sponsible for receptor activation and give rise to signals that promote the formation of new vessels. We investi gated VEGF receptors using electron microscopy, Xray crystallography and small angle solution scattering tech niques. The structural and functional information gained in this project was used to develop a comprehensive mo lecular model of receptor function that was essential for further progress in the development of new receptor an tagonists for future medical applications.
Based on the newly gained insights into the activation mechanism of VEGF receptors, we developed new anti bodylike molecules to block receptor activation. In a fol low up study we are now investigating the potential of these new molecular tools to block tumour growth in an animal model.
Project coordinator Prof. Dr. Kurt BallmerHofer
Laboratory of Biomolecular Research Molecular Cell Biology
Paul Scherrer Institut Bldg. OFLC 102 CH5232 VilligenPSI Phone +41 (0)56 310 41 65 Fax +41 (0)56 310 52 88 [email protected]
BallmerHofer Kurt | Structural and functional analysis of ligand-mediated activation of VEGF receptor 2; identification and characterization of structural motifs for the development of new receptor inhibitory drugs for anti-vascular tumor therapy (OCS 02100082007)
Vascular Endothelial Growth Factors (VEGFs) constitute a family of proteins that regulate blood and lymphatic ves sel development. Vessel formation and the maintenance of proper vessel organization are absolutely required for sustaining organ function in higher organisms. Aberrant vessel formation is associated with various diseases, such as arteriosclerosis, retinopathies, lymphoproliferative or rheumatoid disease, and in tumour growth where newly formed vessels allow cancer cells to grow more rapidly and to disseminate into the entire body. Tumour vascular ization is a hallmark of many types of highly aggressive malignancies, such as breast, colon and stomach cancer. VEGFs bind to receptors expressed on the surface of cells and thereby activate their target cells to migrate, prolifer ate and ultimately to form new vessels. VEGF binding to cell surface exposed receptors induces changes in recep tor structure that lead to receptor activation thereby giv ing rise to the generation of signals transmitted across the cell membrane to the intracellular milieu of the cell. We investigated the molecular mechanism of VEGF recep tor activation with the aim to develop new receptor inhib itors applicable for the treatment of disease associated with aberrant vessel formation. Studying the structure of VEGF receptors and VEGFreceptor complexes, we found that VEGF binding drastically alters the threedimensional structure of the receptor. These structural changes are re
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BentiresAlj Mohamed | Role of GAB2/SHP2 and 11q13 amplification in breast cancer
(OCS 01922082006)
Each year 1.1 million new cases of breast cancer will occur among women worldwide, and 400,000 women will die of this disease. Although progress has been made in un derstanding breast tumour biology, most of the relevant molecules and pathways remain undefined. Their delinea tion is critical to a rational approach to breast cancer ther apy.
This project focuses on the roles of the signalling proteins GAB2/SHP2 (part 1) and on amplification of the chromo somal region 11q13 (part 2) in breast cancer. To address these questions, we used a combination of 3D cultures and xenograft models. In the first part, we found that in hibition of SHP2 dramatically reduces proliferation and re verses the invasiveness of transformed breast cells grown in 3D cultures. Moreover, SHP2 knockdown after tumour formation blocks tumour progression. In the second part, we found two highly defined genomic regions of 11q13 amplification in breast tumours and identified 8 genes that are coamplified and cooverexpressed in these tu mours. Taken together, our studies revealed potential therapeutic targets for the treatment of breast cancer.
Project coordinator Dr. Mohamed BentiresAlj Friedrich Miescher Institut für biomedizinische Forschung (FMI) Maulbeerstrasse 66
CH4058 Basel
Phone +41 (0)61 697 40 48 Fax +41 (0)61 697 39 76 [email protected]
Brunner Thomas | Characterization and role of extra-adrenal glucocorticoid synthesis in colorectal cancer (OCS 02025022007)
Glucocorticoids are important immunoregulatory ster oids, produced mainly in the adrenal glands. However, our past research demonstrated that the epithelial cells of the intestinal crypts are capable of producing glucocorticoids in response to immune cell activation and that intestinal glucocorticoids contribute to the control of local immune responses. The aim of this study was to investigate whether colon carcinoma can produce glucocorticoids, how tumour glucocorticoid synthesis is regulated and whether tumourderived glucocorticoids actively suppress immune cells.
