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«Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der ...»

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zur Erlangung der Würde eines Doktors der Philosophie vorgelegt

der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel



aus Esslingen am Neckar, Deutschland

Dissertationsleiter: Prof. Dr. Jan Hofsteenge

Friedrich Miescher Institut für Biomedizinische Forschung Basel, 2009 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Dieses Werk ist unter dem Vertrag „Creative Commons Namensnennung-Keine kommerzielle Nutzung-Keine Bearbeitung 2.5 Schweiz“ lizenziert. Die vollständige Lizenz kann unter creativecommons.org/licences/by-nc-nd/2.5/ch eingesehen werden.

Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Jan Hofsteenge Prof. Dr. G. Christofori Prof. Dr. Nancy E. Hynes Basel, den 8. Dezember 2009 Prof. Dr. Eberhard Parlow Dekan Namensnennung-Keine kommerzielle Nutzung-Keine Bearbeitung 2.5 Schweiz

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Die Commons Deed ist kein Lizenzvertrag. Sie ist lediglich ein Referenztext, der den zugrundeliegenden Lizenzvertrag übersichtlich und in allgemeinverständlicher Sprache wiedergibt. Die Deed selbst entfaltet keine juristische Wirkung und erscheint im eigentlichen Lizenzvertrag nicht. Creative Commons ist keine Rechtsanwaltsgesellschaft und leistet keine Rechtsberatung. Die Weitergabe und Verlinkung des Commons Deeds führt zu keinem Mandatsverhältnis.

Quelle: http://creativecommons.org/licenses/by-nc-nd/2.5/ch/ Datum: 3.4.2009 “I believe there exists, and I feel within me, an instinct for the truth, or knowledge or discovery, of something of the same nature as the instinct of virtue, and that our having such an instinct is reason enough for scientific researches without any practical results ever ensuing from them.”

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Breast cancer is the most prevalent form of cancer in females: one of nine women develops breast cancer during her lifetime and it is predicted that one in 27 women will die as a result of this disease. Moreover, it is anticipated that with almost 30 % of females affected, breast cancer will be the most frequently diagnosed cancer in 2009 (www.cancer.org). Given these facts, much time and resources have been provided to research in the breast cancer area.

The ErbB2 receptor tyrosine kinase is one of the most-studied oncogenes in breast cancer as amplification and overexpression of the ERBB2 gene is known to occur in up to 25 % of all affected patients and is correlated with a highly aggressive disease and poor patient prognosis.

Our study focused on signaling molecules interacting with the C-terminal regulatory region of the ErbB2 receptor. We used T47D breast cancer cells metabolically labeled with SILAC to identify binding partners of the pTyr1248 site of ErbB2. Using a peptide affinity pull-down approach followed by quantitative mass spectrometry, we identified Copine III as a novel interaction partner of ErbB2-pTyr1248.

Copine III belongs to a family of Ca2+-dependent phospholipid binding proteins that is conserved from plants to humans. All copines carry two C2 domains followed by an A domain, similar to the von Willebrand A domain of integrins, in their C-terminus. Although Copine III is ubiquitously expressed, to date it has not been assigned a function downstream of ErbB2.

In this study we first analyzed the biochemical properties of Copine III and its interaction with ErbB2. We show that Copine III is a cytoplasmic protein that localizes to the nucleus and the plasma membrane in a Ca2+-dependent manner and upon stimulation of the cells with the ErbB ligand heregulin (HRG). We used FRET acceptor photobleaching to show that Copine III and ErbB2 not only co-localize in HRG-stimulated breast cancer cells, but also interact at the plasma membrane. This co-localization is blocked when the cells are treated with the ErbB2 inhibitor AEE788, implying that Copine III only interacts with phosphorylated active ErbB2.

The second goal of my studies was to place Copine III within a signaling pathway downstream of ErbB2. For this, we again used SILAC together with quantitative mass spectrometry and identified the scaffolding protein RACK1 as a binding partner of Copine III. We were able to show that Copine III, RACK1 and the adaptor molecule Shc form a complex with ErbB2 in HRGstimulated cells. RACK1 has been implicated in focal-adhesion mediated cell migration and here we demonstrate that Copine III localizes to focal adhesions and is required for ErbB2dependent cell migration. Moreover, knock-down of Copine III affects Src kinase activity and the subsequent phosphorylation of focal adhesion kinase, resulting in the observed defects in cellular migration. Thus Copine III is an important effector molecule in ErbB2-mediated cell migration.


Finally, we analyzed Copine III expression in the broader context of cancer, looking at carcinomas of the breast, prostate and ovary. In a set of 49 breast cancer tumor samples, 10 of the 11 cases with ERBB2 amplification display elevated levels of Copine III. This connected well with the protein expression levels of Copine III in a panel of breast cancer cell lines that also correlated with ErbB2 amplification. In published ovarian and prostate transcriptome studies, Copine III mRNA levels are upregulated in cancer as compared to normal tissue. Based on these findings, we performed immunohistochemistry (IHC) stainings of Copine III on breast, prostate and ovarian tissue microarrays. While some Copine III staining was evident in normal breast, normal prostate and ovarian tissues have very low levels of Copine-III. Strikingly, tumors of all three types showed higher Copine III levels.

To summarize, we present Copine III here for the first time as an interaction partner of the ErbB2 receptor. Copine III interacts with ErbB2 in a Ca2+- and HRG-dependent manner and is required for tumor cell migration. Furthermore, Copine III levels were found to be upregulated in tissue microarrays of breast, ovarian and prostate tumor tissue as compared to normal tissue.

