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CNIO Cancer Conference

Small GTPases in human carciogenesis

Madrid, June 16-18, 2003SPANISH NATIONAL CANCER CENTRE

Organisers:

Juan C. Lacal, Channing Der, Shuh Narumiya

CNIO Auditorium Centro Nacional de Investigaciones OncológicasMelchor Fernández Almagro, 3E-28029 Madridwww.cnio.es

CNIO CCC Staff: Dept. External Relations and Communications, [email protected] Ferreiro, Xiomara Guédez, Alma Izquierdo and Amanda Wren

Contents

Foreword

Programme Overview

Detailed Programme

Abstracts-sessions

Session 1: Small GTPases in cancer 15

Session II: Intracellular Signalling 20

Session III: Cell cycle and proliferation by Rho GTPases 26

Session IV: Adhesion, Migration and Invasion by small GTPases 30

Session V: Tumour phenotypes and therapeutic strategies 35

Abstracts-posters

In alphabetical order of presenting author

Invited Speakers’ portfolio

A compilation of short biographies of each speaker organised in accordance with the order

of the scientific programme.

List of Speakers and Participants

Acknowledgements

Notes

Forthcoming CNIO activities

Contents

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Foreword

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Dear Colleague,

We would like to welcome you to the CNIO Cancer Conference (CCC) on Small GTPases in Human Carcinogenesis.

This conference is following the trend being established through the CCC´s: to bring together the very top scientists in agiven field related to cancer and to provide the best possible forum for discussion. We are confident that collectively, theCNIO Cancer Conferences will be of great interest to the scientific community and attract international attention to theCNIO. At these conferences, we emphasise a workshop ambience, thus CNIO scientists will be able to follow the confe-rence from our seminar rooms in order to keep the number of participants to the necessary minimum.

This CCC will integrate recent findings in small GTPases and their role and mechanisms in human carcinogenesis. In relationto this, two main branches of the large family of small GTPases have been linked to cancer onset in humans. The Ras branch,first discovered as oncogenes, has for the last 20 years provided a wealth of information regarding their relevance in tumour

formation. The second one, Rho GTPases, has recently gained its place as a legitimate oncogenic driver. Although rho genesare not mutated in human tumours as compared to the high incidence of mutations in the ras genes, their overexpressionor deregulated activation is frequently observed in many types of cancer. Additionally the activity of several transcrition fac-tors has been linked to Rho-mediated transformation. Special focus will be placed upon the emerging role of the family ofRho GTPases and the underlying molecular connections mediated by their deregulation and activation of downstream effec-tors to promote carcinogenesis as well as in drugs that affect Rho signalling that have been developed with promising in vivo

antitumoural activity.

We very much hope that you enjoy this exciting conference.

With our personal thanks,

Juan C. Lacal Channing J. Der Shuh Narumiya

Instituto de Investigaciones Biomédicas, CSIC University of North Carolina at Chapel Hill University of KyotoMadrid, Spain Chapel Hill, USA Kyoto, Japan

As the newest member of the European Cancer Community, the CNIO is very much open to establishing strong collaborations with youand your respective organisations. We would therefore be delighted to inform you about our Scientific Strategic Plan for the next fewyears (the CNIO will not be fully staffed until 2005), and to show you our facilities. Should you wish to meet with any member of our

Faculty, please let us know and we will be delighted to introduce you.

Programme overview

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Lunches: Lunch will be provided, courtesy of the CNIO, in the main dining room of the building.

* CNIO Building Tour: following directly on from the close of Monday´s sessions, a short Tour will be conducted by CNIOstaff for participants interested in CNIO activities. The meeting point will be at the Registration Desk.

Small GTPases in Human CarcinogenesisAuditorium of the Spanish National Cancer Centre, CNIO (Melchor Fernández Almagro, 3, E-28029 Madrid)June 16-18, 2003

Monday, June, 16 Tuesday, June, 17 Wednesday, June, 18

Welcome Address / 9:30 - 9:45h III. Cell cycle and proliferation V. Tumour phenotypes and

Mariano Barbacid. Madrid (Spain) by Rho GTPases / 9:30 - 12:30h therapeutic strategies / 9:30 - 12:50h

I. Small GTPases in cancer / 9:45 - 13:10h Chair: Channing J. Der Chair: Dafna Bar-Sagi

Chair: Chris Marshall Shuh Narumiya. Kyoto (Japan) Harry Mellor. Bristol (UK)

Dafna Bar-Sagi. New York (USA) Richard Assoian. Philadelphia (USA) Kenneth L. van Golen. Ann Arbor (USA)

Linda Van Aelst. Cold Spring Harbor (USA) Coffee Break / Poster viewing (10:50-11:30h) Coffee Break (10:50-11:20h)

Carmen Guerra. Madrid (Spain) Christoph Moroni. Basel (Switzerland) Adrienne Cox. Chapel Hill (USA)

Coffee Break / Poster viewing (11:20-11:50h) John G. Collard. Amsterdam (The Netherlands) Gilles Favre. Toulouse (France)

Xosé Bustelo. Salamanca (Spain) Lunch Break (12:30-14:30h) Poster prize announcement and

Larry Feig. Boston (USA) IV. Adhesion, Migration and Invasion concluding remarks / 12:40 - 12:50

Lunch Break (13:10-14:30h) by small GTPases / 14:30 - 17:50h

II Intracellular Signalling / 14:30 - 17:45h Chair: Shuh Narumiya

Chair: Johanes Bos Johannes Bos. Utrecht (The Netherlands)

Channing Der. , Chapel Hill (USA) Marc Symons. Manhasset (USA)

Chris Marshall. London (UK) Rosa María Guasch. Valencia (Spain)

Juan Carlos Lacal. Madrid (Spain) Coffee Break / Poster viewing (16:05-16:30h)

Coffee Break / Poster viewing (16:20-16:50h) Miguel del Pozo. La Jolla (USA)

Vijay Yajnik. Boston (USA) Jeffrey Settleman. Charlestown (USA)

Silvio Gutkind. Bethesda (USA)

*

Detailed Programme

Monday, June 16, 2003Welcome address9:30-9:45 Mariano Barbacid / Director, Centro Nacional de Investigaciones Oncológicas

Madrid, Spain

Session I: Small GTPases in cancerChair: Chris Marshall

9:45-10:25 Dafna Bar-Sagi / State University of New York at Stony Brook, USARas Signaling and Growth Control

10:25-11:05 Linda Van Aelst / Cold Spring Harbor Laboratory, Cold Spring Harbor, USAThe role of Ras and Rap signaling in morphogenesis and cell growth control

11:05-11:20 Carmen Guerra / Centro Nacional de Investigaciones Oncológicas, Madrid, SpainShort talk Wide expression of an endogenous K-ras oncogene induces lung adenomas,

but has no detectable consequences in other tissues

11:20-11:50 Coffee-Break/Poster viewing

11:50-12:30 Xosé Bustelo / Centro de Investigación del Cáncer-CSIC-USAL, Salamanca, SpainVav mediates Ras stimulation by direct activation of the GDP/GTP exchange factor Ras GRP1

12:30-13:10 Larry Feig / Tufts University School of Medicine, Boston, USARegulation and Function of Ral GTPases

13:10-14:30 Lunch-Break

Session II: Intracellular Signalling Chair: Johanes Bos

14:30-15:10 Channing Der / University of North Carolina at Chapel Hill, Chapel Hill, USAAberrant Rho GTPase regulation and signalling in oncogenesis

15:10-15:40 Chris Marshall / Cancer Research UK Centre for Cell and Mol. Biology, London, UKInteractions Between Small GTPase Signalling Pathways in Cell Transformation

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2003 CNIO CCC´s. Small GTPases in human carciogenesis

Detailed Programme

9

15:40-16:20 Juan Carlos Lacal / Instituto de Investigaciones Biomédicas -CSIC-, Madrid, SpainRho signals involved in transcription regulation: mechanisms and biological effects


16:50-17:05 Vijay Yajnik / Massachusetts General Hospital, Harvard Medical School, Boston, USAShort talk DOCK4, a novel member of the CDM family of proteins, is an activator of Rap1 small

GTPase signaling pathway

17:05-17:45 Silvio Gutkind / National Institutes of Health, Bethesda, USARho GTPases link heterotrimeric G proteins to the nucleus and beyond

Tuesday, June 17, 2003

Session III: Cell cycle and proliferation by Rho GTPasesChair: Channing J. Der

9:30-10:10 Shuh Narumiya / Kyoto University Faculty of Medicine, Kyoto, JapanCdc42-mDia3 Pathway in Mitosis; Implications in Chromosome Instability

10:10-10:50 Richard Assoian / University of Pennsylvania School of Medicine, Philadelphia, USALinks between Rho GTPases and G1 phase control


11:30-11:45 Christoph Moroni / University of Basel, Basel,SwitzerlandShort talk Mechanism of ocogenic ras-induced mRNA stabilization involving the PI3-kinase -PKB pathway

11:45-12:25 John G. Collard / The Netherlands Cancer Institute, Amsterdam, The NetherlandsRho-family proteins and tumorigenesis

12:30-14:30 Lunch Break

Session IV: Adhesion, Migration and Invasion by small GTPases Chair: Shuh Narumiya

14:30-15:10 Johannes Bos / University Medical Centre Utrecht, Utrecht, The NetherlandsThe small GTPase Rap1, the cAMP target Epac and integrin-mediated cell adhesion

15:10-15:50 Marc Symons / North Shore-Long Island Jewish Research Institute, Manhasset, USADissection of Rac-mediated functions in human glioblastoma cell lines using RNA interference

15:50-16:05 Rosa María Guasch / Instituto de Investigaciones Citológicas (Fundación Valenciana de Investigaciones Biomédicas), Valencia, Spain

Short talk Effect of the small GTPase RhoE in U-87 glioblastoma cellular proliferation.


16:30-17:10 Miguel del Pozo / The Scripps Research Institute, La Jolla, USACaveolin and lipid rafts in Integrin control of Rac targeting and signaling

17:10-17:50 Jeffrey Settleman / Harvard Medical School, Charlestown, USARegulated expression of a cell surface protease by the Rho-LIM-Kinase pathway is requiredfor tissue morphogenesis during Drosophila development

Wednesday, June 18, 2003

Session V: Tumor phenotypes and therapeutic strategies Chair: Dafna Bar-Sagi

9:30-10:10 Harry Mellor / University of Bristol, School of Medical Sciences, Bristol, UKFarnesyltransferase inhibitors, RhoB and receptor traffic

10:10-10:50 Kenneth L.. van Golen / University of Michigan Comprehensive Cancer Center, Ann Arbor, USAModulation of breast cancer phenotypes by RhoC function

10:50-11:20 Coffee-Break

11:20-12:00 Adrienne Cox, / North Carolina Clinical Cancer Center, Chapel Hill, USARho family proteins as targets of prenyltransferases for anticancer treatment

12:00-12:40 Gilles Favre / Institut Claudius Regaud- INSERM, Toulouse, FranceRhoB a suppressor gene of cell transformation?*

12:40-12:50 Poster prize announcement and concluding remarks

* Abstract and scientific biography unavailable

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Posters (in alphabetical order of presenting author)Note: Only those that have not been included in the oral sessions are listed

ROCK and NFkappaB-dependent activation of COX2 by Rho GTPases: effects over tumor growth and the-rapeutic consequencesSalvador Aznar-Benitah, Instituto de Investigaciones Biomedicas-CSIC, Madrid, Spain

Differential activation of H-Ras in endomembranes by the guanine nucleotide exchange factors RasGRF 1 and 2Piero Crespo, Instituto de Investigaciones Biomedicas, Unidad de Biomedicina de la Universidad de Cantabria, Santander, Spain

STAT5a activation mediates the epithelial to mesenchymal transition induced by oncogenic RhoACarolina Espina, Instituto de Investigaciones Biomedicas-CSIC, Madrid, Spain

Regulation of Stat3 transcription factors by RhoGTPases. Implication in RhoA-Mediated neoplastic transformationPilar Fernández-Valerón, Centro de Ciencias de la Salud, Las Palmas de Gran Canaria, Spain

Light Chain 2 (LC2) : a novel partner for RhoBIsabelle Lajoie-Mazenc, Institut Claudius Regaud, Toulouse, France

RhoB tumor suppressive activity in human lung cancerJulien Mazières, Institut Claudius Regaud, Toulouse, France

Characterization of a VNTR sequence in the human rhoB promoter that negatively regulatestranscriptional activityDaniel Tovar, Institut Claudius Regaud, Toulouse, France

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Detailed Programme

Abstracts-Sessions

Ras Signaling and Growth Control

Dafna Bar-SagiDepartment of Molecular Genetics and Microbiology, Stony Brook University, New York, USA

It is well established that the effector pathways controlled by Ras are key determinants in the regulation of both normal cellproliferation and oncogenic transformation. In the past several years increasing number studies have reported that the bio-logical outcome of Ras signaling can be dramatically altered as a result of changes in the intensity of Ras-dependent signals.We have been interested in identifying the molecular sensors for the strength of Ras signaling as well as the compensatorymechanisms used by cells in response to different levels of Ras signaling particularly as they pertain to the process of tumo-

rigenicity. To this end, we have established experimental systems that enable us to modulate the levels of RasV12 expres-sion. We have utilized these systems to assess differences in gene expression profiles as a function of intensity of Ras signa-ling. This analysis has revealed a tight correlation between the levels of expression of RasV12 and the chemokine IL-8.Investigation of the signaling pathways by which Ras controls the expression of IL-8 has demonstrated differential utilizationof Ras effector pathways with varying levels of Ras expression. IL-8 has been shown to act as a proinflammatory and pro-angiogenic factor and as such is likely to contribute to tumor growth and metastasis. Therefore, we have tested the func-

tional significance of IL-8 expression for the tumorigenicity of RasV12 expressing cells. The results of this analysis will be dis-cussed.

