ALKYL PCDFS--INHIBITION OF MAMMARY CANCER

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Previous studies have demonstrated that 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) inhibits a broad spectrum of estrogen-induced responses in the female rat uterus and in MCF-7 human breast cancer cells. TCDD also inhibits mammary tumor formation in rats and in mice transplanted with MCF-7 cells. 6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) and related alkyl polychlorodibenzofurans (PCDFs) are relatively non-toxic analogs of TCDD which exhibit comparable in vitro and in vivo antiestrogenic and antitumorigenic activities. Thus, the alkyl PCDFs represent a new class of antiestrogens which act through the aryl hydrocarbon (Ah) receptor signal transduction pathway and thus may have clinical potential as chemotherapeutic agents for treatment of breast cancer. TCDD and MCDF, are potent antiestrogens in T47D and MCF-7 human breast cancer cells (ER- and AhR-positive, ER+AhR+) but exhibit minimal activity in ER-AhR- MDA-MB-231 cells. The in vitro or in vivo effects of alkyl PCDFs or TCDD on ER-AhR+ or ER+AhR- cells have not been previously investigated due to the unavailability of these cell lines. Experiment 1 of this project will thoroughly characterize a series of ER-AhR+ and ER+AhR- variant breast cancer cell lines. ER+AhR- cells will be isolated by culturing MCF-7 and T47D cells in 1 micromole benzo[a]pyrene (BaP) and the ER+AhR- variant cell lines will be fully characterized. ER-AhR+ variant cells will be isolated from high passage T47D cells and from MDA-MB-231 cells (ER-AhR-) stably transfected with the human arnt gene which restores Ah- responsiveness in this cell line. The resultant stable transfectants will represent an ER-AhR+ phenotype which hitherto has not been described. Moreover, since MDA-MB-231 cells are resistant to endocrine and cytotoxic drug therapy, the ER-AhR+ cells will serve as a model for assessing the antitumorigenic and antiproliferative effects of AhR agonists (e.g. alkyl PCDFs) in these cells. Although the growth of MDA-MB-231 cells are estrogen-independent, various growth factors (IGF-l, EGF, TGFalpha, insulin) act as mitogens and, therefore, growth factor-induced proliferation and a number of other characteristics of the ER-AhR+ stable transfectant cell lines will be thoroughly.investigated. Immune deficient mice transplanted with breast cancer cells will be utilized in Experiment 2 to (a) determine the factors which control the development of tumors in athymic mice transplanted with the wild-type and variant breast cancer cell lines characterized in Experiment 1 and (b) assess the relative potencies of TCDD and the alkyl PCDFs as chemotherapeutic agents in this in vivo model. In parallel studies, the relative potencies of the alkyl PCDF as antiestrogens will also be determined in the female rat uterus. These short-term studies will provide critical relative potency data for this series of alkyl PCDFs in a well-established estrogen-responsive target organ and prioritize their use in in vivo studies (Experiment 4) on the antitumorigenicity of selected congeners in the DMBA-induced rat tumor model. These proposed studies will provide critical new data on the mechanism of action of AhR agonists as antiestrogens and antitumorigenic agents in two in vivo models and determine which alkyl PCDFs should be further investigated as potential chemotherapeutic agents for treatment of mammary cancer.