EGFR/Ras-signaling-dependent CCL20 production in tumor cells critically contributes to angiogenesis and tumor progression [Elektronische Ressource] / vorgelegt von Anne Schorr

Andreas Hippe

Description

EGFR/Ras-signaling-dependent CCL20 production in tumor cells critically contributes to angiogenesis and tumor progression Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Anne Schorr, M.Sc. aus Düsseldorf Düsseldorf, 2010 1 Abbreviations Aus der Hautklinik, Forschungslabor für Dermato-Immunologie und Onkologie der Heinrich-Heine-Universität Düsseldorf Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. Bernhard Homey Korreferent: Prof. Dr. Dieter Willbold Tag der mündlichen Prüfung: 18.01.2011 2 Abbreviations Meinen Eltern und meinem Freund 3 Abbreviations Acknowledgment I would like to express my sincere gratitude to my supervisor Prof. Dr. Bernhard Homey for giving me the opportunity to carry out this interesting research and for the committed guidance. I am also grateful for the financial support and for introducing me already at very early stages to high-caliber scientists in- and outside of the Heinrich-Heine-University research community. My special thanks are also due to Prof. Dr. Dieter Willbold for co-advising this work. I am very grateful to Dr.

Sujets

• Biologie

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2011
• Nombre de lectures: 41
• Langue: English
• Poids de l'ouvrage: 28 Mo

Extrait

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EGFR/Ras-signaling-dependent CCL20

production in tumor cells critically contributes

to angiogenesis and tumor progression

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Inaugural-Dissertation

zur

Erlangung des Doktorgrades

der Mathematisch-Naturwissenschaftlichen Fakultät

der Heinrich-Heine-Universität Düsseldorf

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vorgelegt von

Anne Schorr, M.Sc.

aus Düsseldorf

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Düsseldorf, 2010

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 Aus der Hautklinik,

Forschungslabor für Dermato-Immunologie und Onkologie

der Heinrich-Heine-Universität Düsseldorf

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Gedruckt mit der Genehmigung der

Mathematisch-Naturwissenschaftlichen Fakultät der

Heinrich-Heine-Universität Düsseldorf

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Referent:

Korreferent:

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Prof. Dr. Bernhard Homey

Prof. Dr. Dieter Willbold

Tag der mündlichen Prüfung: 18.01.2011

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 Acknowledgment

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I would like to express my sincere gratitude to my supervisor Prof. Dr. Bernhard Homey for giving me the opportunity to carry out this interesting research and for the committed guidance. I am also grateful for the financial support and for introducing me already at very early stages to high-caliber scientists in- and outside of the Heinrich-Heine-University research community.

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My special thanks are also due to Prof. Dr. Dieter Willbold for co-advising this work.

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I am very grateful to Dr. Andreas Hippe who guided me through all steps of this project with patience. He provided me with advice, valuable suggestions and continuous encouragement. Thank you for your help!

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I also would like to thank all those who supported me over the years in one way or the other and in particular:

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Attila Antal, Virginia Bardeli, Erich Bühnemann, Bettina Alexandra Buhren, Marcel Castrogiovanni, Thomas K. Hoffmann, Anke van Lierop, Sabine Kellermann, Robert Kubitza, Jumana Saleh, Uta and Jochen Schorr

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Table of contents

Table of contents

1SUMMARY ................................................................................................................. 14

2ZUSAMMENFASSUNG ............................................................................................. 15

3INTRODUCTION ........................................................................................................ 16

3.1 17 .....................................................The epidermal growth factor receptor (EGFR) 3.1.1Signaling by MAPK pathway ......................................................................... 17 3.1.2The proto-oncogene Ras ............................................................................... 19 3.2................................02................................egeniss........................................oignA 3.2.1What is angiogenesis? .................................................................................. 20 3.2.2 ...................................................................................Morphology of vessels 20 3.2.3Molecular mechanisms of angiogenesis........................................................ 22 3.2.4 ................................................................................... 23The angiogenic switch 3.3Chemokines ......................................................................................................... 24 3.3.1The chemokine superfamily........................................................................... 24 3.3.2Angiogenic chemokines................................................................................. 26 3.3.3Chemokines in tumor-associated angiogenesis ............................................ 27 3.3.4Angiostatic chemokines ................................................................................. 27 3.4Aim of the thesis................................................................................................... 29

