THE NETHERLANDS CANCER INSTITUTE SCIENTIFIC ANNUAL REPORT 2005

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1 THE NETHERLANDS CANCER INSTITUTE SCIENTIFIC ANNUAL REPORT 2005 Mitogen starvation p53 Apoptosis S G 2 G 2 arrest Cyc B1 Cdk1 Cyc A Cdk2 E2F p27 p21 p130 prb p107 Cyc E Cdk2 G 1 Cyc D1 Cdk4/6 M Pocket-protein dependent G 1 arrest Mitogen starvation

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3 SCIENTIFIC ANNUAL REPORT

4 4 Patron HM The Queen Beatrix

5 5 SCIENTIFIC ANNUAL REPORT 2005 THE NETHERLANDS CANCER INSTITUTE CANCER RESEARCH LABORATORY AND CANCER HOSPITAL

6 6 COPYRIGHT Scientific Annual Report 2005 Illustrations and unpublished data in these reports may not be used without permission of the author. Copyright : The Netherlands Cancer Institute Antoni van Leeuwenhoek Hospital Plesmanlaan CX Amsterdam The Netherlands Phone Fax ISSN

7 7 CONTENTS Board Members 8 Research Divisions 9 Introduction 11 Education in Oncology 15 I Division of Cell Biology 16 II Division of Molecular Carcinogenesis 23 III Division of Cellular Biochemistry 31 IV Division of Immunology 40 V Division of Molecular Biology 48 VI Division of Tumor Biology 55 VII Division of Molecular Genetics 66 VIII Division of Experimental Therapy 74 IX Division of Radiotherapy 86 X Division of Medical Oncology 100 XI Division of Surgical Oncology 112 XII Division of Psychosocial Research and Epidemiology 121 XIII Division of Diagnostic Oncology 131 Biometrics Department 142 Clinical Trials 146 Invited Speakers 161 Projects 163 Personnel Index 176

8 8BOARD MEMBERS President of Board of Governors WF Duisenberg (until July 31) President of Board of Governors W Kok (from September 12) BOARD MEMBERS INTERNATIONAL SCIENTIFIC ADVISORY BOARD JR Bertino, Professor of Medicine and Pharmacology, The Cancer Institute of New Jersey, New Brunswick, USA (until July) T De Lange, Leon Hess Professor, Rockefeller University, New York, USA (from July) RA Flavell, Professor of Immunobiology, Yale-University School of Medicine, New Haven, USA S Hellman, AN Pritzker Distinguished Service Professor, The University of Chicago, Chicago, USA WGJ Hol, Professor of Biochemistry and Biological Structure, University of Washington, Seattle, USA J Mendelsohn, President, MD Anderson Cancer Center, The University of Texas, Houston, USA P Nurse, President, The Rockefeller University, New York, USA R Nusse, President, The Rockefeller University, New York, USA HL Ploegh, Professor of Immunopathology, Harvard Medical School, Boston, USA (until September); from September: Whitehead Institute of Biomedical Research, Cambridge, USA K Vousden, Director, Beatson Institute for Cancer Research, Glasgow, UK (from February 2006) RA Weinberg, Professor of Biology, Massachusetts Institute of Technology, Whitehead Institute, Cambridge, USA C Weissman, Professor of Biomedical Research and Molecular Biology, Scripps Florida, Jupiter, USA (until July) BOARD OF DIRECTORS AJM Berns, chairman and director of research S Rodenhuis, director clinical research and development WH Van Harten, director organization and management BOARD OF GOVERNORS WF Duisenberg, president (until July 3 1) HCJ Van der Wielen, vice-president PJ Kalff, treasurer W Kok, president (from September 12) D Sinninghe Damsté GNJ Tytgat PF Van der Heijden J Van der Meer GP Vooijs MWM Vos-Van Gortel SCIENTIFIC ADVISORY COUNCIL AJM Berns, chairman W Moolenaar, secretary S Rodenhuis R Medema J Neefjes S Rodenhuis L Van t Veer NATIONAL SCIENTIFIC ADVISORY BOARD LA Aarden, Professor of Molecular Immunology, Amsterdam SWJ Lamberts, Professor of Internal Medicine, Rotterdam B Löwenberg, Professor of Hematology, Rotterdam CJLM Meijer, Professor of Pathological Anatomy, Amsterdam CJM Melief, Professor of Immunohematology, Leiden HM Pinedo, Professor of Clinical Oncology, Amsterdam FH Schröder, Professor of Urology, Rotterdam GNJ Tytgat, Professor of Gastroenterology, Amsterdam AJ Van der Eb, Professor of Fundamental Virology, Leiden PC Van der Vliet, Professor of Physiological Chemistry, Utrecht

9 9 RESEARCH DIVISIONS RESEARCH DIVISIONS I Cell Biology Arnoud Sonnenberg (head) John Collard Kees Jalink Ed Roos Natasja Borsje-Maeyer (office manager) II Molecular Carcinogenesis René Bernards (head) Roderick Beijersbergen Anastassis Perrakis Titia Sixma Suellen Boonen (office manager) III Cellular Biochemistry Wouter Moolenaar (head) Nullin Divecha Fred Van Leeuwen Huib Ovaa Wim Van Blitterswijk Marcel Verheij Suzanne Corsetto (office manager) IV Immunlogy Jannie Borst (head) John Haanen Heinz Jacobs Ton Schumacher Florry Vyth-Dreese José Overwater (office manager) V Molecular Biology Hein Te Riele (head) Piet Borst (honorary staff member) Sabine Linn Henri Van Luenen Jos Jonkers Tom De Knegt (office manager) Linda Römer (secretary) VI Tumor Biology Jacques Neefjes (head) Reuven Agami Maarten Fornerod John Hilkens Rob Michalides Peter Peters Marieke Van der Velde (office manager) VII Molecular Genetics Maarten Van Lohuizen (head) Anton Berns Paul Krimpenfort Daniel Peeper Margriet Snoek Bas Van Steensel Gerdien De Kuijer (office manager) Marie Anne Van Halem (secretary) VIII Experimental Therapy Alfred Schinkel (head) Adrian Begg Petra Nederlof Jan Schellens Fiona Stewart Marc Van de Vijver Laura Van t Veer Thea Eggenhuizen (office manager) IX Radiotherapy Harry Bartelink (head) Berthe Aleman José Belderbos Iaïn Bruinvis Eugene Damen Roel De Boer Katrien De Jaeger Luc Dewit Rick Haas Guus Hart Wilma Heemsbergen Frank Hoebers Edwin Jansen Joos Lebesque Corrie Marijnen Harry Masselink Ben Mijnheer Luc Moonen Floris Pos Coen Rasch Peter Remeijer Nicola Russell Govert Salverda Christoph Schneider Joep Stroom Jacqueline Theuws Bart Van Bunningen Marcel Van Herk Corine Van Vliet Marcel Verheij Conny Vrieling Frits Wittkämper Patricia Haye-Fewer (office manager) X Medical Oncology Sjoerd Rodenhuis (head) Joke Baars Paul Baas Evert Bais Jos Beijnen Willem Boogerd Henk Boot Sjaak Burgers Annemieke Cats Jan Paul De Boer Bert De Gast Leo Gualtherie van Weezel John Haanen Alwin Huitema Martijn Kerst

