Abstract book

WELCOME

Dear participants, welcome to the 2010 International PhD Students Cancer Conference here at the IFOM-IEO Campus in Milan! We have tried to organize this conference at our best, hoping it will be an excellent opportunity to discuss about science, to meet new and interesting people and to broad our knowledge. We are very glad to host such an international meeting, with students coming from institutes all across Europe; moreover, this year we are very pleased to host students from the National Centre for Biological Sciences, Bangalore, India.

We do hope you will find this conference really exciting and that you will have a great time here in Milan!

Thank you all for coming!! The Organizers

FOR ORGANIZATION REASONS, YOU ARE KINDLY REQUESTED TO ALWAYS WEAR/SHOW THE CONFERENCE BADGE!

THANK YOU VERY MUCH FOR YOUR COLLABORATION!

Federica Castellucci: [email protected] Francesca Milanesi: [email protected] Gianmaria Sarra Ferraris: [email protected] Chiara Segrè: [email protected] Gianluca Varetti: [email protected]

International PhD Student Cancer Conference

International PhD Student Cancer Conference 19th – 21st May 2010 IFOM-IEO Campus, Milan, Italy

This is the fourth annual conference that is hosted and organized by students from the European School of Molecular Medicine (SEMM).

The Conference will cover many topics related to cancer, from basic biology to clinical aspects of the disease. All attendees will present their research, by either giving a talk or presenting a poster.

This conference is an opportunity to introduce PhD students to top cancer research institutes across Europe.

International PhD Student Cancer Conference

ORGANIZATION

SEMM PhD student conference organizing committee

Federica Castellucci Francesca Milanesi Gian Maria Sarra Ferraris Chiara Segré Gianluca Varetti

Supported by:

Francesco Blasi (SEMM PhD Program Coordinator) Deborah Agostini (web and graphic design) Sabrina Frata (SEMM events coordinator)

International PhD Student Cancer Conference

PARTICIPATING INSTITUTES

CORE PARTICIPANTING INSTITUTES

European School of Molecular Medicine (SEMM) – IFOM-IEO Campus, Milan

Cancer Research UK Institutes: ▪▪ Beatson Institute for Cancer Research (BICR), Glasgow ▪▪ Cambridge Research Institute (CRI), Cambridge, UK ▪▪ MRC Gray Institute of Radiation Biology (GIROB), Oxford ▪▪ London Research Institute (LRI), London ▪▪ Paterson Institute for Cancer Research (PICR), Manchester

The Netherlands Cancer Institute (NKI), Amsterdam

INVITED INSTITUTES

European Molecular Biology Labs (EMBL), Heidelberg & European Bioinformatics Institute (EMBL EBI), Hinxton

German Cancer Research Centre (DKFZ), Heidelberg

Medical Research Council (MRC) National Institute for Medical Research (NIMR), London

National Centre for Biological Sciences (NCBS), Bangalore

Spanish National Cancer Centre (CNIO), Madrid

Swiss Institute for Experimental Cancer Research (ISREC-EPFL) and Department of Biochemistry of the Lausanne University (DB-UNIL), Lausanne

International PhD Student Cancer Conference

SPONSORS

A very “Special Thanks” to our kind Sponsors

International PhD Student Cancer Conference

TABLE OF CONTENTS

Scientific Program

May 19, 2010 – Keynote lecture, lecture, oral presentations and poster session I May 20, 2010 – Lectures, poster session II and oral presentations May 21, 2010 – Keynote lecture and oral presentations

Keynote Speakers

Special Sessions

Authors’ Index

Important Addresses

Know before you go - Milan

International PhD Student Cancer Conference

Scientific Program

SCIENTIFIC PROGRAM MAY 19, 2010

h. 12.00/14.00 Registration, poster session I set up, sandwiches and hotel check in

h. 14.00/14.15 Opening remarks Prof. Pier Giuseppe Pelicci (IEO, Milan)

h. 14.15/14.45 Special session I Dr. Giuseppe Testa (IEO, Milan) Priming the epigenome in differentiation and cancer

SESSION I: Cellular Differentiation and development Chairperson: Guillermo Menendez (LRI, London)

h. 14.45/15.05 Matteo Marzi (IFOM, Milan) E1A regulated micrornas couple differentiation and cell cycle exit

h. 15.05/15.25 Joerg Hoeck (LRI, London) Fbw7 controls neural stem cell differentiation and progenitor apoptosis by antagonising Notch and JNK/c-Jun signalling

h. 15.25/15.45 Shefali Talwar (NCBS, Bangalore) Spatiotemporal analysis of higher order chromatin assembly during cellular differentiation

h. 15.45/16.05 Feng Wang (CRI, Cambridge) The Xenopus orthologue of TGFBI is required for early embryogenesis through regulation of canonical Wnt signalling

h. 16.05/16.35 Coffee break

International PhD Student Cancer Conference 15 Scientific Program

h. 16.35/17.45 Poster session I

h. 17.45/18.30 Keynote lecture I: Dr. Peter Friedl (Nijmegen Centre for Molecular Life Sciences, Radboud University, Nijmegen; The Netherlands) Plasticity of cancer invasion and implications for therapy

h. 18.30/18.45 Set up poster session II (removal posters session I)

h. 18.45/21.00 “Pizza party” at the IFOM-IEO Campus bar

h. 21.00 Transfer by private bus from IFOM-IEO Campus to Ripamonti apartment hotel

International PhD Student Cancer Conference 16 Scientific Program

MAY 20, 2010

h. 8.30 transfer by private bus from Ripamonti apartment hotel to IFOM-IEO Campus

SESSION II: Cell migration & Immunology Chairperson: Meiling Gao (BICR, Glasgow)

SESSION IIa: Cell migration h. 9.30-9.50 Anne Lempens (BICR, Glasgow) ERK2 but not ERK1 contributes to invasive migration of tumour cells

h. 9.50-10.10 Roheet Bantval Rao (CRI, Cambridge) Huntingtin interacting protein 1 (HIP1) induces Epithelial-to-Mesenchymal Transition (EMT) in prostate cancer cells

h. 10.10-10.30 Melda Tozluoglu (LRI, London) Multiscale Modelling of Cancer Cell Motility

SESSION IIb: Immunology h. 10.30-10.50 Tim Schnyder (LRI, London) Spatially resolved negative regulation of B cell receptor signalling

h. 10.50-11.10 Eva Schlecker (DKFZ, Heidelberg) Control of anti-tumor immune responses by myeloid-derived suppressor cells

h. 11.10-11.30 Coffee break

h. 11.30-12.00 Special session II: Prof. Pier Paolo Di Fiore (IFOM, Milan) Endocytosis and recycling at the crossroad of signaling, attenuation, execution of polarized functions and cell fate determination

h. 12.00-12.30 Participating Institute presentations

International PhD Student Cancer Conference 17 Scientific Program

h. 12.30-13.30 Lunch at the IFOM-IEO Campus bar

SESSION III: Modeling and large scale analysis in cancer research Chairperson: Willem-Jan Keune (PICR, Manchester)

h. 13.30-13.50 Christiaan Klijn (NKI, Amsterdam) Finding co-occurrence and mutual exclusiveness of DNA copy number changes

h. 13.50-14.10 Xiaofan Li (LRI, London) How does a protein find its mates in a crowded cell?

h. 14.10-14.30 Gilda Nappo (IFOM, Milan) A SILAC Proteomic Approach Identified Novel Candidate Players in the Non-Clathrin Endocytic Pathway of the EGFR

h. 14.30-14.50 Rebecca Burrell (LRI, London) Using Integrative Genomics To Investigate Mechanisms of Chromosomal Instability

h. 14.50-16.00 Poster session II and soft drinks

SESSION IV: Genome instability and cancer Chairperson: Magdalena Zajac (CNIO, Madrid)

h. 16.00-16.20 Marieke Peuscher (NKI, Amsterdam) An shRNA screen to identify novel factors involved in telomere damage induced genome instability

h. 16.20-16.40 Tanja van Harn (NKI, Amsterdam) Loss of RB proteins causes genomic instability in the absence of mitogenic signaling

International PhD Student Cancer Conference 18 Scientific Program

h. 16.40-17.00 Dhaval Varshney (BICR, Glasgow) Silent assassins: DNA methylation does not inhibit SINE transcription, but suppressed their recombination

h. 17.00-17.20 Sietske Bakker (NKI, Amsterdam) Fancm-deficient mice reveal unique features of Fanconi anemia complementation group M

h. 17.20-18.05 Special session III: Bioethics round table Silvia Camporesi & Alessandro Blasimme (IFOM-IEO, Milan) World-wide human embryonic stem cell policies: who decides and on what grounds?

h. 18.10 Transfer to Ripamonti apartment hotel by private bus

h. 20.00 Transfer from Ripamonti apartment hotel to the Skyline Restaurant by private bus

h. 20.30: Social Dinner at Skyline restaurant

h 24.00 Transfer by private bus from the Skyline restaurant to Ripamonti apartment hotel

International PhD Student Cancer Conference 19 Scientific Program

MAY 21, 2010

h. 9.30 Transfer by private bus from Ripamonti apartment hotel to IFOM-IEO Campus

SESSION V: Signal transduction & Cancer genetics and drug resistance Chairperson: Gordana Halec (DKFZ, Heidelberg)

SESSION Va: Signal transduction h. 10.00-10.20 Andrea Longatti (LRI, London) Rab-GTPases and Rab-GAPs Involved in Autophagy

h. 10.20-10.40 Francesca Montani (IEO, Milan) The mitotic exit oscillator at work

h. 10.40-11.00 Lakshmi Revathi Perumalsamy (NCBS, Bangalore) A non-canonical Notch cascade converges on mitochondria for the regulation of cell survival

SESSION Vb: Cancer genetics and drug resistance h. 11.00-11.20 Rinske Drost (NKI, Amsterdam) The impact of defined Brca1 mutations on tumor development, drug response and acquired resistance

h. 11.20-11.40 Charlotte Ng (CRI, Cambridge) Genomic analysis of genetic heterogeneity and evolution in high-grade serous ovarian carcinoma

h. 11.40-12.00 Conference photo

h. 12.00-12.45 Tour of the IFOM-IEO-CAMPUS

h. 12.45-13.45 Lunch at the IFOM-IEO Campus bar

International PhD Student Cancer Conference 20 Scientific Program

h. 13.45-14.30 Keynote lecture II : Prof. Andreas Trumpp (Divison of Stem Cells and Cancer, Deutsches Krebsforschungszentrum (DKFZ) and Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Heidelberg, Germany) Dormancy in normal and malignant stem cells

h. 14.30-14.45 break with soft drinks

SESSION VI: Biology of stem cells in development and cancer Chairperson: Paola Brescia (IEO, Milan)

h. 14.45-15.05 Margherita Yayoi Turco (IEO, Milan) The RALP adaptor protein modulates mouse embryonic stem cell differentiation

h. 15.05-15.25 Fernando Flores-Guzman (DKFZ, Heidelberg) Potential cancer stem cells in ret transgenic mouse model of spontaneous melanoma

h. 15.25-15.45 Stephen Goldie (CRI, Cambridge) FRMD4A is a potential marker of cancer stem cells in human head and neck squamous cell carcinoma (HNSCC)

h. 15.45-16.05 Cristina Elisabetta Pasi (IEO, Milan) Regulation of self-renewal in mammary stem cells

h. 16.05-onward Closing remarks Francesco Blasi (IFOM, Milan)

International PhD Student Cancer Conference 21

May 19, 2010 Keynote lecture, lecture, oral presentations and poster session I

SESSION I: CELLULAR DIFFERENTIATION AND DEVELOPMENT

International PhD Student Cancer Conference

May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

Keynote lecture Peter Friedl Nijmegen Centre for molecular Life Sciences (NCMLS), Nijmegen

Lecture Giuseppe Testa European Institute of Oncology, Milan

Oral presentations Matteo Marzi The FIRC Institute of Molecular Oncology Foundation, IFOM, Milan

Joerg Hoeck London Research Institute, London

Shefali Talwar National Centre for Biological Sciences, Bangalore

Feng Wang Cambridge Research Institute, Cambridge, UK

International PhD Student Cancer Conference 25

May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

Plasticity of cancer invasion and implications for therapy Peter Friedl Microscopical Imaging of the Cell, Nijmegen Centre for Molecular Life Sciences, Radboud University, Geert Grooteplein 28, 6525GA Nijmegen; The Netherlands; Phone: +31.24.3614329 (secretary), +31.24.3610907 (office), Fax: +31.24.3615317 E-mail: [email protected]