4 ................................................................................... 30MATERIALS AND METHODS

4.1Buffers and solutions............................................................................................ 30 4.2 .................................................................................................... 31Biopsy samples 4.3Mice...................................................................................................................... 31 4.4Cell culture ........................................................................................................... 32 4.5Total RNA isolation .............................................................................................. 33 4.6 34OD measurement ................................................................................................. 4.7Complementary DNA (cDNA) synthesis .............................................................. 34 4.8Quantitative real-time PCR (qRT-PCR) analysis ................................................. 35 4.9Ras activity assay ................................................................................................ 37 4.9.1Lysis of cells .................................................................................................. 37 4.9.2Protein measurement .................................................................................... 37 4.9.3Affinity precipitation of activated Ras............................................................. 38

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Table of contents

4.9.4SDS-PAGE Gel Electrophoresis.................................................................... 38 4.9.5Semi-dry transblotting.................................................................................... 38 4.9.6 ..................................................... 39Immunodetection / Western Blot Analysis 4.10Immunohistochemistry ...................................................................................... 39 4.10.1 .......................................................................................... 39Frozen sections 4.10.2 ......................................................................................... 40Paraffin sections 4.11Immunofluorescence......................................................................................... 41 4.12In vitromonolayer wound healing assay........................................................... 42 4.13Tube formation assay ....................................................................................... 43 4.14Chemotactic cell migration ................................................................................ 43 4.15Enzyme-linked immunosorbent assay (ELISA)................................................. 44 4.16Flow cytometric analysis (FACS) ...................................................................... 44 4.17 ........................................................................................... 45Matrigel plug assay 4.18 .......................................................... 45Murine syngeneic tumor model B16/F10 4.19Statistic analysis ............................................................................................... 46

5RESULTS ................................................................................................................... 47

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5.1 .......................................................... 47CCL20 is highly induced by Ras activation 5.2The EGFR/Ras signaling pathway regulates CCL20 gene and protein  expression............................................................................................................ 48 5.3CCL20 expression is induced in several cancer types......................................... 50 5.4CCL20 expression of tumor tissues correlates with ERK activation,  advanced cancer staging and poor prognosis ..................................................... 52 5.5Endothelial cells express the CCL20-specific chemokine receptor CCR6 ........... 54 5.6CCL20 promotes the migration of microvascular endothelial cells and tubule  formationin vitro................................................................................................. 56

5.7CCL20 enhances vascularization of Matrigel plugs and tumors .......................... 58 5.7.1CCL20 recruits CD31-positive vessels into Matrigel plugs in vivo................. 58 5.7.2in CCR6-deficient mice are smaller and less vascularized thanTumors  tumors in wild-type mice ................................................................................. 59

DISCUSSION ............................................................................................................. 62

REFERENCES ........................................................................................................... 70

PUBLICATIONS ......................................................................................................... 87

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8.1

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Table of contents

Poster ................................................................................................................... 87

Talks..................................................................................................................... 88

9................................................89.......LARADEC...TION................................................

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 Abbreviations

A A ADF AEC AL AR AR  B BAS B cell BEC bFGF Bis-Tris BPE BSA BTC 

C C C CA C57BL/6 CD cDNA CCL

CCR CCX-CKR cm cm CO 2  CpG C T  CXCL CXCR CX 3 CL CX 3 CR  D Δ 3D Da DAPI DARC

 absorbance Arbeitsgemeinschaft dermatologischer Forschung 3-amino-9-ethyl-carbazol Alabama amphiregulin Arkansas   bovine serum albumin B lymphocyte human dermal microvascular blood endothelial cell basic fibroblast growth factor

bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane bovine pituitary extract bovine serum albumin betacellulin 

 cysteine degree Celsius California C57 black 6 cluster of differentiation complementary DNA CC-motif chemokine ligand

