E-cadherin mutations in cancer [Elektronische Ressource] : impact on cell motility and signal transduction / Anja Bremm

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• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 10 Mo

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TECHNISCHE UNIVERSITÄT MÜNCHEN

Lehrstuhl für Ernährungsphysiologie





E-cadherin mutations in cancer: impact on cell
motility and signal transduction


Anja Bremm



Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan
für Ernährung, Landnutzung und Umwelt der Technischen Universität München
zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.





Vorsitzender: Univ.-Prof. Dr. D. Haller

Prüfer der Dissertation: 1. Univ.-Prof. Dr. H. Daniel
2. Priv.-Doz. Dr. B. Luber






Die Dissertation wurde am 14.07.2008 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 12.11.2008 angenommen. Table of contents

1 Introduction ____________________________________________________________ 4
1.1 Gastric cancer ______________________________________________________ 4
1.1.1 Tumour development and survival ___________________________________ 4
1.1.2 Tumour classification _____________________________________________ 5
1.1.3 Gastric cancer predisposition _______________________________________ 5
1.1.4 Molecular alterations in gastric cancer ________________________________ 6
1.2 Cadherins__________________________________________________________ 6
1.2.1 The cadherin superfamily __________________________________________ 7
1.2.2 Cadherin-mediated cell adhesion ____________________________________ 8
1.2.3 The cadherin-catenin complex ______________________________________ 9
1.2.4 E-cadherin in tumourigenesis 10
1.2.5 E-cadherin mutations in gastric cancer _______________________________ 11
1.3 The ErbB family of receptor tyrosine kinases (RTK) _____________________ 11
1.3.1 Receptor activation ______________________________________________ 13
1.3.2 Signalling pathways activated by EGFR______________________________ 14
1.3.3 Compartmentalization of EGFR signalling____________________________ 16
1.3.4 Attenuation of EGFR signalling ____________________________________ 16
1.3.5 Crosstalk between EGFR and E-cadherin_____________________________ 17
1.3.6 EGFR and cancer _______________________________________________ 18
1.4 Matrix metalloproteinases ___________________________________________ 19
1.4.1 Reciprocal interaction between E-cadherin and MMPs __________________ 19
1.4.2 MMPs and cancer 20
1.5 Aim of this study ___________________________________________________ 21
2 Materials and Methods 23
2.1 Cell culture________________________________________________________ 23
2.1.1 Cell culture conditions ___________________________________________ 23
2.1.2 Cell lines and growth media _______________________________________ 23
2.1.3 Passaging of cells _______________________________________________ 24
2.1.4 Preparation of frozen stocks 24
2.1.5 Starting culture from frozen stocks __________________________________ 24
2.1.6 Positive selection of E-cadherin expressing cells _______________________ 24
2.1.7 Detection of Mycoplasma in cultured cells____________________________ 25
2.2 Western blot analysis _______________________________________________ 26
2.2.1 Cell lysis ______________________________________________________ 26
2.2.2 Calcium-depletion procedure ______________________________________ 27
2.2.3 Protein quantification (based on the method of Bradford) ________________ 27
2.2.4 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) __________________ 28
2.2.5 Immunoblotting_________________________________________________ 29
2.2.6 Immunological detection of immobilized proteins ______________________ 29
2.2.7 Stripping 29
2.2.8 Antibodies _____________________________________________________ 30
2.2.9 Buffers and solutions for Western blot analysis ________________________ 31
2.3 Immunoprecipitation _______________________________________________ 32
2.4 GST-Raf1-RBD pulldown assay ______________________________________ 32
2.5 Quantitative indirect immunofluorescence assay_________________________ 33
2.6 Flow cytometry ____________________________________________________ 33
2.7 Immunohistochemistry______________________________________________ 34
2.8 Invasion assay _____________________________________________________ 35
2.9 Zymography ______________________________________________________ 35
2.9.1 Buffers for zymography __________________________________________ 36
2.10 List of providers ___________________________________________________ 38
3 Results________________________________________________________________ 39
3.1 Receptor crosstalk between EGFR and mutant E-cadherin________________ 39
3.1.1 Effect of E-cadherin mutations on EGFR expression ____________________ 39
3.1.2 Enhanced activation of EGFR caused by somatic E-cadherin mutations _____ 40
3.1.3 Kinetic of EGFR activation________________________________________ 42
3.1.4 Activation of EGFR is sensitive to treatment with EGFR kinase inhibitor
Tyrphostin AG 1478 _____________________________________________________ 44
3.1.5 EGF-induced EGFR activation is cell density dependent_________________ 45
3.1.6 Activation of EGFR downstream signalling ___________________________ 46
2
3.1.7 Mutant E-cadherin increases the level of active Ras_____________________ 49
3.1.8 EGFR forms a complex with wt and del 8 E-cadherin ___________________ 50
3.1.9 Mutant E-cadherin shows decreased cell surface localization _____________ 51
3.1.10 Internalization of EGFR is affected by E-cadherin______________________ 52
3.1.11 Expression and localization of E-cadherin and phosphorylated EGFR in gastric
cancer ______________________________________________________________53
3.2 Mutant E-cadherin and its influence on further tyrosine kinases ___________ 58
3.2.1 Activation of hepatocyte growth factor receptor (Met) in MDA-MB-435S-
transfectants ___________________________________________________________ 58
3.2.2 Mutant E-cadherin and the non-receptor tyrosine kinase Src ______________ 59
3.3 Matrix Metalloproteinase 3 (MMP-3)__________________________________ 60
3.3.1 Secreted MMP-3 is active in MDA-MB-435S-transfectants ______________ 60
3.3.2 Upregulation of MMP-3 in del 9 E-cadherin promotes cell invasion ________ 62
4 Discussion_____________________________________________________________ 64
4.1 Crosstalk between mutant E-cadherin and EGFR _______________________ 64
4.1.1 Mechanism of EGFR activation by somatic E-cadherin mutations _________ 64
4.1.2 EGFR activation in tumours _______________________________________ 67
4.1.3 EGFR as a target for gastric cancer therapy? __________________________ 68
4.2 Src-independent changes in cell adhesion and motility ____________________ 69
4.3 MMP-3 in cell invasion and motility ___________________________________ 70
4.3.1 An integrated model for mutant E-cadherin enhanced cell motility _________ 72
4.4 Significance of EGFR activation by E-cadherin mutations for tumour
progression______________________________________________________________ 74
4.5 Outlook___________________________________________________________ 75
5 Summary 76
6 References ____________________________________________________________ 78
7 Abbreviations __________________________________________________________ 88