Molecular programs controlling metastatic cancer progression are diverse in different cancers as well as within the microenvironment of a single lesion. These include amoeboid, mesenchymal and collective invasion processes, followed by different mechanisms to seed and condition the metastatic site for secondar y sur vival and grow th. Cancer plasticity may further supported as side-effect of therapeutic interference, prompting cellular and molecular adaptation programs. Examples for unexpected adaptation programs suited to overcome molecular interference are the mesenchymal- amoeboid transition after interference with surface proteases or surface integrins, the amoeboid-mesenchymal transition after interference with Rho/ROCK pathways, and the collective–to-amoeboid transition leading to the dissociation of multicellular lesions followed by amoeboid single-cell dissemination. Thus, an understanding of cellular plasticity of invasion programs will be important to better target cancer progression. Multiphoton microscopy (MPM) has become the method of choice for investigating cell structure and function in tissues and organs, including the invasion and progression of cancer lesions. Using a novel approach of infrared-excited (IR-)MPM at wavelengths above 1080 nm that enhances deep tissue microscopy in orthotopic fibrosarcoma xenografts, we here show deep collective invasion strands of several hundred connected cells. These multicellular units proliferate and simultaneously move with velocities of up to 200 µm per day along pre-existing blood vessels but not tumor- induced neovessels and proliferate (“invasive growth”). These perivascular tumor cell strands further maintain invasion and robust survival during otherwise regression- inducing experimental radiation therapy. Both, primary growth and survival and increased resistance to experimental therapy were ablated by interfering with beta1 and beta3 integrins, implicating integrin-mediated signals as microenvironmental denoinators of response to therapy. In conclusion, intravital deep tumor imaging by IR-MPM identifies the tumor-vessel interface as preferred niche of invasive growth, radioresistance and enhance preclinical anti-cancer therapy.

International PhD Student Cancer Conference 27 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

Priming the epigenome in differentiation and cancer Giuseppe Testa Istituto Europeo di Oncologia, Via Ripamonti 435, 20141 Milan, Italy

Our lab investigates the epigenetic mechanisms that enable lineage commitment and their aberrations in cancer. Understanding phyisiological and physiopathological cell fate transitions requires the elucidation of how genomic programs are progressively deployed and what are the chromatin regulatory mechanisms that coordinate their deployment. Among these, over the last decade it has emerged that the methylation of histone H3 on lysine tails 4 and 27, respectively mediated by the Trithorax (Trx) and Polycomb (PcG) protein families, is central to the programming of genomes that underlies the establishment and maintenance of differentiated cell states. Not surprisingly, aberrations in these pathways have also emerged as important determinants or modulators of tumors, hinting at common regulatory circuits that preside over stem cell physiology and that are perturbed or hijacked in oncogenesis. Finally, changes in these posttranslational modifications are also prominent in the epigenetic rewiring that underlies the reacquisition of pluripotency from differentiated cells through nuclear transfer or the expression of few pluripotency factors. Hence, we present here complementary lines of research that use conditional mutagenesis to investigate PcG and Trx function in: i) the physiology of genome programming during differentiation; ii) the aberrant genome programming that accompanies tumorigenesis; and iii) the controlled genome reprogramming that mediates induced pluripotency.

International PhD Student Cancer Conference 28 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

E1A REGULATED microRNAs COUPLE DIFFERENTIATION AND CELL CYCLE EXIT (Marzi Matteo1), Puggioni Eleonora2, Dall’íolio Valentina3, Bernard Loris3, Crescenzi Marco2, Bianchi Fabrizio1, Nicassio Francesco1 and Di Fiore Pier Paolo 1, 4, 5 1 IFOM-IEO Campus, via Adamello 16, Milan, Italy 2 Istituto Superiore di Sanità, viale Regina Elena 299, Rome, Italy 3 Cogentech, Via Adamello, 16, Milan, Italy 4 Dipartimento di Medicina, Chirurgia ed Odontoiatria, Università degli Studi di Milano, Via di Rudini’ 8, 20122 Milan, Italy 5 Istituto Europeo di Oncologia, Via Ripamonti 435, 20141 Milan, Italy

Cell cycle reentry and dedifferentiation are two hallmarks of cancer. We exploit the ability of the adenoviral oncogene E1A to overcome the proliferative block of terminally differentiated myotubes. This model was previously used to identify a class of E1A-induced cancer-associated genes capable of predicting the metastatic risk in independent datasets of breast carcinoma (Nicassio F et al. JCI 2005). Now we show that the integration of mRNA and miRNA expression profiling data reveals a deep interaction between transcriptional and post-transcriptional control of gene expression to rule the post-mitotic state. E1A action has been largely linked with its interference with the growth suppression function of Rb-family proteins. Surprisingly, we found that most miRNAs that accumulate during terminal differentiation and then are down-regulated by E1A during cell cycle re-entry , are controlled through a Rb-independent mechanism. These microRNAs act as negative regulators of proliferation since their accumulation in terminally differentiated cells prevents the re-expression of proliferative Rb-dependent genes. This two-sided mechanism outlines a regulatory loop in the control of post-mitotic state under physiological and pathological conditions. We further show that these microRNAs are biologically relevant in the maintenance of post-mitotic state, since their knock- down increased, and their over-expression reduced, E1A induced cell-cycle reentry.

International PhD Student Cancer Conference 29 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

Fbw7 controls neural stem cell differentiation and progenitor apoptosis by antagonising Notch and JNK/c-Jun signalling (Joerg Hoeck), Axel Behrens Cancer Research UK, London Research Institute, London, United Kingdom

Neural stem/progenitor cells (NSCs/NPCs) give rise to neurons, astrocytes, and oligodendrocytes. However, the mechanisms underlying the decision of a stem cell to either self-renew or differentiate are not completely understood. Fbw7 (also known as Fbxw7, hCdc4, hSel-10) is a member of the F-box family of proteins, which function as substrate recognition adaptors for SCF (complex of SKP1, CUL1 and Fbox protein)-type ubiquitin ligases. SCF(Fbw7) targets several important oncoproteins, including c-Myc, c-Jun, CyclinE1, and Notch, for ubiquitin-dependent proteolysis. Fbw7 mutations have been detected in a variety of human cancers, indicating that Fbw7 is a tumour suppressor. In my PhD project, I have demonstrated that Fbw7 is a key regulator of NSC/ NPC viability and differentiation. The absence of Fbw7 in NSCs/NPCs caused severely impaired stem cell differentiation and increased cell death. Fbw7-deficient neurospheres showed accumulation of the SCF(Fbw7) substrates active Notch1 and phosphorylated c-Jun. Genetic and pharmacological rescue experiments identified c-Jun as a key substrate of Fbw7 in controlling progenitor cell viability, whereas inhibition of Notch signalling alleviated the block in stem cell differentiation. Thus Fbw7 controls neural stem cell function by antagonising Notch and JNK/c-Jun signalling.

International PhD Student Cancer Conference 30 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

Spatiotemporal analysis of higher order chromatin assembly during cellular differentiation. 1. Shefali Talwar affiliated to NCBS, Bangalore 2. Dipanjan Bhattacharya affiliated to Raman Research Institute(RRI), Bangalore 3. Aprotim Mazumder affiliated to NCBS, Bangalore

Cellular differentiation and developmental programs require changing patterns of gene expression. Recent experiments have revealed that chromatin organization is highly dynamic within living cells suggesting possible mechanisms to alter gene expression programs. Importantly emerging evidence suggests that mechanical cues, as seen in physiology, impinge on cellular differentiation and developmental programs yet the mechanistic principles are unknown. We contrast the differences in the dynamic organization of nuclear architecture between undifferentiated mouse embryonic stem (ES) cells and terminally differentiated primary mouse embryonic fibroblasts (PMEF). Live-cell confocal tracking of nuclear lamina and chromatin assembly evidences highly flexible nuclear architecture within ES cells as compared to PMEF. Further probing histone dynamics during the development of the Drosophila melanogaster embryo revealed an intriguing transition in the chromatin assembly dynamics synchronous with cellular differentiation. In order to examine, if higher order chromatin can be portrayed as epigenetic landscape which gets tuned with onset of differentiation, the spatio- temporal dynamics of chromatin assembly was explored using anisotropy imaging to study the compaction states of chromatin. Future work is aimed at understanding if this fluid to solid like transition of chromatin is coupled to fluctuations in gene expression and the underlying molecular mechanisms facilitating such switching.

International PhD Student Cancer Conference 31 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

The Xenopus orthologue of TGFBI is required for early embryogenesis through regulation of canonical Wnt signalling (Feng Wang)1, Jian Xian1, Shin-ichi Ohnuma2*, James Brenton1* 1 Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK 2 Institute of Ophthalmology, UCL, 11-43 Bath Street, London EC1V 9EL

Tgfbi, a fasciclin family ECM protein, has been shown as a versatile molecule playing various roles in different context, i.e. from development of corneal dystrophies to cancer. No detailed in vivo functional study into the mechanism of this protein during vertebrate development has been reported. Here, we have examined the role of Tgfbi using Xenopus as a model. Our expression analysis showed that Tgfbi is expressed at developmentally important tissues such as the dorsal marginal zone, notochord and floorplate of the neural tube during Xenopus early embryogenesis. Knockdown of Xtgfbi by anti-sense morpholinos in Xenopus causes bent anterio-posterior axis, smaller eyes and tail fins morphologically, as well as defective induction of organizer, patterning and differentiation of muscle, neuron and neural crest cells, which are associated with repressed Wnt signaling. Exogenous activation of the Wnt pathway is strongly inhibited by depletion, while Xtgfbi activation by itself at the conditions without endogenous Wnt activity cannot trigger the Wnt activity. Furthermore, biochemical analysis using mammalian cell lines indicated that Tgfbi activates the Wnt pathway through stabilizing unphosphorylated β-catenin. All observations indicate that Tgfbi is a novel essential regulator of the canonical Wnt pathway and is required for multiple Wnt-mediated processes in embryonic development.

International PhD Student Cancer Conference 32 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

POSTER SESSION I A1 Federica Alberghini A2 Seema Alexander A3 Veronica Algisi A4 Ajoeb Baridi A5 Julian Blaser A6 Helene Bon A7 Vanessa Borges A8 Paola Brescia A9 Katja Butterbach A10 Marieta Caganova A11 Marìa Carretero A12 Federica Castellucci A13 Adrian Charbin A14 Avradip Chatterjee A15 Filippo Ciceri A16 Catherine Cowell A17 Matteo D’Antonio A18 Amrita Dasgupta A19 Andrew Davidson A20 Sahra Derkits A21 Ivana Dokic A22 Sara Donnelly A23 Sarah Dowding A24 Charlotte Durkin A25 Mike Fletcher A26 Giulia Fragola A27 Daniele Frangioni A28 Meiling Gao A29 Ernst Geutjes A30 Gordana Halec A31 Willaim Harris A32 David Hobson A33 Charlotte Hodson A34 Hung Yi Kristal Kaan A35 Mirjam Ketema A36 Guy Kingham A37 Ulrike Krebs A38 Serena Lunardi A39 Cerys Manning A40 Stefano Marchesi A41 Sara Mari A42 Andrzej Mazan

International PhD Student Cancer Conference 33

May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A1 The role of the Polycomb group protein Bmi-1 in B cell development and adaptive immunity (Alberghini Federica), Casola Stefano

Bmi-1 is a Polycomb group member. Polycomb group proteins are a heterogeneous group of transcriptional repressors that act through covalent histone modification which play pivotal roles in processes such as embryo development, cell lineage specification and cell-cycle regulation. Furthermore, Bmi-1 is implicated in homeostasis of the hematopoietic compartment in adult mammals, where it regulates hematopoietic stem cell self-renewal and controls early B and T cell lymphopoiesis. Bmi-1-deficient mice display severely reduced B and T cell numbers, accounting for a crucial role for Bmi-1 in early lymphoid development. Additionally, recent studies reporting Bmi-1 differential expression in peripheral B cells and its deregulation in lymphoma cells suggest a role for the protein in peripheral B cell development. The present study provides an analysis of the distribution of mature B cell subsets in Bmi-1-deficient animals. Additionally, we estimated the efficiency of germinal center responses in mutant animals following immunization with T cell-dependent antigens. Our results indicate a severe impairment in the development of follicular, B-1 and marginal zone B cells upon Bmi-1 inactivation. Moreover, while germinal center B cell development and immunoglobulin somatic hypermutation were unaffected, an impaired selection of high-affinity mutants was observed in germinal centers of Bmi-1-deficient animals.