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Session I: Small GTPases in cancer..

Rap1 and AF-6/Canoe function in epithelial morphogenesis

Linda Van AelstCold Spring Harbor Laboratory, Cold Spring Harbor, USA

Several studies suggest a relationship between loss of epithelial cell polarity and loss of growth control. Studies in my labo-ratory using mammalian epithelial cells have suggested a role for Rap1, a member of the Ras family, and the junctional pro-tein AF-6, which associate with GTP-bound Rap, in the regulation of epithelial cell shape changes and cell adhesion. Due tothe finding that AF-6 is largely refractory to both loss- and gain-of-function analyses, we decided to explore this hypothesisin the genetically more tractable Drosophila system.

In Drosophila, the process called dorsal closure (DC) relies on elongation and migration of epithelial cells and has proved tobe an excellent model system for the study of the molecular basis underlying epithelial cell shape changes. In collaborationwith U. Gaul (Rockefeller University), we recently provided biochemical and genetic evidence that Canoe (Drosophila ortho-logues of AF-6) acts as a Drosophila Rap1 effector in the process of dorsal closure. To date, no other protein has been repor-ted to act as a mediator or direct effector of the small GTPase Rap1 in a physiological context. Furthermore, we obtained

evidence that Canoe participates in DC through two cellular pathways: one is controlled by DRap1 and is independent ofthe JNK cascade, while the second is not activated by DRap1 and feeds into JNK signaling. We are currently setting out toidentify additional Canoe-interacting proteins to obtain further insights into Canoe’s mode of action in dorsal closure. Tocomplement these studies, we are planning to assess a role for the mammalian counterparts of these Drosophila proteins inmammary epithelial polarity and invasive behavior. These studies will contribute to a better understanding of the fundamentalmechanisms that drive cell shape changes and migration.

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Session I: Small GTPases in cancer.

Wide expression of an endogenous K-ras oncogene induces lung adenomas, buthas no detectable consequences in other tissues

Guerra C., Dhawahir A., Mijimolle N., Campuzano V and Barbacid M.

Molecular Oncology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain

We have generated a K-ras knock-in strain of mice that carries the following engineered modifications at the endogenousK-ras allele: (i) a floxed transcriptional STOP cassette in the first intron, (ii) an oncogenic mutation in codon 12 (Val12) and(iii) a bicistronic IRES-?-geo cassette inserted between the stop codon and the polyA signal. These modifications render theoncogenic allele silent except in those cells that express Cre recombinase that removes the STOP cassette. Moreover,

expression of the K-rasV12 allele results in the concomitant expression of the LacZ marker. Mating K-ras(+/V12) mice withCMV-Cre transgenic mice results in frequent embryonic lethality. However, a significant percentage of mice are bornwithout obvious defects due to limited excision of the STOP cassette during embryogenesis. These mice, have a low butsignificant percentage of K-rasV12 expressing cells in most tissues as determined by X-Gal staining. These mice develop lungadenomas after a latency of 8 months. Time that is significantly reduced in the presence of an oncogenic Cdk4 R24C pro-tein. Surprisingly, most cells expressing either K-Ras V12 alone or in combination with Cdk4 R24C do not show abnor-

mal proliferative characteristics, even mild dysplasia. These observations suggest that most cells are resistant to oncoge-nesis even when they carry more than one endogenous oncogene (vs. highly expressed transgenes). We have also deve-loped a knock-in strain that expresses ubiquitously an inducible CreERT2 recombinase (RERTn mice). Systemic treatmentof K-Ras (+/V12);RERTn(+/ERT) mice with 4-OHT results in postnatal activation of the endogenous K-rasV12 oncogene in multi-ple cell types throughout the body. In spite of widespread expression of the K-rasV12 oncogene, these mice only deve-lop lung adenomas. This new knock-in mouse model will help us to dissect the molecular events necessary for neoplastic

development in physiological settings that closely recapitulate the human disease.

Vav mediates Ras stimulation by direct activation of the GDP/GTP exchangefactor Ras GRP1

María José Caloca, Xosé R. Bustelo

Centro de Investigación del Cáncer-USAL-CSIC, Salamanca, Spain

Here we describe a new signaling cross talk between the Vav/Rac1 and Ras pathways that is established through the stimu-lation of members of the RasGRP family, a group of exchange factors for Ras subfamily GTPases. The activation of RasGRPproteins by Vav and Rac1 requires two signaling steps. One step involves the activation of phospholipase C-g and the subse-quent generation of diacylglycerol. The second step is mediated by the polymerization of actin, a biological response induced

by Vav and Rac1 that facilitates the translocation of RasGRPs to juxtamembrane areas. This cross talk is crucial for Ras acti-vation in lymphoid cells, since this GTPase cannot become activated in the absence of Vav proteins. This signaling defect canbe rescued by tyrosine-phosphorylated Vav, constitutively active Rac1 or, alternatively, by mimicking the activation of phos-pholipase C-g via the stimulation of cells with phorbol esters. Conversely, Ras activation can be blocked in lymphocytes andectopic systems using inhibitors affecting either the catalytic activity of phospholipase C-g or F-actin polymerization. Theseresults indicate that a relay mechanism exists in lymphoid and other cells helping in the generation of robust signaling res-

ponses by the Rac/Rho and Ras pathways upon membrane receptor engagement.

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Session I: Small GTPases in cancer.

Regulation and Function of Ral GTPases

L. FeigTufts University School of Medicine, Boston, USA

There is a growing appreciation that the Ral GTPases may contribute to human oncogenesis. The first evidence came fromstudies showing that activation of Ral-GEFs by Ras initiates one of a set of effector signaling pathways emanating from Ras.Recent data suggest that activated versions of Ral-GEFs can substantially contribute to the transformation of normal cells tothe fully oncogenic state. Our studies on Ral proteins have involved both investigations of how Ral-GEFs are regulated andhow activated Ral GTPases influence cellular function. In this presentation, I will review recent studies that have identified

regulatory events that either negatively or positively regulate the Ral-GEF, Ral-GDS and describe simple mutations that canactivate its GEF activity. I will also review recent data on the mechanism by which Ral proteins stimulate exocytosis in cells.I will also discuss how each of these events may relate to the oncogenic potential of the Ral signaling cascade.

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Aberrant Rho GTPase regulation and signalling in oncogenesis

Channing J. Der1, John M. Lambert1, Michelle A. Booden1, Todd R. Palmby1, Patricia A. Solski1, Rhonda Saunders1, Antione E.Karnoub1, Krister Wennerberg1, Anurag Singh1, Marie-Annick Forget2, Shawn M. Ellerbroek1, William T. Arthur1, John Sondek1,

JoAnn Trejo1, Adrienne D. Cox1, Keith Burridge1, Jeffrey Settleman3 and Steen Hansen2

1University of North Carolina at Chapel Hill; 2Boston Biomedical Research Institute; 3MGH Cancer Center and Harvard Medical School,USA

Evidence for the involvement of Rho GTPases in human oncogenesis is now considerable. However, unlike the mutationalactivation of Ras in human cancers, Rho GTPase function is perturbed by mechanisms that involve altered regulation and

expression. In particular, Rho GTPase function has been shown to be required for the transforming actions of Ras and otheroncogene proteins. A key mechanism that links Rho GTPases with oncogene function involves Dbl family guanine nucleoti-de exchange factors (e.g., Tiam1, LARG). For example, we recently determined that Ras activation of Rac involved Tiam1.We have also found that the invasion-inducing activity of the PAR1 G protein-coupled receptor requires RhoA activation viaLARG. We have also characterized the mechanisms by which other Dbl family proteins (Vav2, Ect2, Tim, Asef) are regulatedand the signalling pathways they activate to promote transformation. Finally, unlike other Rho GTPases, Rnd3/RhoE is cons-

titutively activated and disrupts, rather than promotes, actin organization. We determined that Rnd3 expression is upregu-lated in Ras-transformed human epithelial cells, that it is a target for farnesyltransferase inhibitors, and that it utilizes a RhoGTPase activating protein (p190RhoGAP) as an effortor to cause the inhibition of RhoA function.

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Interactions Between Small GTPase Signalling Pathways in Cell Transformation

Chris Marshall, Erik Sahai, Hugh Paterson, Emmanuel Vial, Paul Frankel, Ana González-GarcíaOncogene Team, Cancer Research UK Centre for Cell & Molecular Biology, The Institute of Cancer Research Chester Beatty

Laboratories, London, UK

Small GTPases of the Ras, Rho and Ral families play important roles in tumour biology. Genetic alterations to small GTPasesunderscore their important role. Ras is mutated in some tumours while RhoA and RhoC are over-expressed particularly inmore aggressive tumours. As well as their individual roles it is emerging that there are significant interactions between smallGTPase signalling pathways. For example Rho signalling is required to suppress CDK inhibitory levels of p21Waf1 induced

by Ras signalling.

Interesting examples of interactions between small GTPase signalling pathways are emerging through studies on invasion/cellmotility. We have delineated two modes of cell motility one dependent on Rho signalling through the ROCK family of Rhodependent kinases the other requires ROCK signalling to be down-regulated to permit Rac dependent lamelliopodiumextension. In tumour cells with high levels of Rho-GTP, down-regulation of ROCK expression can be achieved via Ras depen-

dent activation of ERK signalling. In other tumour cells which use Rac dependent lamellipodium extension, activation of Rhoto the GTP bound state is suppressed by ERK-MAP kinase activation. This mechanism of suppressing Rho activation is a con-sequence of sustained ERK signalling inducing the transcription factor Fra-1 which then leads to inactivation of Beta-1 inte-grin signalling which would normally lead to activation of Rho. This mechanism may account for the long-standing observa-tion that Ras signalling through the ERK-MAP kinase pathway leads to the inactivation of integrin signalling.

Several lines of evidence indicate that the Ral GTPases RalA and RalB may play important roles in the malignant phenotype.In order to elucidate the roles of Ral proteins we have searched for new binding partners and generated knock-out mice forRal-GDS a Ras dependent activator of Ral. We have identified ZONAB as a Ral binding partner that only interacts with Ralin the active GTP bound state. Ral is co-localised with ZONAB at tight junctions, further when Ral-GTP is artificially eleva-ted in cells the effects of ZONAB on transcription are suppressed. Thus activation of Ral through oncogenic Ras may per-turb ZONAB function. Mice in which the Ral-GDS locus is inactivated are viable and fertile. However in skin carcinogene-

sis protocols tumour induction is reduced in the knock-out mice. These experiments provide strong genetic evidence thatRal-GDS is an important effector of Ras oncogenesis in an epithelial tissue.

Session II: Intracellular Signalling..

Rho signals involved in the regulation of transcription: mechanisms and biologicaleffects

S. Aznar, P. F. Valerón, C. Espina and J.C. LacalInstituto de Investigaciones Biomédicas-CSIC, Madrid, Spain

Rho GTPases are oncogenes with the ability to modulate cellular behaviour depending on the cell system and growth con-ditions. Thus, expression of wild type or mutated Rho proteins in model cellular systems induce oncogenic transformation

or apoptosis. In keeping with this, Rho GTPases are overexpressed in a variety of human tumors contributing to both tumorproliferation and metastasis, while Rho-transformed cells induce tumors in mice with a very high apoptotic index. The involvement of Rho GTPases in signal transduction pathways leading to transcription activation is one of the major rolesof this family of GTPases. Thus, the identification of transcription factors regulated by Rho GTPases as well as the unders-tanding of the mechanisms of their activation and its biological outcome is of great interest for a full description of Rho func-tions in vivo. We have investigated further the effect of Rho signaling on several transcription factors (NF-kB and members

of the Stat family) and searched for their biological implications. Our more recent findings are:- Stats (signal transducers and activators of transcription) are latent cytoplasmic transcription factors that upon a specific sti-mulus migrate to the nucleus where they exert their transcriptional activity. A novel signaling pathway has been identifiedwhereby RhoA can efficiently modulate Stat3 transcriptional activity by inducing its simultaneous tyrosine and serine phos-phorylation. Tyrosine phosphorylation is exerted via a member of the Src family of kinases (SrcFK) and JAK2, while the JNKpathway mediates serine phosphorylation. Furthermore, cooperation of both tyrosine as well as serine phosphorylation is

necessary for full activation of Stat3. Induction of Stat3 activity depends on the effector domain of RhoA, and correlates withinduction of both Src kinase-related and JNK activities. Activation of Stat3 has biological implications. Coexpression of anoncogenic version of RhoA along with the wild type, non-transforming Stat3 gene, significantly enhances its oncogenic acti-vity on human HEK cells, suggesting that Stat3 is an essential component of RhoA mediated transformation. In keeping withthis, dominant negative Stat3 mutants, or inhibition of its tyrosine or serine phosphorylation, completely abrogate RhoAoncogenic potential. Taken together, these results indicate that Stat3 is an important player in RhoA-mediated oncogenic

transformation, which requires simultaneous phosphorylation at both tyrosine and serine residues by specific signaling eventstriggered by RhoA effectors

Rho GTPases also regulate other member of the Stat family of transcription factors. RhoA triggers tyrosine phos-phorylation (Y696) of Stat5a via a JAK2-dependent mechanism, and promotes its DNA-binding activity. Contrary toStat3 regulation, RhoA reduces serine phosphorylation of Stat5a at both serine residues S726 and S780, resulting in

a further increase of activity as defined by mutagenesis experiments. Furthermore, serine dephosphorylation of

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Stat5a by RhoA does not take place by downmodulation of either JNK1, MEK1 or p38 MAP kinases, as determinedby transfection experiments or chemical inhibition of both MEK1, p38 and JNK serine kinases. Thus, RhoA regulatesStat5a via tyrosine phosphorylation, and by a yet to be determined novel downmodulating pathway that involves seri-ne dephosphorylation. Stat5a activation has biological consequences, since it is involved in RhoA-induced epithelialto mesenchymal transition (EMT) with concomitant increase in vimentin expression, E-cadherin downregulation and

cell motility. Thus, different members of the Stat family seems to mediate different biological functions of RhoGTPases.

