Results from our laboratory suggest that the expression of the estrogen receptor (ER) in human breast cancer is a complex process involving multiple steps subject to regulation. Although transcriptional events contribute to regulation, our studies indicate that post-transcriptional regulation plays a predominant role in determining the level of ER. Estrogen and TPA regulate ER by destabilization of ER mRNA, whereas, the anti-estrogen, ICI-164,384, decreases ER protein half-life and blocks translation of the ER transcript. Additional studies have identified an ER-positive breast cancer cell line in which treatment with estrogen results in altered regulation of ER expression. The goal of this proposal is to investigate the mechanisms responsible for the post-transcriptional regulation of ER in breast cancer. This study is divided into two major areas of research: 1, to determine the mechanism of regulation of ER mRNA stability; and 2, to determine the mechanism of translational regulation of ER expression. To determine the mechanism of regulation of ER mRNA stability, experiments are designed to: 1, identify the region of the ER transcript which mediates the estrogen response; 2, identify the factor which recognizes the estrogen responsive element in the ER mRNA; and 3, identify the nuclease which mediates the estrogen effect. Several approaches are designed to identify the region of the ER transcript which mediates the estrogen response: 1, tests the role of the 5' UTR, 3' UTR, and the coding region of the ER mRNA in a transfection assay; 2, assays the ability of RNA corresponding to different regions of the transcript to, inhibit ER mRNA degradation; and 3, is a genetic approach. To identify the factor which recognizes the estrogen responsive element in the ER mRNA, three isolation strategies are proposed: 1, conventional chromatography; 2, affinity purification using biotinylated RNA transcripts; and 3, affinity column chromatography using 2'-OH RNA derivatives. The nuclease and its associated factors will be identified using two in vitro RNA degradation assays. To determine the mechanism of translational regulation of ER expression, the role of the 5' untranslated region of the ER transcript in the regulation of translation and in targeting ER mRNA to the endoplasmic reticulum will be conducted. Mutational analysis will determine whether the small 5' open reading frame (ORF) plays a role in regulation and in targeting. Additional studies will determine whether the 5' ORF functions in cis or in trans. A putative, signal consensus sequence, identified in the 5' ORF, will be mutated to determine its role in targeting the ER mRNA to the endoplasmic reticulum.
| Martin, M B; Franke, T F; Stoica, G E et al. (2000) A role for Akt in mediating the estrogenic functions of epidermal growth factor and insulin-like growth factor I. Endocrinology 141:4503-11 |
| Stoica, A; Saceda, M; Fakhro, A et al. (1999) Regulation of estrogen receptor-alpha gene expression by 1, 25-dihydroxyvitamin D in MCF-7 cells. J Cell Biochem 75:640-51 |
| Saceda, M; Lindsey, R K; Solomon, H et al. (1998) Estradiol regulates estrogen receptor mRNA stability. J Steroid Biochem Mol Biol 66:113-20 |
| Saceda, M; Knabbe, C; Dickson, R B et al. (1991) Post-transcriptional destabilization of estrogen receptor mRNA in MCF-7 cells by 12-O-tetradecanoylphorbol-13-acetate. J Biol Chem 266:17809-14 |