International PhD Student Cancer Conference 35 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A2 RAC2 and Asparaginyl Endopeptidase regulates invasion in pre-B acute lymphoblastic leukaemia S Alexander, M Holland, F Castro, S Krishnan, C Dempsey, D Smith, Y Connolly, M Walker, D Bitton, C Miller, N Patel, J Liu, A Masurekar, A Whetton and V Saha

Relapsed disease in childhood acute lymphoblastic leukaemia (ALL) is characterised by recurrence in the central nervous system (CNS). Lymphoblasts are thus able to cross blood-brain and blood-csf barriers. This phenomenon is poorly understood. Exploring the mechanisms by which this occurs, using both gene expression and quantitative proteomic analysis, we have identified two candidate proteins overexpressed in lymphoblasts from high-risk patients, namely RAC2 and asparaginyl endopeptidase (AEP). While AEP-overexpressing ALL cell lines show in vitro invasion; overexpression, inhibition or down-regulation of AEP do not contribute significantly to this phenomenon. On the contrary, RAC2 inhibition or knockdown in AEP+ cell lines almost completely abolishes invasion. RAC2-expressing cells cause CNS disease in mice, which can be inhibited by a RAC2 inhibitor. Microscopy showed active AEP in a discrete compartment at the plasma membrane of AEP+ cells. Induced RAC2 expression leads to reorganisation of the actin cytoskeleton and may regulate the apical localisation of AEP. Our data supports an invasive process by which lymphoblasts may invade across endothelium via a RAC2/ AEP-mediated process. Further work is required to establish the interaction of RAC2 with the lysosomal protease AEP.

International PhD Student Cancer Conference 36 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A3 Role of clathrin endocytic adaptors in controlling EGFR fate Veronica Algisi, Sara Sigismund1, Simona Polo1,2, Pier Paolo di Fiore1, 2,3 1 IFOM, the FIRC Institute for Molecular Oncology, Via Adamello 16, 20139, Milan, Italy 2 Dipartimento di Medicina, Chirurgia ed Odontoiatria, Università degli Studi di Milano, Via di Rudiní 8, 20122 Milan, Italy 3 European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy

Epidermal Growth Factor Receptor (EGFR) can be internalized through different endocytic pathway according to ligand concentration. At low doses of ligand, the receptor (EGFR) is internalized almost exclusively through clathrin-mediated endocytosis (CME), while at higher concentrations of ligand a sizable amount of receptors becomes ubiquitinated and is endocytosed through a non-clathrin endocytosis (NCE), which targets the majority of the receptors to degradation. EGFRs that enter through CME are subjected to different fates. CME is mainly involved in receptor recycling and allows prolonged signaling to occur from the intracellular compartments, but still one third of the receptor is delivered to degradation into the lysosomes. Preliminary data collected in our lab suggest the existence of two distinct populations of clathrin-coated vesicles, regulated by different endocytic adaptors, which link the EGFR to distinct intracellular fates (degradation versus signaling/recycling). To gain insight into this issue, we carried out a complete characterization of the routing, fate and signaling of the EGFR upon RNA interference of the different adaptors. Biochemical assays, molecular genetics and live-imaging techniques were combined in the study in order to design a comprehensive picture. Results will be presented.

International PhD Student Cancer Conference 37 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A4 Deciphering the human prostate epithelial cell hierarchy Ajoeb Baridi, Ian Mills, John Stingl, David Neal. Cambridge University, Department of Oncology. Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Department of Oncology, Cambridgge University, Cambridge, United Kingdom

Deciphering the human prostate epithelial cell hierarchy Ajoeb Baridi, Ian Mills, John Stingl and David Neal. Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Cambridge, United Kingdom Emerging evidence from a variety of tissues demonstrates that tissues are composed of a hierarchy of cells that span from stem to progenitor to differentiated cells. Stem and progenitor cells are of interest because it is perceived that these cells are the initial targets for malignant transformation, and that these cells, when mutated, can function as cancer stem cells. The properties and developmental fates of stem and progenitor cells in the normal human prostate are not well defined. To detect and characterize prostate epithelial stem and progenitor cells, we propose to use fluorescence- activated cell sorting (FACS) in combination with robust functional in vitro and in vivo primary and secondary assays to detect, phenotypically characterize and determine the developmental fates of these cells. Gene expression profiling of purified subsets of cells will then be compared to those obtained from human prostate tumours. This information will be used to interpret the presentation and cellular context of human prostate tumours.

International PhD Student Cancer Conference 38 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A5 A functional shRNAi screen of phosphoinositide modulators in haematopoiesis and leukaemogenesis Julian Blaser, Nullin Divecha, Tim Somervaille, Iman van den Bout,

Self-renewal, proliferation and differentiation are key features of haematopoiesis. The process is complex and tightly regulated by signalling networks, which are still poorly understood. As a result of genetic mutations, some of which occur in signal transduction pathways, normal haematopoietic cells can be converted into leukaemia stem cells to initiate haematological malignancies such as acute myeloid leukaemia. Phosphoinositides are pivotal signalling lipids, which orchestrate proliferation and differentiation in addition to many other physiological aspects of the cell. Although many current cancer studies are focused on the phosphoinositide 3-kinase (PI3K) pathway, a more comprehensive evaluation of other phosphoinositide modulators in haematopoiesis and leukaemogenesis has not been performed. To identify novel phosphoinositide modulators in normal and malignant haematopoiesis a lentivirus short hairpin (shRNAi) library targeting all 90 human phosphoinositide modulators, predominantly consisting of kinases and phosphatases, has been transduced into human leukaemia cell lines as well as normal and leukaemic primary human bone marrow cells. In vitro and in vivo functional screens will potentially identify novel regulators of proliferation, differentiation in normal and malignant haematopoiesis. Negative regulators of the latter could be exploited by kinase or phosphatase inhibitors and thus this work could identify novel chemotherapeutic targets.

International PhD Student Cancer Conference 39 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A6 EXPLORING THE CONTRIBUTION OF CALMODULIN DEPENDENT KINASES TO PROSTATE CANCER [Helene Bon], Ian Mills, Clarlie Massie, David Neal

Androgen Receptor (AR) signalling plays a major role in the development of prostate cancer, but it is still unclear how it is deregulated in this disease. We aim to characterize AR signalling pathway at a protein level using Multiple Reaction Monitoring, a method which allows to measure a high number of proteins in a single experimental procedure. For this assay, a specific set of targets of the network need to be defined. Of the targets which were upregulated in metastatic cancer, we selected Ca2+/Calmodulin-dependent protein kinase II-β (CaMKII-β) which has been characterised as a regulator of cell cycle progression in different malignancies. To explore its role in prostate cancer, we compared CaMKII-β overexpressing LNCaP with the metastatic androgen independent cell line C4-2 and the androgen dependent cell line LNCaP. Using cell proliferation, cell counting and cell cycle analysis, we showed that the overexpression of the kinase promoted LNCaP growth, and that its inhibition with a specific inhibitory peptide induced cell cycle arrest. We performed a comparative protein expression profiling of C4-2, LNCaP and the CaMKII-β overexpressing cell line using mass spectrometry. We identified 9 proteins which were upregulated or downregulated in the CaMKII-β overexpressing cell line and in the androgen independent model cell line C4-2 in comparison with the androgen dependent LNCaP cells. These proteins will need further validation and might be potential candidate biomarkers for the MRM assay.

International PhD Student Cancer Conference 40 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A7 Establishment of sister chromatid cohesion during S phase Vanessa Borges, Chromosome Segregation Laboratory, Cancer Research UK, London Research Institute, Lincoln’s Inn Fields Laboratories, London WC2A 3PX, UK Frank Uhlmann, Chromosome Segregation Laboratory, Cancer Research UK, London Research Institute, Lincoln’s Inn Fields Laboratories, London WC2A 3PX, UK

Establishment of sister-chromatid cohesion during S phase is essential for faithful chromosome segregation. In Saccharomyces cerevisiae, cohesion is mediated by a cohesin complex consisting of Smc1, Smc3, Scc1/Mcd1, and Scc3. Cohesin is loaded onto chromosomes during G1, but cohesion is only established on S-phase upon Smc3 acetylation by the replication fork-associated acetyltransferase Eco1. After remaining relatively constant throughout the period between S phase and M (G2), Smc3 acetylation diminishes again when cells enter in anaphase. However, the Smc3 deacetylase and possible importance of Smc3 deacetylation are still unknow. We discovered that the class I histone deacetylase family member Hos1 is responsible for Smc3 deacetylation. The deacetylation reaction is triggered by separase cleavage of cohesin at anaphase onset. This results complete the description of the Smc3 acetylation cycle and provide insight into the importance of Smc3 acetylation/ deacetylation during sister chromatid cohesion establishment.

International PhD Student Cancer Conference 41 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A8 THE ROLE OF CD133 IN THE IDENTIFICATION AND MAINTENANCE OF CANCER STEM CELLS DERIVED FROM HUMAN GLIOBLASTOMA (Paola Brescia), Cristina Richichi, Elisa Bellani, Davide Riva, Daniela Osti, Barbara Ortensi, Giuliana Pelicci. Department of Experimental Oncology, European Institute of Oncology (IEO), Campus IFOM- IEO, Via Adamello 16, 20139 Milan, Italy

Glioblastomas are among the most biologically aggressive and therapeutically challenging cancers. There are now emerging concepts in tumor biology validating the hierarchical organization of gliomas as abnormal tissues originated from and maintained by cancer stem-like cells (CSCs). The role of CD133 as a CSC marker in brain tumors has been widely investigated, however these studies are not conclusive and hardly anything is known about its function. In this study the efficiency of CD133 on isolation of glioma CSCs has been analyzed, re-examining the methods of purification and the stem behavior of CD133-positive and negative cells in fresh gliomas and glioma sphere cultures. We observed that CD133-negative cells isolated from human GBM-derived neurospheres using antibodies raised against glycosylated epitopes still express the whole protein, have self-renewal capacity in vitro and are tumorigenic in vivo, although there is a delayed mortality of the xeno-transplanted mice. CD133 promoter activity was tracked using the GFP reporter gene, to follow the CD133 expression in vitro and in vivo. The biological effects in vitro and in vivo of CD133 down-regulation in GBM-derived neurospheres was also investigated. Targeting CD133 by lentiviral-mediated short hairpin RNA (shRNA) interference, inhibits the proliferation and alters the cell cycle distribution of glioma stem cells.

International PhD Student Cancer Conference 42 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A9 Association of JAK-STAT pathway related genes with lymphoma risk (Katja Butterbach1), Sabine Behrens1, Silvia de Sanjosé2, Yolanda Benavente2, Nikolaus Becker1, Lenka Foretova3, Marc Maynadie4, Pier Luigi Cocco5, Anthony Staines6, Paolo Boffetta7, Paul Brennan7, Alexandra Nieters8 1) Division of Cancer Epidemiology, German Cancer Research Center, Heidelberg, Germany 2) Unit of Infections and Cancer, Catalan Institute of Oncology, Barcelona, Spain 3) Cancer Epidemiology and Genetics, Masaryk Memorial Cancer Institute, Brno, Czech Republic 4) Registre des Hémopathies Malignes, EA4184 University of Burgundy, Dijon, France 5) Institute of Occupational Medicine, University of Cagliari, Cagliari, Italy 6) School of Nursing, Dublin City University, Glasnevin, Dublin, Ireland 7) International Agency for Research on Cancer, Lyon, France 8) Molecular Epidemiology, Centre of Chronic Immunodeficiency, Freiburg, Germany

Non-Hodgkin Lymphoma (NHL) constitute the seventh most common cancer in Europe. Genetic variants in immunoregulatory factors have been implicated in lymphomagenesis. To further knowledge on genetic susceptibility of lymphomas, a total of 1481 lymphoma cases and 1491 age, sex, and study centre matched controls of the EpiLymph study, a European multi-centric case-control study, were genotyped for 1536 single nucleotide polymorphisms (SNPs) using Illumina GoldenGate BeadArrayTM Technology. Association between SNPs and haplotypes of the JAK-STAT pathway and risk of Hodgkin lymphoma (HL) and NHL were estimated by calculating Odds Ratios (OR) and 95% confidence intervals (CI) using unconditional logistic regression (SAS9.2). Among 220 relevant SNPs, polymorphisms in several genes (STAT3, STAT6, IFNG, STAT5A) were significantly associated with lymphoma risk. Reduced risk for NHL overall and subtypes were seen in association with seven STAT3 SNPs in high linkage disequilibrium and respective haplotypes. Variant rs4103200 conferred an about 20% reduced NHL risk (ORGG 0.78, 95%CI 0.66-0.91, ptrend =0.002). For certain subtypes (DLBCL, FL) a reduced risk with rs4103200 was also evident. A functional variant in STAT6 previously associated with IgE levels (rs324011) was inversely associated with HL risk. Our results implicate a relevant role of the JAK-STAT signalling in the development of lymphoma.

