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Hedayatollah Hosseini Molecular mechanism of early metastatic breast cancer dissemination Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Fakultät für Biologie und Vorklinische Medizin der Universität Regensburg vorgelegt von Hedayatollah Hosseini aus Ahwaz, Iran 2016 Das Promotionsgesuch wurde eingereicht am 14.Dec. 2016 Die Arbeit wurde angeleitet von Herr Prof. Dr. Christoph A. Klein Untershrift: 2 Contents 1 Introduction ........................................................................................................................................................ 7 1.1 Metastasis ..................................................................................................................................................... 7 1.2 Local invasion and migration ....................................................................................................................... 8 1.3 Cancer stem cells in metastasis ..................................................................................................................... 9 1.4 Mammary gland development and breast cancer ........................................................................................ 10 1.5 Early versus late dissemination of cancer cells........................................................................................... 14 1.6 Balb-NeuT as a mouse model for early dissemination ............................................................................... 15 1.7 Aim of work ............................................................................................................................................... 17 2 Materials and Methods .................................................................................................................................... 19 2.1 Methods ...................................................................................................................................................... 19 2.1.1 Animal Experiments .......................................................................................................................... 19 2.1.2 Laser Microdissection and microarray analysis ................................................................................. 21 2.1.3 Cell culture experiments .................................................................................................................... 22 2.1.4 Differentiation of mammosphere cells ............................................................................................... 25 2.1.5 Migration/Invasion assays ................................................................................................................. 25 2.1.6 Proliferation assay .............................................................................................................................. 27 2.1.7 Staining .............................................................................................................................................. 27 2.1.8 PCR .................................................................................................................................................... 29 2.1.9 Viral transduction .............................................................................................................................. 30 2.1.1 Western blot analysis ......................................................................................................................... 33 2.1.10 miRNA analyses and sequencing ....................................................................................................... 35 2.1.11 Analysis of Breast Cancer Patients: ................................................................................................... 36 2.1.12 Bioinformatic and Statistical analysis ................................................................................................ 36 2.2 Materials ..................................................................................................................................................... 37 2.2.1 Reagent .............................................................................................................................................. 37 2.2.2 Consumable ....................................................................................................................................... 39 2.2.3 Kits ..................................................................................................................................................... 40 2.2.4 Devices .............................................................................................................................................. 40 2.2.5 Primers ............................................................................................................................................... 42 2.2.6 Antibodies .......................................................................................................................................... 43 2.2.7 miRNAs ............................................................................................................................................. 43 2.2.8 Cell lines ............................................................................................................................................ 44 2.1.2 Special cell culturing media ............................................................................................................... 46 2.2 Abbreviation ............................................................................................................................................... 47 3 Results ................................................................................................................................................................ 49 3.1 Cancer cell dissemination starts at early stages in Balb-NeuT model .................................................. 49 3.2 Progesterone and Her2 signaling regulate gene expression program of early lesions ........................ 51 3.2.1 Gene expression analyses revealed an exclusive expression profile for early lesions ................. 51 3.2.2 Progesterone as regulator of EL gene expression profile ............................................................. 56 3.2.3 Her2 and progesterone cooperate to generate EL signature ........................................................ 59 3.3 Progesterone signaling regulates the migration program in EL and is linked to side branching and morphogenesis ....................................................................................................................................................... 61 3.3.1 Role of Progesterone in side branching and morphogenesis ........................................................ 61 3.3.2 Progesterone and its paracrine factors regulate in vitro migration of PT and EL cells ............. 63 3.4 Progesterone signaling regulates migration, stemness, and proliferation of cancer cells ................... 64 3 3.4.1 Higher sphere formation ability of young mammary epithelial cells .......................................... 64 3.4.2 Higher expression of PgR in young mammary glands .................................................................. 65 3.4.3 Progesterone-induced paracrine factors regulate stemness of PT and EL cells ......................... 66 3.4.4 Stemness feature of migrating cells ................................................................................................ 67 3.5 Her2 expression levels determine migratory vs. proliferative cellular responses ................... 68 3.5.1 Progesterone-induced paracrine factors act as inducer of stemness and migration for EL-like cells and supressor for PT-like cells ................................................................................................................ 68 3.5.2 Her2 expression level defines types of responses to the progestrone signaling ........................... 71 3.5.3 Her2 and PgR expression in the migratory cells ........................................................................... 72 3.6 In vivo support for the identified mechanism ......................................................................................... 74 3.6.1 Proliferation and metastasis properties of PT and EL ................................................................. 74 3.6.2 Proliferation and metastasis traits are constant phenotypes over the time ................................ 76 3.6.3 Higher metastatic property of EL cells comes from higher dissemination ................................. 77 3.7 PgR is the distinticive environmantal factor in regulation of dissemination ....................................... 78 3.7.1 Impact of mammary gland environment on dissemination of PT and EL cells ......................... 78 3.7.2 PgR expression changes dissemination behaviour of cancer cells ............................................... 81 3.7.3 Pregnancy induces migration of EL and proliferation of PT cells .............................................. 82 3.8 Cell density regulates proliferation and dissemination phenotypes ..................................................... 83 3.8.1 PgR expression in PT cells regulated by cell density .................................................................... 83 3.8.2 PT cells regulate PgR expression via miRNAs .............................................................................. 86 3.8.3 miR9-5p and miR30a-5P regulate PgR
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