Finally, we have further investigated the relationship between Rho GTPases and the transcription factor NF-kB. In anattempt to identify molecular targets under control of Rho GTPases, we have found that RhoA, Rac1 and Cdc42promote the expression of cyclooxygenase-2 (COX-2) at the transcriptional level by a mechanism that is dependent

on the transcription factor NFkB, but not Stat3, a transcription factor required for RhoA-induced tumorigenesis. Withrespect to RhoA, this effect is dependent on ROCK, but not PKN. Treatment of RhoA-, Rac1- and Cdc42-transfor-med epithelial cells with Sulindac and NS-398, two well-characterized non-steroid anti-inflammatory drugs (NSAIDs),results in growth inhibition as determined by cell proliferation assays. Accordingly, tumor growth of RhoA-expressingepithelial cells in syngeneic mice is strongly inhibited by NS-398 treatment. The effect of NSAIDs over RhoA-inducedtumor growth is not exclusively dependent on COX-2 since DNA-binding of NFkB is also abolished upon NSAIDs

treatment, resulting in complete loss of COX-2 expression. Finally, treatment of RhoA-transformed cells with Bay11-7083, a specific NFkB inhibitor, leads to inhibition of cell proliferation. Thus, activation of NF-kB by Rho GTPases hasbiological consequences since it also participates in the oncogenic potential and involves NF-kB –targets genes suchas cox-2. These results suggest that treatment of human tumors that overexpress Rho GTPases with NSAIDs anddrugs that target NFkB could constitute a novel valid antitumoral strategy.

ReferencesOncogene 8, 1285-1292 (1993)Oncogene 15, 3047-3057 (1997)Oncogene 10, 811-816 (1995)Oncogene 11, 2657-2665(1995)Oncogene. 17: 1855-1869 (1998)Mol. Biol. Cell 11:4347-4358 (2000)Genes & Development 11:463-475 (1997)J. Biol. Chem. 273, 12779-12785 (1998)J. Biol. Chem. 274:8506-8515 (1999)Cancer Letters. 165:1-10 (2001)Mol. Biol. Cell 12:3282-3294 (2001)Mol Biol Cell. 14:40-53 (2002)Aznar Benitah S, Fernández-Valerón P, and Lacal J.C. Mol. Biol. Cell (in press) (2003)

Session II: Intracellular Signalling...

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DOCK4, a novel member of the CDM family of proteins, is an activator of Rap1small GTPase signaling pathway

Vijay Yajnik

Massachusetts General Hospital, Harvard Medical School, Boston, USA

DOCK4 gene was identified in a screen for homozygous deletions in tumors driven by the loss of tumor suppressors, NF2and p53 (Cell, 112: 673-84). Tumors in this genetic background are highly aggressive and show distant metastasis. Deletionon mouse chromosome 12 specifically inactivates the DOCK4 gene. In a screen of established human cancer cell lines, threemissense mutations targeting evolutionary conserved residues have been found in five out of forty cell lines ranging from

prostate, ovary, colon and brain cancers. The CDM protein family members, in particular human DOCK180 and DOCK2 havebeen shown to be GEFs for small GTPase Rac1 and are involved in cell movement.We have shown that DOCK4 is an activator of samll GTPase Rap1. Since, Rap1 signaling pathway has been recently shownto be critical in the formation of cellular adherens junctions and loss of adherens junctions is frequently observed in humanmalignancy, we tested the hypothesis that DOCK4 may be a regulator of the formation of adherens junctions and DOCK4signaling pathway is disrupted in tumorigenesis. As expected, DOCK4 expression in mouse osteosarcoma cells with a dele-

tion of the endogenous gene restores the formation of cellular adherens junctions, an effect that is suppressed by co-expres-sion of a dominant negative Rap1. Functional studies on Pro1718Leu, a recurrent mutation in DOCK4 found in prostate andovarian cancer, shows loss of function in that it is unable to activate GTPase Rap1 or restore adherens junction formation.Expression of wild-type, but not mutant, DOCK4 in mouse osteosarcoma cells with a deletion of the endogenous gene sup-presses growth in soft agar and tumor invasion in vivo. Therefore we conclude that DOCK4 and Rap1 mediated signalingpathway is disrupted in tumorigenesis. We are now studying the functional properties of DOCK4 mutations, Lys1105Thr

from colon cancer and Val1887Met from brain tumors with respect to activation of Rho and Ras family GTPase and adhe-rens junction formation. To understand the mechanism of Rap activation by of DOCK4 by identifying DOCK4 associatedproteins. Lastly, we are sequencing DOCK4 in primary tumors to screen for inactivating mutations.

Rho GTPases link heterotrimeric G proteins to the nucleus and beyond

J. Silvio Gutkind Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, HHS, Bethesda, MD, USA

In the last few years we have witnessed a real explosion in the knowledge of how cell surface receptors transmit signals tothe nucleus thereby controlling the expression of genetic programs involved in many cellular processes, including normal andaberrant cell growth. In particular, our laboratory has focused on proliferative signaling through G protein-coupled receptors(GPCRs). We have shown that certain GPCRs can behave as potent ligand-dependent oncogenes and that expression of acti-vated G? q/11 and G? 12/13 can induce malignant transformation. In an effort to dissect the pathway linking transforming

GPCRs to the nucleus, we found that these receptors activate MAP Kinase by PKC and by G protein beta-gamma subunitsacting on Ras. However, we observed that GPCRs induce the expression of c-jun independently of MAP Kinase. This lead tothe finding that GPCRs can stimulate c-Jun N-terminal kinases (JNKs) and contributed to the discovery that Rac1 and Cdc42link many cell surface receptors to this novel enzyme. More recently, we have learned that a unique repertoire of signalingmolecules link transforming GPCRs to a network of newly discovered MAP kinase cascades, in many cases through the acti-vation of small GTP-binding proteins of the Rho family. These observations prompted the search for the underlying mecha-

nism by which G proteins activate Rho GTPases. Of interest, a closely related family of RhoGEFs, that includes p115RhoGEF,PDZ-RhoGEF and LARG exhibits an area of homology to regulators of G protein signaling (RGSs), and recent evidence fromour lab and others suggests that this RGS domain provides a structural feature by which G proteins of the G? 12/13 familycan bind and activate these RhoGEFs, thereby stimulating Rho. Recent work on the regulation of Rho and RGS-containingRhoGEFs by GPCRs and the axon-guiding molecule Plexin B, as well the emerging mechanisms by which Rho regulates geneexpression through PKN/PRK and ROK/ROCK will be presented.

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Session II: Intracellular Signalling.

26


Cdc42-mDia3 Pathway in Mitosis; Implications in Chromosome Instability

Shuh NarumiyaDepartment of Pharmacology, Kyoto University Faculty of Medicine, Kyoto, Japan

Most cancers are aneuploid and show high rates of chromosome losses, gains and rearrangement, a phenomenon termedchromosomal instability of cancer. Such form of genetic instability is apparently caused by abnormality during mitosis.During mitosis, sister chromatids are irreversibly segregated to daughter cells by the action of the mitotic spindle. The mito-tic spindle is a bipolar structure composed of microtubules (MTs) and associated motors, and bind with their plus ends tothe kinetochore complex assembled on the centromere, a chromosome site for spindle attachment. While the composition

of kinetochore proteins and their interaction are being extensively studied, a signaling pathway that regulates kinetochore’scapture of MTs remains unclear. The Rho family small GTPases work as molecular switches in cell morphogenesis by orga-nizing actin cytoskeleton and modulating MT dynamics. While it has been documented that the Rho GTPases such as Rhoand Cdc42 are involved in cytokinesis, the cytoplasmic division, their involvement in nuclear division remains unknown. Herewe report that a Rho GTPase, Cdc42 and its effector, mDia3, regulate this process. Inactivation of Cdc42 in mitotic HeLacells either by treatment with Clostridium difficile toxin B or expression of a dominant negative mutant results in loss of

kinetochore attachment to MTs, disorganized spindle assembly and missegregation of chromosomes. As a consequence,these cells show tetraploid nuclei of abnormal shape. Expression of dominant active Cdc42 or activation of endogenousCdc42 by manipulation of either its exchanger or GAP activity also causes similar chromosomal abnormality. mDia3 thatbinds selectively to the GTP-bound form of Cd42 binds to a kinetochore protein, CENP-A, and co-localizes with it on thecentromeres of mitotic chromosomes. Suppressed expression of mDia3 by RNA interference or expression of its activemutant results, again, in misalignment of metaphase chromosomes and prometaphase delay. These results suggest that the

Cdc42-mDia3 signaling pathway is essential for proper spindle assembly, and deregulation of this pathway may work as oneof the mechanisms of chromosomal instability found in cancers.

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Session III: Cell cycle and proliferation by Rho GTPases.

Links between Rho GTPases and G1 phase control

Richard K. AssoianUniversity of Pennsylvania School of Medicine, Philadelphia, PA, USA

Cell adhesion to the extracellular matrix (ECM) is required for the proliferation of almost all cell types. This anchorage requi-rement reflects the fact that the G1 phase cell cycle events stimulatory events historically attributed to soluble mitogenicgrowth factors actually reflects coordinated signaling between soluble growth factors and the ECM. Changes in the actincytoskeleton that occur as a consequence of cell adhesion and Rho GTPase activation also have a critical role in regulatingcell cycle progression. We have recently been analyzing the relationships between integrin signaling, Rho GTPases, the actin

cytoskeleton, and G1 phase cell cycle control. Recent results will be discussed.

Mechanism of ocogenic ras-induced mRNA stabilization involving thePI3-kinase –PKB pathway

Sabrina Leuenberger1, Georg Stoecklin1, Martin Schmidlin1 Min Lu1,Brigitte Gross1, Marco Colombi1 Daniel Hess2, Brian

Hemmings2 Reto Brem3, Ulrich Certa3 and Christoph Moroni1

1Institute for Medical Microbiology, University of Basel; 2Friedrich Miescher Institute, Basel, 3Roche Research, Basel,Switzerland

mRNA from growth factors, cyclins and many proto-oncogenes contain in their 3‘UTR an AU-rich element (ARE) which isresponsible for the short half-life of corresponding transcripts. Specific human and experimental tumors show stabilization

of ARE-containing transcripts. We provide evidence that activated ras can stabilize ARE-transcripts via activation or the p38MAPK pathway as well as via PI-3 kinase-PKB/akt. To analyze the mechanism, we have recently adopted a functional cloningstrategy to isolate ARE-binding proteins and have identified BRF1, an immediate early gene which promoted in vivo ARE-spe-cific mRNA decay1.

We now report that activated forms of ras, PI-3K and PKB/akt stabilize ARE-mRNA involving phosphorylation of BRF1. By

mass spectrometry, we have identified the major phosphorylation site at S92. In vitro, recombinant (r)BRF1 promoted rapidARE-dependent RNA decay in an exosomal system using a S100 fraction from BRF1-/- slowC cells. Phosphorylation of S92had little effect on ARE-RNA binding, yet abrogated the in vitro decay activity of rBRF1. We provide evidence that ARE-mRNAstabilization by PKB involves phosphorylation BRF1 at S92, which promotes complexing BRF1 to the 14-3-3 protein. This canbe blocked by a phosphopeptide, which may have therapeutic implications for tumors with increased ARE-mRNA stability.

The importance of mRNA stabilization and ras-induced oncogenesis was confirmed in an experimental vH-ras-induced mas-tocytoma model where autocrine IL-3 production is the result of ARE-mRNA stabilization. 6 independent tumors derivedfrom the same clonal normal precursor cell were analyzed by Affimetrx gene chips. Interestingly, BRF1 was downregulated inall tumors and the ARE-stabilizing protein HuR was upregulated. Transfection of tristetraprolin, an ARE-binding protein rela-ted to BRF1 could restore rapid ARE-mRNA decay in an autocrine line and suppress tumor formation in vivo2. Our resultsclarify the mechanism of ARE-dependent mRNA turnover and implicate ARE-mRNA stabilization as an important element of

ras, PI3-K/PTEN and PKB7akt dependent oncogenesis.