International PhD Student Cancer Conference 43 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A10 The role of Ezh2 in B cell development and immune responses. (Marieta Caganova)1, I-hsin Su 2,3, Alexander Tarakhovsky2 and Stefano Casola 1,# 1. IFOM, FIRC Institute of Molecular Oncology Foundation, IFOM-IEO Campus, Via Adamello 16, 20139 Milan, Italy 2. The Rockefeller University, New York, USA 3. presently at: School of Biological Sciences, Nanyang Technological University, Singapore, Republic of Singapore # Corresponding author: [email protected]

Polycomb Group (PcG) proteins act as transcriptional repressors modulating chromatin accessibility of target genes. Methylation of the lysine 27 residue on histone 3 (H3K27me3) is catalyzed by the PcG member Ezh2 and represents a dynamic repressive epigenetic mark linked to cell fate decisions and maintenance. A number of PcG knockout and transgenic mice display immunological defects demonstrating the regulatory role of PcG proteins in haematopoiesis. In the B cell compartment Ezh2 is expressed in pro-B cells, at lower levels in pre-B, immature and mature B-lymphocytes. Upon entry of naïve mature B cells into the germinal center (GC) reaction, triggered in response to antigen recognition through the B cell receptor (BCR), Ezh2 expression is highly induced. In GCs, B cells undergo intensive proliferation, somatic hypermutation and class switch recombination to exit as terminally differentiated high affinity BCR expressing memory B cells or antibodies secreting plasma cells. Ablation of Ezh2 in mature B cells leads to significant reduction of GC B cells. Proliferation and differentiation towards the memory compartment in absence of Ezh2 is normal. Affymetrix microarray analysis of in vitro stimulated B cells showed that Ezh2 controls the expression of genes involved in cell cycle progression regulation, p53 signalling and apoptosis.

International PhD Student Cancer Conference 44 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A11 Mouse models for cohesin regulatory factors Pds5A and Pds5B Carretero RamÍrez M., Barthelemy I., Losada A.

Chromosome segregation is a crucial event in cell proliferation. One of the important mechanism that ensures faithful chromosome segregation is sister chromatid cohesin mediated by cohesin, a protein complex form by four subunits: SMC1,SMC3, Rad21, and SA. Many additional factors modulate the interaction of the cohesin with chromatin throughtout the cell cycle. Besides its function in cohesion, cohesin plays important roles in DNA repair and regulation of gene expression. Mutations in cohesin regulatory factors like SCC2 or PDS5B as well as SMC1 and SMC3 have been related to a congenital disease called Cornelia de Lange syndrome, characterized by growth and mental retardation. So far, it is unknown whether these symptoms are caused by abnormal cell proliferation during development or by transcriptional deregulation on developmental genes. In vertebrates there are two homologs of Pds5, Pds5A and Pds5B. We are generating conditional mouse models for these cohesin regulatory factors. We have found growth retardation and skeletal abnormalities in the Pds5A+/Δ mice that have shown a relation to abnormal bone development. This phenotype seems to be related both with a delay in the ossification process due to a deregulation in the expression of some key genes during the bone forming process and a lower amount of bone progenitors. We think that our work model might represent an important tool to understand the ethiology of the CdLs and also, other roles of the cohesin and their regulatory factors.

International PhD Student Cancer Conference 45 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A12 SUMO protease mediated regulation of chromosome integrity (Federica Castellucci), Daniele Fachinetti, Andrea Cocito and Dana Branzei IFOM FIRC Institute of Molecular Oncology, Milan, Italy

Sumoylation has been implicated in different processes promoting genome integrity, such as checkpoint maintenance, DNA repair and in the modulation of cell cycle transitions. Our lab previously demonstrated that a subpathway of sumoylation is required to prevent the pathological accumulation of recombinogenic structures during replication of damaged templates, thus pointing out sumoylation as an important regulatory pathway promoting genome integrity in the face of intra-S damage. Yet, how DNA damage activates the SUMO events required to restore the integrity of damaged forks and which are the key targets involved in this process are questions that largely remain to be answered. Sumoylation can be regulated by different mechanisms, one of which may involve controlling the activity of desumoylating enzymes. In budding yeast, desumoylation events are mediated by two SUMO proteases, Ulp1 and Ulp2/Smt4. Using the yeast S. cerevisiae as a model organism, we have found that Ulp2 is phosphorylated in response to cell cycle transitions and different types of replication stress, and that distinct kinases are implicated in these phosphorylation events. We are currently investigating the role of Ulp2 and its phosphorylation in promoting replication fork integrity and DNA repair. The new results will be presented and discussed.

International PhD Student Cancer Conference 46 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A13 How does condensin promote chromosome resolution? (Adrian A. Charbin)[1] & Frank Uhlmann[1] [1] Chromosome Segregation Laboratory, Cancer Research UK, London Research Institute, London, United Kingdom

Proteins of the SMC (structural maintenance of chromosomes) family promote chromosome stability. In particular, the condensin complex mediates chromosome condensation and resolution, which is crucial in allowing the successful segregation of DNA in mitosis. While better known for its role in condensation, condensin also drives chromosome resolution, important for resolving persistent catenation between sister chromatids before entry into anaphase. Indeed, in budding yeast, chromosome compaction is only relevant to shorten the longest chromosomes, but all chromosomes fail to segregate without condensin. Genetic analysis has shown that condensin’s condensation and decatenation functions can be separated. It has already been demonstrated that in condensin mutants, anaphase bridges are ubiquitously observed, but the mechanism behind these remains unclear. It has been speculated that persistent catenation could be the cause, but this has not been directly tested. Using Saccharomyces cerevisiae as a model, we are currently addressing how sister chromatid catenation is resolved during the cell cycle, with a focus on the interaction between condensin and topoisomerase II, the cell is main enzyme capable of resolving topological linkages. To achieve these goals, condensin will be studied in both in vivo and in vitro systems, and initial work has yielded the development of an accurate and reliable catenation assay that is crucial in assessing the role condensin plays in chromosome resolution during the yeast cell cycle.

International PhD Student Cancer Conference 47 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A14 Structural analysis of sister chromatid cohesion (Avradip Chatterjee) London Research Institute, London, UK

Sister chromatid cohesion is a process that ensures faithful segregation of the chromosomes during cell division. It is brought about by a ring-shaped complex called cohesin, which entraps the two sister chromatids. Cohesion is established during the S phase, when the DNA replicates and continues till the anaphase of mitosis when the chromosomes start segregating. The timing of cohesion starting from S phase to the M phase (anaphase) is critical and requires the recruitment of additional proteins. Important among these are two regulatory proteins, Eco1 and Rad61 (in S. cerevisiae). Eco1 is an acetyltransferase, which activates cohesin by acetylating it, while Rad61 has been proposed to form an antiestablishment complex with two other proteins, Pds5 and Scc3, the effect of which is overcome by Eco1. The importance of these proteins for the entire process makes it necessary to understand their functional mechanism, which is not quite clear yet. Insights gained from structural analyses coupled with complementary biochemical studies of these proteins might provide valuable insights into the process and as a first step to achieve this goal, we are trying to elucidate the crystal structures of Eco1 and Rad61 proteins.

International PhD Student Cancer Conference 48 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A15 Cell Cycle Control in Human Acute Myeloid Leukemia (Filippo Ciceri), T.C.P. Somervaille Paterson Institute for Cancer Reasearch, Manchester, United Kingdom

Cellular quiescence may be an important mechanism by which leukaemic stem cells evade chemotherapy and trigger relapse of acute myeloid leukaemia. One way in which the effects of cell cycle specific chemotherapy might be enhanced is through inducing LSCs to enter cell cycle prior to administering chemotherapy. However, currently little is known of the mechanisms that regulate the cell cycle status of human LSCs. To address this question, analyses of the cell cycle status of primary human AML cells have been performed using Ki67, PCNA and propidium iodide. Cells from patients are in G0 because they are Ki67 negative, PCNA negative and G0/G1 on propidium iodide staining. After 24 hours in culture with growth factors all became positive for Ki67 as they enter the G1 phase of the cell cycle. Treatment with PI3K and MAPK inhibitors showed that up regulation of Ki67 during G1 phase entry is PI3K, but not MAPK dependent. Finally we plan to sort human AML blasts in G0 and G1 phases and perform comparative transcriptional profiling to determine which genes and pathways are responsible for the G0/G1 transition. The role of those genes in regulation of quiescence and proliferation will be further confirmed by genetic knockdown and over expression.

International PhD Student Cancer Conference 49 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A16 Identification of genes important in resistance to EFGR inhibition in lung cancer. (Catherine F Cowell)1, Elza de Bruin1, Michael Howell2 , Julian Downward1 1 Signal Transduction Laboratory, Cancer Research UK London Research Institute, London, UK. 2 High Through-put Screening, Cancer Research UK London Research Institute, London, UK.

The Epidermal Growth Factor Receptor (EGFR) gene is mutated in 20% of non-small cell lung carcinomas resulting in constitutively active EGFR signalling. Treatment with EGFR tyrosine kinase inhibitors (such as erlotinib) prolongs patient survival in these cases, however most patients eventually develop resistance. In approximately half of all resistant tumours this is caused by a secondary EGFR mutation (T790M) or to a lesser extent by amplification of the tyrosine kinase receptor MET, yet the cause of resistance in the remainder of patients is largely unknown. In order to identify potential mechanisms of acquired resistance to erlotinib we performed a Genome-wide siRNA screen examining cell viability in the presence and absence of the drug. We have identified several genes that differentially effect cell survival in response to erlotinib treatment, and that validated in a subsequent deconvolution screen. We are currently examining a selection of these targets in more detail to unravel the underlying mechanisms of resistance and signalling pathways involved. Using this approach we aim to identify new drug targets that may increase the effectiveness of EGFR targeted therapy.

International PhD Student Cancer Conference 50 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A17 Systems-level properties of cancer genes in evolution Matteo D’Antonio, Francesca D. Ciccarelli IFOM-IEO-Campus, Via Adamello, 16, 20139 Milan, Italy

Our lab has recently shown that cancer genes tend to be singletons and to code for highly connected and interconnected proteins of the human protein-protein interaction network (PIN). Surprisingly, in the entire human PIN, singleton genes encode proteins that are generally less connected than those encoded by duplicable genes. Thus, cancer genes have evolved differently from other human singletons and display peculiar genetic and network properties. In this project, we aim at understanding the reasons of this peculiarity and the potential contribution to cancer development. As a first analysis, we measured the appearance of cancer genes during evolution. When compared to the rest, cancer genes display a significant burst with the appearance of Metazoans while they are depleted within genes born with Mammals and Primates. We further investigated this finding, in order to understand whether a relationship exists between the age of a gene, its duplicability status and its network properties. The results of our study show that older genes are more connected and more central than younger genes. Furthermore, different relationships between duplicability and network properties are found for genes appeared at different evolutionary levels. In this context, cancer genes show significant discrepancies at all evolutionary levels, demonstrating that specific evolutionary constraints have acted on them.