The expression and induction of transcription factors and enzymes involved in the synthesis of cortisol from choles terol in colon carcinoma cell lines as well as primary tu mours was investigated using quantitative PCR and lucif erase reporter assays. The role of the transcription factor LRH1 (liver receptor homolog1) in the regulation of tu mour glucocorticoid synthesis was assessed using overex pression and RNA interference. Cortisol production was measured using radioimmunoassay, thin layer chromatog
Beermann Friedrich | In vivo screening of candidate
genes in melanoma (OCS 01500022004)
Melanoma is a malignant tumour arising from melano cytes, which are pigment cells derived from the neural crest. In the mouse, melanoma does not occur spontane ously, and, thus transgenic mouse models are used to ad dress genetic changes leading to melanomagenesis or to study melanocyte development. In this project we wanted to address the role of candidate genes, first in melanocyte biology and homeostasis and later in melanoma develop ment. We used transgenic mouse models to analyze the Notch signalling pathway in normal melanocyte biology, and to address the lack of the oncogene cMyc during melanocyte development. Moreover, we addressed the relevance of bcatenin directly in a mouse model of mel anoma.
Notch signalling: Notch has been shown to be expressed in mouse melanocytes and human melanoma. Using transgenic mouse models, we showed that deletion of Notch signalling in the melanocyte lineage induces preco cious hair greying, the intensity of which depends on the number of deleted alleles of Notch1 and Notch2. Further histological analysis showed that this is due to a loss of melanocyte precursors as well as melanocyte stem cells after birth. This confirms that Notch signalling affects hair pigmentation and melanoblast population in a gene dos agedependent manner. When we overexpressed Notch in melanocytes, we could not prevent normal hair greying or induce melanoma tumours, indicating that Notch sig nalling by itself is not sufficient for melanoma formation.
The c-Myc oncogene: cMyc is expressed in many tumours, including melanoma. To understand its role in melano cytes, we removed it specifically in a transgenic mouse model. Removal of cMyc leads to a hair greying pheno type that is not due to an effect in stem cells. In contrast to Notch, the phenotype is caused by a problem in prolif eration during midgestation. These results indicated that cMyc is an important player in melanocyte biology, and we plan to address its role in a mouse model of melanoma. b-catenin: This protein is part of the Wnt signalling path way and has been reported to be mislocalized in human melanoma. In collaboration with Lionel Larue’s group (Orsay, France), we used mice that express a stable form of bcatenin that leads to repression of the tumour sup pressor p16 by binding to its promoter. Double transgenic animals expressing both an activated NRas oncogene and an activated bcatenin had a high incidence of mela noma with a short latency period. Histopathological anal ysis suggested that most melanomas arose from melano cytes located in the hair follicles. The results thus reveal that synergy between the Wnt and mitogenactivated protein (MAP) kinase pathways (due to activated NRas) may represent an important mechanism underpinning the genesis of melanoma.
Project coordinator Dr Friedrich Beermann
Institut suisse de recherche expérimentale sur le cancer (ISREC)
Faculté Sciences de la vie EPF de Lausanne (EPFL) Station 19, Bâtiment SV CH1015 Lausanne Phone +41 (0)21 693 07 27 [email protected]
Project coordinator Prof. Dr. Thomas Brunner
Lehrstuhl für biochemische Pharmakologie Fachbereich Biologie Universität Konstanz D78457 Konstanz Deutschland Phone +49 (0)7531 88 53 70 thomas.brunner@unikonstanz.de
raphy and bioassay. The suppressive and apoptosisinduc ing activity of tumour glucocorticoids was assessed on ac tivated T cells and thymocytes.
Our study could show that colon carcinoma cell lines as well as primary tumours express all enzymes required for synthesis of glucocorticoids and secrete bioactive cortisol. Similar to primary intestinal epithelial cells, LRH1 was found to play a critical role in the regulation of tumour glucocorticoid synthesis and to be overexpressed in many primary colon carcinomas. Cortisol produced by colon carcinomas potently suppressed T cell activation and in duced apoptosis in glucocorticoidsensitive thymocytes. This is the first demonstration of glucocorticoid synthesis in tumours not derived from steroidogenic organs, and it suggests that tumourderived glucocorticoids could play an important role in the regulation of antitumour im mune responses. Whereas the glucocorticoid synthesis in primary epithelial cells contributes to intestinal immune homeostasis and prevents intestinal inflammation, gluco corticoid synthesis in colon carcinoma cells may represent a tumourpromoting factor.
