Investigation of the role of CUZD1-STAT5 signaling in mammary gland development and breast cancer

In the mammary gland, genetic circuits controlled by the hormones, estrogen, progesterone and prolactin (PRL), act in concert with pathways regulated by the epidermal growth factor (EGF) family to control the growth and morphogenesis of this tissue during puberty, pregnancy and lactation. However, the precise molecular mechanisms that integrate these signaling pathways are unclear. In this study, we identifed CUZD1 (CUB and zona pellucida-like domain containing protein- 1) as a novel mediator of PRL signaling in steroid hormone-primed mouse mammary gland and undertook an examination of its role in growth and differentiation of this tissue during pregnancy. CUZD1 expression is markedly induced in steroid-primed mammary epithelial cells in response to PRL treatment. Cuzd1-null mice exhibited a striking impairment in ductal branching and alveolar development during pregnancy, resulting in a subsequent defect in lactation. Interestingly, phosphorylation and activation of STAT5, a transcription factor that mediates PRL signaling, was absent in Cuzd1-null mammary tissue during pregnancy and lactation. We also noted that the expression of epiregulin (EREG), an EGF family growth factor regulated directly by STAT5, is suppressed in Cuzd1-null mammary gland. Protein interaction studies, using flag-tagged CUZD1 expressed in HC11 mouse mammary epithelial cells, revealed that CUZD1 associates with a multi-protein complex containing JAK1/JAK2 and STAT5. Elevated expression of CUZD1 in HC11 cells stimulated phosphorylation and nuclear translocation of STAT5. Chromatin immunoprecipitation experiments indicated that STAT5 and CUZD1 co-occupy the same regulatory region of the Ereg gene. Over-expression of CUZD1 in mammary epithelial HC11 cells induced tumorigenic characteristics, such as substrate independent growth and migration. Furthermore, HC11-Cuzd1 cells formed mammary tumors in vivo following orthotopic injection into nude and Balb/C mice. Mammary tumor cells derived from these animals showed elevated levels of phosphorylation and nuclear localization of STAT5 and consequent activation of the EGF signaling pathway. Blockade of JAK/STAT5 signaling through the use of a STAT5 inhibitor markedly reduced the production of the EGF family growth factors and inhibited PRL-induced tumor cell proliferation in vitro. It also impaired the progression of CUZD1-driven mammary tumorigenesis in vivo. Collectively, our findings suggest that CUZD1 plays an important role in mammary epithelial cell proliferation during mammary gland development and in tumorigenesis by facilitating JAK-STAT5 signaling and subsequent production of growth factors, such as EREG. CUZD1, therefore, emerges as a critical mediator of PRL action that controls mammary alveolar development during pregnancy and lactation and cell proliferation during tumorigenesis