(1) Stoecklin et al., EMBO J, 21,4709.2002(2) Stoecklin et al. Oncogene, 2003, in press

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29

Rho-family proteins and tumorigenesis

John G. Collard The Netherlands Cancer Institute, Division of Cell Biology, Amsterdam, The Netherlands

Rho-family proteins control signaling pathways that regulate a wide range of biological processes. Similarly to Ras proteins,Rho GTPases are guanine nucleotide binding proteins that cycle between an active GTP-bound and an inactive GDP boundstate. The activity of Rho proteins is controlled by guanine nucleotide exchange factors (GEFs) and GTPase activating pro-teins (GAPs). GEFs activate small GTPases by promoting the exchange of GDP for GTP, whereas GAPs enhance the intrin-sic rate of hydrolysis of bound GTP to GDP, leading to inactivation. In cells, Rho-GTPases exist predominantly in their inac-

tive GDP-bound form in complex with GDP dissociation inhibitors (GDIs).

Activation of Rho GTPases results in binding to various effector molecules that elicit downstream responses. Since Rho-pro-teins control a wide range of signaling pathways that regulate various biological processes, it is not surprising that dysregu-lation of their activities can result in diverse aberrant cellular phenotypes found in different diseases. Evidence implicatingaberrant Rho-signaling in cancer has been obtained from in vitro studies that focussed on specific aspects of tumor cell bio-

logy and to a lesser extent from mutations found in genes encoding Rho signaling components. More recently, in vivo studiesusing recombinant mice lacking or overexpressing Rho signaling proteins have provided direct evidence for the involvementof Rho proteins in cancer. Rho signaling pathways cross-talk with different oncogenic signaling cascades and contribute tovarious aspects of tumorigenesis, including survival, growth, and progression of tumor cells.

Session III: Cell cycle and proliferation by Rho GTPases..

The small GTPase Rap1, the cAMP target Epac and integrin-mediated celladhesion

Johannes L. Bos

Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands

We have identified two novel cAMP targets, Epac1 and 2, which are guanine nucleotide exchange factors for Rap1. The pro-teins have a C-terminal catalytic domain and an N-terminal regulatory domain (RD). The RD of Epac1 has a DEP domain thatanchors the proteins to membranes and a single cAMP domain. Epac2 has in addition a second low affinity cAMP domain. Incollaboration with A. Wittinghofer (Dortmund) we have determined the crystal structure of the RD of Epac2 in the absen-

ce of cAMP. Comparing this structure with published cAMP domains of PKA, which were determined in the presence ofcAMP, revealed a molecular mechanism of activation by cAMP1.

By rational drug design we developed a novel cAMP analog, 8CPT-2’OMe-cAMP that efficiently activates Epac but not pro-tein kinase A (PKA) (2). In various cell lines this Epac-specific cAMP analog was able to induce Rap1 activation, but not thephosphorylation of the PKA substrate CREB. Using this analog we could show that cAMP-induced regulation of ERK and

activation of the small GTPase Rap1 are independent processes2. In addition, we found that 8CPT-2’OMe-cAMP could indu-ce integrin-mediated cell adhesion to fibronectin. This result is compatible with our previous finding that Rap1 is involved inthe regulation of integrins. Moreover, we found that stimulation of cells with isoproterenol, which activates the Gs-coupledb2-adrenergic receptor, also induces integrin-mediated cell adhesion to fibronectin. This induction is independent of PKA, butdependent of Rap1. Thus, b2-adrenergic receptors can regulate integrin activation through the activation of Epac and Rap13.Very recently, in collaboration with G. Thomas (Basel) we found that Tsc2, a putative Rap1GAP is a GAP for the Ras-like small

GTPase Rheb. Indeed, Rheb functions in the Tcs1/2-mTor-S6 kinase pathway4.

(1) Nature Struct. Biol. (2003) 10, 26-32(2) Nature Cell Biol., (2002). 4, 901-906 (3) J Cell Biol. (2003). 160, 487-493(4) Garami, A., Zwartkruis, F.J.T.,Nobukuni, T., Joaquin, M., Roccio, M.,Stocker, H. Kozma, S.C., Hafen, E., Bos, J.L. and Thomas, G, (2003) submitted

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Dissection of Rac-mediated functions in human glioblastoma cell lines using RNAinterference

Amanda Y. Chan1, Ya-yu Chuang1, Salvatore J. Coniglio3 and Marc Symons1,2,3,4

1Center for Oncology and Cell Biology; 2Department of Surgery, North Shore-Long Island Jewish Research Institute, Manhasset, NY;3Department of Anatomy and Structural Biology; 4Department of Surgery, Albert Einstein College of Medicine, Bronx, NY, USA

Members of the Rho family of small GTP-binding proteins have been shown to be involved in tumorigenesis and metastasis.

The molecular mechanisms that are responsible for these activities largely remain to be elucidated. Studies using dominantnegative and constitutively active versions of Rho family members have led to the notion that these proteins can regulate alarge number of functions, including the organization of the actin cytoskeleton, regulation of cell motility, lipid metabolism,gene transcription and vesicle trafficking. However, dominant negative versions of Rho proteins lack specificity, as they mayaffect the function of multiple, especially closely related, family members. We therefore set out to dissect the functions thatare mediated by members of the Rac subfamily of GTPases using RNA interference. We focused on human glioblastoma cell

lines, since previous studies had shown that adenoviral-mediated expression of dominant negative Rac1 very potently inhi-bits the survival of glioblastoma cells, but not of astrocytes, the normal cell type from which glioblastoma cells originate(Senger DL, 2002, Cancer Res. 62, 2131).

In this study we used two glioblastoma cell lines, SNB19 and U87MG. We employed two independent small interfering RNAduplexes (siRNA), resulting in a specific decrease in Rac1 protein expression by approximately 75% for both duplexes. In bothcell lines, siRNA-mediated depletion of Rac1 protein expression only has a minor effect on cell proliferation and cell survival.Rac1 depletion however causes a 5-fold decrease in Matrigel invasion compared to control-treated cells. In addition, Rac1

siRNA-treated SNB19 cells fail to extend lamellipodia in respond to serum stimulation and scrape wound healing assays revea-led that Rac1 siRNA-treated cells migrate approximately three-fold slower than control cells. These results suggest that Rac1-mediated organization of the actin cytoskeleton and cell migration contribute to the role of Rac1 in glioblastoma cell invasion.Current efforts are geared toward delineating the signaling cascades that are mediated by Rac to regulate cell invasion. Interestingly, siRNA-mediated depletion of Rac3 only has minor effects on glioblastoma cell invasion and proliferation. Thus,one explanation for the discrepancy between the results obtained with dominant negative Rac1 and Rac1-directed siRNA is

that dominant negative Rac1 inhibits the function of more distantly related Rho GTPases. Another possibility however is thatoverexpression of dominant negative Rac obliterates all Rac activity and that the low amount of Rac1 that remains in theRac1 siRNA-treated cells performs functions that are essential for the survival and proliferation of glioblastoma cells.

In summary, our studies demonstrate that RNA interference provides a powerful new tool for the elucidation of Rho GTPasefunctions.

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Session IV: Adhesion, Migration and Invasion by small GTPases.

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Effect of the small GTPase RhoE in U-87 glioblastoma cellular proliferation

Guasch RM, Miñambres R, Pérez-Roger I and Guerri CInstituto de Investigaciones Citológicas (Fundación Valenciana de Investigaciones Biomédicas), Valencia, Spain

Rho family proteins are involved in regulating several cellular events as cytoskeletal organisation and cell motility. Differentmembers of the family induce distinct changes to the actin cytoskeleton, including stress fiber formation and extension offilopodia and lamellipodia, which are essential for the movement of the cell. RhoA, the main member of the family, stimulatesactomyosin-based contractility through activation of its downstream effector ROCK. We cloned and characterised RhoE, amember of the family that lacks GTPase activity (Guasch et al., Mol Cell Biol 18:4761, 1998). In contrast to RhoA, overex-

pression of RhoE in epithelial cells induces stress fiber disassembly and stimulates cell migration. The effect of RhoE on theactin cytoskeleton is due to its binding to and inhibition of ROCK I–induced stress fiber formation and phosphorylation ofits downstream target myosin light chain phosphatase (Riento et al., Mol Cell Biol., in press). Therefore, it seems that RhoEacts as an antagonist of the cellular activities of RhoA. There is a clear implication of Rho proteins in cancer: activated Rhomutants are able to transform fibroblasts and inhibitory mutants block the transformation induced by Ras. As Rho proteinsare involved in cellular movement and this event is crucial in invasion, modulation of Rho activity can promote invasion and

metastasis. Rho proteins also affect the cell cycle machinery, by increasing the expression of cyclin D1 and reducing the CDKinhibitors expression. Downregulation of p27Kip1 seems to require RhoA activity, although this could be an indirect effect.Active RhoA reduces the levels of p21Cip1, which is crucial for Ras induced cell cycle entry (rewieved in Sahai and Marshall,Nat Rew Cancer 2:133, 2002). Given that RhoA induces cell cycle entry and progression through the G1 phase of the cellcycle, and that RhoE is an antagonist of RhoA, we studied the involvement of RhoE in the regulation of the U-87 glioblasto-ma cell cycle. Our results indicate that overexpression of RhoE reduces the proliferating capabilities of the cells. Flow cyto-

metry analysis shows an increase of the number of cells in G1 and G2 and a reduction of the S phase of the cell cycle.Quiescent cells restimulated with serum show a transient induction of p21Cip1 expression, which is necessary for theassembly of active cyclin D-CDK complexes in early G1. However, cells overexpressing RhoE are unable to induce p21Cip1expression and therefore RhoE blocks cell cycle entry. Our results suggest that RhoE is involved in abnormal cellular proli-feration in U-87 glioblastoma cells.

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Caveolin and lipid rafts in Integrin control of Rac targeting and signaling

Miguel A del PozoThe Scripps Research Institute, La Jolla, USA

Activation of Rac1 induces its translocation to the plasma membrane, an essential step for activation of effectors 1. Previousstudies have shown that Rac1 recruitment to the plasma membrane requires integrin-mediated adhesion 2,3 and that acti-vated Rac1 is enriched in cholesterol-rich raft domains 4. We now show that loss of integrin-mediated cell adhesion causesa coordinate loss of the lipid raft marker GM1 and caveolae from the cell surface, accumulation in an intracellular compart-ment and inhibition of Rac1 recruitment to the plasma membrane. Active Rac1 binds preferentially to low density, choleste-

rol-rich membranes, and the binding sites are provided at least in part by lipids. Preventing GM1 internalization in non-adhe-rent cells retains Rac1 targeting and effector activation. Caveolin-1 expression is required for internalization of GM1 and lossof Rac1 targeting in non-adherent cells. These results show that integrin-dependent recruitment of Rac1 to the plasma mem-brane is mediated by a caveolin-dependent membrane trafficking pathway.

(1) J. Trends Cell Biol 10, 415-9. (2000).(2) Embo J 19, 2008-2014. (2000).(3) Nature Cell Biol. 4, 232-239 (2002).(4) J. Biol. Chem. 274, 21430-21436 (1999).

Session IV: Adhesion, Migration and Invasion by small GTPases.

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Regulated expression of a cell surface protease by the Rho-LIM-Kinase pathway isrequired for tissue morphogenesis during Drosophila development

J. Settleman

Harvard Medical School, Charlestown, USA

The Lim kinases transduce signals that promote F-actin assembly in response to the activation of Rho, Rac, and Cdc42GTPases. We have identified the single Drosophila orthologue of the mammalian Lim kinases, designated Dlimk, that is expres-sed widely in developing imaginal discs. Altering levels of Dlimk activity in larval tissues disrupts normal morphogenesis.Excessive Dlimk activity in developing leg and wing imaginal discs results in a substantial accumulation of F-actin and conse-

quent adhesion defects. Genetically, this phenotype can be modified specifically by mutations in Rho pathway components,indicating that Rho-mediated morphogenesis of developing tissues requires Limk-induced F-actin assembly. We also find thatRho and Dlimk regulate the expression of the Stubble gene, which encodes a transmembrane serine protease that is alsorequired for leg morphogenesis. Moreover, reducing levels of Stubble modifies Rho- and LimK-induced morphogenesisdefects. These results suggest that an important function of the Rho-Lim kinase signal in the proper formation of adult tis-sues is to regulate expression of cell surface protease activity. In addition to its role in development, it appears that the Rho-

Lim kinase pathway may also contribute to tumorigenesis. In a large fraction of human prostate cancers, a closely relatedStubble orthologue, called Hepsin, is reportedly substantially up-regulated in expression. Moreover, Rho and Lim-kinase appe-ar to influence the progression of prostate tumors to an invasive state. Our preliminary results suggest that the Rho-Limkinase pathway may also regulate Hepsin expression in prostate cells, indicating that this pathway might similarly influencetumor progression by affecting cell surface protease expression.

