The Function And Mechanism Of Numb And Numbl On Mammary Gland Development And Lactogenesis

The mammary gland is a unique organ, which is predominantly developed postnatally, displaying a ductal-tree structure. Mammary epithelium is comprised of an inner layer of luminal epithelial cells and an outer layer of contractile myoepithelial cells. Elucidating the mechanisms that govern the balance between basal and luminal cells can help uncover the underlying principles of mammary gland development. In mammals, there are two homologs of conserved Numb, Numb and Numblike (Numbl). Numb and Numbl proteins are considered to have both overlapping and distinct functions. Numb and Numbl are considered as well-known suppressors of the Notch signaling pathway. The Notch signaling pathway plays an important role in mammary stem cell (MaSC) self-renewal and luminal cell commitment. However, the in vivo role of Numb and Numbl in mammary gland development during puberty and pregnancy has not been explored.In this study, we utilized Numb/Numbl double knockout mouse, combined with in vitro cells culture method to investigate the role of Numb and Numbl in mammary development and lactogenesis. The analysis of FACS and qRT-PCR shows that Numb and Numbl are enriched in mammary myoepithelial cells, with their expression peaking during pregnancy. Loss of NumblNumbl resulted in impairment of mammary ductal branching, as well as reduction in basal cells and increase in luminal cells, suggesting the importance of Numb and Numbl in maintaining the balance of basal and luminal epithelial cells. In molecular mechanism, this mammary phenotype is resulted from activation of Notch signaling. In addition, knockout of Numb/Numbl lead to downregulation of p21/p53, and upregulation of Cyclin D1, resulting in proliferation of luminal cells. Our findings further shows that loss of Numbl promoted epithelial-to-mesenchymal transition (EMT) and overexpression of Numbl repressed cell migration, suggesting an important role of Numb and Numbl in maintaining the balance of epithelial and mesenchymal cell fate. Further, this study showed that Numbl knockout female mice failed to lactate and raise their pups after birth. Numbl knockout female mice displayed expanded alveoli, loss of contractile myoepithelial layer and apoptotic bodies in lumen at late pregnancy. In molecular mechanism, we demonstrated that STAT5 and PRLR signaling were inactivated and STAT3 was abnormally activated in Numb/Numbl double knockout mice at late pregnancy by immuno-histochemistry, immunoflourescence and qRT-PCR. Loss of Numb/Numbl promoted upregulation of p63 and activation of Notch signaling. Moreover, in this study, we demonstrated that loss of Numbl represses lung metastasis, using MMTV-PyVT breast cancer mouse model.Taken together, we demonstrates that Numb/Numbl regulate the balance between basal and luminal cells via the Notch signaling pathway and p53-p21 axis in puberty. Numb/Numbl maintain p63 activity in basal cells by repressing Notch signaling at late pregnancy, which is required for normal lactogenic differentiation via activation of STAT5 and repression of STAT3 signaling pathway. These findings highlight the importance of Numb and Numbl in the control of myoepithelial cell fate determination, epithelial identity, and lactogenesis, broadening our understanding of the regulation network of mammary gland development and lactogenesis. The study provides new insight and potential strategies for mammary diseases and breast cancer for therapeutic intervention.