Mechanisms underlying cell-fate determination in the mammary gland development

Notch signaling pathway is an important developmental pathway and has been implicated in both mammary gland development and tumorigenesis. Several studies investigating Notch signaling in mouse mammary gland implied that Notch signaling is an important factor in the determination of the luminal cell type. Clinical studies on breast tumors as well as studies in transgenic mouse models overexpressing active Notch receptors suggested an oncogenic function of Notch in the mammary gland connecting it to the poor breast cancer prognosis. However the physiological role of Notch signaling and its downstream mechanisms remain unclear. Analysis of Transgenic-EGFP Notch reporter mouse mammary epithelium revealed that Notch signaling is active specifically in a subset of hormone receptor positive cells. More sensitive FACS analysis consistently shows EGFP expression in HR+ luminal cells and reveals a weaker signal in a subset of basal (CD24lo) cells. Additionally mRNA analysis revealed that Notch active luminal cells are expressing Wnt4 ligand. To unveil the role of the Notch signaling in the Wnt4 expressing HR+ cells, we conditionally deleted RBP-Jκ, Notch signaling mediator, in Wnt4 expressing subpopulation of hormone receptor positive cells via Wnt4Cre. Abrogation of RBP-Jκ resulted in loss of progesterone receptor expression in 90% of cells bearing the RBP-Jκ deletion while estrogen receptor expression remained intact, implicating RBP-Jκ in regulation of progesterone receptor expression. To test whether Notch signaling regulates PR expression in the entire HR+ population, we analyzed mammary epithelium in which RBP-Jκ has been conditionally deleted via MMTV-Cre in progenitor cells revealing the presence of a subpopulation of luminal hormone receptor positive cells that differentiate independently of Notch signaling. Time directed deletion of RBP-Jκ via intraductally injected Adeno-Cre virus into the adult mouse mammary ductal system through the nipple additionally confirmed that RBP-Jκ is required for progesterone receptor expression. Chromatin immunoprecipitation assay showed that RBP-Jκ binds two out of four putative RBPJκ binding sites in the progesterone receptor promoter. Inhibition of Notch signaling in vivo via intraductal injection of γ-secretase inhibitor, DAPT, resulted in reduced progesterone receptor expression strongly suggesting that Notch-related RBP-Jκ signaling is responsible for PR expression. In this project we are proposing that there are two different populations of HR+ cells: one defined as Wnt4 expressing subpopulation of HR+ cells in which PR expression is dependent of Notch signaling, and another one defined as Wnt4 non expressing subpopulation of HR+ cells which is independent of Notch signaling. Since Notch signaling is able to replace ER signaling and activate ER target genes in the endocrine therapy resistant cell lines, Wnt4 expressing population of HR+ luminal cells might play a crucial role in the acquiring of the resistance.