Farnesyltransferase inhibitors, RhoB and receptor traffic

Harry MellorUniversity of Bristol, School of Medical Sciences, Bristol, UK

Farnesyltransferase inhibitors (FTIs) are a novel class of anticancer therapeutics designed to target the Ras GTPase. Whilethese compounds are clearly capable of reversing Ras-induced transformation, there has been considerable speculation as towhether Ras is the sole target for their actions. One potential secondary target of FTI action is the RhoB GTPase. RhoBcan be either farnesylated or geranylgeranylated in cells and FTI treatment leads to a loss of the farnesylated RhoB (RhoB-F) and a gain of geranylgeranylated protein (RhoB-GG). Prendergast and co-workers have suggested that this gain of RhoB-

GG mediates at least some of the broad spectrum of FTI actions on transformed cells.

Little is known of the normal cellular functions of the RhoB GTPase. In our studies we have attempted to dissect the roleof RhoB in controlling intracellular receptor traffic. RhoB is localised to two cellular compartments: the plasma membrane,and the bounding membrane of late endocytic vesicles. FTI treatment causes a loss of RhoB at the cell surface, with a con-sequent gain in the endosomal pool. In keeping with the role of endosomal RhoB in regulation of intracellular traffic, FTI tre-

atment profoundly disrupts the endocytic sorting of the EGF receptor in a RhoB-dependent fashion. A combination of con-focal microscopy and ultra-structural studies allows a precise definition of this trafficking defect.

Recent work from many laboratories has uncovered evidence for links between receptor trafficking pathways and signallingoutputs. The key question is whether the effects of FTIs on receptor traffic have a part to play in the clinical actions of thesedrugs. Preliminary work on the functional consequences of RhoB-mediated regulation of receptor traffic in FTI treated and

unperturbed cells will be discussed.

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Session V: Tumour phenotypes and therapeutic strategies..

36


Modulation of breast cancer phenotypes by RhoC function

K. L. van GolenUniversity of Michigan Comprehensive Cancer Center, Ann Arbor, USA

Overwhelming, the majority of literature on inflammatory breast cancer (IBC) describes the clinical characteristics of thisdisease. IBC is a particularly aggressive form of locally advanced breast cancer that affects approximately 6% of breast can-cer patients annually. Tumor emboli invade the dermal lymphatics of the skin overlying the breast. Because of this nearly allwomen have lymph node involvement and 36% have gross distant metastasis at the time of diagnosis. Many women progressafter 12 months of diagnosis due to outgrowth of occult metastases. Taking this into account it is not surprising that IBC

carries with it a poor prognosis, with <45% disease-free survival rate. Until recently, very little was known about the gene-tic mechanisms involved in IBC progression. In a study aimed at identifying genetic alterations specific for IBC, mRNA fromthe SUM149 IBC cell line was compared with mRNA from two normal human mammary epithelial (HME) cell lines and thatpatients own lymphocytes. Using a modified differential display technique, 17 differentially expressed transcripts were iden-tified. To determine which of these transcripts were specific for IBC, in situ hybridization was performed on 29 IBC and 19stage-matched, non-IBC patient samples. Two genes, RhoC GTPase and LIBC (Genbank #AF143679; aka. Wisp3 or IGFBP-

rP9), were concordantly altered in a significant number of the archival IBC specimens.

RhoC GTPase is a member of the Ras-superfamily of small GTP binding proteins and is involved in cytoskeletal reorganiza-tion leading to cellular motility. Like Ras, RhoC is a putative oncogene implicated in the progression of aggressive cancerssuch as pancreatic adenocarcinoma, and metastatic melanoma. To determine the effect of RhoC on the IBC, stable HME-RhoC overexpressing clones were generated. Compared with controls, the HME-RhoC cells had significantly increased

anchorage independent growth, motility, invasion, production of angiogenic factors, and formed tumors and metastases whenorthotopically injected into nude mice. Recently, we have demonstrated that RhoC expression can be used as a prognosticmarker for identifying breast cancers with metastatic ability. In particular, we have identified RhoC expression in small bre-ast cancer (< 1cm) that have metastasized. RhoC expression was not limited to IBC specimens. These data have given usclues as to the role of RhoC in aggressive breast cancer progression.

Rho family proteins as targets for anticancer treatment

Patricia L. Joyce, Anastacia C. Berzat and Adrienne D Cox Departments of Radiation Oncology and Pharmacology; Lineberger Comprehensive Cancer Center. University of North Carolina at

Chapel Hill, Chapel Hill, NC, USA

Considerable evidence suggests a role for aberrant function of Rho family GTPases in cancer, suggesting that Rho proteinsmay be novel targets for anti-cancer agents. The enzyme geranylgeranyltransferase I (GGTase I) catalyzes the addition of ageranylgeranyl (GG) isoprenoid lipid to the C-terminus of Rho proteins, that is critical for Rho membrane association andfunction. Several GGTIs have been shown to exhibit anti-proliferative activity in vitro and anti-tumor activity in preclinical

animal models, but the specific GG-modified proteins that account for this activity have not been defined. Since many RhoGTPases are both known substrates of GGTase I and are known to promote cell proliferation, we evaluated the possibilitythat the inhibition of Rho lipid modification contributes to the inhibitory actions of GGTIs. We postulated that for physio-logically relevant targets of GGTIs, genetically-engineered variants that no longer require modification by GGTase I for func-tion should render cells less responsive to GGTI treatment. We generated structural mutants of Rac1 and Rac3 that aremodified by the addition of a farnesyl lipid (designated Rac1-F and Rac3-F). We first verified that activated forms of Rac1 and

Rac3 retained signaling and transforming activity equivalent to that of their authentically GG-modified counterparts, inde-pendent of the type of isoprenoid lipid modification, and that the functions of Rac1-F and Rac3-F were resistant to GGTItreatment. Importantly then, ectopic expression of either Rac1-F or Rac3-F reduced the ability of GGTI treatment to inhi-bit cellular proliferation in monolayer culture or in soft agar. However, human cancers do not have oncogenically mutatedRho GTPases. but increased activity of upstream Rho regulators. Therefore, we also developed an assay to test the ability ofwild type (not constitutively GTP-bound) Rac1-F and Rac3-F to cause resistance to GGTIs. Cells expressing Rac1 or Rac3

respond to PDGF stimulation by membrane ruffling that can be inhibited by GGTI, but cells expressing Rac1-F and Rac3-Fare resistant to such inhibition. Taken together, these observations support the possibility that the anti-tumor activity ofGGTIs is mediated, in part, by inhibition of Rac GTPases. We also evaluated other, less studied Rho GTPases as candidatetargets for GGTIs. We found that, like other RhoGTPases, Wrch-1 (Wnt-regulated homolog of Cdc42-1) possesses C-ter-minal sequences important for association with the plasma membrane. However, although the Wrch-1 C-terminus predic-ted that it would be a substrate for GGTase I, we found that Wrch-1 membrane association was not perturbed by GGTI tre-

atment. Thus, although a role for Wrch-1 in oncogenesis has been described (via the Wnt signaling pathway), GGTI antitu-mor activity is not due to inhibition of Wrch-1 function. Further study of such Rho GTPases will help to define the specificfamily members that are critical to GGTI action as anticancer therapy.

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Session V: Tumour phenotypes and therapeutic strategies.

Abstracts-posters(Only those that have not been included in the oral session are listed)

In alphabetical order of presenting author

ROCK and NFkappaB-dependent activation of COX2 by Rho GTPases: effectsover tumor growth and therapeutic consequences

Aznar Benitah S, Fernandez Valerón P, Lacal JC

Molecular Oncology Group, Instituto de Investigaciones Biomédicas (IIB-CSIC), Madrid, Spain.

Rho GTPases are overexpressed in a variety of human tumors contributing to both tumor proliferation and metastasis.

Recently, several studies demonstrate an essential role of transcriptional regulation in Rho GTPases-induced oncogenesis.Here we demonstrate that RhoA, Rac1 and Cdc42 promote the expression of cyclooxygenase-2 (COX-2) at the transcrip-tional level. Furthermore, whereas the endogenous level of COX-2 in human colorectal cell line HT29 is completely abolis-hed by expression of dominant negative mutant Cdc42, RhoA induces a potent expression of COX2 in DLD-1 cells whichcompletely lack COX-2 expression, indicating that Rho GTPases might be important for COX-2 expression in colorectal car-cinomas. Expression of COX-2 by all three Rho GTPases is dependent on the transcription factor NFkB, but not Stat3, a

transcription factor required for RhoA-induced tumorigenesis. With respect to RhoA, this effect is dependent on ROCK, butnot PKN. Treatment of RhoA-, Rac1- and Cdc42-transformed epithelial cells with Sulindac and NS-398, two well-characteri-zed non-steroid anti-inflammatory drugs (NSAIDs), results in growth inhibition as determined by cell proliferation assays.

Accordingly, tumor growth of RhoA-expressing epithelial cells in syngeneic mice is strongly inhibited by NS-398 treatment.The effect of NSAIDs over RhoA-induced tumor growth is not exclusively dependent on COX-2 since DNA-binding of NFkB

is also abolished upon NSAIDs treatment, resulting in complete loss of COX-2 expression. Finally, treatment of RhoA-trans-formed cells with Bay11-7083, a specific NFkB inhibitor, leads to inhibition of cell proliferation. We suggest that treatment ofhuman tumors that overexpress Rho GTPases with NSAIDs and drugs that target NFkB could constitute a valid antitumo-ral strategy.

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Abstracts-Posters.

Differential activation of H-Ras in endomembranes by the guanine nucleotideexchange factors RasGRF 1 and 2

Imanol Arozarena1, David Matallanas1, María T. Berciano2, Fernando Calvo1, Victoria Sanz-Moreno1, María T. Muñoz3, Gustavo

Egea3, Miguel Lafarga2 and Piero Crespo1

1Instituto de Investigaciones Biomédicas, CSIC, Madrid, Spain; 2Departamento de Biología Celular, Universidad de Cantabria,Santander,Spain; 3Departamento de Biología Celular, Universidad de Barcelona, Barcelona, Spain

Recent findings indicate that, in addition to the peripheral membrane, H-Ras is also found in endomembranes like the endo-plasmic reticulum and the Golgi complex. In these locations H-Ras is functional and can efficiently engage downstream effec-tors, but it is not yet know how H-Ras activation is regulated in these environments. Herein, we show that RasGRF familyexchange factors, endogenous or ectopically expressed, are present in the endoplasmic reticulum but not in the Golgi appa-ratus. With the aid of H-Ras constructs specifically tethered to the plasma-membrane, reticulum and Golgi we demonstrate

that RasGRF1 and 2 can activate plasma-membrane and reticular, but not Golgi-associated H-Ras. We also show that RasGRFDH domain is required for the activation of H-Ras in the reticulum but not in the plasma-membrane. Furthermore, wedemonstrate that RasGRF mediation preferentially sensitizes reticular H-Ras to lysophosphatidic acid treatment. Contrarily,plasma-membrane H-Ras is more responsive to stimulation by ionomycin. Overall, our results provide the initial insights intothe regulation of H-Ras activation in endomembranes

42


STAT5a activation mediates the epithelial to mesenchymal transition induced byoncogenic RhoA

Espina C, Aznar Benitah S, Valeron PF, Lacal JC


The involvement of Rho GTPases in signal transduction pathways leading to transcription activation is one of the major rolesof this family of GTPases. Thus, the identification of transcription factors regulated by Rho GTPases as well as the unders-tanding of the mechanisms of their activation and its biological outcome is of great interest. We provide here evidence thatRho GTPases modulate Stat5a, a transcription factor of the family of Signal Transducers and Activators of Transcription. RhoA

triggers tyrosine phosphorylation (Y696) of Stat5a via a JAK2-dependent mechanism, and promotes DNA-binding activity ofStat5a. Tyrosine phosphorylation of Stat5a is also stimulated physiologically by LPA in a Rho-dependent manner.Simultaneously, RhoA reduces serine phosphorylation of Stat5a at both serine residues S726 and S780, resulting in a furtherincrease of activity as defined by mutagenesis experiments. Furthermore, serine dephosphorylation of Stat5a by RhoA doesnot take place by downmodulation of either JNK1, MEK1 or p38 MAP kinases, as determined by transfection experimentsor chemical inhibition of both MEK1, p38 and JNK serine kinases. Thus, RhoA regulates Stat5a via tyrosine phosphorylation,

and by a yet to be determined novel downmodulating pathway that involves serine dephosphorylation. At last we provideevidence for a role of Stat5a in RhoA-induced epithelial to mesenchymal transition (EMT) with concomitant increase invimentin expression, E-cadherin downregulation and cell motility.

43

Abstracts-Posters.

44


Regulation of Stat3 transcription factors by RhoGTPases. Implication in RhoA-mediated neoplastic transformation

Aznar Benitah S, Valeron PF, Lacal JC.


Stats (signal transducers and activators of transcription) are latent cytoplasmic transcription factors that upon a specific sti-mulus migrate to the nucleus and exert their transcriptional activity. Here we report a novel signaling pathway wherebyRhoA can efficiently modulate Stat3 transcriptional activity by inducing its simultaneous tyrosine and serine phosphorylation.Tyrosine phosphorylation is exerted via a member of the Src family of kinases (SrcFK) and JAK2, while the JNK pathway

mediates serine phosphorylation. Furthermore, cooperation of both tyrosine as well as serine phosphorylation is necessaryfor full activation of Stat3.