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tracellular domain to act as a low affinity receptor for chemokines and growth factors. The elucidation of podo planin’s mode of action in inducing collective cell invasion provides important new insights into the molecular mech anisms of cancer cell invasion and metastasis formation.
Project coordinator Prof. Dr. Gerhard Christofori Institut für Biochemie und Genetik Departement Biomedizin Universität Basel Mattenstrasse 28 CH4058 Basel Phone +41 (0)61 267 35 62 Fax +41 (0)61 267 35 66 [email protected]
Citi Sandra | The role of tight junction proteins in epithelial morphogenesis and differentiation
(OCS 01916082006)
The majority of cancers originate from epithelial cells and tissues, which cover all surfaces and cavities of the body (skin, gastrointestinal, respiratory and urinary tracts, etc.) and constitute glands (breast, kidney, liver, pancreas). Normal epithelial cells proliferate in a controlled fashion, are closely bound together and have a characteristic “po larized” shape, with apical, basal and lateral sides. When epithelial cells become cancerous, they “dedifferentiate” and lose some or most of these properties. For example, they may lose their ability to adhere to one another, lose polarity, become more motile, migrate elsewhere in the body and start to divide in an uncontrolled way, thus forming metastases. Our research focuses on the role of proteins, which constitute specialized “cellcell junctions”. Junctions are structures that control adhesion between cells and are also implicated in signalling mechanisms that regulate epithelial cell growth and proliferation. There are different types of junctions, and we are focusing our at tention on “tight” and adherens junctions, which are lo calized at the apicolateral border of epithelial cells. Tight junctions are involved in the maintenance of the polarized shape and also serve to form a barrier that controls the passage of molecules and pathogens across sheets of ep ithelial cells. Adherens junctions play a major role in cell cell adhesion and signalling mechanisms that regulate morphogenesis and cell sorting.
The goal of our project was to understand the role of spe cific proteins of tight junctions (cingulin and paracingulin) in the establishment and maintenance of the differenti ated state and signalling properties of epithelial cells, us ing as the main experimental approaches studies on cells in culture and on human epithelial lung cancer tissues. We made important discoveries about the role of cingulin and paracingulin in controlling the activities of RhoA and Rac1, molecular switches that regulate the assembly of the cytoskeleton and also in controlling proliferation and gene expression in epithelial cells. The activities of RhoA
Christofori Gerhard | Podoplanin-mediated signalling and its role in collective cell invasion and metastasis formation (OCS 01932082006)
Tumour invasion is the first step in the formation of me tastases, which is the actual cause of death in most can cer fatalities. Originally, tumour cell invasion was thought to be dependent on the loss of cellcell adhesion and on single cell migration. However, we showed previously that cancer cells can also invade the surrounding tissue in a cell collective. We identified the small mucinlike transmem brane protein podoplanin as a key regulator of collective cell migration and invasion, and we investigated the func tional role of podoplanin during tumour progression. For example, expression of podoplanin in tumour cells of a transgenic mouse model of pancreatic cancer caused tumour cell invasion in the absence of any loss of cellcell adhesion. Moreover, forced expression of podoplanin in breast cancer cell lines also leads to increased cell migra tion and invasion without loss of cellcell adhesion. Finally, podoplanin expression correlates with tumour invasion in squamous cell carcinomas (SCC) of various organs. Together, the results indicate that podoplanin employs a novel molecular pathway of inducing collective tumour cell invasion. However, the regulation of podoplanin ex pression in the invading cancer front as well as the molec ular mechanisms by which podoplanin confers its promi gratory and proinvasive function have remained elusive. In the past years, we investigated how the curious expres sion of podoplanin in the invasive cancer front is regulated and how podoplanin mediates collective cancer cell mi gration. We isolated the podoplaninexpressing invading cells of squamous cell carcinoma by laser capture micros copy of tissue sections and employed gene expression profiling to determine the differences between podopla ninexpressing and podoplaninnegative cancer cells. The podoplaninexpressing cells exhibited a significant upreg ulation of inflammatory signalling pathways, including the interferong, tumour necrosisfactora (TNFa) and trans forming growth factorb (TGFb) pathways. Subsequent studies showed that indeed podoplanin expression is in duced by the treatment of cancer cells with these inflam matory cytokines. We are currently ablating these signal ling pathways in squamous cell carcinoma cells in culture and in mouse models to study the effect of the lack of these inflammatory signalling pathways on podoplanin expression and collective cell invasion.
