DOMINANT NEGATIVE ESTROGEN RECEPTORS AND BREAST CANCER

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We will develop and investigate a novel method for functional inactivation of estrogen receptor (ER) in estrogen-dependent human breast cancer cells. Our approach is based on dominant negative mutants. These are ER mutants which are inactive on their own, but are able to suppress the activity of wild-type ER when they are co-expressed in the same cells. Our Specific Aims are: (1) To generate more powerful dominant negative mutants of ER; (2) To determine the molecular mechanisms responsible for the effectiveness of the mutants; (3) To use the dominant negative mutants to block estrogen-dependent growth of breast cancer cells in vitro and in vivo in nude mice. In preliminary studies we generated three effective and ER-specific dominant negative mutants. These mutants appear to function by inactivation of the C-terminal transactivation domain of ER. We will generate even more potent mutants by replacing both the C- and N-terminal transactivation domains. We will use the P22 challenge phage system to generate ER mutants with enhanced binding to the estrogen response element. These mutations, which lead to increased ability to compete for binding to the estrogen response element, will be introduced into dominant negative mutants with inactive transactivation domains to produce extremely potent dominant negative mutants. We will determine the relative contributions of competition for binding to the estrogen response element, formation of heterodimers, and interference with ER-specific components of the transcription apparatus to the activity of our most effective dominant negative mutants. The ability of our most powerful dominant negative mutants to block estrogen-dependent growth of MCF-7 human breast cancer cells will be evaluated. Stably transfected cells producing the dominant negative mutant ERs will be tested for suppression of estrogen-stimulated growth in and for suppression of the expression of some growth factors and proteins thought to be important in ER-stimulated growth and tumorigenicity. We will use stably transfected MCF-7 cells expressing a potent dominant negative mutant from a regulated promoter to initiate estrogen-dependent growth of MCF-7 tumors in athymic nude mice. Then we will activate production of the dominant negative mutant, and determine if growth of the tumors is blocked or reversed. These studies should provide new insights into the molecular mechanism of ER action and the proliferation and tumorigenicity of breast cancer cells, and allow evaluation of a novel approach for the suppression of estrogen-dependent breast cancer growth.