Induction of Stat3 activity depends on the effector domain of RhoA, and correlates with induction of both Src Kinase-rela-ted and JNK activities. Activation of Stat3 has biological implications. Coexpression of an oncogenic version of RhoA alongwith the wild type, non-transforming Stat3 gene, significantly enhances its oncogenic activity on human HEK cells, suggesting

that Stat3 is an essential component of RhoA mediated transformation. In keeping with this, dominant negative Stat3 mutants,or inhibition of its tyrosine or serine phosphorylation, completely abrogate RhoA oncogenic potential. Taken together, theseresults indicate that Stat3 is an important player in RhoA-mediated oncogenic transformation, which requires simultaneousphosphorylation at both tyrosine and serine residues by specific signaling events triggered by RhoA effectors.

45

Light Chain 2 (LC2): a novel partner for RhoB

Isabelle Lajoie-Mazenc*, Daniel Tovar*, Florence Moujenot, Catherine Jean-de Coster and Gilles FavreINSERM-Département “Innovation Thérapeutique et Oncologie Moléculaire”. Institut Claudius Regaud, Toulouse, France* These authors contributed equally and should both be considered first authors

RhoB is a member of the Rho family of small GTPases that has been implicated in actin regulation, cell growth control, geneexpression and cellular transformation. Although closely related to RhoA, its better-studied relative, RhoB differs in severalaspects that indicate it has unique cellular functions. First, RhoB is located in early endosome and nuclear membranes andhas a specialized role in intracellular receptor trafficking. Second, unlike most small GTPases, RhoB is short lived and its levels

are elevated by a variety of stimuli including EGF, PDGF and UV irradiation. Lastly, RhoB is unusual in that it is normally eitherfarnesylated or geranygeranylated. In an attempt to precisely define the role of RhoB in intracellular pathways, we performeda yeast two-hybrid screening of a human adult brain cDNA library using a bait construct encoding activated form of RhoB(RhoBv14). From the positive colonies that were isolated and sequenced, 4 independent clones encoded full length LC2, asubunit of the microtubule-associated protein MAP1A. LC2 or Light Chain 2 is a 28kDa protein, generated by proteolyticcleavage of the MAP1A polyprotein precusor. In the two-hybrid system, LC2 interacts specifically with RhoB, while no inter-

action was found with RhoA, RhoC or other Rho GTPases tested.

Furthermore, no interaction is found between LC2 and a RhoB form deleted of its last 18 amino acids, suggesting a role forthe hypervariable region of RhoB. We confirmed this interaction by GST-pull down assays as well as by co-immunoprecipa-tion experiments. As LC2 is able to establish a link between microtubules and microfilaments, the specific binding of LC2 tothe activated form of RhoB suggest that RhoB could control interactions between microtubule and actin cytoskeleton.

Abstracts-Posters.

46


RhoB tumor suppressive activity in human lung cancer

Julien Mazieres, Ghislaine Daste, Anne Pradines and Gilles FavreDépartement Innovation Thérapeutique et Oncologie Moléculaire, INSERM, Institut Claudius Regaud,Toulouse, France

While most Rho proteins have been shown to have positive role in proliferation and malignant transformation processes, thespecific role of RhoB appears more divergent. RhoB was initially shown to promote cell proliferation and transformation inmurine models and there is now overwhelming evidence that demonstrates RhoB suppressive activity in some humantumors.

To clarify the role of RhoB in lung carcinogenesis, we investigated the expression of RhoB protein by immunohistochemistryin normal or inflammatory lung tissue, hyperplastic, dysplastic lesions or preinvasive carcinoma, invasive carcinoma of low-grade and high-grade of malignancy in different anatomopathological subtypes. We showed that the expression of RhoBdecreases with lung cancer progression. The loss of expression of RhoB is correlated with tumor stage and appears to be alate event in lung carcinogenesis. We also addressed the question of a putative correlation of RhoB and Bcl-2, p53 and Ki-67expression. No correlation can be done between RhoB and p53 or Bcl-2 expression but the loss of expression of RhoB is

correlated with the proliferative index Ki-67. To directly assess the inhibitory role played by RhoB, we transfected adeno-carcinoma cell lines (A549) with RhoB and observed an inhibition of cell proliferation, of anchorage-independent growth anda suppression of xenograft tumor growth in nude mice. Moreover, we demonstrated in this model that RhoB inhibited bothMAPK and PI3K/AKT pathways. These results reinforce the hypothesis of an antioncogenic activity of RhoB. Moreover theloss of expression of RhoB should be considered as a critical event in lung carcinogenesis and thus raises its potential inte-rest in anticancer therapy.

47

Abstracts-Posters.

Characterization of a VNTR sequence in the human rhoB promoter thatnegatively regulates transcriptional activity

Daniel Tovar, Jean-Charles Faye and Gilles Favre

Département Innovation Thérapeutique et Oncologie Moléculaire. Institut Claudius Regaud, Inserm, Toulouse, France

rhoB is an immediate-early gene implicated in cell growth control, cytoskeletal organization and neoplastic transformation.Although the murine rhoB promoter has already been described and its regulation largely studied, the human promoter is

still unknown. Using an in silico approach, we identified the human RhoB gene from the human genome. We cloned the humanrhoB 5’-flanking region using PCR amplification with human peripheral blood lymphocyte genomic DNA as template. By per-forming successive deletions we characterized its promoter region. Although the overall structure of the human promoteris similar to the murine counterpart, the human promoter shows several striking differences, particularly the presence of avariable number of tandem repeats sequence (VNTR) with a 34-bp repetitive unit, between positions -1124 and -821. Wedemonstrated by luciferase activity assays that the presence of this VNTR sequence is able to significantly decrease the trans-

criptional activity of the rhoB promoter, as well as the activity of the heterologous SV40 promoter. PCR amplification of theVNTR sequence, using genomic DNA extracted from several human cell lines as templates, revealed that this sequence ispolymorphic with the existence of at five alleles containing different numbers of the 34-bp repetitive units. Our data descri-bes the human rhoB promoter and several important differences which exist between human and murine rhoB promoterstructures. Furthermore, it describes a negative regulatory role for the VNTR sequence in the human promoter. These datashow the way for further investigations into the link beetween the VNTR polymorphism in the human rhoB promoter and

the biological functions of RhoB.

Human rhoB – Promoter – VNTR

Invited Speakers’ Portfolio A compilation of short biographies of each speaker organised in accordance with the

order of the scientific programme

Dafna Bar-Sagi, Ph. D.Professor, Department of Molecular Genetics and Microbiology,State Univesity of New York at Stony Brook, USA

Dafna Bar-Sagi was born in Tel-Aviv, Israel. She obtained herPh.D. degree in 1984 from

Stony Brook University, New York. Forher postdoctoral training (1984-1987)she joined the group of J. Feramisco atCold Spring Harbor Laboratory, USA,where she became interested in theregulation of growth control by Rasproteins. Her work during this periodled to the identification of two newbiological activities of Ras proteins oneinvolving the capacity to promote neu-ronal differentiation and the other theability to stimulate cytoskeletal rea-rrangements. In 1987, Bar-Sagi wasappointed Junior Staff Investigator atCold Spring Harbor and then promo-ted to Senior Staff Investigator in 1991.She relocated her laboratory to StonyBrook University where she iscurrently Professor and Chair of the

Department of Molecular Genetics andMicrobiology. Bar-Sagi’s research hasremained focused on the mechanismsof signal transduction by Ras proteins.She has authored 95 peer-reviewedpublication and her accomplishmentshave been recognized by several awardsincluding the Catacasino Cancer Rese-arch Award, Baldwin Breast CancerResearch Award and NIH Merit Award.

I. Small GTPases in Cancer.

51


52

Linda Van Aelst, Ph.D.Associate ProfessorCold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA

Linda Van Aelst was born in Aars-chot, Belgium. She obtained herPh.D. degree in Molecular Biology

at the Catholic University Leuven, Bel-gium (1991), for work in J. Thevelein´slaboratory on the regulation of adeny-late cyclase in yeast. She then becameinterested in Ras signaling in mamma-lian cells and joined M. H. Wigler´sgroup at Cold Spring Harbor Labora-tory (CSHL), New York (1992). Hermost seminal work was the discoveryof the first Ras effector, theserine/threonine kinase Raf, mediatingthe effects of Ras on transformation inmammalian cells. She was subsequentlypromoted to Staff Associate (1994)and appointed to Assistant Professor in1996 at CSHL. She expanded herresearch into the Rho family of smallGTPases and provided importantinsights into downstream effector

pathways of the Rac1 GTPase. In1998, she was promoted to AssociateProfessor at CSHL. Her current rese-arch continues to focus on defining therole and mechanisms by which the Rasand Rho GTPases exert their effectson specific aspects of tumorigenesisand neuronal development. Van Aelst isalso a faculty member of the Geneticsand Molecular and Cellular BiologyProgram at SUNY, Stony Brook, NY,USA.

Van Aelst’s work has been recogni-zed through several honors andawards at all stages of her academiccareer. She was granted a BelgiumNational Foundation for ScientificResearch Pre-doctoral award and aCollen Research Foundation Postdoc-toral fellowship. Since her appoint-ment to Staff at CSHL, she has recei-ved additional awards, including the V

foundation for Cancer ResearchAward, The Sidney Kimmel CancerResearch Award, and the Neurofibro-ma foundation and Dana FoundationAwards.

Van Aelst has authored more than40 peer-reviewed publications in inter-national scientific journals and invitedreviews and book chapters. She servesas reviewer for numerous scientificjournals as well as various grantingagencies and is on the editorial boardof Experimental Cell Research.

I. Small GTPases in Cancer.

53

Xosé R. Bustelo, Ph.D. Tenured ScientistCentro de Investigación del Cáncer, USAL-CSIC, Salamanca, Spain

Born in Iria Flavia (Galicia, Spain),Xosé R. Bustelo received hisPh.D. from the University of San-

tiago of Compostela in 1990 for workat M. Freire’s laboratory on the invol-vement of prothymosin ? in cell proli-feration and thymic development.Soon after, Bustelo moved to the Uni-ted Sates where he was a ResearchStaff Scientist at the Brystol-MyersPharmaceutical Research Instituteunder the supervision of M. Barbacid.

By 1996, he occupied an AssistantProfessorship position at the Depart-ment of Pathology, Stony Brook Univer-sity Medical School, NY, USA. In 2000,Bustelo joined the Centro de Investiga-ción del Cáncer (CIC) of Salamancathrough a tenured position at the Spa-nish National Research Council (CSIC).Since that time he has also been appoin-ted as CIC Vice Director and Directorof the CIC Genomics and Proteomics

Programme. This year, Xosé Bustelo hasbeen appointed as one of the NationalCoordinators of the Genomics andProteomics Programme of the SpanishNetwork of Cancer Centres.

The main field of Bustelo’s researchhas been the functional characteriza-tion of the vav oncoprotein and relatedproteins. His research led to the dis-covery of the implication of this onco-protein on tyrosine kinase-dependentpathways, the identification of all Vavfamily members, and to important con-tributions to the understanding of themechanism of regulation of those pro-teins during normal signal transductionand oncogenesis. More recently, hisresearch interests have expanded intorelated fields, including the study of theregulation of Ras GRP/CalDAG-GEFproteins, genomics and proteomics ofcancer, and the characterisation of newmembers of the Rho/Rac family.

Bustelo’s honors include a numberof research awards such as the Williamand Florence Catacosinos Award forCancer Research (1996), the Sinshei-mer Award for Cancer Research(1997), and the Carol M. BaldwinAward for Breast Cancer Research(1997).

Larry Feig, Ph.D. Professor of Biochemistry, Tufts University School of Medicine,Boston, USA

Born in New York, Larry Feigreceived his BS degree fromColumbia University, his MS

degree from the Massachusetts Institu-te of Technology (MIT), and his Ph.D.from Harvard University. He then con-ducted his postdoctoral work with G.Cooper at the Dana Farber CancerInstitute.

In 1987 he became Assistant Pro-fessor of Biochemistry at Tufts Univer-sity School of Medicine and eventuallybecame full Professor in 1997.

Feig has worked on many aspects ofRas signaling, focusing primarily onGuanine Nucleotide Exchange Factorsfor Ras Family GTPases.

He has served on study sections atNIH at various private funding organi-sations and is on the Editorial Board ofMolecular and Cellular Biology.


54

55

.IIIII. Intracellular Signalling..

Channing J. Der, Ph.D.Professor, Department of PharmacologyThe University of North Carolina at Chapel Hill, Chapel Hill, USA

Channing J. Der received his B.A.from the University of Califor-nia at Los Angeles in 1975 and

his Ph.D. from the University of Cali-fornia at Irvine in 1981, where he wor-ked with E. J. Stanbridge on tumoursuppressor genes. He first discoveredactivated ras genes in human cancersduring his postdoctoral studies with G.J. Cooper at Harvard Medical Schoolthe Dana-Farber Cancer Institute in1982. He joined the faculty at theBurnham Institute in 1985, then thefaculty at the University of NorthCarolina in 1992, becoming a full Pro-fessor in 1995. His lab is focused onunderstanding the role of Ras and RhoGTPases in signal transduction andoncogenesis.

