent genetic event in breast cancer, and the candidate tumor suppressor gene, ATBF1, has been implicated in breast cancer by genomic deletion, transcriptional down-regulation, and association with better prognostic parameters. In addition, estrogen receptor (ER)-positive breast cancer expresses a higher level of ATBF1, suggesting a role of ATBF1 in ER-positive breast cancer. In this study, we examined whether and how ATBF1 affects the ER function in breast cancer cells. We found that ATBF1 inhibited ER-mediated gene transcription, cell growth, and proliferation in ER-positive breast cancer cells. In vitro and in vivo immunoprecipitation experiments revealed that ATBF1 interacted physically with the ER and that multiple domains in both ATBF1 and ER proteins mediated the interaction. Furthermore, we demonstrated that ATBF1 inhibited ER function by selectively competing with the steroid receptor coactivator AIB1 but not GRIP1 or SRC1 for binding to the ER. These findings not only support the concept that ATBF1 plays a tumor-suppressive role in breast cancer, they also provide a mechanism for how ATBF1 functions as a tumor suppressor in breast cancer.

FIGURE 1.

Expression of ATBF1 protein in ER-positive and ER-negative breast cancer cell lines. Names of cell lines are at the top, and protein names are at the left. Expression of ATBF1 protein in MCF-7 cells served as the positive control in the panel of ER-negative cell lines and β-actin as the loading control.

FIGURE 2.

Effect of ATBF1 on ER-mediated cell growth and proliferation in ER-positive breast cancer cell lines MCF-7 and T-47D treated with or without E₂. A and B, from DNA synthesis assays; C and D, from SRB staining assays. In MCF-7 cells where ATBF1 is highly expressed, ATBF1 was knocked down by RNAi, whereas in T-47D cells, which express little ATBF1, expression plasmids were transfected to express ATBF1. ATBF1 expression in transfected cells was confirmed by Western blotting in both cell lines (E and F). Transfection conditions are indicated in each panel. The final concentration of E₂ in the media was 1 μm. n.s., not significant. *, p < 0.05; **, p < 0.005. SiLuc, negative control siRNA against the luciferase gene; siATBF1, ATBF1 siRNA.

FIGURE 3.

Inhibitory effect of ATBF1 on ER-mediated transcriptional activity. A–C, knockdown of ATBF1 in ER-positive MCF-7 cells, which was confirmed by Western blotting (B), significantly increased the transcription of ER target genes (A) and the estrogen-responsive element-mediated reporter activity (C) caused by E₂ treatment. SiLuc, siControl, and siATBF1, siRNAs against the luciferase gene, negative control siRNA (purchased from Dharmacon), and siRNAs against ATBF1, respectively. D–F, overexpression of ATBF1 in T-47D cells, as verified by Western blotting (E), significantly inhibited E₂-driven transcription of ER target genes (D) and estrogen-responsive reporter activity (F). FLAG-ATBF1 plasmid was used to restore ATBF1 expression with FLAG-pcDNA3 as the control. E₂ treatment was at 1 μm, and its vehicle alcohol was used as the control. n.s., not significant. *, p < 0.05; **, p < 0.005.

FIGURE 4.

Detection of protein-protein interactions between ATBF1 and ER by both IP and GST pulldown assays. A, ATBF1 and ER double-negative 22Rv1 cells were cotransfected with FLAG-ATBF1 and pCMV-ER plasmids, and cell lysates were precipitated with anti-FLAG affinity gel and immunoblotted (IB) with anti-ER antibody. B, T-47D cells, which express endogenous ER, were transfected with FLAG-ATBF1, and cell lysates were precipitated with anti-FLAG affinity gel and blotted with anti-ER antibody. C, cell lysates from MCF-7 cells, which express both ATBF1 and ER, were precipitated with anti-ATBF1 antibody against amino acid residues 1–160 and blotted with anti-ER antibody. Cells were treated with 1 μm E₂ or its vehicle alcohol. The expression of ATBF1 and ER in cell lysate was confirmed as shown in the lower two panels in A–C. D, in vitro translated ³⁵S-labeled ER protein was pulled down by GST-ATBF1D3 recombinant protein in a GST pulldown assay. ATBF1D3 is one of the deletion mutants of ATBF1 protein that interacts with ER protein.

FIGURE 5.

Mapping of interacting domains in ATBF1 (A) and ER (B) proteins by co-IP assays. A, summary of the mapping results for different deletion mutants of ATBF1. Full-length ATBF1 protein (residues 1–3703) is shown at the top, with the consensus LXXLL NR motif marked by a black box and 31 LXXXL variants marked by gray boxes. B, summary of mapping results for ER deletion mutants interacting with ATBF1. Full-length ER protein is shown at the top with different domains indicated. Black and white bars indicate positive and negative interactions with ER, respectively, with the name of each mutant and the residues spanned shown to the right of each bar. The results of co-IP and Western blotting for both panels are shown in supplemental Figs. S1 and S2.

FIGURE 6.

Competition of ATBF1 with AIB1 but not GRIP1 or SRC1 for binding to ER. A, expression of ATBF1 decreased the amount of AIB1-bound ER (left panel) but not GRIP1- or SRC1-bound ER (middle and right panels). Plasmids for SRCs, ER, and ATBF1 were cotransfected into 22Rv1 cells, and co-IP and immunoblotting (IB) were performed with affinity gels as indicated. Input, indicates samples without co-IP. B, knockdown of ATBF1 in MCF-7 cells increased and expression of ATBF1 in T-47D cells decreased the binding of AIB1, but not that of GRIP1, SRC1 or ER, to promoters of two ER target genes, CATD and EBAG9, as detected by ChIP assays. Cell line names are indicated at the left, gene names at the right, antibody names and cell treatments at the top, and names of molecules examined at the bottom of the panels. Normal IgG was used as the negative control for antibodies against AIB1 (aAIB1), GRIP1 (aGRIP1), SRC1 (aSRC1), and ER (aER). Alcohol was used to dissolve E₂ and as the negative control for E₂. H₂O was used as the negative control for PCR. Human β-actin gene was used as the negative control for ER target genes. The C indicates negative controls for transfection of siRNA against luciferase gene in MCF-7 cells or FLAG-pcDNA3 vector control plasmid in T-47D cells, and T indicates transfection of siRNA against ATBF1 in MCF-7 cells or FLAG-ATBF1 plasmid in T-47D cells. Input indicates cell lysates not subjected to ChIP. E₂ treatment was at 1 μm. C, expression of ATBF1 inhibited AIB1-enhanced ER activity (left), but not that by GRIP1 (middle) or SRC1 (right), as determined by luciferase reporter assays in T-47D cells transfected with the indicated plasmids. D, ATBF1 inhibited AIB1·GRIP1 (left)- or AIB1·SRC1 (middle)- but not GRIP1·SRC1-enhanced (right) ER activity. n.s., not significant. **, p < 0.005.