The LIM protein Ajuba recruits DBC1 and CBP/p300 to acetylate ERα and enhances ERα target gene expression in breast cancer cells

Abstract

Estrogen/ERα signaling is critical for breast cancer progression and therapeutic treatments. Thus, identifying new regulators of this pathway will help to develop new therapeutics to overcome chemotherapy resistance of the breast cancer cells. Here, we report Ajuba directly interacts with ERα to potentiate ERα target gene expression, and biologically Ajuba promotes breast cancer cell growth and contributes to tamoxifen resistance of these cells. Ajuba constitutively binds the DBD and AF2 regions of ERα, and these interactions can be markedly enhanced by estrogen treatment. Mechanistically, Ajuba recruits DBC1 and CBP/p300 and forms a ternary complex to co-activate ERα transcriptional activity and concomitantly enhances ERα acetylation. Moreover, components of this complex can be found at endogenous promoters containing functional ERα responsive elements. Taken together, these data demonstrate that Ajuba functions as a novel co-activator of ERα and that Ajuba/DBC1/CBP/p300 ternary complex may be a new target for developing therapeutics to treat breast cancer.

Figure 1.

Ajuba interacts with ERα independent of the conserved NR boxes. (A) The three conserved NR-boxes in Ajuba were shown on the upper panel. Plasmids were transiently transfected into 293T cells and the co-IP assay was carried out with Myc antibody. * IgG-heavy chain. (B) Flag-ERα and Myc-Ajuba plasmids were transfected into 293T cells and co-IP assay was performed by using Flag-M2-beads. (C) The preLIM and LIM regions of Ajuba were illustrated on the upper panel. The full length or truncations of Ajuba were transiently co-transfected into 293T cells together with Flag-ERα plasmid. The co-IP assay was performed by using Flag antibody. (D) The functional domains of ERα were shown on the upper panel. The interaction between full-length or truncations of ERα with Ajuba in 293T cells was detected by co-IP assay and western blotting. The relative amount of co-eluted Myc-Ajuba was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled. (E) The plasmids encoding Ajuba, LIMD1, Zyxin, Wtip and Lpp were respectively co-transfected with Flag-ERα plasmid into 293T cells and co-IP assay was performed. (F) The plasmids encoding Flag-ERα and Myc-Ajuba were transfected into 293T cells, and the resulting cells were cultured in phenol-red free media containing 5% charcoal stripped FBS for 2 days and then treated with E2 (20 or 100 nM) or ethanol for 12 h. The co-IP assay was performed by using Flag-M2 beads. The relative amount of immunoprecipitated Myc-Ajuba was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled. (G) GST-ERα and His-Ajuba proteins were expressed in E. coli BL21, and GST-pulldown assay was performed in the presence of E2 (100 nM) or ethanol. The relative amount of pulled-down His-Ajuba was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled. (H) T47D cells treated with 100 nM E2 or ethanol for 12 h were harvested and co-IP assay was performed by using ERα antibody or IgG control. The relative amount of immunoprecipitated Ajuba was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled.

Figure 2.

Ajuba enhances ERα transcriptional activity. (A) The pGL3.0-ERE-Luc reporter contains three tandem ERE elements and were transiently transfected into 293T cells. The resulting cells were treated with 20nM E2 or ethanol for 12hrs and were prepared for the luciferase reporter assay. The luciferase activity was normalized to the value of β-gal activity (three repeats, * means P < 0.05). (B and C) Wild-typed (B) or mutated (C) TFF1 promoter driven luciferase reporter plasmids were co-transfected with ERα and/or Ajuba plasmids into 293T cells. The transfected cells were treated with 20 nM E2 before being harvested for luciferase reporter assay and the value was normalized to β−gal (Three repeats, * means P < 0.05, NS means no significance). (D–F) The location of ERE in the promoter or enhancer of ERα target genes was shown in the upper panel. The T47D cells were treated with 100 nM E2 or ethanol for 1 h and prepared for the ChIP assay. The relative enrichment of Ajuba and ERα on the promoter or enhancer of TFF1 (D), Greb1 (E) and SGK3 (F) was detected by qPCR and normalized to input (three repeats, * means P < 0.05).

Figure 3.

Ajuba enhances ERα target gene expression and promotes breast cancer cell growth. (A) Ajuba was stably expressed in T47D cells and western blot assay showed the expression level of Ajuba. (B–D) The T47D-Ajuba and T47D-vector cells were cultured in phenol-red free media containing 5% charcoal stripped FBS for 2 days and then treated with 20 nM E2 or ethanol for 12 h and the relative mRNA expression of TFF1 (B), Greb1 (C) and SGK3 (D) was detected by qRT-PCR and normalized by the expression of β-actin (three repeats, * means P < 0.05). (E) Ajuba was stably expressed in MCF7 cells. (F–H) The expression of TFF1 (F), Greb1 (G) and SGK3 (H) in MCF7-Ajuba and vector-control cells was detected by qRT-PCR (three repeats, * means P < 0.05). (I) Ajuba was stably knocked-down in T47D cells (shLuc stands for sh-luciferase). (J–L) The expression of TFF1 (J), Greb1 (K) and SGK3 (L) in T47D-shAjuba and sh-Luc control cells was detected by qRT-PCR (three repeats, * means P < 0.05). (M–N) CCK8 assays were carried out to examine the cell growth in Ajuba overexpressed or knocked-down T47D cells respectively (three repeats, * means P < 0.05).

Figure 4.

