ERS has a profound inhibitory effect on mammary carcinogenesis, a phenomenon recognized for almost a century, yet little is known about the mechanism(s) that accounts for this inhibitory activity. We hypothesize that ERS exerts its effects on carcinogenesis via a concomitant increase in adrenal cortical steroid secretory activity and a decrease in insulin and insulin- like growth factor (IGF) signaling activity. Evidence obtained on this project indicates that three consequences of these endocrine changes are: 1) alterations of the cell cycle regulatory machinery including p27 and cyclin D1 resulting in a reduction in the number of cells in the proliferative pool; 2) deletion of transformed cells from the mammary gland; and 3) a decrease in neo-angiogenesis critical for lesion progression. The goal of this application is to further investigate these provocative observations. The following questions will be addressed: 1) What is the role of adrenal cortical steroids and insulin-like growth factors (IGFs) in mediating the effects of ERS? We propose carcinogenesis studies in which classical ablation and/or replacement approaches will be used to determine if effects on glucocorticoids and/or IGFs account for the cancer inhibitory activity of ERS via mechanisms to be investigated in aims 3 and 4; 2) Does ERS induce the deletion of transformed cells from the mammary gland and what is the mechanism? Proposed studies include carcinogenesis and transplantation studies to assess for evidence of "sustained protection" following a period of ERS via a cell deletion mechanism; 3) Does ERS mediate its cancer inhibitory activity via regulation of cell cycle transit at the level of cyclin dependent kinase inhibitors? We propose to systematically evaluate the potential role of cyclin dependent kinase inhibitors in accounting for the cancer inhibitory activity of ERS mediated by inhibition of proliferation, induction of apoptosis and/or by cell cycle arrest, and to determine if effects are mediated via glucocorticoids and/or IGFs. The role of p27 as an effector of ERS effects on cell cycle machinery will be investigated; 4) Does suppression of neo- angiogenesis account for the apparent block in the progression of intraductal proliferations (IDP) to carcinomas observed in response to ERS? We hypothesize that an anti-angiogenic effect of ERS accounts, at least in part, for its cancer inhibitory activity. We will examine the microvessel density of pre- malignant and malignant mammary gland lesions to establish that neo-angiogenesis is indeed decreased by ERS and then will proceed to determine if ERS inhibits angiogenesis by decreasing VEGF production. The intent of the proposed experiments is to define the mechanism(s) by which ERS exerts its protective effect against cancer. This knowledge could ultimately provide new opportunities to circumvent the practical problem of implementing a program of chronic energy restriction in human populations, yet still achieve the wide-ranging health benefits of such a program using a chemically-defined approach that targets specific genetic cascades.