1 Declaration .................................................. 3
2 Abstract ..................................................... 4
3 Contents ..................................................... 6
4 Introduction ................................................. 9
4.1 Preface ................................................. 9
4.2 Tumor progression and metastasis ....................... 11
4.3 Epithelial-mesenchymal transition in cancer ............ 14
4.3.1 Markers of EMT .................................. 16
4.3.2 Regulators of EMT ............................... 17
4.4 TGF-β .................................................. 20
4.4.1 TGF-β signaling pathway ......................... 21
4.4.1.1 TGF-β activation ....................... 21
4.4.1.2 Canonical TGF-β/Smads signaling
pathway ................................ 22
4.4.2 Specificity in TGF-β signal transduction ........ 24
4.4.3 TGF-β signaling crosstalk ....................... 25
4.4.3.1 Smad-independent TGF-β signaling ....... 25
4.4.3.2 Regulation of Smad activity by kinase
pathways ............................... 27
4.4.4 TGF-β in cancer ................................. 27
4.4.4.1 TGF-β acts as a tumor suppressor ....... 28
4.4.4.2 TGF-β promotes tumor progression and
EMT .................................... 28
4.5 Cadherins .............................................. 30
4.5.1 Stucture and function ........................... 31
4.5.2 VE-cadherin ..................................... 33
4.5.2.1 Role of VE-cadherin in signal
transduction ........................... 34
4.5.2.2 Control of VE-cadherin expression in
endothelial cells ...................... 35
4.5.3 Cadherin switch in tumor progression and EMT .... 36
4.6 Aims of the study ...................................... 38
4.7 Experimental model ..................................... 39
5 Results ..................................................... 42
5.1 VE-cadherin is expressed in aggressive tumor cells ..... 42
5.1.1 VE-cadherin is expressed in aggressive tumor
cells in vitro .................................. 42
5.1.2 VE-cadherin expression is induced during
TGF-β1-mediated EMT in vitro .................... 44
5.1.3 VE-cadherin is expressed in aggressive tumor
cells in vivo ................................... 47
5.1.4 VE-cadherin is expressed in human mammary
tumor cells ..................................... 49
5.2 VE-cadherin expression influences tumor cell behavior
in vitro as well as in vivo ............................ 51
5.2.1 VE-cadherin expression promotes proliferation
of Ep5ExTu cells ................................ 51
5.2.2 VE-cadherin expression promotes cord-like
structures formation by Ep5ExTu cells ........... 54
5.2.3 VE-cadherin expression promotes Ep5ExTu cell
adhesion to endothelial cells ................... 55
5.2.4 VE-cadherin expression affects N-cadherin
localization in Ep5ExTu cells ................... 55
5.2.5 Isolation of tumor cell lines with stable
knock-down of VE-cadherin ....................... 57
5.2.6 VE-cadherin suppression reduces Ep5ExTu cell
proliferation in vitro .......................... 60
5.2.7 VE-cadherin suppression reduces tumor growth
in vivo ......................................... 61
5.2.8 Stable suppression of VE-cadherin affects
Ep5ExTu cell morphology and expression of
EMT-related genes ............................... 61
5.3 Characterization of the molecular mechanism by which
VE-cadherin affects tumor cell behavior ................ 64
5.3.1 Stable suppression of VE-cadherin
downregulates Smad2 phosphorylation and TGF-β
target gene expression in Ep5ExTu cells ......... 64
5.3.2 Inhibition of the TGF-β pathway reduces
Ep5ExTu cell proliferation ...................... 68
5.4 Summary of results ..................................... 69
6 Discussion .................................................. 70
6.1 VE-cadherin is expressed in aggressive tumor cells ..... 70
6.1.1 VE-cadherin expression during tumor
progression and EMT ............................. 70
6.1.2 VE-cadherin is expressed in tumor cells in
vivo ............................................ 73
6.2 VE-cadherin expression influences tumor cell
behavior ............................................... 75
6.3 Characterization of the molecular mechanism by which
VE-cadherin affects tumor cell behavior ................ 81
6.3.1 VE-cadherin promotes Smad2 phosphorylation and
TGF-β target gene expression .................... 82
6.3.1.1 Smad2 phosphorylation .................. 82
6.3.1.2 Snail and Slug expression .............. 82
6.3.1.3 PAI-1 expression ....................... 83
6.3.1.4 MMP-9 expression ....................... 83
6.3.1.5 Evaluation of the possible
contribution of other signaling
pathways ............................... 84
6.3.2 The effects of VE-cadherin suppression on
tumor growth and cell morphology are
consistent with an inhibition of the TGF-β
pathway ......................................... 85
6.3.3 Inhibition of the TGF-β pathway reduces
Ep5ExTu cell proliferation ...................... 85
6.4 VE-cadherin as a target for anti-tumor therapies ....... 87
6.5 Conclusion ............................................. 88
7 Material and methods ........................................ 90
7.1 Cell culture ........................................... 90
7.2 Generation of lentiviral vectors ....................... 90
7.2.1 Vector design and cloning ....................... 92
7.2.2 Lentiviral vector production .................... 94
7.2.3 Replication test ................................ 94
7.2.4 Titer determination ............................. 95
7.3 Lentiviral transduction of target cells and
generation of stable cell lines ........................ 95
7.4 Mouse tumor model ...................................... 96
7.5 Microarray analysis .................................... 97
7.6 Transcient transfection with siRNA ..................... 97
7.7 RNA isolation and Reverse Transcriptase (RT)-PCR
analysis ............................................... 98
7.8 Preparation of cell lysates ............................ 99
7.9 Determination of protein concentration ................. 99
7.10 Immunoblot analysis .................................... 99
7.11 Immunoprecipitation ................................... 100
7.12 Fluorescence activated cell sorting (FACS) analysis ... 101
7.13 Immunofluorescence staining of tissue culture cells ... 102
7.14 Immunohistochemistry and immunofluorescence on
frozen tumor sections ................................. 102
7.15 Immunohistochemistry on paraffin-embedded tumor
sections .............................................. 103
7.16 Proliferation assay ................................... 103
7.17 Cord-like structure formation in collagen gel ......... 104
7.18 Adhesion assay ........................................ 104
7.19 Invasion assay ........................................ 105
7.20 Zymography ............................................ 105
7.21 Statistics ............................................ 106
8 Acknowledgements ........................................... 107
9 Publication ................................................ 108
10 References ................................................. 110
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