Breast cancer is a significant cause of death among women. The long- range goal of this program is to understand the molecular basis of breast cancer development. The general approach is to determine the status and function of cellular tumor suppressor genes in normal and preneoplastic mouse mammary epithelial cells. The murine mammary tumor model system with its wealth of background information and its well- defined, intermediate stage (preneoplastic lesions) provides an unparalleled opportunity to examine the molecular basis of suppressor gene involvement in breast cancer. As early preneoplastic disease would be the most effective target for diagnostic and therapeutic approaches, we have focused our program on that phase of breast cancer development.
Specific aims are the following: (1) To establish whether specific changes in the p53 suppressor gene are associated with defined stages of mammary tumorigenesis. The status of p53 protein [mutant, wild type (WT)] will be determined in established mammary epithelial cell lines (MECL), in preneoplastic HAN outgrowths derived in vivo from the MECL, and in tumors arising from the outgrowths. Specific p53 mutations will be identified using polymerase chain reaction. Significance of p53 mutations will be assessed in functional assays (ras co-transformation, alteration of normal MECL to induce HAN outgrowths). (2) To determine if inherited changes in p53 predispose to breast cancer development. We have made mice with disrupted p53 alleles in the germ line [p53(+/-) and p53(-/-)]. They will be tested for increased susceptibility breast cancer induced by chemical carcinogen (DMBA), virus (MMTV), or oncogene (wnt-1). Dysplasias and tumors in the heterozygotes will be examined for status of the remaining p53 allele (activated vs. inactivated). We propose to develop a mouse by gene targeting with a point mutation in one p53 allele (mimicking the LiFraumeni syndrome) to extend this model. (3) To identify tumor suppressor genes important in early stages of breast cancer development. Somatic cell hybridization between MECL that induce normal or HAN outgrowths will reveal the dominant phenotype in the mammary gland. (4) To analyze whether tumor suppressor genes inhibit mammary tumor formation. WT p53 under the control of an inducible promoter will be introduced into MECL containing different p53 mutations. WT p53-expressing cells will be transplanted in mammary fat pads to assay for reversion of phenotype. This program builds on our years of experience with the murine mammary tumor system, the ability to experimentally manipulate preneoplastic mammary lesions both in vivo and in vitro, and our familiarity with the p53 system. It takes advantage of our recent success at establishing mouse MECL in culture, as well as our recent development of p53-deficient mice. These tools allow us to experimentally address molecular genetic mechanisms responsible for early stages of breast disease, which we believe will have profound implications toward understanding the development of human breast cancer.
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