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Brustdrüse ths Prof. Dr. Elsässer Hans-Peter Elsässer, Hans-Peter (Prof. Dr.) English Function of the Myc-binding protein Miz1 in the mouse mammary gland 2014-10-01 application/pdf Funktion des Myc bindenden Proteins Miz1 in der Brustdrüse der Maus monograph ppn:348037503 Medical sciences Medicine Medizin Medizin Miz1 Stat5 Miz1 stat5 mammary gland Die Analyse von Proteinen, die für die embryonale Entwicklung sowie für Erkrankungen relevant sind, stellen wertvolle Informationen für die Entwicklung neuer therapeutischer Konzepte zur Verfügung. Das Protooncogen Myc ist zum Beispiel ein vielversprechendes Zielprotein in einer Reihe von unterschiedlichen Tumoren einschließlich des sogenannten „triple negativen“ Brustdrüsenkarzinoms. In der vorliegenden Arbeit wurde zum ersten Mal die Funktion des Transkriptionsfaktors Miz1 in der Brustdrüse der Maus untersucht. Hierzu wurde ein konditionelles Miz1 knockout Mausmodell verwendet. Bei diesem Modell wird die POZ Domäne deletiert. Diese ist essentiell für die Ausbildung von Tetrameren, die dann stabil an das Chromatin binden können. Die Deletion wurde mit zwei unterschiedlichen Cre-Stämmen durchgeführt. Ein MMTV-Cre Stamm wurde verwand, um die POZ Domäne in nulliparen Mäusen zu deletieren, wobei die postnatale Morphogenese der Drüse, sowie die Brustdrüsenstammzellen untersucht wurden. Ein Wap-Cre Stamm wurde eingesetzt, um die Alveologenese und die Brustdrüsendifferenzierung während der Schwangerschaft und der anschließenden Laktation zu analysieren. Die Deletion der POZ Domäne mit MMTV-Cre erfolgt bereits im embryonalen Gewebe und führte zu einer Verzögerung der postnatalen Ausbildung und Verzweigung des Drüsenganges und einem zellärmeren Gangepithel. Weiterhin wurde eine von Myc unabhängige Anreicherung von Stamm- bzw. Vorläuferzellen in Miz1ΔPOZ Tieren beobachtet. Unterschiede in der Expression von luminalen und myoepithelialen Markern traten nicht auf. Die Verzögerung der postnatalen Gangentwicklung war in zwei Monate alten Tieren nicht mehr zu beobachten. Mit Hilfe der Immunhistochemie und mit Western Blots konnte gezeigt werden, dass die Expression von endogenem Miz1 in normalen Tieren während der Laktation stark erhöht ist, während am Ende der Schwangerschaft nur wenig Miz1 nachgewiesen werden konnte. Die Miz1 Expression wird also während bzw. kurz nach der Geburt sprunghaft erhöht. Wird die Involution eingeleitet, fällt die Miz1 Expression innerhalb von 48 Stunden auf Werte zurück, wie sie vor der Laktation beobachtet wurden. Die Deletion der POZ Domäne während der Schwangerschaft mit Hilfe von Wap-Cre erzeugte einen Laktationsdefekt während der ersten und der zweiten Schwangerschaft. Drüsen mit mutiertem Miz1 zeigte eine reduzierte Alveologenese, sowie eine verminderte Proliferation und Differenzierung. Dies konnte in der HC11 Zelllinie, die aus einer Maus-Brustdrüse isoliert wurde, verifiziert werden. HC11 Zellen mit geringer Miz1 Expression proliferieren langsamer und expremieren weniger β-Casein, wenn sie mit einem „laktogenen Hormoncocktail“, der u.a. Prolactin enthält, behandelt werden. Dabei zeigt sich weder in vivo noch in vitro eine Änderung der Apoptoserate , wenn die Miz1 POZ Domäne deletiert oder Miz1 ausgeschaltet wird. Brustdrüsen mit mutiertem Miz1 haben geringere Mengen an Stat5, was zu einer Reduktion der Expression entsprechender Zielgene wie α-Casein, β-Casein or whey acidic protein (Wap) führt. Negative Regulatoren des Jak2/Stat5 Signalwegs wie Socs (Socs1, Socs2, Socs3) oder Caveolin-1 (Cav1) waren in ihrer Expression nicht verändert. Im Gegensatz dazu wurde eine Verringerung des Prolactinrezeptors und von ErbB4 beobachtet. Beide Proteine sind wichtig für die Phosphorylierung von Stat5. Allerdings konnte mit Hilfe von CHIP-Seq Experimenten eine direkte Bindung von Miz1 an die Gene dieser beiden Proteine nicht gezeigt werden. Jedoch bindet Miz1 an verschiedene Gene, die für die Regulation des vesikulären Transportes, der Endozytose und der Autophagie entscheidend sind. Ein Modell, in dem die Störung des vesikulären Transportes der beiden Rezeptorproteine im Mittelpunkt steht, wird vorgeschlagen. 2014-03-10 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. 2014 doctoralThesis https://doi.org/10.17192/z2014.0285 opus:5462 Sanz Moreno, Adrian Sanz Moreno Adrian