10 10 RESEARCH DIVISIONS Sabine Linn Anne Lukas Herman Neering Jan Schellens Jan Schornagel Marianne Smits Babs Taal Wim Ten Bokkel Huinink Jaap Van der Sande Marchien Van der Weide Nico Van Zandwijk Mariëlle De Kwant (office manager) Annemieke Hoogland (office manager) XI Surgical Oncology Bin Kroon (board; head) Fons Balm (board) Omgo Nieweg (board) Axel Bex Dick Buitelaar Matthé Burger Marcel Copper Guus Fons Steven Gonggrijp Joris Hage Frans Hilgers Simon Horenblas Hans Huitink Christiaan Keijzer Houke Klomp Frans Kroon Peter Lohuis Lottie Lubsen Wim Meinhardt Hester Oldenburg May Ronday Emiel Rutgers Peter Schutte Michael Scrámek Marian Slot Ludi Smeele Tino Stoppa Bing Tan Julia Ten Cate Adriaan Timmers Marc Van Beurden Frits Van Coevorden Michiel Van den Brekel Henk Van der Poel Willemien Van Driel Arjen Van Turnhout Vic Verwaal Marie-Jeanne Vrancken Peeters Leonie Woerdeman Frans Zoetmulder Joke Van der Veen (office manager) XII Psycosocial Research and Epidemiology Neil Aaronson (head) Matti Rookus Frits Van Dam Wim van Harten Flora Van Leeuwen Marian Chin-A-Kwie (office manager) XIII Diagnostic Oncology Marc Van de Vijver (head) Tanja Alderliesten Philippe Baars Peter Besnard Hans Bonfrer Daphne De Jong Kenneth Gilhuijs Cees Hoefnagel Frans Hogervorst Irma Kluijt Wim Koops Robert Kröger Claudette Loo Saar Muller Petra Nederlof Willem Nooijen Frank Pameijer Renée Van Pel Hans Peterse Warner Prevoo Michiel Sinaasappel Ferida Sivro Jelle Teertstra Renato Valdes Olmos Hester Van Boven Olaf Van Tellingen Laura Van t Veer Loes Van Velthuysen Senno Verhoef Christine Arkes (secretary) Carla Van Tiggelen (secretary) Biometrics Department Otilia Dalesio (head) Harm Van Tinteren Financial Administration Christel Deckers General Facilities Petra Tuyp Personnel Department Eric De Wilde General Research Coordination Wouter Moolenaar, laboratory research coordinator Henri Van Luenen, laboratory research manager

11 11 INTRODUCTION Director of Research Ton Berns INTRODUCTION I am pleased to present our Scientific Annual Report It provides an overview of the scientific activities at The Netherlands Cancer Institute Antoni van Leeuwenhoek Hospital (NKI-AVL). Additional information can be found on the websites of the various divisions, as well as at The NKI-AVL is a Comprehensive Cancer Center, combining hospital and research laboratories under one roof in a single independent organization. The hospital comprises 180 beds, an outpatient clinic and a large radiotherapy department. Facilities for clinical research include a large patient database, clinical data management, many diagnostic facilities, a pharmacy with a production unit for experimental drugs, and active research groups in pharmacy, epidemiology and psychosocial oncology. The laboratory covers all major areas of cancer research, with special emphasis on cellbased screens, mouse tumor models, cell biology, structural biology, immunology and translational research. There is close collaboration between clinical and basic scientists. This scientific report deals mainly with the basic and clinical science in the NKI-AVL. Information on patient-care is described in our General Report. On July 3 1, Wim Duisenberg, Chairman of our board of Governors, died unexpectedly. We have not only lost a committed and influential supporter but also a very kind and pleasant person, who was able to reconcile diverging views in a superb manner. We will miss him and wish his wife, children and family strength in dealing with this tragic loss. At the same time we are very grateful that Wim Kok, former prime minister of The Netherlands, has consented to step into Wim Duisenberg s shoes. With him, we again have a exceptionally skilled chairman of exquisite stature. The renovation of our research premises has started in 2005 with the auditorium. In the meanwhile most of the old hospital building has been vacated. Three research divisions have moved to temporary facilities. This wellcoordinated action has caused only minor disruptions. Our clinical activities expanded further, with most growth occurring in the Divisions of Surgery and Radiotherapy. In the latter division the growth was more substantial because the department added new linear accelerators, permitting cone beam CT guided radiotherapy. In 2005 the hospital budget was under more strain than in the previous years, due to only partial compensation of rising expenses. Our research also suffered from financial restrictions due to the imposed 10% reduction in core funding by the Ministry of Health that was implemented in 2004 and to a lower contribution of the Dutch Cancer Society as a consequence of lower revenues from legacies. However, the revenues of the Dutch Cancer Society are again increasing and their contribution to the NKI will follow suit. We now have to convince the government to agree on a funding arrangement that does justice to the contribution of the NKI-AVL to the public cause. In December we signed a new agreement with the Dutch Cancer Society, which specifies that the NKI will continue to receive 15% of its revenues over the next five years. We also agreed to found a joint research fund that will specifically raise funds for translational research at the NKI. We expect that this foundation will be able to raise additional funds. This should allow us to invest in new projects in the coming years. For the time being we will ease the pain of financial constraints by using some of our reserves trusting that in the future both the contribution of the government and the Dutch Cancer Society will increase to absorb these costs. In 2005 two staff retreats were held, one with our junior staff members and one with the heads of the divisions and members of the Advisory Research Council. The constructive discussions identified a number of areas of research and marked specific support facilities that are worth expanding. In the course of 2006 these discussions will be converted to actual plans and action resulting in additional investments in personnel and facilities. Highlights The scientific output of the institute was again comfortingly high in It was again a successful year Table 1 Research funding NKI-AVL from the different sources in period in million Euros. Year Cancer Society Ministry of Health Project Grants Other income Total An additional 2.5 million Euro/year is received from the Stichting Fondsen of the NKI for specific investments in equiment.