Channing J. Der is currently a Pro-fessor at the Department of Pharma-cology at the University of NorthCarolina at Chapel Hill and member of

the Lineberger Comprehensive Can-cer Center, Chapel Hill USA.

He is author of over 200 publica-tions and has been funded by theNational Cancer Institute since 1989.His other appointments include jour-nal editorial boards (Molecular CellBiology, Cancer Research), NIH PathBStudy Section, and he has served as aconsultant for various pharmaceuticaland biotechnology companies.


56

Christopher J. Marshall, FRS, Ph.D.Director, Cancer Research UK Centre for Cell and Molecular Biology Institute of Cancer Research, Cancer Research UK, London, UK

Chris Marshall obtained hisD.Phil. at Oxford followed bypost-doctoral work at the

Imperial Cancer Research Fund in Lon-don, UK and the Dana-Farber CancerInstitute in Boston, USA. In 1980, hemoved to the Institute of CancerResearch in London and became inte-rested in trying to identify humanoncogenes by transfection resulting inthe identification of N-Ras, a novelmember of the Ras family of smallGTPases.

His subsequent work has focusedon delineating the roles that smallGTPases play in tumour cells. Thesestudies include the identification ofRaf-ERKMAP kinase signalling pathwaydownstream of Ras, and the elucida-tion of the signals that target Ras tothe plasma membrane. Currently hislaboratory is investigating the interplay

between Ras and Rho signalling andthe role of Rho family proteins intumour cell invasion.

.IIIII. Intracellular Signalling.

57

Juan Carlos Lacal, Ph.D.Professor of Research and Group LeaderInstituto de Investigaciones Biomédicas, CSIC, Madrid, Spain

Juan Carlos Lacal received his Ph.D.in 1982 from the Universidad Autó-noma de Madrid, Spain. From 1983-

88 he served as an EMBO Postdocto-ral Fellow, then as a PostdoctoralFellow of the Fogarty InternationalCenter (NIH) and then as VisitingAssociate in the Laboratory of Cellularand Molecular Biology, National Can-cer Institute (Bethesda Maryland,USA). In 1989 he joined the SpanishResearch Council as Investigator at theInstituto de Investigaciones Biomédicas(IIB), Madrid. From 1991-94 he waselected as Secretary General of theSpanish Association for Cancer Rese-arch/ASEICA, then as Vice-President(1995-1996), and President (1997-1998). He served as Member of theExecutive Committee of the SpanishFederation of Cancer Societies/FESEO(1991-1994 and 1997-1998), as Mem-ber of the Executive Committee of the

European Association for CancerResearch/EACR (1996-2000) and asMember of the Council of the Federa-tion of European CancerSocieties/FECS (1997-1999).

His research interest has focusedon the identification of signal transduc-tion pathways altered after oncogenictransformation, towards the aim ofidentifying new molecular targets anddesigning novel antitumour strategies.His major contribution has been madein the field of small GTPases within theRas superfamily, and in particular theRas and Rho families. Importantly hiswork was pioneering in identifyingenzymes involved in phospholipidmetabolism activated by Ras oncoge-nes such as phospholipase D and cho-line kinase, which have been the basisfor the design and synthesis of newantitumour drugs. Furthermore, hiswork on the oncogenic, apoptotic, and

metastatic properties of Rho GTPaseshas been pioneering in this field. At last,the identification of specific transcrip-tion factors activated by the family ofRho GTPases, has been of great rele-vance in defining the biological proper-ties of Rho GTPases. He has published130 articles in specialised journals andbooks.

He has been the recipient of severalawards for his research accomplis-hments, such as the Fundación Salud2000 Research Award in 1992, and theFundación Dr. Antonio Esteve Rese-arch Award in 1999.

He is Member of the EditorialBoard of Cancer Letters, The LancetOncology, Oncology Reports, Hema-tología, and since 1999 serves as Exe-cutive Editor of Revista de Oncología(Rev. Oncol.), the official journal ofFESEO and the Mexican National Can-cer Institute.


58

J. Silvio Gutkind, Ph.D.Senior Investigator, National Institute of Dental and CraniofacialResearch Chief, the Oral and Pharyngeal Cancer Branch,National Institutes of Health, Bethesda, USA

J.Silvio Gutkind received his Ph.D.in Pharmacy and Biochemistryfrom the University of Buenos

Aires, Argentina. He joined the Natio-nal Cancer Institute, USA (1988),where he carried out research on thenormal and oncogenic function of non-receptor protein-tyrosine kinases. Hesubsequently moved to the NationalInstitute of Dental and CraniofacialResearch, where he established a newprogram addressing the molecularbasis of cancer by studying normal andaberrant functions of molecules invol-ved in the transduction of proliferativesignals. His laboratory has made semi-nal contributions to the field, and hel-ped elucidate some of the basic mole-cular mechanisms whereby cell surfacereceptors regulate the nuclear expres-sion of growth promoting genes. In1998, he was appointed as the Chief of

the Oral and Pharyngeal CancerBranch. He also heads a multi-institu-tional effort aimed to elucidate themolecular mechanisms that contributeto the evolution of squamous cell car-cinomas, and to use this knowledge todevelop molecular markers of diseaseprogression and novel therapeuticapproaches in oral malignancies. Gut-kind has been the recipient of nume-rous awards including the National Ins-titute of Dental Research Director’sExemplary Service Award and theNational Institutes of Health MeritAward. He has published over 180scientific papers, and contributednumerous review articles and bookchapters addressing both basic mole-cular mechanisms and oral cancerresearch.

.IIIIII. Cell cycle and proliferation by RhoGTPases.

59

Shuh Narumiya, M.D., Ph.D.Professor, Department of PharmacologyKyoto University Faculty of Medicine, Kyoto, Japan

Shuh Narumiya was born in Shiga,Japan and obtained his M.D.(1973), and Ph.D. in Biochemistry

(1979) from Kyoto University, Japan. From 1979-1981 Narumiya was a

Postdoctoral Fellow at the WellcomeResearch Laboratories in Beckenham,UK. He then returned to Japan asAssistant Professor at the Kyoto Uni-versity Faculty of Medicine. By 1992he was appointed as Chairman andProfessor, Department of Pharmaco-logy at the same University.

In 1986 he discovered a novel exo-enzyme that ADP-ribosylates a 22 kDaprotein in mammalian cells, knownnow as botulinum C3. His identifica-tion of its substrate as a Rho proteinand the ADP-ribosylation site as itsAsn41 residue, opened the way to dis-sect the functions of Rho proteins inthe cell, and led to the discovery thatRho regulates the actin cytoskeleton.

Narumiya also found that Rho regula-tes neurite outgrowth and retraction,works critically in cytokinesis and inthe G1-S progression of cell cycle. In1995-1997, his group identified anessential series of effector moleculesfor Rho.

In 1997, he reported a syntheticcompound, Y-27632, as a specificROCK inhibitor, which has been widelyused to study the physiological func-tions of the Rho-ROCK signaling path-way.

Narumiya’s current interest is howthe Rho actions are integrated in timeand space in the cell, and how theseactions are utilized to tissue morpho-genesis in vivo in the body. Alongsidehis study on Rho signaling, he has alsocontributed to the study on prosta-glandins.

Narumiya has published more than250 original papers in international

scientific journals and more than 30invited reviews and book chapters. Hiswork has been recognised by severalawards, the most recent being theLorenzini Gold Medal (Italy, 2000) andthe Uehara Prize (Japan, 2002).


60

Richard Assoian, Ph.D.Professor of Pharmacology, University of Pennsylvania School ofMedicine, Philadelphia, USA

Richard Assoian received his BAfrom John Hopkins and his Ph.D.in Biochemistry from the Uni-

versity of Chicago. His post-doctoraltraining was with M. Sporn at theNational Cancer Institute, where hereported the isolation and purificationof TGF-beta from human platelets. Hejoined the Department of Pharmaco-logy at Penn in 1998 after facultyappointments the Department of Bio-chemistry and Molecular Biophysics inthe College of Physicians and Surgeonsat Columbia University and theDepartment of Cell Biology at the Uni-versity of Miami School of Medicine.He served on the CDF3 study sectionat NIH from 1997-2001 and is pre-sently an editorial board member forMolecular and Cellular Biology, anassociated editor for Molecular Bio-logy of the Cell, and an Editor for Jour-nal of Cell Science. For the last seve-

ral years, his research has focused onthe interplay between growth factorsand the extracellular matrix in regula-ting the signal transduction cascadesthat control the G1 phase cyclin-dependent kinases.

.IIIIII. Cell cycle and proliferation by RhoGTPases.

61

John G. Collard was awarded withhis Ph.D. from the University of Nij-megen, The Netherlands by 1976 for

his thesis entitled: A comparative studyof the normal and transformed cellsurface with the lectin Concanavalin A.

The Netherlands Cancer Institutein Amsterdam has been key toCollard´s scientific career, where hehas been appointed since 1976, initiallyat the Department of ExperimentalCytology, and since 1979 at the Divi-sion of Cell Biology, where he directsthe Group “Genetic control of invasionand metastasis”.

In 1977 he was a Visiting Scientist atthe Weizmann Institute of Science,Department of Membrane Biology,Israel.

Research at Collard’s Laboratory isfocused on the role that oncogenesand tumour suppressor genes play in

the escape of growth control as well asin aspects of tumour progression. Mostrecently, he moved into the field ofmetastasis using function-based scree-ning assays to identify genes involved inprocesses of invasion and metastasis oftumour cells. These studies have high-lighted the role of Rho-like GT Pasesand in particular Rac in processes ofinvasion and migration of normal andtumour cells in vitro.

Recent studies with mutant miceclearly demonstrate that Rho-like pro-teins play an essential role in differentaspects of tumorigenesis in vivo.

Key publications of Collard´s workhave been published in high impactinternational journals. Collard is aMember of the International Metasta-sis Research Society, the AmericanAssociation for the Advancement ofScience, the Dutch Society for Cell

Biology and the Dutch Cancer Society.He is called upon regularly as Reviewerfor several Boards and scientific jour-nals of prestige.

John G. Collard, Ph.D.Group Leader, The Division of Cell Biology, The NetherlandsCancer Institute, Amsterdam, The Netherlands


62

Johannes L. Bos, Ph.D.Professor, Physiological Chemistry, University Medical CentreUtrecht, Utrecht, The Netherlands

Johannes L. Bos studied Biology atthe Free University, Amsterdam, andreceived his Ph.D. in Molecular Bio-

logy from the University of Amsterdamin 1980 where he studied mitochon-drial RNA from yeast in the laboratoryof P.Borst. For his post-doctoral trai-ning he joined the group of A.van derEb at the University of Leiden to workon oncogenic transformation by adeno-virus E1. In 1985 he started his work onthe analysis of Ras mutations in humantumours and the function of Ras inoncogenic transformation. After a sab-batical year with F. McCormick inEmeryville, CA, USA, he joined the Uni-versity Medical Center Utrecht in 1991as Professor in Physiological Chemistry,where he continued to work on smallGTPases of the Ras family.

In 2000 he became Director of theGraduate School for Developmental

Biology in Utrecht. Bos participates intwo Dutch networks of excellence, theCentre of Biomedical Genetics and theCancer Genomics Consortium of theNational Genomics Initiative (2001). In1999 he also co-founded Semaia Phar-maceuticals that strives to developantibodies against novel cancer drugs.

.IIIIV. Adhesion, Migration and Invasion by small GTPases.

63

Marc Symons, Ph.D.Associate InvestigatorHead, Laboratory of Tumor Cell Biology, North Shore-Long IslandJewish Research Institute, Manhasset, USA

Marc Symons received his Ph.D.in Biophysics from the FreeUniversity of Brussels, Bel-

gium, in 1980. During post-doctoraltraining at the Weizmann Institute,Israel, working on time-resolved lumi-nescence and fluorescence photoblea-ching techniques, he became interestedin cell motility and actin cytoskeletaldynamics. Subsequently, he joined thelaboratory of T. Mitchison at the Uni-versity of California at San Francisco,where he developed new methodolo-gies to study the control of actin poly-merization. In 1992, he established agroup at Onyx Pharmaceuticals tostudy the role of Rho family GTPasesin malignant transformation. In 1998,Marc Symons moved to the PicowerInstitute for Medical Research, nowNorth Shore-Long Island Jewish Rese-arch Institute, NY, USA, where he is

Associate Investigator and directs theLaboratory of Tumor Cell Biology.Current focus of the laboratory is onthe elucidation of signaling cascadesthat are regulated by Rac GTPases tocontrol of tumor cell proliferation andinvasion.


64

Miguel A. del Pozo, M.D., Ph.D.Assistant ProfessorDepartments of Immunology and Cell Biology,The Scripps Research Institute, La Jolla, California, USA

Miguel A. del Pozo was born inSan Sebastián, Spain, andobtained his M.D. degree at

the Universidad de Valladolid in 1991.He then completed a medical speciali-zation in Immunology at the Hospitalde la Princesa (Madrid, 1995) and aPh.D. in Biochemistry at the Universi-dad Autónoma de Madrid (1997),through work at F. Sánchez’s labora-tory. At that time he showed thatcytoskeletal polarization and migrationof blood cells require the integrationof signals transduced by both integrins(extracellular matrix receptors) andchemotactic factors. He then becameinterested in the regulation of cellmigration by Rho family GTPases, andconsequently joined M. A. Schwartz´sgroup, at Scripps, La Jolla, California(1998). He showed that integrinslocally regulate membrane targeting of

the Rho family GTPases Rac andCdc42, defining a novel mechanism forspatial control of efficient GTPasedownstream signaling. He was appoin-ted Assistant Professor in the Depart-ments of Immunology and Cell Biologyat Scripps (2002).