CC-motif chemokine receptor chemocentryx chemokine receptor square centimeter cubic centimeter

carbon dioxide deoxycytidyl-deoxyguanosine

threshold cycle CXC-motif chemokine ligand CXC-motif chemokine receptor CX 3 C-motif chemokine ligand CX 3 C-motif chemokine receptor   delta, difference three-dimensional Dalton 4,6-diamidino-2-phenylindole duffy antigen/chemokine receptor

Abbreviations

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DC DE DEPC dH 2 O DMEM DMSO DNA DNAse dNTP DTT  E EC ECL EDTA EGF EGFR EGM-2  18S ELISA ELR EPR ErbB Erk ESDR ESE 1 - EST et al. Ets  F FACS FCS fg Fig. fpVCT FSC  G g g GAP GDP GEF Glu-Leu-Arg GPCR

dendritic cell Delaware diethylpyrocarbonate distilled water Dulbeccos modified Eagles medium dimethyl sulfoxide deoxyribonucleic acid deoxyribonuclease deoxyribonucleotide triphosphate dithiothreitol   endothelial cell enhanced chemiluminescence solution ethylenediaminetetraacetic acid epidermal growth factor EGF receptor endothelial cell growth medium-2 18 svedberg enzyme-linked immunosorbent assay glutamic acid-leucine-arginine

epiregulin erythroblastic leukemia viral oncogene extracellular-signal regulated kinase European Society for Dermatology Research epithelium –specific Ets expressed sequence tags et alii, and others E-twenty six   flow cytometric analysis fetal calf serum femtogram figure flat-panel VCT forward scatter

  gram gravitational acceleration GTPase activating protein guanosine-5-diphosphate

guanine nucleotide exchange factor glutamic acid-leucine-arginine G-protein-coupled receptor

Abbreviations

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G-protein Grb2 GST GTP  H h HaCaT HB-EGF HCl HER HGF HIMEC HIV HMEC HNSCC H 2 O H-ras H 2 SO 4 HRP HUVEC  I i.e. IgG IGF-1 IL 

J JaK 

K KC kDa K-ras 

L λ LARC LC LEC L-Glutamate log  M µ

Abbreviations

guanine nucleotide-binding-proteins growth factor receptor-bound protein 2 glutathione-S-transferase guanosine-5-triphosphate   hour human adult skin keratinocytes propagated under low Ca2+ and elevated temperature  heparin-binding EGF hydrogen chloride human epidermal growth factor receptor hepatocyte growth factor human intestinal microvascular endothelial cell

human immunodeficiency virus human microvascular endothelial cell head and neck SCC hydrogen oxide, water Harvey-ras sulfuric acid horseradish peroxidase human umbilical vein endothelial cell   id est, that is immunoglobulin G insulin like growth factor interleukin 

 Janus kinases/signal transducers and activators of transcription   primary mucosal keratinocytes kilo Dalton Kirsten-ras   lambda, wavelength liver and activation regulated chemokine langerhans cell human lymphatic endothelial cell laevus-glutamate logarithm   micro

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M MA MALT MAPK MD MEGM µg MgCl 2  MGM-4 MIP-3a µl µm µM mM MEM min ml MMP MN MO mRNA MOPS  N n NaCl NJ ng nm nM NP-40 N-ras NSCLC NY 

O OD oligo(dt)  P P p90RSK PDGFB PDGFBRβ PE pERK

molar Massachusetts mucosa-associated lymphatic tissue mitogen-activated protein kinase

Maryland mammary epithelial cell growth medium

microgram magnesium chloride melanocyte growth medium-4 macrophage inflammatory protein-3a microliter micrometer micromolar millimolar minimal essential medium minute milliliter matrix metalloproteinase Minnesota Missouri messenger RNA 3-(N-morpholino)propanesulfonic acid   number sodium chloride New Jersey nanogram nanometer nanomolar nonyl phenoxypolyethoxylethanol-40 neuroblastoma-ras

non-small cell lung cancer cell New York   optical density oligo(deoxythymidylic acid)   probability p90 ribosomal S6 kinase platelet-derived growth factor B PDGF receptorβ phycoerythrin phosphorylated ERK

Abbreviations

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