Research in this laboratory has identified a novel mechanism of transcriptional activation of genes by E2 in breast cancer cells and this involves interaction of Eralpha/Spl with selected GC-rich Spl binding sites in target gene promoters. We hypothesize that hERalpha/Spl action through GC-rich elements play an important role in hormonal regulation of gene expression and growth in breast cancer cells. Genes upregulated through Eralpha/Spl are potential chemotherapeutic targets, and the proposed studies will investigate key coactivators required for ERalpha/Spl action in breast cancer cells. ERalpha/Spl-mediated gene expression is dependent, in part, on activation function 1 (AF1) of ERalpha and preliminary studies show the p160 steroid receptor coactivators (SRCs) do not enhance Eralpha/Spl action from GC-rich promoters. In contrast, several vitamin D receptor interacting proteins (DRIPs) enhance hormone-dependent transactivation in breast cancer cells transfected with GC-rich promoter-reporter constructs, and Aim 1 will characterize the molecular mechanisms of ERalpha/Spl coactivation by members of the DRIP family of mediator proteins. Based on preliminary studies, Aim 2 will investigate the molecular mechanisms of Era/Spl coactivation by SNURF and further enhanced coactivation by DRIP protein coactivators. Preliminary studies have shown that Brahma-related gene 1 (Brg-1) and protein inhibitor of activated STAT3 (PIAS3) also coactivate Eralpha/Spl in breast cancer cells, and the molecular mechanisms of coactivation will be investigated in this aim.
Aim 3 will focus on the assembly of Eralpha/ Spl and other coregulatory proteins on GC-rich E2- responsive cathepsin D and other gene promoters in MCF-7 and ZR-75 cells. Hormone- and time dependent cycling of nuclear transcription factors on and off promoters will be determined using endogenous, stably-transfected and transiently transfected promoters and multiple antibodies for obtaining immunoprecipitable crosslinked DNA-protein complexes. These mechanistic studies will identify new coactivators required for hormone action in breast cancer cells and thereby delineate potential targets for development of chemotherapeutic drugs for treatment of breast cancer.
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