The promigratory function of podoplanin is at least in part mediated by the remodelling of the actin cytoskele ton of cancer cells. We have set out to identify binding partners of podoplanin, which potentially play critical roles in transmitting the promigratory signals of podopla nin. However, various biochemical analyses did not iden tify proteins that interact with podoplanin in a specific and robust manner. From these experiments we conclude that podoplanin exerts its promigratory and invasive function via pathways that do not include a direct interaction with other signalling proteins. Based on this and other evi dence, we are currently testing the possibility that podo planin exerts its signalling functions by clustering on the cell surface and employing its highly glycosylated ex
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pressed in breast cancer, we developed a CD1dantiCEA fusion protein specific of a tumour antigen overexpressed in colon cancer, as well as a CD1dantiVEGFR3 fusion protein targeting a marker of tumour neovascularization. Sustained iNKT activation and inhibition of tumour growth by these three CD1d bifunctional proteins were evidenced in several tumour models. 2) At the cellular level, in vivo and in vitro experiments demonstrated that iNKT cell activation by soluble CD1d proteins prevented the negative retrocontrol of PD1/PDL1 interaction that normally occurs during cellcell interaction between iNKT and APCs, leading to their subsequent unresponsiveness. The attenuated PD1 upregulation largely explains the sustained iNKT cells obtained with recombinant CD1d proteins, which is optimal for a systemic treatment. 3) Several in vivo experiments have shown that the adjuvant effect of CD1dmediated therapy on the adaptive im mune response remains limited by immunosuppressive mechanisms developed in the periphery and at the tu mour site. In particular, the expansion of myeloidderived suppressor cells (MDSCs) induced by the tumour environ ment is known to inhibit the antitumour immune response and remains a challenge for cancer immunotherapy. MD SCs act by various mechanisms, among which are several enzymatic activities such as arginase 1, which depletes ar ginine essential for the activity of T lymphocytes. We could demonstrate the inhibitory effect of arginase 1 on CD1dmediated therapy in a mouse model in which argi nase 1 has been eliminated in the myeloid compartment. In these mice, the antitumour effect of CD1dantitumour treatment was indeed far more potent than in mice with active arginase 1.
In view of these results, we are testing chemotherapeutic agents able to deplete preferentially MDSCs, such as 5fluorouracil. The next step is to combine CD1dmedi ated immunotherapy with chemotherapy, and these protocols will be tested in two transgenic mouse models developing spontaneous tumours, representative of mela noma tumours and prostate cancer in humans. Combina tion of multiple anticancer therapies is more and more considered, as it can result in a synergistic tumour inhibi tion and limit tumour escape.
Project coordinator Dr Alena Donda
Département de biochimie Université de Lausanne Chemin des Boveresses 155 CH1066 Epalinges Phone +41 (0)21 692 58 57 [email protected]
and Rac1 are frequently altered in cancer tissues, which could account for their altered migratory properties. Therefore, our studies are directly relevant to understand ing the molecular mechanisms of regulation of signalling pathways that are altered in cancer. We further studied the dynamics of cingulin and paracingulin in living cells and clarified differences in their behaviour and localiza tion, which will help to clarify their redundant and nonre dundant functions. Using antibodies that we developed in our laboratory, we studied the expression of cingulin in different types of human lung tumours in collaboration with the Department of Clinical Pathology at Geneva University Medical School. These results, and results ob tained using several other antibodies against tight junc tion proteins and reversetranscription polymerase chain reaction to assess gene expression, allowed us to propose a new molecular classification of lung carcinomas based on expression of specific tight junction proteins. Recently, we identified a novel adherens junction protein, PLEKHA7, against which we developed specific monoclonal antibod ies, and which we would like to use to examine its expres sion in human cancer, which has not been done so far. In summary, the results that we obtained have provided new information on the function of certain tight junction proteins, their role in the process of differentiation and cancer formation and their possible use in cancer diagno sis. In addition, our studies led to the discovery and pre liminary characterization of a new adherens junction pro tein.
Project coordinator Dr Sandra Citi
Département de biologie moléculaire Sciences III Université de Genève 4, boulevard d’Ivoy CH1205 Genève Phone +41 (0)22 379 61 82 Fax +41 (0)22 379 68 68 [email protected]
Donda Alena | CD1d-antitumor bifunctional molecules to redirect the innate and adaptive immune responses