The expression of estrogen receptor (ER) has a profound influence on the biology of breast carcinoma. Patients with ER-positive tumors tend to have a better prognosis than patients with carcinomas which lack receptor expression. However, little is known about mechanisms which control ER expression. By utilizing a cell line model of breast cancer, preliminary studies have determined that ER expression is controlled through transcriptional regulation. No previous studies have defined the ER promoter.
The specific aim of this application is to characterize the ER promoter and define important transcriptional control regions which are functional in breast carcinoma. These results will then be used to determine the mechanism responsible for the lack of ER expression in ER- negative carcinomas. The ER promoter will be functionally mapped using a luciferase reporter system. A human genomic lambda library will be screened to identify clones containing genomic DNA from the 5' end of the ER gene. Various regions of DNA upstream of the transcriptional start site of the ER gene will be cloned into luciferase reporter plasmids. The first intron from the ER gene is 20 kbp and this region will also be examined for enhancer activity using the luciferase reporter system. These constructs will be transfected into MCF-7 (ER positive) and MDA-MB-231 (ER negative) cells to define what region of the ER gene are necessary for ER expression. One of two possible results seems most likely; either these constructs will function the same in both cell lines or the MCF-7 cell line will demonstrate expression while MDA-MB-231 will not. These results will be useful to determine if the lack of expression in MDA-MB-231 is due to a cis-effect defined by DNA mutations or methylation or a trans-effect possibly due to a different transcription factor(s) functioning in these two cell lines. It is hoped that these experiments will define a mechanism responsible for the varied expression found in these breast cancer cell lines. Finally, the universality of these mechanisms will be explored by examining a variety of other cell lines and fresh cancer isolates.
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