12 12 INTRODUCTION with important new findings and scientific publications that received wide recognition. It is always difficult to estimate the impact of research when the results are still fresh. The research highlights summarized here serve primarily as a sampling of interesting work currently on-going in the Institute. A more complete and detailed account of specific projects can be found in the reports of individual group leaders in this SAR and on the website ( Sander Mertens in the group of John Collard showed that GTPase Rac plays a key role in the establishment of cell polarity. They found that the Rac activator Tiam 1 controls tight junction biogenesis and polarity in epithelial cells by association with and activation of the Par3/Par6/aPKC polarity complex. Kevin Wilhelmsen and Sandy Litjens in the group of Arnoud Sonnenberg have identified a novel outer nuclear membrane protein, nesprin-3, that can associate with the intermediate filament cytoskeleton through the cytoplasmic protein plectin. This is the first time that a protein scaffold has been described that can connect the nucleus to the intermediate filaments. This discovery will increase our understanding of nuclear events, such as positioning, structure and breakdown during mitosis. Miriam Epping and colleagues in the group of René Bernards identified the human tumor antigen PRAME as a dominant repressor of retinoic acid signaling. The data suggest that over-expression of PRAME, which is frequently observed in human cancers, confers proliferation or survival advantages by antagonizing retinoic acid signaling. In a systematic loss-of-function genetic screen, Sebastiaan Nijman and colleagues identified the deubiquitinating enzyme USP1 as a novel regulator of the Fanconi anemia pathway. His data indicate that USP 1 forms a physical complex with the FANCD2 protein and can remove mono-ubiquitin side chains from FANCD2, thereby recycling the protein. Wim Van Blitterswijk and Marcel Verheij and collaborators found that short-chain sphingolipids in a liposomal formulation of doxorubicin facilitate the uptake of the drug by tumor cells. The data look promising for possible clinical application. The group of Wouter Moolenaar, in close collaboration with Jos Jonkers, discovered that the metastasisenhancing enzyme autotaxin, which produces the growth factor lysophosphatidic acid (LPA), is essential for blood vessel formation during mouse embryonic development. This finding will lead to a better molecular understanding of the role of LPA in angiogenesis and its importance in tumor progression. Huib Ovaa, David Egan and Wouter Moolenaar were successful in the first NKI high-throughput screen of a chemical library with the metastasis-enhancing enzyme autotaxin as a target. Using a robust fluorescence-based assay, they identified a number of small-molecule inhibitors of autotaxin, which may serve as potential lead compounds for the development of novel targeted anticancer drugs. Using RNAi screens, Ingrid Kolfschoten in the Agami lab identified a transcription factor PITX that suppressed RAS activity and tumor formation. Mathijs Voorhoeve and Carlos le Sage in the Agami group established a mirna library. By screening for mirnas that cooperated with oncogenes in cell transformation to tumors, they identified two mirnas that act as oncogenes in testicular tumors. These mirnas were found overexpressed in human testicular germline tumors that still contained wild-type p53. The group of Peter Peters showed using EM that phagosomes are devoid of ER material. This is a critical finding for understanding the endosomal pathway. Helen Pickersgill in Fornerod s lab visualized the location of DNA at the nuclear matrix. Using DamID technology (in collaboration with Bas Van Steensel), they showed that the nucleus is subspecialized with relatively silent areas of chromatin located to the outside boundaries. Joost Neijssen in the Neefjes lab showed that cytosolic peptides were not restricted to the cell of origin but could diffuse through gap junction to surrounding cells. They showed that the peptides could also diffuse into specialized antigen presenting cells for crosspresentation. This pathway is blocked in tumor cells and possibly an explanation for the poor immune responses to tumor cells. Wilbert Zwart and Alex Griekspoor from the Neefjes/ Michalides lab showed how MHC class II molecules functioned in living cells using FRET technology (in collaboration with Kees Jalink). They showed that MHC class II molecules did not meet their chaperone on endosomal subdomains where they were not functional. This fact was used by intracellular pathogens to avoid recognition by CD4 + T cells. The group of Hein Te Riele has found that genes in mouse embryonic stem cells can be modified by the use of short single-stranded DNA oligonucleotides. However, oligo targeting only works effectively in the absence of DNA mismatch repair. They therefore developed a procedure to transiently down regulate the central mismatch repair gene MSH2. Oligo targeting provides a rapid and easy alternative to current gene modification procedures and has already been used to generate several mutant mouse lines. The Te Riele group also uncovered a novel cell cycle checkpoint that operates in the G 2 phase of the cell cycle and prevents cells from proliferating in the absence of mitogens. This G 2 restriction point is particularly important to restrict proliferation of cells that have lost proper cell cycle control in the G 1 phase, as is the case in the majority of cancer cells. Contamination of milk with drugs, pesticides and other xenotoxins can present a major health risk to breast-fed infants and dairy consumers. The group of Alfred Schinkel found that the drug transporter breast cancer resistance protein (BCRP/ABCG2) is highly induced in the mammary gland during pregnancy and lactation, where it is responsible for the active secretion of many clinically and toxicologically important substrates into milk. Several dietary carcinogens, and anticancer, antiulcerative, antibiotic and antiviral drugs were found to be concentrated in milk by BCRP. These insights will be very useful in the use and development of drugs and

13 13 INTRODUCTION pesticides that are applied during lactation in humans and dairy animals. For instance, drugs that are good BCRP substrates have a high chance of being concentrated into milk, and should thus usually be avoided during lactation. The Molecular Pathology department of the NKI participated in an International EU 6th frameworkfunded project TRANSBIG, where the prognostic power of NKI s 70-gene expression profile was confirmed for node-negative breast cancer patients. This validation led to the start of the EORTC breast group MINDACT trial (coordinator Emiel Rutgers, NKI) that will prospectively validate the signature in a 6000-patient randomized trial. The group of Daniel Peeper has obtained evidence that Oncogene-Induced Senescence (OIS), a process previously thought to occur in cultured cells only, represents a physiologically relevant process in humans. OIS is induced when cells are exposed to activated oncogenes, and results in cessation of proliferation. In collaboration with Wolter Mooi (VUmc), they found that human melanocytic nevi ( moles ) display the critical hallmarks of senescent cells: a mutation in an oncogene (BRAF), stable growth arrest, and induction of tumor suppressors (p 16) and a typical senescence marker. These results suggest that, in addition to apoptosis (programmed cell death), OIS constitutes a physiological response that protects against the growth-stimulating action of mutated cancer genes. The Epidemiology group coordinated a nationwide collaboration of Clinical Genetic Centers and brought together 139 families, comprising family members, with pathogenic mutations in the BRCA2 gene. They assessed the risk of cancers other than breast or ovarian cancer in this database and observed that risk compared to the general population was increased for four cancer sites: pancreas (Relative risk (RR)=5.9), prostate (RR=2.5), bone (RR=14.4) and pharynx (RR=7.3). Families with mutations outside the previously defined ovarian cancer cluster regions tended to have a higher cancer risk. The group of Aaronson reported the results of a nationwide observational study of 846 women at increased risk of hereditary ovarian cancer, approximately half of whom had opted for prophylactic oophorectomy (PO) and half for periodic gynecologic screening (GS). PO was associated with significantly reduced cancer worries and cancer risk perception, but significantly increased levels of endocrine and sexual symptoms. Balanced information about the pros and cons of PO versus screening will help clinicians and high-risk women make informed decisions about the optimal preventive health strategy. Harry Bartelink and colleagues clinically implemented Cone beam CT (CBCT) guided radiotherapy for a large group of patients in our institute and elsewhere. This has led to several new treatment approaches, for example to image guided radiotherapy for lung cancer patients. This new approach was made possible by software for image reconstruction and analysis, developed by Marcel van Herk and coworkers. This software has been licensed to industry, and is currently being installed and used in dozens of institutes worldwide. The first outcome results of a large trial in prostate cancer patients demonstrated that delivering a 10 Gy higher radiation dose resulted in a significantly reduction of recurrent disease. This radiation dose escalation was made possible by using sophisticated treatment techniques with conformal or intensity modulated radiotherapy. In this multicenter Dutch randomized phase III trial 669 patients were enrolled. Quality of research The quality of research can be monitored in several ways. Our scientific productivity as based on bibliometric parameters (citations and impact of scientific articles published by the NKI staff) has shown a steady increase since the beginning of the 1980 s, although in recent years the number of citations and impact has shown some fluctuation (Table 2). It is satisfying to see that the institute has acquired such an internationally prominent position in cancer research. The quality of our research was independently confirmed in a recent national survey that scores citations and a range of other output parameters in The Netherlands. A survey on science and technology indicators (NOWT) identified the NKI as one of the 2 institutes with an exceptionally high scientific impact. The other Institute was the FOM Institute for Atomic and Molecular Physics. In the area of Fundamental Life Sciences and Clinical Experimental Medical Sciences the NKI scored far better than all other institutions and universities in The Netherlands. On average Dutch performance is significantly better than the international average, illustrating that science in The Netherlands is still at a high level although many investigators fear that it will be very difficult to maintain this level in the current funding climate and with ever increasing bureaucracy. While a high citation score is gratifying, it is only one measure of scientific productivity. The quality of our work should also be gauged by the invitations of our staff to present at prestigious scientific meetings, and from our success in obtaining competitive grant awards. We score very well on all those items. We remain relatively successful in securing grant support, scoring on average twofold better than our colleagues in The Netherlands. Another measure of quality is based on external site visits, in which international leaders in a particular field of research review the work of a division or research groups with a similar theme, on a quinquennial basis. These site visits require that group leaders prepare a report and reflect on their research activities. This by itself is a useful exercise that helps to focus the research. In 2005 the work of the Division of Immunology was evaluated in a one-day site visit by a site visit team consisting of Hidde Ploegh (Harvard Medical School & Whitehead Institute, Cambridge USA), Richard Flavell (Yale University, Yale, USA) and Antonio Lanzavecchia (Belinzona, Switzerland). The site visitors were overall very positive about the activities of the division but also made a number of recommendations to be considered by the individual groups.

