ERα interacts with coactivator or corepressor complexes at target genes. The balance between coactivator and corepressor is primarily directed by the nature of the ligand bound to the LBD, but many other factors are involved. Components of the corepressor complexes may significantly affect this equilibrium by influencing phosphorylation, ribosylation and acetylation of several components, or by changing the stability of key subunits of the complexes. The protein TAB2, known as a facultative component of the NCoR complex, was shown to shuttle NCoR to the cytoplasm when phosphorylated in response to inflammatory signals, causing resistance to antiandrogen in prostate and antiestrogen in breast cancer cells (Zhu et al., 2006, Cell 124: 615-29). We addressed the question whether TAB2 has a more general role in Tamoxifen resistance in breast cancer. First, we studied the localization of TAB2 and NCoR in TAM-resistant cell clones (MCF-7/TAMR-1) derived from MCF7 by continuous exposure to the drug. In all studied clones that retained ERα expression, NCoR was aberrantly localized in the cytoplasm, similarly to what is observed in wild-type Tamoxifen-sensitive MCF7 cells after treatment with IL-1β. Following these observations, we directly assessed the role of TAB2 in NCoR delocalization, by down-regulating TAB2 in TAMR cells using specific siRNA. Reduction of TAB2 protein was indeed sufficient to recover the correct nuclear localization of NCoR to cell nuclei, as demonstrated by immunofluorescence and cell fractionation studies and, importantly, restored Tamoxifen sensitivity in TAMR cells. Similar results were obtained using an additional breast cancer cell line, BT474, that overexpresses ERBB2. Gene expression profiling was then carried out using one TAMR cell clone, after TAB2 down-regulation by siRNA, by means of the 44K Agilent oligo-glass microarray system. The data obtained suggest that TAB2 may be involved in the regulation of a number of genes important for cell proliferation and estrogen response. On the other side, one important question is whether TAB2 may regulate recruitment of NCoR by estrogen-bound ERα at several genes that are repressed by estrogen. To address this point, we first obtained a list of potential targets (i.e. estrogen down-regulated genes) by crossing data from different available microarray databases and data of ERα genomic occupancy by ChIP-on-chip, ChIP-PET and ChIP-Seq analysis in MCF7 cells. ERα binding sites were then individually studied by ChIP using ERα, NCoR and TAB2 immunoprecipitation. This work is now in progress, but data are available concerning the binding of TAB2 to some of the addressed genomic locations, among which the E-Cadherin promoter (Cardamone et al., PNAS 2009, 106(18):7420-25). In parallel, the activity of TAB2 is being studied by gene expression profiling, following down-regulation of TAB2 by RNA intereference, in MCF7 cells.
TAB2 as a regulator of NCoR engagement at ERα target genes
RICCI, LAURA;GROSSO, Enrico;REINERI, STEFANIA;CUTRUPI, SANTINA;DE BORTOLI, Michele
2009-01-01
Abstract
ERα interacts with coactivator or corepressor complexes at target genes. The balance between coactivator and corepressor is primarily directed by the nature of the ligand bound to the LBD, but many other factors are involved. Components of the corepressor complexes may significantly affect this equilibrium by influencing phosphorylation, ribosylation and acetylation of several components, or by changing the stability of key subunits of the complexes. The protein TAB2, known as a facultative component of the NCoR complex, was shown to shuttle NCoR to the cytoplasm when phosphorylated in response to inflammatory signals, causing resistance to antiandrogen in prostate and antiestrogen in breast cancer cells (Zhu et al., 2006, Cell 124: 615-29). We addressed the question whether TAB2 has a more general role in Tamoxifen resistance in breast cancer. First, we studied the localization of TAB2 and NCoR in TAM-resistant cell clones (MCF-7/TAMR-1) derived from MCF7 by continuous exposure to the drug. In all studied clones that retained ERα expression, NCoR was aberrantly localized in the cytoplasm, similarly to what is observed in wild-type Tamoxifen-sensitive MCF7 cells after treatment with IL-1β. Following these observations, we directly assessed the role of TAB2 in NCoR delocalization, by down-regulating TAB2 in TAMR cells using specific siRNA. Reduction of TAB2 protein was indeed sufficient to recover the correct nuclear localization of NCoR to cell nuclei, as demonstrated by immunofluorescence and cell fractionation studies and, importantly, restored Tamoxifen sensitivity in TAMR cells. Similar results were obtained using an additional breast cancer cell line, BT474, that overexpresses ERBB2. Gene expression profiling was then carried out using one TAMR cell clone, after TAB2 down-regulation by siRNA, by means of the 44K Agilent oligo-glass microarray system. The data obtained suggest that TAB2 may be involved in the regulation of a number of genes important for cell proliferation and estrogen response. On the other side, one important question is whether TAB2 may regulate recruitment of NCoR by estrogen-bound ERα at several genes that are repressed by estrogen. To address this point, we first obtained a list of potential targets (i.e. estrogen down-regulated genes) by crossing data from different available microarray databases and data of ERα genomic occupancy by ChIP-on-chip, ChIP-PET and ChIP-Seq analysis in MCF7 cells. ERα binding sites were then individually studied by ChIP using ERα, NCoR and TAB2 immunoprecipitation. This work is now in progress, but data are available concerning the binding of TAB2 to some of the addressed genomic locations, among which the E-Cadherin promoter (Cardamone et al., PNAS 2009, 106(18):7420-25). In parallel, the activity of TAB2 is being studied by gene expression profiling, following down-regulation of TAB2 by RNA intereference, in MCF7 cells.
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