New insights into S100A4-induced colon cancer metastasis [Elektronische Ressource] : the role of exo- and endogenous inhibitors / Ulrike Sack. Gutachter: Achim Leutz ; Matthias Selbach ; Ulrike Stein

Ulrike Sack

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• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2011
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 9 Mo

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New insights into S100A4-induced colon cancer metastasis:
The role of exo- and endogenous inhibitors




D i s s e r t a t i o n
zur Erlangung des akademischen Grades
d o c t o r r e r u m n a t u r a l i u m
(Dr. rer. nat.)
im Promotionsfach Biologie
eingereicht an der


Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin
von


MSc in Mol-Biol. Ulrike Sack


Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Jan-Hendrik Olbertz

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Andreas Herrmann


Gutachter: 1. Prof. Dr. Achim Leutz
2. Prof. Dr. Matthias Selbach
3. Prof. Dr. Ulrike Stein


Tag der mündlichen Prüfung: 30.03.2011
























This study was conducted at the Max Delbrück Centrum for Molecular Medicine Berlin-
Buch supervised by Prof. Dr. Ulrike Stein in the research group of Prof. Dr. Dr. h.c.
Peter M. Schlag.






















Für meine Familie





INDEX OF CONTENTS
INDEX OF CONTENTS

ABBREVIATIONS 8
ABSTRACT 10
ZUSAMMENFASSUNG 11

1. INTRODUCTION 13
1.1. Colon cancer
1.1.1. Colon cancer epidemiology
1.1.2. Risk factors and causes for colon cancer 13
1.2. The development of metastatic colon cancer 14
1.2.1. The normal colon crypt 14
1.2.2. The adenoma-carcinoma cascade 16
1.2.3. Metastasis formation 17
1.2.4. Molecular pathways involved in colon cancer metastasis 19
1.3. The canonical Wnt/ β-catenin pathway 20
1.3.1. Components of the Wnt pathway
1.3.2. Wnt signaling antagonists 22
1.3.3. Wnt pathway in metastasis formation 23
1.4. S100A4 24
1.4.1. S100A4 gene structure and transcription 24
1.4.2. S100A4 protein structure 25
1.4.3. S100A4 loosens cell adhesion 26
1.4.4. S100A4 increases cell migration
1.4.5. ll invasion 29
1.4.6. S100A4 enhances angiogenesis
1.4.7. S100A4 and cell growth 30
1.4.8. S100A4 drives metastasis formation in vivo 31
1.4.9. S100A4 expression correlates with metastasis in colon cancer patients 31
1.5. Inhibition of S100A4 expression for therapeutic intervention 32
1.6. Identification of the molecular mechanism underlying
S100A4-driven metastasis 34

2. AIM 35
4 INDEX OF CONTENTS
3. MATERIALS AND METHODS 36
3.1. Cloning
3.1.1. S100A4 cDNA expression vector
3.2. Cell culture 37
3.2.1. Colon cancer cell lines
3.2.2. Transfections
3.2.3. HCT116 derivative cells 38
3.3. Drugs and treatments 39
3.3.1. RNA interference
3.3.2. Small molecules
3.3.3. Recombinant DKK-1 protein
3.3.4. Cytotoxicity assay
3.4. Gene expression analysis 40
3.4.1. RNA isolation
3.4.2. Reverse transcription
3.4.3. Quantitative real-time PCR
3.4.4. Restriction fragment length polymorphism analysis 42
3.4.5. Protein extraction 42
3.4.6. Protein quantification 43
3.4.7. Western blot analysis
3.4.8. Enzyme-linked immunosorbent assay 44
3.5. Functional in vitro Assays 45
3.5.1. Migration assay
3.5.2. Invasion assay
3.5.3. Wound healing assay
3.5.4. Proliferation assay 46
3.5.5. Colony formation assay
3.6. Wnt/β-catenin pathway analysis
3.6.1. TOP/FOPflash reporter assay
3.6.2. Electrophoretic mobility shift assay 47
3.6.3. Chromatin immunoprecipitation assay 48
3.7. In vivo metastasis formation and bioluminescence imaging 49
3.7.1. Intrasplenal tumor transplantation 49
3.7.2. In vivo dose-finding for niclosamide
3.7.3. Analysis of metastasis formation in vivo
5 INDEX OF CONTENTS
3.7.4. In vivo bioluminescence imaging 50
3.7.5. Human S100A4 expression in murine xenograft tissue 50
3.8. Statistical analysis