14 14 INTRODUCTION Honors and appointments The NKI-AVL cannot award university degrees. However, many of our staff members hold special part-time chairs at Dutch universities. This facilitates awarding degrees to graduate students receiving their training at The Netherlands Cancer Institute. In 2005, Ton Schumacher was appointed as Professor of Immune Technology at the University of Leiden, bringing the total number of full professors to 27. René Bernards received the Spinoza premium, which provides 1.5 million euros for research. In addition, he received the prestigious Pezcoller Award, and the Ernst W. Bertner Award for Cancer Research, M.D. Anderson Cancer Center for his contributions to cancer research. Simon Horenblas received the Folke Edsmyr Award 2005 for outstanding contributions to the progress of cancer research in Urology from the Karolinska Institute. Harry Bartelink received the Antoine Béclère medal from the International Society of Radiology, Neil Aaronson received the President s Award from the International Table 2 Short-term citations and impact of scientific articles published by the NKI research staff Publication year Citations* Impact** * *** * Citations in the two years after publication, excluding self-citations. Starting 1989 citation analysis has been carried out on line. This allows detection (and elimination) of all self-citations. Before 1989 this pruning was limited to first authors. ** The impact factor is the average number of citations per year of an article in a given journal. The total impact is the sum of the impact of all articles published that year. *** As from 1997 publication year of articles is the criterion, instead of Scientific Report-year listing. Society for Quality of Life Research and Frans Hilgers was awarded a Honorary Corresponding Membership of the Deutsche Gesellschaft für Plastische und Wiederhers tellungschirurgie (German Society for Plastic and Reconstructive Surgery). He received this award for his long-term contributions to the field of postlaryngectomy voice, pulmonary and olfactory rehabilitation. The AvL-prize 2005 for young, promising NKI-scientists was awarded to Adriaan Bins for his work on the development of an effective tattoo DNA vaccination method. There are also changes to report in our scientific staff. Two AvL fellows, Reuven Agami and Daniel Peeper were promoted to associate professor. Lodewijk Wessels was appointed as head of our new bioinformatics group and Michael Hauptmann as biostatistician. He will succeed Guus Hart who will retire in Staff of the NKI-AVL fulfilled numerous functions in national and international organizations, on scientific boards of scientific journals, as members of study sections, and as organizers or co-organizers of scientific meetings, workshops and congresses. Outlook and acknowledgements I am confident that research at the NKI-AVL will continue to flourish, especially now that we have decided to utilize part of our reserves to strengthen critical research areas and to further upgrade support facilities. Obviously, we need to secure more funds to invest in promising new areas of research as well. This is a recurrent theme that becomes increasingly important now that research developments are so dependent on complex approaches that require highly skilled experts, expensive equipment and a large budget for consumables. Flexibility is key and it is of critical importance that we retain the capacity to respond to new developments and opportunities. The Board of Directors will step up its efforts to find new funding sources for our research. The joint Research Fund with the Dutch Cancer Society is one of the initiatives in this direction. But there should also be a public interest in a dedicated internationally renowned cancer institute in The Netherlands. Research is the only way to improve cure rates and the quality of life of cancer patients. There is a broadly held belief that breakthroughs in cancer diagnosis and treatment can be realized with approaches that are now broadly applied in the NKI. We are particularly grateful to those who continue to support us: The Dutch Cancer Society, the most reliable and significant sponsor of our research; the Ministry of Health, which despite of recent budget cuts, still provides a substantial core grant to the Institute; and, last but not least, the many individuals who support us directly with their gifts and also with their moral and practical support. Only with their help can we continue to develop new ideas that will lead to the prevention, early detection, and more effective treatment of cancer. We hope they will feel encouraged by this report. Ton Berns Director of Research

15 15 EDUCATION IN ONCOLOGY EDUCATION IN ONCOLOGY In the Netherlands Cancer Institute, student training takes place at various levels, from Master, PhD to postdoctoral level and involves medical workers, technical personnel as well as scientists. Research and clinical staff as well as their group members are involved in theoretical and practical training. A number of staff members have joint appointments as professors at Dutch universities and also contribute to the regular curriculum at these universities. The research divisions attract students from universities throughout the country. The NKI has a formal affiliation with the Science faculty of the University of Amsterdam (UvA) and is committed to contributing education for Master students. The institute participates in the Oncology Graduate School Amsterdam, together with the medical faculties of the UvA and the Free University (VU). All educational activities are supervised by the Teaching Committee, which consists of J Borst (president and dean undergraduate school), H te Riele (general affairs), R Beijersbergen (undergraduate course), J Hilkens, J Collard (HLO students and publicity), T Sixma (dean graduate) and F Balm (clinical teaching). UNDERGRADUATE STUDENTS The undergraduate program in Experimental Oncology attracts Master students of all national universities. Students generally have a background in (Medical) Biology, Chemistry, Pharmacology, Medicine or Psychology. The undergraduate program offers combined practical and theoretical training in various aspects of experimental oncology. Practical training includes participation in ongoing research projects for a minimum of 4 months. In 2005, 27 university students completed a placement of 5-12 months at the biomedical research divisions. The majority (16) of these students came from the Amsterdam Universities (UvA and VU), 7 from Utrecht University and others from Wageningen, Leiden, Groningen and Nijmegen. The institute also provides practical training opportunities to trainee technicians. In 2005, we received, as in the preceding Table 1 Course in Experimental Oncology, fall 2005 three years, a number of Bachelor students in Medical Biology of the UvA for a 7-week rotation. Many of these ministage students subsequently do a long-term placement. The core element of theoretical training is the course in Experimental Oncology, given twice yearly (Table 1). This course includes lectures and tutorials. Information for undergraduate students and their supervisors can be found at under research and education. For the graduate student education the NKI-AVL participates in the Oncology Graduate School Amsterdam (OOA) together with the oncology departments of VUMC and AMC. This education program is an essential part of the PhD program and consists of a series of topical courses, an annual retreat and some skills courses, that are aimed at scientific thinking or writing. The combination with VUMC and AMC allows for a broad spectrum in courses. The NKI has 110 graduate students participating in the joint education program. In the past year we contributed 3 general courses, on chromatin structure and gene regulation (Bas Van Steensel and Maarten Van Lohuizen), on breast cancer (organized by Marc Van de Vijver) and on functional genomics (Roderick Beijersbergen and Rene Bernards). A course on writing and presenting in English was given twice, due to the high demand. The annual retreat is run almost entirely by the graduate students and focusses completely on research. First year students present a poster and older students present their research in short presentations and chair the sessions. There is a lively scientific discussion during breaks and after the sessions. In this manner the retreat not only trains important skills in presentation and interaction but also provides an overview of the research within the OOA at an early stage of the student s career. In practice this means that new collaborations can be forged at a time where the student can still benefit from them. It also presents a wonderful opportunity for the basic scientists and the more clinical graduate students to interact. That kind of interaction will benefit translational research, not only at short notice but also in the longer term of the careers of this generation of students. Table 2 OOA courses in 2005 Epidemiology D Voskuil Surgery F Zoetmulder Radiodiagnostics R Valdes Olmos Pathology* D de Jong Molecular diagnostics** A Velds, H Van Boven Drug therapy J Schornagel Radiotherapy* M Verheij DNA damage response and apoptosis** A Begg, J Borst Signal transduction** W Moolenaar, N Divecha Cell cycle** R Bernards, D Peeper Cell senescence and genomic instability** R Beijersbergen, H Te Riele Cell adhesion** E Roos Angiogenesis M Gebbink (guest) Mouse models of cancer** J Jonkers Gene therapy H Van der Poel Immunology and immunotherapy** T Schumacher, J Haanen Analysis of protein structure T Sixma Rational drug development and drug delivery** J Beijnen, A Schinkel * including tour ** including tutorial Jan 31 Feb 12 May 30 June 1 June 20 July 1 Sept Nov 9 11 Chromatin structure and gene regulation B Van Steensel, M Van Lohuizen Viral Oncogenesis CJLM Meijer, J Middeldorp, PJ Snijders Breast cancer M Van de Vijver Functional genomics R Bernards, R Beijersbergen Annual Retreat TK Sixma, MK Van der Velde

