The P.I. is investigating a novel method for the functional inactivation of estrogen receptors (ERs) in estrogen- dependent human breast cancer cells based on the use of potent dominant negative (DN) ER mutants. The P.I. has generated several potent DN-ERs and shown that they inhibit estrogen-stimulated gene expression and proliferation of breast cancer cells. The present proposal focuses on two critical advances made during this work: 1 the identification by 2-hybrid interaction cloning of a novel corepressor protein, denoted REA for repressor of estrogen action. REA selectively enhance the potency of DN-ERs, while having very little effect on wild type ER and no effect on other nuclear receptors. 2) The development of a system for generating targeted DN-ERs which bind with high affinity and selectivity to specific hormone response elements.
The Specific Aims are: 1) To analyze the molecular mechanisms by which the corepressor REA is recruited by DN-ERs and potentiates their activity. Physica and functional mapping of DN-ER/REA interaction will be carried out using GST pull-down methods, mammalian 2-hybrid transactivation assays and mutational analyses. Using antibodies to REA, and antisense methodology, intracellular RE will be neutralized/eliminated and the functional importance of the DN-ER/REA interaction will be defined in intact cells. The P.I. will identify additional REA interaction partners which may potentiate corepressor activity, and characterize the effect of REA on ER cellular distribution. 2) To search for other dominant negative corepressors, the P.I. will use 2-hybrid interaction cloning with the two most potent DN-ERs. 3) To optimize receptor-corepressor interaction, 2-hybrid screening with REA will be used to screen ER mutant libraries for mutants exhibiting enhanced corepressor binding. 4) To assess the roles of the c-myc, TGFa and cathepsin D genes in the proliferation and invasiveness of ER positive breast cancer cells, the modified P22 challenge phage system will be used to create DN-ERs that bind selectively and with high affinity to the different non-consensus EREs found in each of these genes. The P.I. will introduce the gene- selective DN-ERs into cells using her efficient adenovirus system, determine their effects on gene expression, and examine their importance in ER regulated breast cancer cell proliferation and invasiveness.
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