4. RESULTS 51
4.1. Small molecules interfere with cell viability 51
4.2. Small molecules restrict S100A4 expression 52
4.2.1. Inhibition of S100A4 expression is concentration-dependent 52
4.2.2. ression is time-dependent 54
4.2.3. Exogenous expression of S100A4 is not affected by small molecules 56
4.3. Small molecules decrease cell migration, invasion and proliferation 57
4.3.1. Small molecules reduce cell migration of colon cancer cells 57
4.3.2. Small molecules impair cell invasion 59
4.3.3. Small molecules decrease cell proliferation 60
4.3.4. Small molecules arrest colony formation 61
4.4. Structural changes on niclosamide reduce its inhibitory efficiency 63
4.5. Small molecules interfere with the Wnt pathway 64
4.5.1. Small molecules inhibit the constitutively active Wnt pathway 65
4.5.2. Calcimycin inhibits the β-catenin expression 67
4.5.3. Niclosamide interferes with the β-catenin/TCF-complex 68
4.6. Niclosamide inhibits metastasis formation in colon cancer
xenograft mice 70
4.6.1. Evaluation of an in vivo applicable niclosamide concentration 70
4.6.2. In vivo metastasis can be visualized by bioluminescence imaging 71
4.6.3. Niclosamide restricts metastasis formation in mouse xenografts 73
4.7. Relation of the DKK-1 and S100A4 expression in colon cancer cells 75
4.7.1. DKK-1 and S100A4 expression is inversely correlated in cells with
mutated or non-mutated β-catenin 75
4.7.2. DKK-1 and S100A4 expression is negatively correlated in human
colon cancer cell lines. 76
4.8. S100A4 is a negative regulator of DKK-1 expression 78
4.8.1. Exogenous overexpression of S100A4 inhibits DKK-1 expression 78
4.8.2. Reduction of S100A4 expression recovers DKK-1 expression 79
4.9. DKK-1 inhibits S100A4 expression 81
6 INDEX OF CONTENTS
5. DISCUSSION 83
5.1. Inhibition of S100A4 transcription inhibits S100A4-induced
cell motility
5.1.1. Small molecules inhibit S100A4 expression 83
5.1.2. Small molecules restrict S100A4-induced cell motility 84
5.2. The small molecules inhibit colon cancer cell proliferation 86
5.3. Both small molecules interfere with constitutively active Wnt pathway 86
5.3.1. Calcimycin inhibits β-catenin expression 87
5.3.2. Niclosamide inhibits β-catenin/TCF complexation
5.3.3. and calcimycin inhibit constitutively active Wnt pathway 88
5.4. Specificity and potential adverse effects of the small molecules 88
5.4.1. Targeting the S100A4-promoter is most efficient 89
5.4.2. Advantage of using small molecules to inhibit S100A4 expression 89
5.5. Niclosamide as novel anti-metastatic treatment 90
5.5.1. Niclosamide is a favorable inhibitor to be applied in vivo
5.5.2. Non-invasive bioluminescence imaging visualized S100A4-induced
metastasis 91
5.5.3. Intrasplenic xenograft model revealed anti-metastatic function of
niclosamide 92
5.5.4. Niclosamide as anti-metastatic drug for colon cancer patients 93
5.6. The inhibitor of the inhibitor – S100A4 and DKK-1 95
5.6.1. S100A4 inhibits DKK-1 expression 95
5.6.2. DKK-1 is an endogenous inhibitor for S100A4 expression 96
5.7. The new roles for small molecules, S100A4 and DKK-1 in the
Wnt pathway 97

6. OUTLOOK 100

REFERENCES 101
ERKLÄRUNG 115
ACKNOWLEDGMENT 118

7 ABBREVIATIONS
ABBREVIATIONS

AP-1 activating protein-1
BCA bicinchoninic acid
BSA bovine serum albumin
CBF core-binding-factor
CCN3 cystein-rich 61-connective tissue growth factor-nephroblastoma
overexpressed-family member 3
CDC4 cell division control protein 4
CIN chromosomal instability
CK-1 α casein kinase-1 α
DKK dickkopf
DMSO dimethylsulfoxide
DTT dithiothreitol
dvl disheveled
EGFR epidermal growth factor receptor
ELISA enzyme-linked immunosorbent assay
EMT epithelial mesenchymal transition
EMT-TF EMT-specific transcription factor
ErbB2 erythroblastosis oncogene B B2
F-actin filamentous actin
FAP familial adenomatous polyposis
FDA Food and Drug Administration
GSK-3 β glycogen synthase kinase -3 β
HNPCC hereditary nonpolyposis colorectal cancer
HTS high throughput screening
IFN- γ interferon-γ
LEF-1 lymphocyte enhancer factor-1
LRP-5/-6 low density lipoprotein receptor-related protein-5 or -6
MAP microfibrill-associated glycoprotein
MAPK mitogen-activated protein kinase
MetAP2 methionine aminopeptidase 2
MMP matrix metalloproteinase
NF-κB nuclear factor κB
PBS phosphate buffered saline
PI3K phosphatidylinositol-3 kinase
8 ABBREVIATIONS
PP2A protein phosphatase 2A
PTEN phosphatase tensine homolog
RAGE receptor for advanced glycation end products
RT reverse transcription
S100A4 soluble in 100% ammonium sulphate solution-family member A4
SDS-PAGE sodiumdodecylsulphate polyacrylamide gel electrophoresis
sFRP secreted frizzled-related protein
shRNA short-hairpin RNA
TCF T-cell factor
TGFRII TGF- β receptor 2
TGF- β tumor growth factor- β
TNF- α tumor necrosis factor α
UTR untranslated