International PhD Student Cancer Conference 51 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A18 Probing the nature of free energy barriers that slow down the initial collapse reaction during the refolding of two small proteins Amrita Dasgupta and Jayant B. Udgaonkar, NCBS, TIFR, Bangalore, India

The ultrafast compaction of the unfolded state of a protein (a hydrophobic collapse) channels it to the unique native structure by reducing its configurational entropy. The free energy barrier encountered by the collapsing polypeptide chain slows down the rate of this collapse reaction. On the other hand, it can be negligible if the experimental conditions strongly favor the native state. In such cases the transition is purely downhill. Two model proteins have been used to investigate the nature of the free energy barriers that limit the collapse reaction. The refolding of the SH3 domain of PI3 kinase was shown to commence via the rapid formation of a compact form in a transition that is gradual and purely solvent-driven. Attempts were made to capture the formation of early collapsed species using a microsecond mixing device which can mix solutions in 150 µseconds using two spectroscopic probes. These results highlight the polymer nature of the unfolded polypeptide chain. To investigate the robustness of the free energy barriers encountered by the collapsing polypeptide chain, the refolding of Barstar was investigated. Folding conditions were perturbed to favour the native state using tryptophan fluorescence and ANS fluorescence as probes. Preliminary results indicate that the folding landscape of the protein changes significantly with a perturbation of the folding environment.

International PhD Student Cancer Conference 52 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A19 Identification of the functional elements of the SCAR/WAVE complex members [ANDREW DAVIDSON], ROBERT INSALL

During metastasis, cancer cells induce dramatic remodeling of their actin cytoskeletons in order to become highly motile. Recruitment and activation of the Arp2/3 complex, an actin nucleator, to the cellís leading edge induces directed actin polymerization that promotes the formation of cellular protrusions, which in turn drive cell locomotion. The heteropentameric SCAR/WAVE complex is an Arp2/3 activator that couples chemoattractant detection and subsequent intracellular signaling to actin polymerization. Analogous to the fundamental cell cycle research conducted using budding yeast, our lab seeks to use the genetically tractable Dictyostelium discoideum to elucidate the role of the SCAR/WAVE complex in chemotaxis and cell motility, with the aim of applying this to further our understanding of cancer metastasis. Here a deletion series of the largest member of the SCAR/WAVE complex, Pir121, has been undertaken in order to identify the functional domains of this protein. In PirA null cells, as in nulls in any of the other complex members, the entire SCAR/WAVE complex is degraded. Actin protrusions are also almost entirely absent. By assaying for intact SCAR protein using Western blotting and by utilizing DIC microscopy to analyze cell morphology, Pir121 truncations that rescue as well as have dominant positive and negative affects on actin protrusion generation have so far been identified.

International PhD Student Cancer Conference 53 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A20 FAK deletion accelerates tumourigenesis in a chemical induced mouse model of hepatocellular carcinoma (HCC) (Sahra E. Derkits1), Gabrielle H. Ashton1, Rachel A. Ridgway1 and Owen J. Sansom1 1 The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD, UK

Hepatocellular carcinoma is the third most common cause of cancer mortality worldwide (Yau et al., 2009). Several studies have shown that high levels of Focal Adhesion Kinase (FAK) are associated with poor prognosis in Hepatocellular carcinoma (Itoh et al., 2004, Jan et al., 2009). FAK is a non-receptor kinase which promotes cell motility, cell cycle progression and cell survival(Hanks and Polte, 1997, Schaller et al., 1992). Diethlynitrosamine (DEN) induced mouse models of liver cancer recapitulate the Ras pathway up-regulation found in human HCC, and are therefore widely used to study murine HCC(Calvisi et al., 2006). To investigate if the loss of FAK reduces the onset of DEN induced liver carcinogenesis, we crossed mice containing conditional FAK (McLean et al., 2004) alleles to AhCre expressing mice (Ireland et al., 2004) and administered a single dose of DEN. We found that mice which lost both copies of FAK had a much more rapid onset of liver cancer as well as a higher tumour burden compared to wt mice treated with DEN (6 and 9 month time points respectively). To investigate if FAK deficiency also increased survival and proliferation of hepatocytes in another setting we then went on to investigate the role of FAK in chemical induced fibrosis. We observed that mice which were lacking FAK had less collagen deposit and hence less fibrotic tissue than their wt littermates. We are currently investigating the potential mechanisms behind the increased regeneration and tumourigenesis caused by FAK deficiency

CALVISI, D. F., LADU, S., GORDEN, A., FARINA, M., CONNER, E. A., LEE, J. S., FACTOR, V. M. & THORGEIRSSON, S. S. 2006. Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterology, 130, 1117-1128. HANKS, S. K. & POLTE, T. R. 1997. Signaling through focal adhesion kinase. Bioessays, 19, 137-45. IRELAND, H., KEMP, R., HOUGHTON, C., HOWARD, L., CLARKE, A. R., SANSOM, O. J. & WINTON, D. J. 2004. Inducible Cre-mediated control of gene expression in the murine gastrointestinal tract: effect of loss of beta-catenin. Gastroenterology, 126, 1236-46. ITOH, S., MAEDA, T., SHIMADA, M., AISHIMA, S., SHIRABE, K., TANAKA, S. & MAEHARA, Y. 2004. Role of expression of focal adhesion kinase in progression of hepatocellular carcinoma. Clin Cancer Res, 10, 2812-7. JAN, Y. J., KO, B. S., HSU, C., CHANG, T. C., CHEN, S. C., WANG, J. & LIOU, J. Y. 2009. Overexpressed focal adhesion kinase predicts a higher incidence of extrahepatic metastasis and worse survival in hepatocellular carcinoma. Hum Pathol, 40, 1384-90. MCLEAN, G. W., KOMIYAMA, N. H., SERRELS, B., ASANO, H., REYNOLDS, L., CONTI, F., HODIVALA- DILKE, K., METZGER, D., CHAMBON, P., GRANT, S. G. & FRAME, M. C. 2004. Specific deletion of focal adhesion kinase suppresses tumor formation and blocks malignant progression. Genes Dev, 18, 2998-3003. SCHALLER, M. D., BORGMAN, C. A., COBB, B. S., VINES, R. R., REYNOLDS, A. B. & PARSONS, J. T. 1992. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci U S A, 89, 5192-6. YAU, T., CHAN, P., EPSTEIN, R. & POON, R. T. 2009. Management of advanced hepatocellular carcinoma in the era of targeted therapy. Liver Int, 29, 10-7

International PhD Student Cancer Conference 54 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A21 Control of microglia cytotoxic activities by glioma cells (Ivana Dokic), Renate Geibig, Anne Régnier-Vigouroux INSERM U701, German Cancer Research Centre, Program Infection and Cancer, INF 242, 69120 Heidelberg, Germany

Glioblastoma are malignant brain tumors highly infiltrated by microglia, the resident brain macrophages. Activated microglia are highly cytotoxic against glioma cells in vitro, but not in vivo. Glioma cells thus likely regulate and control microglia activation. New strategies for glioma treatment may arise from research on the nature of this regulation. Microglia capability for activation under conditions of co-culture with glioma cells was investigated. The read out for their level of activation upon treatment with LPS and IFN-gamma (LI) was the production of TNF-alpha and reactive oxygen species (ROS) which are hallmarks of microglia activation. Primary cultures of mouse microglia cells were cultured alone, in direct contact with glioma cells, or in indirect contact using transwell cultures. Microglia treatment with LI induced their high production of TNF-alpha and ROS. This production was significantly decreased when microglia were co-cultured directly with glioma cells. When cells were co-cultured in a transwell system, we observed no effect on microglial production of TNF-alpha and ROS. These observations indicate that the TNF-alpha and ROS responses of microglia are controlled by glioma cells through a direct cell-to-cell contact. Gap junctions may be involved in this type of microglia-glioma communication, a possibility that we are currently investigating.

International PhD Student Cancer Conference 55 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A22 Nck and N-WASP Signalling Networks and the Regulation of Arp2/3 dependent Actin Polymerisation (Sara Donnelly) Cancer Research UK London Research Institute, Cell Motility Laboratory, 44 Lincoln’s Inn Fields, London, WC2A 3PX UK Ina Weisswange Department of Developmental Biology, Institute for Zoology, Unversity of Heidelberg, INF 230, 69120 Heidelberg, Germany Michael Way Cancer Research UK London Research Institute, Cell Motility Laboratory, 44 Lincoln’s Inn Fields, London, WC2A 3PX UK

Actin polymerisation is required for a variety of biological processes including cell migration, endocytosis and pathogen spread. In cancer, regulation of many signalling networks including those that control actin polymerisation is perturbed. This can lead to defects in tumour cell adhesion and migration resulting in cancer metastasis. Understanding the molecular basis of these signalling networks is crucial to the development of new drug targets and treatments. Our lab uses vaccinia virus as a model to understand how signalling networks promote Arp2/3 dependent actin polymerisation. A vaccinia virus protein, A36, mimics receptor signalling at the plasma membrane and recruits a complex of cellular proteins including Nck, N-WASP, WIP and Grb2, which activates Arp2/3 to induce actin polymerisation. Previous studies have shown that this complex is highly dynamic and that rate of actin-based motility is dependent on the rate of N-WASP turnover. Recruitment of N-WASP is dependent on Nck, however interestingly Nck turns over 3.5 times faster than N-WASP. We have used Nck and N-WASP mutants and a variety of imaging techniques including FRAP to further elucidate the role of Nck in regulating actin polymerisation via N-WASP and Arp2/3.

International PhD Student Cancer Conference 56 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A23 A novel method for characterising p53-protein interactions in situ (Sarah Dowding), Theodoros Kantidakis and Robert J. White The Beatson Institute for Cancer Research, Glasgow, UK

Key to the wide variety of potential outcomes of p53 activation is the capacity of p53 to interact with an extensive range of proteins and for these interactions to change upon p53 modification. Our understanding of p53 relies, therefore, upon our ability to elucidate these interactions. We are optimising the use of proximity ligation assays (PLA) for this purpose. PLA is a relatively novel technique that allows the visualisation of interacting endogenous proteins in situ and has the potential to be especially useful where weak or transient interactions result in the failure of more invasive conventional methods such as coIP. Samples are fixed and incubated with primary antibodies against the two proteins of interest and, following a series of additional steps, a fluorescent signal is visible where the two proteins are in close proximity (<40nm). So in addition to supplying a method to view unstable interactions between endogenous proteins, PLA also tells us where in the cell these interactions take place and, as each signal is thought to be composed of a single protein-protein interaction, we can accurately quantify and compare binding events between different p53-activating conditions.

Soderberg, O., et al. (2006) Nat Methods 3, 995-1000

International PhD Student Cancer Conference 57 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A24 Using vaccinia virus to understand Rho GTPase signalling networks Durkin C, Cordeiro J, Way M Cell Motility Group, Cancer Research UK, London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK

During infection with vaccinia virus, host cells round, contract and bleb before re- spreading and migrating. Previous work in this lab has shown that a vaccinia protein, F11, is required for virus induced migration. F11 stimulates migration by binding and inhibiting RhoA signalling, through a motif with homology to ROCK1. Paradoxically, we have now found that vaccinia induced contraction is dependent on both F11 expression and RhoA activity (due to its sensitivity to Tat-C3). However, this activity is not due to a direct interaction between F11 and RhoA. Through drug treatments and siRNA approaches we hope to determine how F11 signals to RhoA to cause contraction. This study offers the potential to identify new cellular targets of F11 and therefore understand how a vaccinia protein can temporally both activate and inhibit the same signalling pathway. By studying how vaccinia manipulates RhoA we can further our understanding of RhoGTPase signalling networks.

International PhD Student Cancer Conference 58 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A25 FGFR2 expression networks and susceptibility to breast cancer (M Fletcher), K Meyer, B Ponder Cancer Research UK Cambridge Research Institute. Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE United Kingdom

A large proportion of the genetic risk of breast cancer is due to unknown causes. It is suggested that low-risk loci or polygenic effects may play a role. Fibroblast Growth Factor Receptor 2 (FGFR2) was the top hit from a recent genome-wide association study (GWAS). The investigation of downstream FGFR2 signalling targets offers new insights into the biology of risk. FGFR2-associated expression networks may lead to identification of novel risk loci, functional genes, and so a deeper understanding of the underlying genetic factors causing breast cancer.

