Breast cancer is one of the leading causes of morbidity and mortality among women in America and other industrialized countries; latest statistics show that there is an increased incidence of breast cancer among women above 60 years of age. Natural and environmental estrogens appear to be involved at several stages of the carcinogenetic process, especially during menopause and postmenopausic periods. Estrogens regulate the proliferation of their target cells through a two-step mechanism. In the first step, estrogens mediate an increase of the cell number; in the second step, they inhibit cell proliferation (shutoff). Failure to stringently respond to this latter normal fail- safe mechanism may eventually result in breast tumorigenesis. Although this Step-II is less understood than Step-I, it provides the theoretical explanation for the success of DES treatment in advanced breast cancer. The stated purpose of this application is to understand the mechanisms involved in the estrogen-induced proliferative shutoff. Using MCF7 cells, we have succeeded in segregating fixed phenotypes expressing only one of the two estrogen-mediated steps: a) variant E8CASS expresses only the shutoff (Step- II) and b) variant A2E8CASS expresses only Step- I. In addition to the above mentioned relevance to the carcinogenetic process in the breast, these variants provide a model to study progression from estrogen sensitivity to resistance. This represents an important milestone in our quest to explore the regulation of cell proliferation under normal conditions and into the initial and progressive paths toward malignancy.
The specific aim of this proposal is to isolate unique mRNA sequences that mediate the estrogen-induced inhibition of the proliferation of breast cancer cells. Flow cytometry will be used to characterize the kinetics of the proliferative shutoff; this is required to find the appropriate conditions to evoke a proliferative shutoff. Unique mRNA sequences responsible for the shutoff will be isolated using two complementary approaches: a) a subtraction library/genetic screen method (Wang & Brown), and b) a differential display method (Liang & Pardee). Candidates thus obtained will be cloned into mammalian expression vectors controlled by an inducible promoter. Their expression and ability to evoke a shutoff response will be tested in cell culture and in nude mice. Knowledge gathered from this experimental design will contribute to devise better prognostic and therapeutic tools to fight breast cancer, especially the estrogen receptor-positive variety in older women.
