Co-regulators SRC-1 and SMRT interact with ER-alpha and ER-beta in human breast cancer
FJ Fleming1,2, ADK Hill1,2, EW Mc Dermott1,2, N O'Higgins1,2 & LS Young1,2
ER-alpha and ER-beta function as transcription factors to modulate expression of target genes. Both interact with nuclear regulatory proteins to enhance or inhibit transcription. We hypothesized that these co-regulators are expressed in breast cancer tissue and may be differentially regulated by estrogen and tamoxifen.
ER-alpha, ER-beta, the co-activator SRC-1, and the co-repressor SMRT were localized within breast tissue by immunohistochemistry, and the spatial co-expression assessed by immunofluoresence. The ability of beta-estradiol and 4-hydroxytamoxifen (4-HOT) to modulate the protein expression of ER-alpha, ER-beta, SRC-1, and SMRT in primary cell cultures, and MCF-7 and T47-D cell lines was assessed by western blotting.
ER-alpha and ER-beta were found to be co-expressed with the co-regulators SRC-1 and SMRT in the nuclei of breast cancer epithelial cells. Partial and complete response elements for the ER were identified on the promotor region of ER-alpha, ER-beta, SRC-1 and SMRT. Beta-estradiol induced an upregulation in the protein expression of both Estrogen receptors and the co-regulator proteins. Furthermore, beta-estradiol induced a translocation of ER-alpha from the cytoplasm to the nucleus. 4-HOT induced an increase in the expression of ER-alpha and the repressor protein SMRT, whereas it decreased the protein expression of ER-beta and the activator protein SRC-1. Using mobility shift assays, increased binding of ER-alpha and ER-beta to the estrogen response element (ERE) was seen in the presence of beta-estradiol and 4-HOT. The presence of both SRC-1 and SMRT at the ER-alpha and ER-beta-DNA binding site was observed using monoclonal antibodies against the co-regulatory proteins.
ER-alpha, ER-beta, SRC-1 and SMRT can be differentially regulated by beta-estradiol and 4-HOT. The co-localisation of SRC-1, SMRT with ER and the presence of the co-regulators at the ER-ERE binding site, provides evidence that co-regulators and ER function together to modulate target gene transcription in breast cancer.