16 16 CELL BIOLOGY I DIVISION OF CELL BIOLOGY RECEPTORS FOR MATRIX ADHESION Division head, Group leader Arnoud Sonnenberg Arnoud Sonnenberg PhD Group leader Erik Danen PhD Post-doc Karine Raymond PhD Post-doc Johan De Rooij PhD Post-doc Kevin Wilhelmsen PhD Post-doc Sandy Litjens MSc Graduate Student Iman Van den Bout MSc Graduate Student Norman Sachs MSc Graduate Student Stephan Huveneers MSc Graduate Student Maaike Kreft Technical staff Ingrid Kuikman Technical staff Abadir Abdulle Undergraduate student Annelies Van Angelen Undergraduate student Francisco Caiado Undergraduate student Anja Richter Undergraduate student Hoa Truong Undergraduate student Figure I.1: Model depicting how the nesprins can link the nucleus to the actin and IF cytoskeleton systems. Nesprin-3 at the ONM and the integrin b4 subunit at the cell surface are shown bound to the N-terminal ABD of plectin ABD. The C-terminus of plectin is free to make associations with Ifs, thus linking the nucleus to the hemidesmosomes at the cell surface. In an analogous was, nesprin-1/2 and talin, bound to the cytoplasmic tail of b1 integrins, interact with the actin cytoskeleton, which link the nucleus to focal contacts. ER, endoplasmic reticulum; IF, intermediate filament; MT, microtubule; MTOCT, MT-organizing centre. NPC, nuclear pore complex. Integrins connect the extracellular matrix (ECM) with the cell interior and transduce signals by interactions of their cytoplasmic domain with cytoskeletal and signaling molecules. In our group we study the mechanisms by which integrins provide signals for the regulation of important cellular processes such as cell migration and proliferation. In particular we are interested in how integrins contribute to the process of transformation. Role of the integrin a6b4 in tumorgenesis Increased expression of a6b4 has been correlated with a poor prognosis in squamous cell carcinomas. However, little is known about the role of a6b4 in the early stages of tumorigenesis. We have isolated a mouse tumor stem cell line (mtsc) deficient for both p53 and Smad4, and carrying conditional alleles of b4. After subcutaneous injection, these cells produced tumors showing differentiation towards the three-epidermal lineages, indicating that they have retained features of multipotent stem cells and were in an early stage of cell transformation. Inactivation of the b4 gene revealed a suppressive effect of a6b4 on tumor growth. Reconstitution experiments with b4-chimeras indicated that this effect is independent of its adhesion capacity but requires the binding of b4 to the cytoskeletal linker protein plectin. On the other hand, the cooperative effect of a6b4 with oncogenic Ras in stimulating tumor growth, previously observed in human squamous neoplasia also occurred in mtscs. These results show for the first time that a6b4 is endowed with an adhesion-independent tumor-suppressive and that this integrin can either have a tumor-suppressive or -promoting effect, depending on which other lesions are present. Role of avb3 in tumorigenesis Activation of the proto-oncogene c-src involves priming through the disruption of SH2 and SH3 mediated intramolecular interactions followed by activation through transphosphorylation in the kinase domain. In human cancer, a primed state of c-src can be stabilized through binding of overexpressed growth factor receptors to the SH2 domain or through mutations in the SRC gene causing the regulatory C-terminus to be truncated. This frequently leads to high levels of c-src activity in human cancer through an as yet poorly understood mechanism. We have found that activation of primed c-src like wildtype c-src is regulated by avb3. Increased levels of avb3 support Src Y530F -mediated tumor formation in fibroblasts and epithelial cells. avb3 acts upstream of Stat3 and it does not regulate transformation by the viral oncogene v-src, which is constitutively active due to multiple additional mutations compared to Src Y530F. This indicates that avb3 may promote the active conformation of Src Y530F. Indeed, expression of the b3 cytoplasmic tail in the context of a functional integrin is required and sufficient for phosphorylation of Src Y530F in its kinase domain and for Src Y530F -mediated survival, proliferation, and tumor formation. This cooperation between avb3 and c-src may represent a relevant therapeutic target in cancer treatment. Regulation of MacMARCKS expression by the b3 cytoplasmic domain We have previously shown that overexpression of the b3 subunit in b 1 deficient GE 1 1 cells causes the downregulation of MacMARCKS (MRP) and have identified a critical motif in the b3 cytoplasmic tail that is essential for this downregulation. Since MRP downregulation is correlated with a loss of epithelial cell polarization and disruption of cell-cell contacts, we have focused our attention on the regulation of MRP localization within cells. MRP binds to the plasma membrane via insertion of a myristoylation moiety and electrostatic interactions of the basic effector domain (ED) with negative phospholipids. We show that phosphorylation of the ED leads to a re-localization of MRP to vesicles in the perinuclear region. Interestingly, the ED itself is not involved in this targeting event. We are currently investigating the vesicle targeting motif and possible interacting proteins that may be of importance in this process. Additionally, we observed that mutational inhibition of phosphorylation of