Together, these findings imply a biological function for Copine III in cancer progression and suggest that further studies into the functions of Copine III are merited.

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SUMMARY………………………………………………………….……………...……..I TABLE OF CONTENTS………………………………………………………………….…...III 1 INTRODUCTION

1.1 Cell signaling and cancer

1.2 The ErbB family of RTKs

1.2.1 Evolution of the ErbB signaling network

1.3 The human ErbB receptor/ligand network

1.3.1 Ligand induced receptor activation

1.3.2 Signal transduction of ErbB receptors

1.3.3 Transactivation of ErbB receptors

1.3.4 Endocytosis, sorting and recycling of ErbBs

1.4 ErbB receptors in development and disease

1.5 ErbB receptors in cancer – aberrant signaling, treatment and resistance

1.5.1 ErbB2 in breast cancer

Copines – a conserved family of Ca2+-dependent, phospholipid binding proteins.. 23 1.6 1.6.1 General aspects, discovery, homology and conservation

1.6.2 Domain architecture

1.7 Biological roles of Copine family members

1.7.1 Copine in Arabidopsis thaliana

1.7.2 Copines in Caenorhabditis elegans

1.7.3 Copines in Dictyostelium discoideum

1.8 Human Copines

1.8.1 Phenotypes, biological roles


–  –  –


3.1 Introduction

3.2 Materials and methods

3.3 Results

3.4 Discussion


–  –  –


5.1 Abstract

5.2 Introduction

5.3 Materials and Methods

5.4 Results

5.5 Discussion

5.6 Acknowledgements


6.1 Introduction

6.2 Materials and Methods

6.3 Results

6.4 Outlook



8.1 Abbreviations

8.2 List of figures and tables




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Each cell receives a plethora of external signals that it must integrate in order to initiate, maintain and attenuate cellular signaling pathways, thereby regulating such diverse processes as protein synthesis, cell growth, cell cycle progression and cell movement. Most cellular signaling pathways are involved in regulating several cellular functions, and in addition, can interact and cross-regulate other pathways thus generating a complex signaling network.

Proper functioning of this network is essential for maintaining cell growth and homeostasis.

Cancer cells are characterized by the acquisition of mutations leading to deregulated cell growth and homeostasis. In the past years it has become apparent that different types of cancer are characterized by the different mutations they acquire. Furthermore, the majority of cancers display disruption not only in one but several pathways, which results in the effects observed in cancer progression. The different alterations occurring in cancer can be grouped into six categories: self-sufficiency in growth signals, insensitivity to growth-inhibitory (antigrowth) signals, evasion of programmed cell death (apoptosis), limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis (Figure 1-1) (Hanahan and Weinberg, 2000).

–  –  –

Figure 1-1 Acquired traits of cancer It was suggested that most, if not all, types of cancer have acquired the same set of capabilities during their development.

Three of these traits, namely growth signal autonomy, insensitivity to antigrowth signals and resistance to apoptosis, lead to an uncoupling of intracellular signaling from extracellular cues.

1. Introduction | The ErbB family of RTKs The proteins that receive these extracellular signals and convert them into information to guide intracellular responses are designated transmembrane receptors. Therefore, signaling pathways downstream of transmembrane receptors should be studied carefully in order to elucidate, which functional mutations give rise to specific cancer traits.

1.2 The ErbB family of RTKs

Certain classes of signaling proteins, such as molecules governing extracellular growth, differentiation and developmental signals are targeted much more frequently by oncogenic mutations than others. Good examples for this phenomenon are the receptor tyrosine kinases (RTKs), a subclass of transmembrane receptors carrying an intrinsic, ligand-stimulated kinase activity (Blume-Jensen and Hunter, 2001).

(Blume-Jensen and Hunter, 2001) Figure 1-2 Human receptor tyrosine kinases Depicted are the 20 families of human receptor tyrosine kinases. The prototypic receptor for each family is indicated above the receptor and the known members are listed below. EGFR, epidermal growth factor receptor; InsR, insulin receptor; PDGFR, platelet-derived growth factor receptor; VEGFR; vascular endothelial growth factor receptor; FGFR, fibroblast growth factor receptor; KLG/CCK, colon carcinoma kinase;

NGFR, nerve growth factor receptor; HGFR, hepatocyte growth factor receptor, EphR, ephrin receptor; Axl, a Tyro3 PTK; TIE, tyrosine kinase receptor in endothelial cells; RYK, receptor related to tyrosine kinases; DDR, discoidin domain receptor; Ret, rearranged during transfection; ROS, RPTK expressed in some epithelial cell types; LTK, leukocyte tyrosine kinase; ROR, receptor orphan; MuSK, muscle-specific kinase; LMR, Lemur.

Other abbreviations: AB, acidic box; CadhD, cadherin-like domain; CRD, cysteine-rich domain; DiscD, discoidin-like domain; EGFD, epidermal growth factor-like domain;

FNIII, fibronectin type III-like domain; IgD, immunoglobulin-like domain; KrinD, kringle-like domain; LRD, leucine-rich domain. The symbols α and β denote distinct subunits. Members in bold and italic type are implicated in human malignancies. An asterisk indicates lack of intrinsic kinase activity.

1. Introduction | The ErbB family of RTKs

RTKs belong to a larger family consisting of 90 genes encoding for protein tyrosine kinases in the human genome. Of these, 58 encode transmembrane RTKs, which are distributed among 20 subfamilies (Figure 1-2).

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