Function of the Myc-binding protein Miz1 in the mouse mammary gland
The study of the expression and function of proteins important for human health in normal development provides valuable information for the design of therapeutical opportunities in the context of disease. Myc is one of the current most promising targets for a number of cancer types including trip...
Klinische Zytobiologie und Zytopathologie
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|Zusammenfassung:||The study of the expression and function of proteins important for human health in normal development provides valuable information for the design of therapeutical opportunities in the context of disease. Myc is one of the current most promising targets for a number of cancer types including triple-negative breast cancer and Miz1 has been shown to play an important role in Myc-mediated tumorigenesis. In the present work, the function of the Myc-binding protein Miz1 in the mammary gland is investigated for the first time using two different lines of transgenic mice expressing Cre-recombinase to conditionally knockout the POZ domain of Miz1 in the murine mammary gland. Deletion of this evolutionary-conserved region impedes multimerization and stable association of Miz1 with chromatin. MMTV-Cre mediated deletion was used to investigate Miz1 function in the virgin gland, considering branching morphogenesis and mammary stem/progenitor biology. Ablation under the Wap-Cre promoter provided information about alveologenesis and mammary differentiation. The mammary gland is a very suitable organ for stem cell and developmental studies as rounds of proliferation, differentiation and apoptosis occur after each pregnancy. POZ domain deletion using MMTV-Cre (Line A), already active in the embryo, led to a delayed ductal tree formation, less cellularity in knockout ducts and a Myc-independent accumulation of stem/progenitor cells in virgin mammary glands of Miz1DPOZ animals. No differences in the expression of luminal and myoepithelial markers were observed between control and Miz1DPOZ virgin mice. In addition, the delay in the development of the mammary ductal tree in knockout mice is rescued at around two months of age. Endogenous Miz1 expression in the mammary gland of control animals was found to be highly boosted during lactation by immunohistochemistry and Western blotting. Very low Miz1 levels were detected at the end of pregnancy, which increased after parturition and diminished upon cessation of pup suckling at around 48 hours of forced involution. Miz1 POZ domain ablation in luminal alveolar mammary cells during pregnancy using the WAP-Cre transgenic line resulted in a lactation defect in mutant dams during the first two pregnancies analysed. Mutant lactating glands display a reduced alveologenesis as a result of a diminished mammary cell proliferation and differentiation. These data were also confirmed in vitro using the HC11 murine mammary cell line after retroviral infection for stable knockdown of Miz1. HC11 cells with low levels of Miz1 show a reduced proliferation and a decreased expression of ß-casein after inducing differentiation by addition of a lactogenic hormone cocktail containing prolactin. Apoptosis is unaffected after either Miz1 POZ domain ablation in vivo or stable knockdown of Miz1 in vitro. Mutant glands display lower levels of activated Stat5 which lead to a reduced expression of its transcriptional targets, mainly genes which code for milk proteins like a-casein, b-casein or whey acidic protein (WAP). Gene expression of negative regulators of the Jak2/Stat5 pathway like Socs (Socs1, Socs2 and Socs3) or Caveolin-1 (Cav1) is not upregulated in Miz1DPOZ lactating glands. In contrast, the expression of receptors important for a proper phosphorylation of Stat5, like the prolactin receptor or ErbB4, is decreased in lactating mutant glands. ChIP-Seq experiments revealed that genes encoding the prolactin receptor and ErbB4 are not direct targets of Miz1. Rather, Miz1 binds to genes which regulate vesicular transport and thus alters processes like endocytosis and autophagy in mammary gland cells. A model in which the vesicular transport of these receptors in mutant glands could be disrupted is proposed. In conclusion, this work shows for the first time that Miz1 is important for mammary stem/progenitor cell regulation in the virgin gland and for a proper proliferation and differentiation in the lactating mammary gland.|