17 17 CELL BIOLOGY the serine residues within the ED results in a decreased ability of PMA stimulation to induce loss of polarization of epithelial cells. This suggests that MRP is involved in the maintenance of cell morphology through an, as yet unknown, mechanism. Lastly, we are investigating the mechanism through which b3 exerts its effect on cellular morphology by expressing different deletion mutants of b3. To this end, we have expressed and analyzed the effects of these mutants. The results show that there is a small region in the cytoplasmic tail that is directly responsible for the downregulation of cell-cell contacts in our model cell line. Regulation of cell-cell adhesion during metastasis A key step in tumor metastasizing is the loss of the strong epithelial cell-cell adhesions. This can occur through mutation or silencing of genes encoding adhesion proteins (E-cadherin) or through alteration in the signaling pathways or structural networks that regulate cell-cell adhesion. We are interested in the latter two possibilities and study these using cell lines that exhibit a loss of cell-cell adhesion in response to growth factors that are involved in tumor metastasizing. This process in cell lines is called scattering. We are developing software to automatically measure and describe this process in live cells, which enables us to investigate the influence of manipulations on this process (drugs, RNAi, etc). When we used this technique to investigate the influence of the ECM on the loss of cell-cell adhesion we found that contractility in the actomyosin cytoskeleton contributes to efficient loss of cell-cell adhesion. ECMs inducing strong contractility promote scattering compared to ECMs that do not induce contraction. As an explanation, we find that E-cadherin dependent adhesions are regulated by cytoskeletal tension, which implies that these structures can sense and respond to force applied by the connecting actin bundles. Interestingly, it has been shown recently by independent groups that tumor tension promotes invasion in vivo. Currently, we are investigating the underlying mechanism and aim to identify the factors involved. Analysis of the function of CD151 CD 15 1 is a tetraspanin that forms stable complexes with the laminin-binding integrins a3b 1, a6b 1 and a6b4, thereby strengthening the adhesion of cells to the basement membrane. In humans, mutations in the CD 15 1 gene cause end-stage hereditary nephritis associated with the skin-blistering disease pretibial epidermolysis bullosa a phenotype thought to be due to the ablated interaction with the a3 and a6 subunits. In order to assess the function of CD 15 1 we have generated conditional and total knockout mice for its gene. We are currently isolating keratinocytes from these mice to use them in cell biological experiments regarding cell adhesion, migration, invasion and integrin trafficking. To study the role of CD 15 1 in tumorigenesis we will subject these mice to various carcinogenetic treatments. Nesprin-3, a novel outer nuclear membrane protein, associates with plectin Very few proteins involved in nuclear positioning have been identified. Previous work in other laboratories has identified two related outer nuclear membrane proteins, nesprin- 1 and -2, which are known to make direct connections with the actin cytoskeleton through their N-terminal actin-binding domain (ABD). We have recently isolated a third member of the nesprin family that lacks an ABD and instead binds to plectin, which can associate with the intermediate filament (IF) cytoskeleton. In our studies, we provide evidence that nesprin-3 is co-localized with both plectin and the keratin-6 or keratin- 14 IF system at the nuclear perimeter. Further characterization shows that the region responsible for the high affinity binding to plectin is located in the N-terminus of nesprin-3. In fact, we have identified a minor splice variant of nesprin-3, which lacks this region and is unable to associate with plectin in cells. In addition, nesprin-3 was found to be ubiquitiously expressed in all the cell lines and tissues we tested. We believe that this is an important finding because it for the first time reveals a mechanism through which the nucleus is attached to the IF cytoskeleton. We are currently investigating the exact role for nesprin-3 in nuclear positioning, structure and breakdown during mitosis. Publications Danen EHJ. Integrins: Regulators of tissue function and cancer progression. Curr Pharm Des 2005; 11: Danen EHJ, Van Rheenen J, Franken W, Huveneers S, Sonneveld P, Jalink K, Sonnenberg A. Integrins control motile strategy through a Rho-cofilin pathway. J Cell Biol 2005; 169: Descargues P, Deraison C, Bonnart C, Kreft M, Elias P, Barrandon Y, Zambruno G, Sonnenberg A, Hovnanian A. Spink5 deficient mice mimic Netherton syndrome through desmoglein-1 degradation by epidermal protease hyperactivity. Nature Genet 2005; 37: Gontier Y, Taivainen A, Fontao L, Sonnenberg A, Van der Flier A, Carpen O, Faulkner G, Borradori L. The Z-disc proteins myotilin and FATZ interact with each other and are potentially connected to the sarcolemma via muscle-specfic filamins. J Cell Sci 2005; 118: Hamelers IHL, Olivo C, Mertens AEE, Pegtel DM, Van der Kammen RA, Sonnenberg A, Collard JG. The Rac activator Tiam1 is required for a3b1- integrin-mediated laminin-5 deposition and spreading. J Cell Biol 2005; 171: Litjens SHM, Wilhelmsen K, De Pereda JM, Perrakis A, Sonnenberg A. Modeling and experimental validation of the binary complex of the plectin actin binding domain and the first pair of FNIII repeats of the b4 integrin. J Biol Chem 2005; 280: Raymond K, Kreft M, Janssen H, Calafat J, Sonnenberg A. Keratinocytes display normal proliferation, survival and differentiation in conditional b4 integrin knockout mice. J Cell Sci 2005; 118: Wilhelmsen K, Litjens SHM, Kuikman I, Tshimbalanga N, Janssen H, Van den Bout I, Raymond K, Sonnenberg A. A novel outer nuclear envelope protein, nesprin-3, associates with the cytoskeletal linker protein plectin. J Cell Biol 2005; 171:

18 18 CELL BIOLOGY Group leader John Collard John G Collard PhD Group leader Audrey Gerard PhD Post-doc Michiel Pegtel PhD Post-doc Kristin Strumane PhD Post-doc Saskia Ellenbroek MSc Graduate student Amra Hajdo-Milasinovic MSc Graduate student Sander Mertens MSc Graduate student Tomasz Rygiel MSc Graduate student Marlon Van der Plas Undergraduate student Babet Van der Vaart Undergraduate student Rob A Van der Kammen Technical staff GENETIC CONTROL OF INVASION AND METASTASIS The aim of our research is to identify and characterize genes that play an essential role in the acquisition of an invasive and metastatic phenotype of tumorigenic cells. Insight into the signaling pathways involved in the formation and progression of tumors may lead to the development of novel diagnostic tools or improved therapeutic strategies. Function-based screens for invasion-inducing genes In earlier studies we have identified the invasion-inducing gene Tiam 1 by retroviral insertional mutagenesis in combination with in vitro selection of invasive T-lymphoma cells. Additional functional screens for genes involved in T-lymphoma invasion revealed a number of novel genes. These genes include the a5- subunit of the a5b 1 integrin, the LPA2 receptor and Rap, which have been implicated in integrin-mediated cell-matrix adhesion and/or signaling pathways mediated by Rho GTPases. For these novel screens, retroviral cdna libraries were used in combination with in vitro selection of invasive T-lymphoma cells. Currently, the molecular mechanisms are being studied how these genes are involved in the acquisition of an invasive phenotype. Interestingly both Tiam 1 and Rap are implicated in cell polarization and cell migration of lymphoid cells. Rho family proteins control dynamic cytoskeletal changes The previously identified invasion-inducing Tiam 1 gene encodes a guanine nucleotide exchange factor (GEF) or activator for the Rho-like GTPase Rac. Rho-like GTPases, which include Cdc42, Rac and RhoA, control a wide range of biological processes such as adhesion, polarity, and motility of cells. In particular, they act in signaling pathways that regulate the reorganization of the actin cytoskeleton in response to receptor stimulation. The principal regulators of the activities of Rho proteins are the GEFs and GTPase activating proteins (GAPs). GEFs induce activation of the small GTPases by exchanging GDP for GTP, whereas GAPs inactivate the small GTPases by enhancing the intrinsic rate of hydrolysis of bound GTP to GDP. Also Guanine nucleotide dissociation inhibitors (GDIs) control the activity of Rho proteins. For instance, in the cytoplasma Rac is kept in its inactive GDP-bound form by association with RhoGDI. RhoGDI binds and masks the hydrophobic C-terminal region of Rac, the same region that is responsible for targeting Rac to the plasma membrane. We found that calcium signaling regulates this association between RhoGDI and Rac. Both increased intracellular [Ca 2+ ] and the activation of PKC induces phosphorylation of RhoGDI leading to its dissociation from Rac. As a consequence, dissociated cytosolic Rac translocates to the plasma membrane where it can be activated by membrane associated GEFs such as Tiam 1. Figure I.2: Lack of Tiam1 impairs intestinal tumor formation and growth in APC mutant Min/+ mice. (a) Average number of total intestinal lesions per mouse as found in Tiam1 +/+ //Min/+ and Tiam1 -/- //Min/+ mice. Y-error bars show standard error of the mean. The reduction in lesions in Tiam1 -/- //Min/+ mice is statistically highly significant (p<0.005; student t-test). (b) Percentage of tumors larger than 1mm. The reduction in tumor size in Tiam1 -/- //Min/+ mice is statistically highly significant (p<0.005; ANOVA). The activation of GEFs and downstream signaling Apart from increased or decreased expression, GEFs are basically regulated at four different levels in the cell, i.e. by intra-molecular inhibition, changes in intracellular localization, post-translational modifications and interaction with other proteins. All of these regulatory mechanisms seem to be involved in Tiam 1 regulation. In particular, complex formation with various proteins appears to be an important mechanism to determine signaling towards Rac and signaling downstream of activated Rac. Oncogenic Ras was shown to mediate Rac signaling through the direct interaction of Ras with the RBD domain of Tiam 1. The fact that we found that Tiam 1-deficient mice are resistant to Ras-induced skin tumors underlines the physiological relevance of the connection between Ras, Tiam 1 and Rac. By interacting with various effector or scaffold proteins Tiam 1 may also determine the downstream signaling of the Tiam 1-Rac signaling complex. We found recently that Tiam 1 is indispensable for a3b 1-mediated Rac activation, leading to organized laminin-5 deposition and subsequent cell spreading and cell migration of keratinocytes. In contrast to wild-type keratinocytes, Tiam 1-deficient keratinocytes are unable to adhere to and spread on a glass substrate, because they are unable to deposit their own laminin-5 substrate. Both Tiam 1 and constitutive activated V 12Rac 1 can rescue the adhesion and spreading defect of Tiam 1-deficient keratinocytes on glass, indicating that this defect is the result of impaired Rac

19 19 CELL BIOLOGY signaling. Tiam 1-deficient cells are unable to activate Rac upon adhesion to a premade laminin-5 substrate, whereas other integrin-mediated signaling pathways are intact. Moreover, Tiam 1-deficiency impairs keratinocyte migration in vitro and re-epithelialization of excision wounds in mouse skin. Tiam 1 is thus a key molecule in a3b 1-mediated activation of Rac, which is essential for proper production and secretion of laminin-5, a requirement for spreading and migration of keratinocytes. Tiam1-Rac signaling and cell polarity Metastasis of carcinomas is associated with reduced E-cadherin-mediated cell-cell adhesion. Tiam 1 localizes to adherens junctions and ectopic expression of Tiam 1 in epithelial cells inhibits HGF-induced cell scattering by increasing E-cadherin-mediated adhesion. Further support for a role of Tiam 1 in the formation and maintenance of E-cadherin-mediated cell-cell adhesions comes from gene knockdown studies using short interference (si) RNA. Introduction of Tiam 1-specific sirna in epithelial MDCK cells leads to loss of cell-cell adhesions and epithelial-mesenchymal transition. Moreover, keratinocytes derived from Tiam 1-deficient mice are impaired in the formation of junctions. Similar to wild-type keratinocytes, Tiam 1-deficient cells establish primordial E-cadherin-based adhesions but the subsequent polarization step and maturation of the tight junctions (TJs) are severely impaired. Both Tiam 1 and V 12Rac 1 can rescue the TJ maturation defect in Tiam 1-deficient cells, indicating that this defect is the result of impaired Tiam 1/Rac signaling. The expression of several TJ molecules, such as Occludin and ZO- 1 is downregulated in Tiam 1-deficient keratinocytes. However, re-introduction of these proteins in Tiam 1-deficient cells is not sufficient to restore TJ maturation, suggesting that the downregulation of these components is not causal in the lack of cell polarization. Interestingly, we found that Tiam 1 interacts with at least two components of the conserved polarity complex, PAR3 and the atypical PKC, PKCz, which are activated by initial E-cadherin intercellular contacts and are known to play a pivotal in the maturation of the TJ. Rac is activated upon the formation of primordial adhesions in wild-type but not in Tiam 1-deficient cells, suggesting that Tiam 1 is required for triggering biogenesis of the TJ upon the formation of primordial adhesion through local activation of Rac and apkcz. Our data indicate that Tiam 1-mediated activation of Rac in primordial adhesions controls TJ biogenesis and polarity in epithelial cells by association with and activation of the Par3/Par6/ apkc polarity complex. Preliminary data indicate that Tiam 1 also plays a role in cell polarization associated with directional migration as observed in keratinocytes, astrocytes, primary neuronal cells, lymphocytes and fibroblasts. Tumorigenicity in Tiam1 mutant mice We have generated Tiam 1-deficient (Tiam1 -/- ) mice to investigate the consequences of changes in Tiam 1/Rac signaling in tumorigenesis. Although Tiam 1 is expressed during embryonic development, Tiam1 -/- mice develop, grow, and reproduce normally. In mouse skin, Tiam 1 is present in basal and suprabasal keratinocytes of the interfollicular epidermis and in hair follicles. Tiam1 -/- mice display a dramatic resistance to DMBA/TPA induced skin tumor formation. Although the number and growth of tumors is strongly reduced in Tiam1 -/- mice, Tiam 1-deficiency increases the frequency of malignant conversion of the Ras-induced skin tumors that do develop. Currently we are studying the role of Tiam 1 in tumorigenesis induced by different oncogenic signaling pathways i.e. Ras, Wnt, Myc, Neu and PI3-kinase. We found that Tiam 1 is a Wnt-responsive gene and that its expression is regulated b-catenin/tcf signaling. As a consequence, Tiam 1 protein levels are increased in intestinal tumors produced in APC mutant mice as well as in human colon tumors. Interestingly, the initiation and growth of intestinal tumors is reduced in Tiam 1-deficient APC mutant mice, suggesting that Tiam 1 plays a role in Wnt-b-catenin-TCF-induced intestinal tumor formation. In contrast, we do not find differences in T-lymphomagenesis and progression induced by constitutive activation of the PI3-kinase pathway in a Tiam 1-deficient background. For the latter studies, we used conditional PTEN KO mice that have increased PI3-kinase activity in their T-cells by the tissue-specific knockout of the lipid phosphatase PTEN, which normally counteracts PI3-kinase activity. Together, these studies indicate that Tiam 1 contributes to the formation and progression of tumors induced by various -but not all-oncogenic signaling pathways. Publications Hajdo-Milašinović A, Mertens AE, Hamelers IHL, Collard JG. Rho GTPases in cell motility and tumorigenesis. In: Cell Motility in Cancer Invasion and Metastasis; Series Cancer Metastasis - Biology and Treatment. Kluwer, Strumane K, Rygiel TP and Collard JG. The Rac activator Tiam1 and Ras-induced oncogenesis. Methods Enzymol 2006; (in press). Malliri A, Rygiel T, Van der Kammen RA, Song J-Y, Engers R, Hurlstone AFL, Clevers H, Collard JG. The Rac activator Tiam1 is a Wnt-responsive gene implicated in colorectal tumourigenesis. J Biol Chem 2006; 281: Hamelers IHL, Olivo C, Mertens AEE, Pegtel DM, Van der Kammen RA, Sonnenberg A, Collard JG. The Rac activator Tiam1 is required for a3b1- integrin-mediated laminin 5 deposition, cell spreading and cell migration. J Cell Biol 2005; 171: Mertens AE, Rygiel T, Olivo C, Van der Kammen RA, Collard JG. The Rac activator Tiam1 controls tight junction biogenesis in keratinocytes through binding to and activation of the Par polarity complex. J Cell Biol 2005; 170: Brecht M, Steenvoorden ACM, Collard JG, Luf S, Erz D, Bartram CR, Janssen JWG. Activation of GEF-h1, a guanine nucleotide exchange factor for RhoA, by DNA transfection. Int J Cancer 2005; 113: Minard ME, Herynk MH, Collard JG, Gallick GE. The guanine nucleotide exchange factor Tiam1 increases colon carcinoma growth at metastatic sites in an orthotopic nude mouse model. Oncogene 2005; 24:

20 20 CELL BIOLOGY Group leader Ed Roos Ed Roos PhD Group leader Belén Alvarez Palomo PhD Post-doc Agnieszka Masztalerz PhD Post-doc Peter Stroeken PhD Post-doc Joost Meijer MSc Graduate student Yvonne Wijnands Technical staff Rosalie De Bruijn Technical staff Marcel Van Gijlswijk Technical staff Aniek Van der Woude Undergraduate student Gerben Bijl Undergraduate student Figure I.3: Growth rates of liver metastases. For clarity, only one mouse is shown for each cell line. CT26 colon carcinoma cells in which CXCR5 is trapped in the ER (BCA-KDEL) grow initially at a similar rate as control CT26 cells, as indicated by projected lines. CXCR5-CT26 transfectants, that express CXCR5 from day one, grow faster, presumably triggered by the CXCR5 ligand BCA-1 (CXCL13) present in the liver. After 12 days, control cells start growing as fast, probably because they have then upregulated CXCR5, since this acceleration does not occur in the BCA-KDEL cells that have no CXCR5 on the surface. Rather, the growth rate of the latter cells decreased strongly after day 14, and this low rate was maintained for an extended period. MECHANISMS IN METASTASIS Metastasis, the spread of tumor cells to distant sites in the body, is the main cause of cancer lethality. We study the mechanisms of metastasis, with emphasis on lymphomas and carcinomas and on the invasion of tissues by blood-borne tumor cells. Our research on chemokine receptors has revealed a role in outgrowth of metastases, rather than in invasion as was generally assumed. These receptors are therefore potential targets for chemotherapy. Role of the chemokine receptor CXCR4 in carcinoma metastasis We showed previously that CXCR4, the receptor for the chemokine SDF- 1 (CXCL 12) is required for T-lymphoma metastasis, using a method that traps CXCR4 inside the cell. Invasion in vitro was also blocked, indicating that CXCR4 is involved in invasion from the blood, triggered by SDF- 1 in the tissues. Similarly, we showed involvement of CXCR4 in metastasis of a colon carcinoma but, surprisingly, not in invasion. CXCR4 was not expressed in vitro, but upregulated in vivo, after establishment of micrometastases. Using luciferase-transfected cells to measure tumor growth by bioluminescence, we showed that CXCR4 is required for continuation of growth when the micrometastases are cells in size. CXCR4 is not necessary for growth of s.c. tumors from injected tumor cell suspensions. When dispersed as single cells in a s.c. Matrigel plug, however, they stopped proliferation when grown to colonies of cells. Upregulation of CXCR4 could not be induced in monolayer culture by any factors tested so far. Strikingly, however, CXCR4 was induced in these Matrigel tumors. This indicates that CXCR4 upregulation is a response to crowding, perhaps to hypoxia. How CXCR4 is activated is not clear. We could not block growth with inhibitors of the SDF- 1-CXCR4 interaction, released from osmotic pumps. We are now attempting to use a dominant-negative SDF- 1 mutant, transfected into the cells. Another possibility, suggested by recent literature, is that CXCR4 is trans-activated by the insulin-like growth factor receptor or perhaps other growth factor receptors. Role of another chemokine receptor, CXCR5, in carcinoma metastasis, specifically in the liver We also observed a strong induction of a second chemokine receptor, CXCR5, both in tumors and in metastases, and also in the Matrigel plugs. This is surprising since CXCR5 was known to be expressed only by blood cells, in particular B- and some T-lymphocytes. Similarly as CXCR4, CXCR5 was not expressed in monolayers in vitro. To study its function, we transfected CXCR5 into the colon carcinoma cells and found that the ligand, BCA- 1 (CXCL 13) enhanced proliferation, even in the presence of 10% fetal calf serum. BCA- 1 is present in the liver, a major target organ for colon carcinoma metastasis. We also transfected a construct (BCA-KDEL) that traps CXCR5 in the ER, and injected cells into the spleen to generate liver metastases. We expected that the presence of CXCR5 from day one would increase initial growth, that growth of control would be slower but would accelerate later, after induction of CXCR5 expression, and that this increased growth would not occur in BCA-KDEL transfectants. As shown in Fig. I.4, this was exactly what we found. Strikingly, however, the growth rate of liver metastases of CXCR5- suppressed BCA-KDEL cells, rather than remaining constant, strongly decreased after 14 days, and this slow rate was maintained for an extended period. The reason for this is as yet obscure. The effects of CXCR5 are specific for the liver, since the growth rates of s.c. tumors and lung metastases generated from control cells and CXCR5 and BCA-KDEL transfectants were similar. Meanwhile, we have observed CXCR5 expression on colon, bladder, pancreatic and mammary carcinoma cell lines of both mouse and man. In all, CXCR5 was expressed in vivo but in some cell lines also in vitro. For the CXCR5-expressing ESb lymphoma, we also found that BCA- 1 promotes growth, particularly when cultured in Matrigel in vitro. This suggests that also for lymphomas, chemokines have a role in outgrowth of metastases, in addition to invasion. In this respect, it is striking that the ESb lymphoma only metastasizes to liver and spleen, in which BCA- 1 is expressed. A CXCR5 trapping construct has been transfected to test this notion.