The expression of estrogen receptor-alpha (ER() is critical for establishing the hormone responsive phenotype of breast cancers. Functional promoter analysis and hypersensitive site mapping identified the AP2( transcription factor as a regulator of ER( expression and confirmed the location of AP2 regulatory sites in the human ER( gene promoter. Eliminating AP2( expression in ER(-positive cells using siRNA significantly down-regulates the expression of ER( and ER(-target genes. We conclude that AP2( is a key regulator of ER( expression in hormone responsive breast cancer cells. Epigenetic alterations involving methylation and histone acetylation have also been shown to contribute to the control of ER( gene expression. We hypothesize that epigenetic changes involving the AP2 regulatory element may provide an additional mechanism to regulate expression from the ER( promoter. Furthermore, we have identified an interaction between AP2 factors and the tumor suppressor protein p53 and there is substantial evidence showing that overexpression of p53 is capable of inhibiting the transcriptional activity of AP2. Hence, the interaction between AP2 factors and p53 represents another potential mechanism regulating the expression of ER( and may explain the finding that breast cancers with p53 overexpression are usually ER(-negative. Herein we propose to define the mechanisms regulating AP2-mediated activation of ER( and to determine the physiologic processes of hormone response controlled by AP2(. To accomplish this goal we propose to analyze the parameters of hormone response controlled by AP2( in MCF7 cells. We will define the functional domains of AP2 factors that are necessary for regulation of ER(. Using cell models of ER( repression and reactivation, we will determine if epigenetic alterations of the AP2 regulatory region contribute to control of ER( expression. We will investigate the mechanisms of p53-mediated repression of ER( by determining if p53 alters AP2( activity or chromatin interaction. The role of p53 in regulating ER( will be investigated by examining p53 alleles recovered from breast cancers for mutations that disrupt the AP2-interaction domain. We will define residues that are critical for the interaction between AP2 and p53 and generate antagonists as a prelude to developing a specific inhibitor of the AP2-p53 interaction. In the future it may be possible to develop a novel therapeutic to induce hormone response in breast cancers by specifically targeting the AP2 pathway. ? ?
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