PROJECT SUMMARY/ABSTRACT The mammary gland is an exceptionally dynamic organ, undergoing alternating cycles of expansion and regression over the female reproductive lifespan13,21. These highly-regulated cycles of proliferation and cell death are dictated by the ovarian hormones and involve a massive expansion of the mammary epithelium, supported by a transient increase in the number of mammary stem cells (MaSCs)8–10. It is well known that, in patients, both the number of past menstrual cycles and pregnancy (transiently) increase the risk of developing breast cancer1,2. However, the underlying cause of this phenomenon is unclear. Growing evidence, however, suggests that improper involution, the process of tissue regression following lactation, is involved in increasing this risk3,4. Our lab has identified the miR-424(322)/503 cluster as an important regulator of involution6, and I have recently shown that miR-424/503 is lost in ~14% of breast cancers and that, over time, miR- 424(322)/503-/- (KO) female mice develop mammary tumors that are promoted by pregnancy7. My preliminary analysis of human breast tumors has revealed that miR-424(322)/503-deficient breast cancers have accumulation of β-catenin and hyperactivation of the Wnt/β-catenin signaling pathway. Female KO animals show several hallmarks of aberrant canonical Wnt pathway activation, evidenced by the nuclear accumulation of β-catenin as well as Keratin 6 expression. The expansion of the mammary epithelium induced by the ovarian hormones during the estrous cycle and pregnancy is achieved via a transient increase in the number of mammary stem cells (MaSCs) driven by activation of canonical Wnt signaling8–10. Thus, this proposal seeks to elucidate the mechanism of miR-424(322)/503 regulation of mammary epithelial homeostasis via its effect on MaSC expansion. In Aim 1, I will determine whether miR-424(322)/503 regulates various Wnt/β-catenin pathway regulators using biochemical approaches. In Aim 2, I will interrogate the role of miR-424(322)/503 in regulating its targets across various mammary epithelial cell types and stages. Finally, in Aim 3, I will study the effect of miR-424(322)/503 loss on MaSC homeostasis in vivo. These studies seek to reveal a novel mechanism of regulation across reproductive stages and cell types of the mammary epithelium under the umbrella of miR-424(322)/503 regulation.