International PhD Student Cancer Conference 59 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A26 Somatic cell reprogramming in the absence of Ezh2 (Giulia Fragola)1, Alessandro Blasimme1, Gustavo Mostoslavsky2 , Gabriele Bucci3, Giovanni Mazzarol4, Fridolin Gross1, Alexander Tarakhovsky5, Giuseppe Testa6 and Stefano Casola7. 1 European School of Molecular Medicine c/o Campus IFOM-IEO Via Adamello, 16 - 20139 Milan, Italy. 2 Section of Gastroenterology, Department of Medicine, Boston University School of Medicine and Center for Regenerative Medicine of Boston University, Boston, Massachusetts, USA. 3 Consortium for Genomic Technologies (COGENTECH) c/o Campus IFOM-IEO, Via Adamello, 16 - 20139 Milan, Italy. 4 Division of Histopathology, European Institute of Oncology, Milan, Italy. 5 Laboratory of Lymphocyte Signaling, The Rockefeller University, New York, USA. 6 IEO, European Institute of Oncology c/o IFOM-IEO Campus, Milan, Italy. 7 IFOM, the FIRC Institute of Molecular Oncology Foundation, Milano c/o IFOM-IEO Campus, Milan, Italy.

The Polycomb-mediated trimethylation on lysine 27 of histone H3 (H3K27me3), together with trimethylation on lysine 4 of histone H3 (H3K4me3) catalyzed by Trithorax proteins are commonly associated with, respectively, repressed and actively transcribed genes. Different pattern of distribution of these two epigenetic marks on promoters of master genes of differentiation specify different cell states. The ectopic expression of defined transcription factors in somatic cells causes the reacquisition of a pluripotent state in a process called cellular reprogramming. The comparison between the chromatin states of induced pluripotent stem cells and somatic cells uncovered that during transcription factor-induced reprogramming, cells undergo genome- wide epigenetic remodelling that leads to the acquisition of an open chromatin state and to the re-establishment of a pattern of distribution of histone modifications that is typical of embryonic stem cell. These observations suggest that Polycomb group proteins and Trithorax proteins play a critical role in somatic cell reprogramming. Our work is aimed at assessing this hypothesis by focusing on the role of histone H3K27 trimethylation in the reacquisition of pluripotency by somatic cells. We used a conditional allele for the histone methyltransferase Ezh2, in order to define how the removal of this epigenetic mark controls induction of pluripotency.

International PhD Student Cancer Conference 60 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A27 Dysregulation of the SUMO pathway by High-Risk Human Papillomaviruses (Daniele Frangioni1), Rosita Accardi2, Massimo Tommasino2 and Susanna Chiocca1 1 Department of Experimental Oncology, European Institute of Oncology at IFOM-IEO-Campus 20139 Milan, Italy 2 Infections and Cancer Biology Group, International Agency for Research on Cancer, 69008 Lyon, France

Cervical cancer remains the second most common cause of cancer deaths in women world-wide. More than 99% of cervical cancers are directly linked to Human Papillomavirus (HPV) infection with high-risk virus types. To create a suitable environment for surviving in the host cells, viruses hijack components of cellular pathways, among which the ubiquitin-like SUMO pathway. To investigate the possible link between HPV infection and SUMO pathway, we have been studying the effects of over-expressing oncoviral proteins from high risk HPVs in different cell lines. Analyzing the cellular expression of SUMO components, we found a down-regulation of the SUMO-conjugating E2 enzyme (UBC9) protein levels, but quantitative RT-PCR analysis showed no significative differences between transfected and non-transfected cells. Our preliminary data, therefore, suggest a post-transcriptional regulation of SUMO enzymes by high risk HPV viral proteins. In our approach we will try to explain (i) the molecular mechanisms of high risk HPV oncoproteins in the dysregulation of the SUMO pathway; (ii) the relevance of SUMO enzymes in the HPV life cycle using an organotypic raft culure; finally, (iii) how this is relevant for the pathogenesis of cervical cancer.

International PhD Student Cancer Conference 61 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A28 Sprouty2 acts as an inhibitor of EGFR signalling pathway via interfering with EGFR trafficking Meiling Gao, Rachana Patel, Janis Fleming, Jim Norman, Hing Leung

Prostate cancer is the most commonly diagnosed malignancy in men in North America, and second most common cause of cancer related death in UK men. There is emerging evidence that Sprouty proteins may be an important modulator of vital pathways central to the development or progression of cancer, such as cell growth, invasion, migration and cytokinesis. Prostate cancer progression is associated with overexpression of EGFR family members or activation of their downstream signalling pathways, such as PI3K/AKT and MAPK. To test the functional significance of loss of sprouty2 in prostate carcinogenesis, we knocked down Sprouty2 in DU145 and CWR22 cells. The results show increased proliferation with EGF stimulation, and have increase random motility and invasion in the presence of EGF. We used combinations of inhibitors including EGFR and its downstream signaling component such as AKT to define the key signaling events associated with loss of Sprouty2. Given the reported role of sprouty2 as a major regulator of the EGFR signaling cascade, the effect of loss of sprouty2 expression on EGFR trafficking was further examined, and the relevant downstream signaling component studied.

International PhD Student Cancer Conference 62 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A29 DNA demethylation and inhibition of HDAC1 and HDAC2 is sufficient to reactivate epigenetically silenced tumor suppressor genes. [Ernst Geutjes], Rene Bernards, Netherlands Cancer Institute, Amsterdam (NL)

Loss of tumor suppressor gene (TSG) function contributes heavily to both the initiation and progression of cancer and can be the result of mutations in the DNA sequence, but can also be caused by epigenetic disruptions. Promoter hypermethylation and histone hypoacetylation play a key role in epigenetic silencing of TSGs. Indeed, the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (DAC) and the broad spectrum histone deactelyase (HDAC) inhibitor vorinostat (SAHA) are promising anti-cancer drugs which can synergistically reactivate epigenetically silenced TSGs when combined. Although pan-HDAC inhibitors have clear anti-tumor activity in the clinic they induce side effects. Thus, there is great interest to develop selective HDAC inhibitors. However, it is currently unclear which HDAC(s) to hit. We thus screened a HDAC siRNA library for their ability to synergize with DAC in reactivation of silenced TSGs. We found that a combination of HDAC1 and HDAC2 knockdown synergized with DAC treatment in the re-expression of epigenetically silenced TSGs. Interestingly, we found that a compound selectively targeting HDAC1-3 was as effective in inducing cell death as pan-HDAC inhibitors suggesting that HDAC1&2 inhibitors might be as effective as pan-HDACi as anti-cancer drugs.

International PhD Student Cancer Conference 63 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A30 HPV involvement in squamous cell carcinoma of the larynx Authors: (Gordana Halec)1,2, Markus Schmitt2, Gerhard Dyckhoff1, Michael Pawlita2, Franz X. Bosch1 1 Molecular Biology Laboratory, Department of Otolaryngology, Head and Neck Surgery, Heidelberg University, Heidelberg, Germany 2 Division of Genome Modifications and Carcinogenesis, Research Program Infection and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany

Background: Mucosal high-risk human papillomavirus type 16 (HR- HPV16), is recognized as causal factor for majority of cervical squamous cell carcinomas (SCC) and also a subset of oropharyngeal SCC. The involvement of HR-HPV16 in laryngeal (L-)SCC is still an open question. Material and Methods: L-SCC tissues from 105 German patients were analyzed for HPV DNA by multiplex genotyping and for expression of three cellular proteins p53, pRb and p16 by immunohistochemistry (IHC). HPV DNA-positive tumors were analyzed for HR-HPV mRNA by RT–PCR. Results: Eighty-one L-SCC yielded analyzable DNA. Twenty-eight (35%) contained HPV sequences of which 75% were HR-HPV16. Additionally, HR-HPV35,45,56 and low-risk (LR-)HPV11,42,70 were detected. Only three HR-HPV16 DNA+ L-SCC expressed mRNA. Those three HR-HPV16 DNA+/RNA+ and surprisingly the two LR-HPV11 and HPV70 DNA+ L-SCC (RNA analyses pending) showed the expected HPV IHC pattern with down-regulated p53, pRb and up-regulated p16. Conclusions: This study provides evidence for an etiologic role of HR-HPV16 in a very small subset (4%) of German L-SCC. The presence of DNA of LR types HPV11 and HPV70 in combination with an HPV-typical immunohistochemistry pattern raises the question whether LR-HPV types, usually associated with benign anogenital warts and oral papillomas, can have a role in L-SCC development.

International PhD Student Cancer Conference 64 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A31 Aberrant Epigenetic Regulation and Self Renewal in Acute Myeloid Leukaemia Stem Cells (W.J Harris) and T.C.P Somervaille. Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom

Rearrangements involving the mixed lineage leukaemia (MLL) gene can be found in around 10% of human leukaemias and are associated with particularly poor prognosis in childhood ALL and treatment-related secondary leukaemias. These diseases are likely propagated by a deregulated self renewing pool of leukaemia stem cells (LSCs). Characterisation and targeting of the LSC pool represents a key novel strategy for the treatment of AML. It is also becoming increasingly clear that epigenetic regulation of gene expression at the chromatin level is critical for self renewal, proliferation and differentiation decisions in normal stem cells. A recent study (Somervaille et al., 2009) identified a set of genes associated with self renewal in a murine model of human MLL translocation AML. A key chromatin regulatory gene, Cbx5, is critically involved in LSC self renewal. This finding led to the current study which aims to identify other chromatin regulatory genes important for LSC self renewal. An extensive chromatin-annotated RNAi screen is being tested in primary AML patient cells to identify novel targets. An advantage of targeting these genes is that whilst the underlying mutations are genetic and therefore irreversible, the effects they induce are epigenetic and thus may be pharmalogically reversible.

International PhD Student Cancer Conference 65 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A32 Head-on Collision - Characterisation of RNA Polymerase II Elongation (David J. Hobson), Jesper Q Svejstrup Both Cancer Research UK LRI Clare Hall, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, UK

During the production of mRNA, RNA polymerase II encounters many obstacles to its progression. Such obstacles include histones, DNA binding proteins or other elongation complexes. Studies have shown that non-coding RNAs are produced in S. cerevisiae in an opposite orientation to gene open reading frames. Additionally, convergent orientation of genes can result in elongation complex interactions prior to termination. No studies have been published to-date showing whether convergently transcribing elongation complexes collide or bypass. Using in vitro biochemistry to form convergent constructs we show that RNAPII elongation complexes cannot transcribe past each other. However, preliminary data shows that upon incubation with cell extract bypass does occur, suggesting a factor may stimulate bypass in vivo.

International PhD Student Cancer Conference 66 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A33 Determining the Structure of Human FANCL (Charlotte Hodson), Ambrose R Cole, Helen Walden Fanconi Anemia (FA) is a genetic disorder, which predisposes patients to cancers.

The FA pathway recognises DNA interstrand cross-links (ICLs) and recruits the DNA repair machinery. The FA core complex (CC) contains an E3 ubiquitin ligase, FANCL, responsible for the monoubiquitination of FANCI and FANCD2, the essential step in the pathway. Therefore, the FA pathway is a useful biological system to study both ubiquitination and DNA ICL repair. Recently our lab has solved the structure of Drosophila FANCL, which has 20% sequence identity with human FANCL and ~60% sequence similarity. There is currently no core complex evident in the Drosophila system, and sequence comparisons between the organisms do not explain the apparent requirements for the CC in higher eukaryotes, highlighting the importance of gaining structural insights into human FANCL. To date, I have solved the structure of 50% of the human FANCL protein. Based on structural analyses, I am designing functional assays, in order to understand the role of FANCL in the CC, alongside structure determination of the rest of human FANCL. Unveiling the structure of human FANCL will allow a greater understanding of the biochemistry of the FA pathway and the role of E3 ligases in selective monoubiquitination.