Ajuba antagonizes the Tamoxifen-toxic effect in breast cancer cells. (A–C) Ajuba-overexpressed or knocked-down T47D and MCF7 cells were treated with TAM or ethanol for 6 days and cell viability was examined by CCK8 assay. The results were normalized to the normal control cells (ethanol-treated groups) (three repeats, * means P < 0.05, NS means no significance). (D–F) T47D-Ajuba and vector control cells were treated with TAM (100 nM) or ethanol for 4 days, the relative mRNA expression of ERα target genes was detected by qRT-PCR. The results were normalized by ethanol-control group and the inhibitory percent of TAM was labeled (three repeats, * means P < 0.05). (G–I) The effect of TAM (100 nM) on the expression of ERα target genes in MCF7-Ajuba and vector control cells were detected by qRT-PCR. The results were normalized by ethanol-control group and the inhibitory percent of TAM was labled (three repeats, * means P < 0.05). (J–L) Knocking-down Ajuba in T47D cells magnified inhibitory effect of TAM (100 nM) on the expression of ERα target genes. The results were normalized by ethanol-control group and the inhibitory percent of TAM was labeled (three repeats, * means P < 0.05).

Figure 5.

Ajuba recruits DBC1 to enhance ERα transcriptional activity. (A, B) plasmids encoding DBC1 and Ajuba were transfected into 293T cells and co-IP assay was performed by using Myc antibody (A) or Flag-M2-beads (B). (C) The endogenous interaction between DBC1 and Ajuba was detected in T47D cells by co-IP assay. (D) GST-DBC1 and His-Ajuba was respectively expressed in E. coli BL21, and in vitro binding assay was performed. (E) The plasmids of full-length and truncations of Ajuba protein were co-transfected into 293T cells with DBC1 plasmids and co-IP assay was carried out. (F) Increasing amount of plasmids encoding Ajuba were co-expressed along with DBC1 and ERα in 293T cells and co-IP assay showed that Ajuba enhanced the interaction between DBC1 and ERα (the relative amount of immunoprecipitated HA-DBC1 was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled). (G) TFF1 promoter driven luciferase reporter assay was performed in 293T cells and the transfected cells were treated with 20 nM E2 before detecting luciferase activity and the value was normalized to β-gal (three repeats, * means P < 0.05). (H) T47D-Ajuba and vector control cells were treated with 100 nM E2 for 1 h. ChIP assay was performed by using DBC1 antibody and the result was normalized by input (three repeats, * means P < 0.05). (I) MCF7-Ajuba and vector control cells were transfected with siRNA of DBC1 for 48 h, the expression of TFF1 was detected by qPCR and the expression of DBC1 and ERα were analyzed by western blotting (three repeats, * means P < 0.05).

Figure 6.

Ajuba promotes ERα transcriptional activity by recruiting CBP/p300 and increasing the acetylation of ERα. (A) The acetylation of ERα in T47D-shLuc and shAjuba cells was detected by western blotting after the enrichment of the endogenous ERα by using ERα antibody. (B) co-IP assay in transfected 293T cells showed that Ajuba did not bind SIRT1. (C) co-IP assay was performed in transfected 293T cells and the interaction between DBC1and SIRT1 was analyzed. (D) Transfected 293T cells were treated with 100 nM E2 or ethanol for 12 h and co-IP assay showed that E2 enhanced the interaction between Ajuba, CBP and ERα (the relative amount of immunoprecipitated Flag-ERα was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled). (E) Ajuba and E2 could increase ERα acetylation. Plasmids were transfected into 293T cells and the proteins were enriched by Flag antibody, and the acetylation of ERα was detected by using acetyl-Lysine antibody (* means the band of acetylated Ajuba, L means low exposure and H means high exposure). (F) Transfected 293T cells were treated with 20 nM E2 and TFF1-promoter driven luciferase reporter assay was performed and the value was normalized to β-gal (three repeats, * means P < 0.05). (G) shAjuba-T47D and shLuc- T47D cells were treated with 100 nM E2 for 1 h and ChIP assay was carried out by using indicated antibodies. The result was normalized by input and 5–10% as input (three repeats, * means P < 0.05).

Figure 7.

Ajuba, DBC1 and CBP/p300 forms a ternary complex to enhance ERα target gene transcription. (A) Plasmids were transfected into 293T cells and the Co-IP assay was performed by using Flag-M2 beads (the relative amount of immunoprecipitated HA-CBP was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled). (B) Myc-Ajuba, HA-CBP and Flag-DBC1 plasmids were co-transfected into 293T cells and sequential co-IP assays were performed. The first round co-IP assay was carried out by using Flag antibody or normal IgG and then the 3XFlag-peptides eluted product was subjected to the second round co-IP assay by using HA antibody or normal IgG. (C) The endogenous co-IP assay was performed in T47D-PCDH-Vector or T47D-PCDH-Myc-Ajuba cells by using ERα antibody. (D) siRNA-DBC1 was transfected into T47D cells for 48 h, and the co-IP assay was performed by using ERα antibody. (E) The siRNA-NC or siRNA-DBC1 was transfected into T47D cells and the ChIP assay was performed after the E2-treatment and the results were normalized to input and 5–10% as input (three repeats, * means P < 0.05). (F) TFF1-luciferase reporter assays were performed in 293T cells. Transfected cells were treated with 20 nM E2 for 12 h before harvesting and the value was normalized to β-gal (three repeats, * means P < 0.05). (G) The model of Ajuba regulating ERα transcriptional activity. Ajuba forms a complex with DBC1 and CBP to enhance the acetylation and transactivity of ERα.