Stem cells (SCs) are thought to be integral to the development and progression of cancer, and their eradication may be essential for the cure of cancer. Yet, direct proof is lacking due to our poor understanding of the molecular differences between normal and cancer SCs. We will investigate normal and cancer mammary stem cells (MSCs) by focusing on the role of the cell fate determinant Numb in two signaling axes: Numb:Notch and Numb:p53. Numb is a tumor suppressor in human breast cancer. Its expression is lost, through increased degradation, in ~50% of breast cancers. These Numbneg cancers display overall poorer prognosis. Mechanistically, loss of Numb causes increased Notch signaling and decreased p53 signaling. Thus, Numb controls both an oncogenic pathway (the Numb:Notch axis) and a tumor suppressor one (the Numb:p53 axis). We showed that Numb is asymmetrically partitioned at the first division of normal MSCs and hypothesize that loss of Numb affects the kinetics of division and MSC fate. Our specific aims are to: 1. Define the role of the Numb:Notch and Numb:p53 axes in normal and cancer MSCs. We will exploit our capacity to propagate and isolate MSCs to near-purity, for biological, biochemical and omics approaches. In this task, we will integrate knowledge derived from the analysis of real human cancers and of genetically-defined mouse models. 2. Define the broader biological context of Numb impact in stem cell biology, by analyzing the role of endocytosis in MSC fate determination. This is justified by the fact that Numb is an endocytic protein and that data in Drosophila indicate a complex role of endocytosis in cell fate specification. 3. Identify the E3 ligase responsible for Numb degradation in Numbneg breast tumors, in order to obtain druggable targets to restore Numb levels in these tumors. If successful, our work will elucidate major mechanisms of normal and cancer stem cell regulation, and provide tools for SC-specific therapeutic intervention.