International PhD Student Cancer Conference 67 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A34 S-trityl-L-cysteine analogues, investigated in cell lines over-expressing P-glycoprotein, show potential in overcoming multidrug resistance (Hung Yi Kristal Kaan)1, Johanna Weiss2, Dominik Menger2, Venkatasubramanian Ulaganathan1, Christian Laggner4, Florence Popowycz3, Benoît Joseph3, Frank Kozielski1 1 The Beatson Institute for Cancer Research, Switchback Road, Bearsden, Glasgow G61 1BD, Scotland, UK, 2 University Hospital Heidelberg, Department of Clinical Pharmacology and Pharmacoepidemiology, Im Neuenheimer Feld 410, D-69120 Heidelberg, Germany, 3I nstitut de Chimie et Biochimie Moleculaires et Supramoleculaires, UMR-CNRS 5246, Universite de Lyon, Universite Claude Bernard - Lyon 1, Batiment Curien, 43, Boulevard du 11 Novembre 1918, F-69622 Villeurbanne, France. 4 Institute of Pharmacy, Department of Pharmaceutical Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 52c, 6020 Innsbruck, Austria

The mitotic spindle is a validated target for cancer chemotherapy. Drugs such as taxanes and vinca alkaloids specifically target tubulin, interfere with microtubule dynamics, and cause the mitotic spindle to collapse. However, toxicity and resistance are clinical problems associated with these drugs. Thus, alternative approaches to inhibiting the mitotic spindle are currently being pursued. These include targeting mitotic spindle proteins such as Eg5, a human kinesin involved exclusively in the separation of the bipolar spindle in proliferating cells. We previously identified S-trityl- L-cysteine (STLC), an antimitotic agent with antitumour activity, as a potent allosteric inhibitor of Eg5. Here, we report the synthesis of a new series of STLC-like compounds with improved efficacy and in vitro Kiapp in the low nanomolar range. We also performed a multidrug resistance study in cell lines over-expressing P-glycoprotein and showed that some of these inhibitors may have the potential to overcome susceptibility to this efflux pump and hence, overcome resistance associated with tubulin-targeting drugs. Additionally, we determined the structure of a new Eg5-inhibitor complex and described the important interactions between the protein and the ligand. Finally, we performed molecular docking studies of our new series of STLC-like compounds to explain the structure-activity relationship of these compounds.

International PhD Student Cancer Conference 68 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A35 Binding of the nuclear envelope protein nesprin-3 to its interaction partner plectin [Mirjam Ketema] Ingrid Kuikman, Ruben Postel and Arnoud Sonnenberg The Netherlands Cancer Institute, Division of Cell Biology I, Amsterdam, The Netherlands

The nucleus is enclosed by a nuclear envelope (NE), which consists of an inner and an outer nuclear membrane. The outer nuclear membrane is characterized by a specific set of proteins: the nesprin protein family. This family enables the connection of the NE to the cytoskeleton. We have previously demonstrated that nesprin-3 binds to the actin binding domain (ABD) of plectin and that this interaction establishes a link between the NE and the intermediate filament system. Interestingly, the plectin ABD can also bind F-actin and the integrin beta 4 subunit. In fact, we have now demonstrated that the same amino acids in the plectin ABD that were previously shown to be required for the binding to beta 4 are also essential for the interaction with nesprin-3. Furthermore, we narrowed down the plectin binding site on nesprin-3 to seven amino acids in the first spectrin repeat. Deletion of these amino acids completely abolished both the binding to plectin and its recruitment to the NE. In conclusion, our study showed that overlapping sites on the plectin ABD are responsible for binding beta 4 and nesprin-3. This suggests that nesprin-3 and beta 4 bind mutally exculsive and compete for plectin binding.

International PhD Student Cancer Conference 69 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A36 Screening for key players and inhibitors of the homologous recombination DNA repair pathway. (Guy L Kingham), Cecilia Lundin, Thomas Helleday. Helleday Group, Gray Institute of Radiotion Oncology and Biology, Oxford, UK

The homologous recombination (HR) DNA repair pathway mediates the high fidelity repair of DNA double strand breaks, we are currently conducting (a) an RNA interference screen to identify key players in HR and (b) chemical library screens to identify small molecule HR inhibitors. Studies in our lab have suggested that knockdown of proteins involved in homologous recombination is toxic to the Du145 prostate cancer cell line, whilst other labs have shown that compounds active against HR can be used as radiosensitisers. Inhibition of HR has also been shown in our lab to cause selective toxicity to poly-ADP-ribose polymerase 1 (PARP1) knockout mouse embryonic fibroblasts which are deficient in the single strand break DNA repair pathway, suggestive of a synthetic lethal interaction. These observations suggest HR inhibitors may have the potential to selectively kill cancer cells. We aim to use high throughput screening to identify specific small molecule inhibitors of HR to explore this hypothesis. In addition to identification of HR inhibitors, siRNA screens are being employed to characterise the HR network in an effort to identify new targets for inhibition.

International PhD Student Cancer Conference 70 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A37 The junctional protein ARVCF and its association with splicing factors in the nucleus Ulrike Krebs1, Svenja Ternes1,2, Ilse Hofmann1 1 Joint Research Division Vascular Biology of the Medical Faculty Mannheim, University of Heidelberg, and the German Cancer Research Center (DKFZ), Mannheim, Germany 2 Current address: Institute of Molecular Psychiatry, University of Bonn, Bonn, Germany

Protein ARVCF (armadillo repeat gene deleted in velo cardio facial syndrome) is a member of the p120 family of arm-repeat proteins. All members are characterized by their dual localization und multiple functions within the cells: they maintain cellular junctions, but also participate in intracellular processes. The interaction of ARVCF with cadherins in adherens junctions is well known. The present results show a prominent distribution of ARVCF in mitosis: in early stages a diffuse distribution throughout the cytoplasm, in later stages a localization in distinct foci. A similar distribution has also been described for proteins of the splicing/transcription machinery; therefore, we compared the localization of ARVCF with the splicing factor SF2/ASF. Indeed, both are found in structures called mitotic interchromatin granlues (MIGs). Moreover, the formation of MIGs is further enhanced in stressful situations such as oxidative stress. In interphase cells, we found ARVCF in the nucleus again colocalizing with SF2/ASF in nuclear speckles. In addition, ARVCF accumulated in the nucleus after transcription inhibition, which has been described for proteins that are exported from the nucleus through an NES-dependent mechanism. Further analysis of putative interactions of ARVCF with proteins involved in RNA-processing may unravel novel intracellular functions of this protein.

International PhD Student Cancer Conference 71 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A38 Pancreatic Stellate Cells modify the radioresponse of pancreatic cancer Serena Lunardi, Tine Mantoni, Osama Al-assar and Thomas B Brunner Gray Institute for Radiation Oncology and Biology, University of Oxford (UK)

Pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant stromal response. One of the main actors in promoting this desmoplastic reaction is the cell type known as pancreatic stellate cells (PSCs). The aim of our study is to analyse the tumour response to radiation treatment in the presence of PSCs and to determine the stromal and tumour factors involved in this response. PSCs were used in a coculture system with different PDAC cell lines. Their effect on radiation survival was tested using clonogenic survival assays. Panc1 and PSN1 PDAC cell lines showed decreased radiosensitivity when cocultured with PSCs. Coinjection of PDAC with PSCs in nude mice enhanced tumour proliferation. Furthermore, tumour regrowth experiments after irradiation showed that PSCs containing tumours respond to radiation treatment less than cancer cells alone. Consequently, using a proteomic approach we identify three factors that were differentially expressed in the coculture supernatant compared to the monocultures. In conclusion, these data demonstrate that PSCs decrease radiation response and promote tumour growth of PDAC. Further investigations of the role of the factors identified in our screen could lead to a better understanding of pancreatic cancer biology and possibly to new targets for multimodal therapy.

International PhD Student Cancer Conference 72 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A39 Visualising signalling in melanoma metastasis (Cerys Manning), Tumour Cell Biology Laboratory, Cancer Research UK London Research Institute, London, UK Erik Sahai, Tumour Cell Biology Laboratory, Cancer Research UKLondon Research Institute, London, UK

Metastasis is a multi-step process that begins with the acquisition of motility in the primary tumour. Intra-vital imaging of mouse tumours can be used to explore the dynamics of cell motility and has enabled the visualisation of cells moving collectively in groups or chains and single motile cells. It has been hypothesised that the motile cells in a tumour are less differentiated and it is these cells that invade and form metastases. Notch signalling is implicated in the maintenance of the stem cell population in many cell types including melanocytes and therefore I have used intra- vital imaging to study the role of Notch signalling in melanoma. Using a Notch reporter melanoma cell-line I have found that the singly motile cells have higher levels of Notch signalling than non-motile cells and cells moving collectively. Notch signalling is also mutually exclusive with pigment production, an indicator of differentiation, which has recently been associated with actively metastasising cells in vivo. In a mouse lung colonisation assay, cells with actively driven Notch signalling were modestly over- represented compared to control cells 10 days after injection. My work suggests that Notch signalling may maintain melanoma cells in a less-differentiated state and, together with other motility cues, enable them to invade and form metastases.

International PhD Student Cancer Conference 73 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A40 Signaling modulation at G2/M transition: old and new players Stefano Marchesi1, Francesca Montani2, Serena Bologna1,4, Carmela Mazzoccoli1,5, Fabrizio Bianchi1, Francesco Nicassio1 e Pier Paolo Di Fiore1,2,3. 1 Fondazione IFOM, via Adamello 16, Milano. 2 Dipartimento di Oncologia Sperimentale, Istituto Europeo di Oncologia, Via Adamello 16, Milano. 3 Dipartimento di Medicina, Chirurgia ed Odontoiatria, Universita’ di Milano, via Rudin√¨ 8, Milano. 4 Institute of Molecular Cancer Research, University of Zurich, August Forel Street 7, Zurich. 5 Centro di Riferimento Oncologico della Basilicata, via Padre Pio 1, Rionero in Vulture (PZ).

The cell cycle progression is an extremely complex process, which fidelity is monitored by a variety of control systems: the checkpoint mechanisms. Signaling events coming from different routes (i.e. growth factors, cytokines, cell-to-cell contacts or DNA damage) are concerted for ensuring normal cell division by regulating the cell-cycle checkpoints. Among those, the G2/M checkpoint is the most involved in the control of genome integrity, ensuring its preservation before undertaking mitosis; thereby it has a considerable impact on cellular transformation and cancer. Recently we identified DEPDC-1A and DEPDC-1B proteins as new players of the pathway modulating G2/M transition. Depletion of the two proteins inhibits proliferation and causes a delay in mitotic entry in normal and cancer cell lines, suggesting that DEPDC-1A/1B are necessary for a correct G2/M transition. DEPDCs hold a predicted Rho-GAP domain and biochemically interact with RhoA and Rac1, in their active (GTP loaded) conformation, arguing for a role of RhoGTPases in this pathway. Indeed, Rho-GTPases depletion completely rescues the mitotic defect of DEPDC silenced cells, thus revealing a genetic interaction between the two genes and the aforementioned Rho-GTPases. As downstream event, we also identified the MAPK-Erk cascade, that is active from late S to G2/M transition for a correct onset of mitosis. Upon DEPDCs depletion MAPK activity is increased, while pharmacological modulation (by U0126) is sufficient to fully rescue the mitotic phenotype, reinforcing the idea that coordinated signaling events control the G2/M transition. In conclusion, our study proposes a new intriguing role of Rho-GTPases in the modulation of cell cycle progression and identifies DEPDCs, together with RhoA/Rac1 and MAPK, as major players of this G2/M signaling pathway.

International PhD Student Cancer Conference 74 May 19, 2010 – Keynote lecture, lecture , oral presentations and poster session I

A41 REGULATION OF THE E3 LIGASE NEDD4 BY EGFR Sara Mari *, Elena Maspero * and Simona Polo *§ * IFOM, the FIRC Institute for Molecular Oncology, Via Adamello 16, 20139, Milan, Italy § University of Milan Medical School, 20122 Milan, Italy

Stimulation of cells with epidermal growth factor (EGF) results in the activation of the intrinsic tyrosine kinase of the EGFR and the establishment of a complex signalling network where both ubiquitination and phosphorylation regulate protein interactions. Eps15 is one of the endocytic adaptor proteins that undergo coupled monoubiquitination upon EGF stimulation through the action of the E3 ligase Nedd4 1,2. How the signal from EGF is delivered to the Ubiquitination machinery was unknown. We have now evidences that Nedd4 is both ubiquitinated and tyrosine phosphorylated upon EGF stimulation. Using an integrated approach based on pharmacological inhibitors and molecular genetics tools we found that the kinase activity of both, EGFR and Src are required for Nedd4 phosphorylation as well as for eps15 monoubiquitination. To gain insight on a possible regulative role exerted by these post-translation modifications on the ligase activity we set up mass spectrometry approach to identify phosphorylation and ubiquitination sites present in Nedd4 protein upon EGF stimulation. Results will be presented.