3 1 Introduction
1.1 Gastric cancer
Gastric cancer is the fourth most common cancer and the second leading cause of
cancer-related death worldwide (Parkin et al., 2001). The areas with the highest
incidence rates are in Eastern Asia, the Andean regions of South America and Eastern
Europe. Low rates are found in North America, North Europe, and most countries in
Africa and South-eastern Asia (IARC Cancer Mondial Statistical Information System;
http://www-dep.iarc.fr/). In the 1930s, gastric cancer was the most common cause of
cancer-related death in the USA and Europe. A steady decline in the incidence and
mortality rates of gastric carcinoma has been observed worldwide over the past several
decades due to unplanned prevention. The decline took place following the
popularization of refrigerators, which resulted in a decreased intake of salt for meat and
fish preservation and the availability in many countries of fresh fruits and vegetables
throughout the year (Palli, 2000).
Infection by Helicobacter pylori is prevalent in areas with high incidences of gastric
cancers, and increases the risk of gastric tumours. Polymorphisms of proinflammatory
cytokine genes have been shown to associate with risk of H. pylori-related gastric
cancer (El-Omar et al., 2000). However, in some Asian countries, such as India and
Thailand, incidences of gastric carcinomas are not high in spite of the high H. pylori
infection rates, a phenomenon known as the Asian Enigma? (Miwa et al., 2002).
Possible explanations for this include host genetic factors, different virulence among
strains of H. pylori, and dietary factors.
1.1.1 Tumour development and survival
Neoplasia of the stomach is largely composed of adenocarcinomas. The two main