Del Pozo´s group focuses on therelevance of Rac targeting to definedmembrane domains in anchorage-inde-pendent cell growth. He has alsorecently contributed to the Rac signa-ling regulation by the proto-oncogeneVav field.

Miguel A. del Pozo has receivedseveral honors and awards throughouthis career such as the Lady Tata Memo-rial International Award (UK, 2000)and the Special Fellow Award from theLeukemia and Lymphoma Society(USA, 2001). Del Pozo has authored 36peer-reviewed publications in interna-

tional scientific journals and severalinvited reviews and book chapters.

.IIIIV. Adhesion, Migration and Invasion by small GTPases .

65

Jeffrey Settleman, Ph.D.Head of Laboratory, Massachusetts General Hospital CancerCenter, Harvard Medical School, Charlestown, USA

Jeffrey Settleman was born in Balti-more, Maryland, USA. He receivedhis Ph.D. degree in Genetics from

Yale University in 1989. From 1989-1992, Settleman was a postdoctoralfellow at the Whitehead Institute forBiomedical Research at the Massachu-setts Institute of Technology (MIT), inR. Weinberg’s laboratory. His researchthere focused on small GTPases of theRas and Rho families, and during thattime he isolated and subsequently clo-ned the p190 RhoGAP.

In 1992, Settleman joined thefaculty of Harvard Medical School andestablished his laboratory at the Mas-sachusetts General Hospital CancerCenter. His early work at Harvard hel-ped to establish the role of the Rafkinase as a direct binding partner foractive Ras. He was also a pioneer inusing Drosophila genetics to investigatethe role of Rho GTPases in embryonic

development. He continues to use Dro-sophila and mouse genetics to examinethe organization and function of RhoGTPase mediated signaling pathwaysduring the morphogenetic processesof development.

In addition to genetic studies, Set-tleman has used biochemical approa-ches to identify novel components ofRho signaling pathways, including theRho target kinase, PRK2, the un-con-ventional Rho protein, RhoE, and theRac activator, DOCK-180. His studiesof GTPase function in human cancershave led to the identification of theGTPase regulator, DOCK4, as atumor suppressor. In addition, he hasrecently established a novel approachto achieve the specific biochemicalinactivation of oncogenic forms ofRas as a potential therapeutic agentfor tumors that harbor Ras muta-tions.

He has received several prestigiousawards and grants, and has publishedmore than 50 original papers and 12invited review articles and book chap-ters. He is currently serving on the edi-torial boards of Developmental Celland Molecular and Cellular Biology.


66

Harry Mellor, Ph.D.Head of Laboratory, Department of Biochemistry, University ofBristol, School of Medical Sciences, Bristol, UK

Harry Mellor graduated in Bio-chemistry from the Universityof Edinburgh, UK, prior to star-

ting his postgraduate studies with C.Proud in Bristol, looking at proteinkinases involved in protein translation.He continued these studies with J. Jef-ferson at Penn State University, USA, asa Juvenile Diabetes Foundation Fellowbefore moving back to the UK to workwith P. Parker at the Imperial CancerResearch Fund.

This work turned out to be anintroduction to Rho GTPases throughcharacterization of the RhoB signalingprotein. Mellor has now returned tothe Biochemistry Department at theUniversity of Bristol having started hisown laboratory in 1998 through a Well-come Trust Research Career Develop-ment Fellowship. His group is currentlytrying to work out how Rho family

GTPases integrate membrane trafficwith intracellular signaling pathways.

IV. Tumour phenotypes and therapeutic strategies.

67

Kenneth Louis van Golen, Ph.D.Assistant Professor, University of Michigan Comprehensive Cancer Center, Ann Arbor, USA

Kenneth Louis van Golen wasborn in Lansing, Michigan, USAand attended Michigan State

University in East Lansing Michiganfrom 1982-1987 and received a dualdegree in Microbiology and Bioche-mistry. In 1987 van Golen moved toHouston, Texas to work as a ResearchAssistant in the Department of Phar-macology at Baylor College of Medici-ne. He then attended the BiomedicalSciences graduate program at the Uni-versity of Texas Health Science Center,Houston and received his Ph.D. degreein 1996 from the University of TexasM.D. Anderson Cancer Center in thearea of Cell and Cancer Biology. From1996 to 1999 van Golen was a pos-tdoctoral fellow in the Department ofInternal Medicine at the University ofMichigan working on the geneticmechanisms of inflammatory breastcancer.

His work earned him a Susan G.Komen postdoctoral fellowship andseveral other awards. In 1999 he waspromoted to Research Investigator andbegan independent research on therole of RhoC GTPase in locally advan-ced cancers. Van Golen was promotedin 2001 within his department to therank of Assistant Professor.

His laboratory is currently interes-ted in the role and mechanisms of acti-vation of RhoC GTPase in breast, pros-tate and pancreas cancer. He has 32peer-reviewed publications in interna-tionally recognized journals and twoinvited book chapters.


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Adrienne D. Cox, Ph.D.Associate Professor, Departments of Radiation Oncology andPharmacology, North Carolina Clinical Cancer Center, Chapel Hill, USA

Adrienne D. Cox received herB.A. from Pomona College, Cla-remont USA, where she was a

National Merit Scholar, and her Ph.D. ininterdisciplinary Biomedical Sciencesfrom Eastern Virginia Medical School,Norfolk VA, USA. Following a postdoc-toral fellowship at the La Jolla CancerResearch Foundation, she moved to afaculty position at the University ofNorth Carolina at Chapel Hill Schoolof Medicine, Chapel Hill, USA.

Cox currently serves as AssociateProfessor in the Departments of Radia-tion Oncology and Pharmacology atthe University of North Carolina atChapel Hill, and also as member of theLineberger Comprehensive CancerCenter.

Cox is author of over 60 publica-tions and serves on study sections forthe NIH, DOD and Komen Foundation,

and as a consultant for the pharmaceu-tical industry.

Her major research interests areRas superfamily proteins in signaling,transformation and responses to radia-tion, and in targeting such proteins forcancer treatment.

List of Speakers and Participants

71

Invited Speakers List.

Invited Speakers List

Assoian, Richard K. University of [email protected] Philadelphia, USABar-Sagi, Dafne State University of New [email protected] New York ,USABos, Johannes L. University Medical Centre Utrecht

[email protected] Utrecht, The NetherlandsBustelo, Xosé R. Centro de Investigación del Cá[email protected] Salamanca, SpainCollard, John G. The Netherlands Cancer [email protected] Amsterdam, The NetherlandsCox, Adrienne North Carolina Clinical Cancer Center

[email protected] Chapell Hill, USAdel Pozo, Miguel The Scripps Research [email protected] La Jolla, USADer, Channing University of North [email protected] / cjder@ Chapell Hill, USAFavre, Gilles Institut Claudius Regaud

[email protected] Toulouse Cedex, FranceFeig, Larry A. Tufts University School of [email protected] Boston, USAGutkind, J. Silvio National Institutes of [email protected] Bethesda, USALacal, Juan C. Instituto de Investigaciones Biomédicas

[email protected] Madrid, SpainMarshall, Chistopher Cancer Research UK Centre for Cell

and Molecular Biology Institute of Cancer [email protected] London, UKMellor, Harry University of Bristol School of Medical [email protected] Bristol, UK

72


Narumiya, Shuh Kyoto University Faculty of [email protected] Kyoto, Japan

Settleman, Jeffrey Harvard Medical [email protected] Charlestown, USASymons, Marc North Shore-Long Island Jewish Research [email protected] New York, USAVan Aelst, Linda Cold Spring Harbor [email protected] New York, USA

Van Golen, Kenneth L. University of Michigan Comprehensive Cancer [email protected] Ann Arbor, USA

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Participants List.

Participants List

Aznar-Benitah, Salvador Instituto de Investigaciones Biomedicas [email protected] Madrid, Spain

Blasco, Francesc Merck Farma y Química, [email protected] Barcelona, SpainCabañas-Gutierrez, Carlos Instituto de Farmacología y Toxicología [email protected] Madrid, SpainCaloca, María José Centro de Investigación del Cáncer [email protected] Salamanca, Spain

Campos, Ramón Centro Nacional de Investigaciones Oncológicas (CNIO)[email protected] Madrid, SpainCampuzano, Victoria Centro Nacional de Investigaciones Oncológicas (CNIO)[email protected] Madrid, SpainCrespo, Piero Instituto de Investigaciones Biomedicas, -CSIC- Unidad de Biomedicina

asociada a la Universidad de Cantabria

[email protected] Santander, SpainDel Carpio, Alan Instituto de Investigaciones [email protected] Madrid, EspañaEspina, Carolina Instituto de Investigaciones [email protected] Madrid, SpainFernandez-Valeron, Pilar Centro de Ciencias de la Salud (CCS)

[email protected] Las Palmas de Gran canaria, SpainGuasch, Rosa María Instituto de Investigaciones Citológicas

Fundación Valenciana de Investigaciones Biomé[email protected] Valencia, SpainGuerra, Carmen Centro Nacional de Investigaciones Oncológicas (CNIO)[email protected] Madrid, Spain

Lajoie-Mazenc, Isabelle Institut Claudius [email protected] Toulouse, France

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Martínez-Gomariz, Montserrat Instituto de Investigaciones Biomedicas [email protected] Madrid, SpainMazières, Julien Institut Claudius [email protected] Toulouse, FranceMoroni, Christoph Institute of Medical Microbiology

[email protected] Basel, SwitzerlandMoyano, José V. Instituto de Investigaciones Bioló[email protected] Madrid, SpainPradines, Anne Institut Claudius [email protected] Toulouse, FrancePerona, Rosario Instituto de Investigaciones Biomédicas -CSIC

[email protected] Madrid. SpainSánchez-Martín, Lorena Instituto de Framacología y Toxicología [email protected] Madrid. SpainTovar, Daniel Institut Claudius [email protected] Toulouse, FranceVicente-Manzanares, Miguel UAM- Hosp. la Princesa

[email protected] Madrid, SpainYajnik, Vijay Massachusetts General Hospital, Harvard Medical [email protected] Boston, USA

Acknowledgements

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As a non-profit organisation, we would like to thank the support from our CNIO Cancer Conferences (CCC´s) sponsors.

Such contribution helps to ensure that our conferences will continue to establish the CNIO as a point of reference for theinternational cancer research community.

For information surrounding forthcoming exhibition and sponsorship opportunities, please contact: Amanda Wren,

Tel. +34 91 2246985, Fax. +34 91 2246981, Email: [email protected]

Acknowledgements.

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Forthcoming CNIO activities

FORTHCOMING CNIO ACTIVITIES

CNIO CANCER CONFERENCES (CCC)

APOPTOSIS AND CANCER

ORGANISERS: Gabriel Nuñez, Marisol Soengas, Scott LoweDATES: December 1-3, 2003

CNIO MEETINGS

2003 TISSUE MICROARRAYS

ORGANISERS: Miguel A. Piris, Ignacio Casal, Lydia Sánchez.DATES: October 20, 2003

OTHER ACTIVITIES

FIRST CNIO-NCI JOINT MEETING: ADVANCES ON CANCER RESEARCH

ORGANISERS: Miguel Ángel Piris, José Palacios, María J. MerinoDATES: June 26-27, 2003 (main sponsor: DAKO)

ESF EXPLORATORY WORKSHOP ON GENOMIC APPROACHES TO MICROARRAY DATA ANALYSIS

ORGANISERS: JOAQUÍN DOPAZO, RAMÓN DÍAZ-URIARTE, ALVIS BRAZMA

DATES: OCTOBER 30-31, 2003

WORKSHOP ON SNPS ANALYSIS, TOOLS AND APPLICATIONS (FOUNDED BY THE EUROPEAN SCIENCE FOUNDATION)ORGANISERS: JAVIER BENÍTEZ, MERCEDES ROBLEDO, JOAQUÍN DOPAZO

DATES: NOVEMBER 28-29, 2003

SYMPOSIUM ON THE MOLECULAR TAXONOMY OF CANCER

ORGANISERS: KEVIN DAVIES, TODD GOLUB, MIGUEL A. PIRIS

DATES: FEBRUARY 3-6, 2004

ESO INSIDE TRACK CONFERENCE: FAMILIAL CANCER

ORGANISERS: JAVIER BENÍTEZ, ROS EELES, HANS VASEN

DATES: MAY 6-7, 2004

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Forthcoming CNIO activities.

© Fundación Centro Nacional de Investigaciones Oncológicas Carlos III. Madrid, 2003Co-ordinated and compiled by Beatriz Ferreiro ([email protected]) and Amanda Wren ([email protected])Dept. Scientific Events

Design: Mónica Bernabé Print: Gráficas Ave

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