International PhD Student Cancer Conference 75 May 20, 2010 – Lectures, poster session II and oral presentations

A42 A role for Sox7 at the onset of blood specification (Andrzej Mazan), Georges Lacaud, Valerie Kouskoff. Cancer Research UK Stem Cell Haematopoiesis, Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom

We previously demonstrated that haematopoietic cells are derived from haemangioblast through the intermediate formation of a haemogenic endothelium. Recent findings have also revealed that Sox7 is transiently expressed at the onset of haematopoiesis suggesting that Sox7 might be implicated in cell fate specification. To address this question, we will first characterize the cells expressing Sox7 upon haemangioblast specification. This analysis will be performed using a Sox7-GFP knock-in transgenic ES line, which will allow to determine where and when Sox7 is expressed during early blood development. Using the in vitro differentiation of ES cells, we will fully characterize the biological potential of Sox7-expressing cells. Moreover, we will investigate the role of Scl and Runx1, two key transcription factors important in early blood development, in the generation, maintenance and maturation of the Sox7+ subpopulation by using Sox7-GFP Runx1-/- and Sox7-GFP Scl-/- ES cells. The second aim of this project will be to perform loss-of-function studies of Sox7 in vitro and in vivo to assess its impact on blood specification. The outcome of these experiments will define whether Sox7 expression is required for blood specification and at which stage it is critical.

International PhD Student Cancer Conference 76 May 20, 2010 – Lectures, oral presentations and poster session II

SESSION IIa: Cell migration

SESSION IIb: Immunology

SESSION III: Modeling and large scale analysis in cancer research

SESSION IV: Genome instability and cancer

International PhD Student Cancer Conference

May 20, 2010 – Lectures, poster session II and oral presentations

Lectures Pier Paolo Di Fiore Istituto FIRC di Oncologia Molecolare and Istituto Europeo di Oncologia, Milan

Silvia Camporesi and Alessandro Blasimme FOLSATEC PhD Students, European School of Molecular Medicine, Milan Istituto FIRC di Oncologia Molecolare and Istituto Europeo di Oncologia, Milan

Oral presentations Anne Lempens Beatson Institute for Cancer Research, Glasgow

Roheet Bantval Rao Cambridge Research Institute, Cambridge, UK

Melda Tozluoglu London Research Institute, London

Tim Schnyder London Research Institute, London

Eva Schlecker German Cancer Research Centre, Heidelberg

Christiaan Klijn The Netherlands Cancer Institute, Amsterdam

Xiaofan Li London Research Institute, London

Gilda Nappo The FIRC Institute of Molecular Oncology Foundation, IFOM, Milan

Rebecca Burrell London Research Institute, London

Marieke Peuscher The Netherlands Cancer Institute, Amsterdam

Tanja van Harn The Netherlands Cancer Institute, Amsterdam

Dhaval Varshney Beatson Institute for Cancer Research, Glasgow

Sietske Bakker The Netherlands Cancer Institute, Amsterdam

International PhD Student Cancer Conference 79

May 20, 2010 – Lectures, poster session II and oral presentations

Endocytosis and recycling at the crossroad of signaling, attenuation, execution of polarized functions and cell fate determination. Pier Paolo Di Fiore Istituto FIRC di Oncologia Molecolare and Istituto Europeo di Oncologia, Milan, Italy.

Receptor Tyrosine Kinases (RTKs) are internalized following their engagement by cognate ligands, in a process that has been traditionally considered crucial for long- term attenuation of receptor signaling. More recent evidence has pointed also to an important role of endocytosis in conferring spatial and temporal dimensions to signaling (the signaling endosome concept). Once internalized, RTKs are routed to early endosomes where decisions are made whether to commit the receptor for degradation or to recycle it to the cell surface. We will present evidence that recycling of RTKs, and of associated cellular machinery, is also pivotal to the execution of signaling functions, especially those connected with polarized activities, such as cell motility. Of particular interest, in this contention, is an accessory endocytic protein called Numb. Numb is critical for the recycling of certain RTKs to the plasma membrane, and in the organization of remodeling of the actin cytoskeleton leading to motility. In addition Numb is a cell fate determinant that by asymmetrically partitioning at mitosis controls binary cell fate decisions in various developmental systems. In human breast and lung cancers, there is frequent loss of Numb expression, which is due to its enhanced degradation. This causes alterations in two major downstream pathways. On the one hand, lack of Numb allows for unchecked signaling activity of the Notch receptor. On the other, lack of Numb causes attenuation of the p53 signaling pathway. Tumors cells displaying loss-of-Numb expression are addicted to this event and to its molecular consequences. Thus, Numb is a bona fide tumor suppressor gene in breast cancer and it is capable of controlling simultaneously both an oncogenic pathway (Notch) and a tumor suppressor one (p53). Our recent results point to the mammary stem cell (MSC) compartment as the cellular “target” of Numb misregulation in breast tumors. We have developed a technology to cultivate ad purify mammary stem cells both from normal and neoplastic tissues. In normal MSC, Numb is asymmetrically partitioned at mitosis. This in turn dictates the replicative fate, in that the Numb(+) cell remains quiescent (and retains MSC capabilities), whereas the Numb(-) cell acquires a progenitor fate and undergoes rapid symmetric divisions. The control of Numb over MSC fate is executed through the ability of Numb of silencing Notch signaling and maintaining high levels of p53 in the MSC. Lack of Numb in tumor MSC causes a switch form the asymmetric to the symmetric mode of division, thus forcing both daughter cells to assume the same replicative fate. Our understanding of how Numb is mechanistically involved in all these aspects will be discussed.

International PhD Student Cancer Conference 81 May 20, 2010 – Lectures, poster session II and oral presentations

World-wide human embryonic stem cell policies: who decides and on what grounds? Silvia Camporesi and Alessandro Blasimme FOLSATEC PhD Students, European School of Molecular Medicine, Milan Istituto FIRC di Oncologia Molecolare and Istituto Europeo di Oncologia, Milan

Pluripotent cells of embryonic origin are extraordinary tools to model diseases in vitro and to provide tissue to be used for regenerative medicine as well as gametes for reproductive ends. For these reasons human embryonic stem (hES) cells have attracted a great deal of attention well beyond the scientific community, among lay publics, patients’ groups, religious authorities and policy-makers. Although big financial efforts and rigorous scientific work have been already devoted to hES cell research, many more years of study and continued financial support are needed in order to gain enough biological knowledge and technical skills to see appreciable traces of clinical translation. The derivation and use of hES cells, however, are morally contested. The bioethical issues arising from hES cell research range widely, from the moral status of embryos to the commodification of body parts needed to generate hES cells, from disruption of traditional familial lineages to the quest for physical and mental enhancement, from cloning to genetic selection, from participation in clinical trials to exclusive commercial rights on stem cell lines and their clinical applications. Indeed, new democratic solutions have been devised to regulate stem cell research, both at the national level (especially in North America, and in Europe) and internationally (EU, UN). Most notably, science itself reacts to the regulative constraints and the normative urges by reshuffling its epistemic standards and experimental procedures, as exemplified in the case of induced pluripotent stem (iPS) cells. We will analyse how advanced societies cope with the moral, political and financial costs of stem cell research. We will show what models of scientific governance have been established and how those models incorporate different views about the ideal relationship between the scientific community, bioethicists and public decision makers. Finally, we will discuss with the audience both the pragmatic consequences and the ethical foundations of these models.

International PhD Student Cancer Conference 82 May 20, 2010 – Lectures, poster session II and oral presentations

ERK2 but not ERK1 contributes to invasive migration of tumour cells (Anne Lempens)1, Marc Birtwistle2, Oliver Rath1, Jim Norman1, Walter Kolch2 and Alexander von Kriegsheim1 1 Beatson Institute for Cancer Research, Glasgow, United Kingdom 2 Systems Biology Ireland, University College Dublin, Dublin, Ireland

The ERK pathway regulates fundamental cellular processes and is hyperactivated in many human cancers. Since the two predominant isoforms of ERK (ERK1 and ERK2) display common substrate binding domains and have similar spatio-temporal regulation, both are generally believed to be redundant and interchangeable. In contrast to this conventional view, here we show that ERK2 but not ERK1 knockdown significantly inhibits invasive migration in two independent cancer cell lines. Further re-expression of ERK2 but not ERK1 is sufficient to rescue the invasiveness of cells with reduced endogenous ERK2 levels. A detailed quantitative analysis indicates that ERK2 but not ERK1 knockdown decreases migration velocity and increases cellular immobility. Our data emphasise the importance of the MAPK pathway in tumour invasion, and show for the first time that both ERK isoforms play different roles during migration on 3D matrices. To conclude, the data suggest that specific targeting of ERK2 in cancer patients rather than blocking the pathway with MEK inhibitors might be a more selective and effective route to anti-invasive therapy.

International PhD Student Cancer Conference 83 May 20, 2010 – Lectures, poster session II and oral presentations

Huntingtin interacting protein 1 (HIP1) induces Epithelial-to- Mesenchymal Transition (EMT) in prostate cancer cells (Roheet Bantval Rao), David Neal, Ian Mills-CRUK Cambridge Research Institute, Cambridge CB2 ORE, UK Jianghua wang, Micheal M Ittmann Department of Pathology, Baylor College of Medicine and Michael E. DeBakey Veterans Affairs Medical Center, Houston, TX 77030, USA (Rao R B)1, Wang J2, Ittmann MM2 ,Neal D1, Mills IG1 1 CRUK Cambridge Research Institute, Cambridge CB2 ORE, UK 2 Department of Pathology, Baylor College of Medicine and Michael E. DeBakey Veterans Affairs Medical Center, Houston, TX 77030, USA

HIP1, an adaptor protein classically involved in clathrin mediated endocytosis affecting cell signalling, is overexpressed in prostate cancer and drives fibroblast and prostate epithelial cell transformation by perturbing growth factor receptor signalling. To explore its role in prostate cancer we used HIP1 overexpressing (HIP1+) prostate cell lines in Soft agar colony formation, invasion, migration, and adhesion assays. HIP1+ epithelial PNT1a cell lines showed cell transformation. HIP1+ PNT1a and HIP1+ LNCaP showed significantly increased anchorage independent cell growth. HIP1+ LNCaP also showed significantly increased cell adhesion to ECM protein fibronectin, implicated in cancer growth/survival and drug resistance. Epithelial-to-mesenchymal transition (EMT) is associated with increased propensity for cell migration, invasion, and metastasis. Here, we show a >2 fold upregulation of Wnt7b, Snail, and vimentin in HIP1+ cell lines, which have been implicated in EMT. Gene expression arrays showed enrichment of pathways involved in cell-cell signalling, cell movement and metabolic pathways in these HIP1+ cell lines. Furthermore, HIP1+ PNT1a cell lines showed a resistance to paclitaxel treatment in soft agar colony formation assays. In conclusion, HIP1 may contribute to prostate cancer progression by altering cell-cell interaction, migration, and invasion through induction of an EMT-like phenotype.

International PhD Student Cancer Conference 84 May 20, 2010 – Lectures, poster session II and oral presentations

Multiscale Modelling of Cancer Cell Motility [Melda Tozluoglu] London Research Institute, Cancer Research UK, London, UK Erik Sahai London Research Institute, Cancer Research UK, London, UK Paul Bates London Research Institute, Cancer Research UK, London, UK

Cell migration is a concept significant in many biological processes including metastasis in cancer. Inhibiting metastasis is as crucial as minimizing tumour growth for efficient treatment of cancer. In a multi-scale model of cancer cell motility, our primary focus will be amoeboid type cell motility of metastasizing tumour cells. In amoeboid motility, the cells have a flexible morphology and they utilise spherical, pressure driven protrusions, blebs, for motility. We validate our model with the data on dynamics of bleb formation, and we test the movement ability of the cell under changing conditions. The multiscale model of cancer cell motility covers intrinsic cell properties, extracellular conditions, and the relation in between the two. The intrinsic cell properties include stiffness of the cell, membrane-cortex attachment and the dynamics of these properties. The extracellular conditions include ECM density and topology, morphogen gradients affecting cell contractility and membrane-cortex attachment. Our model can be extended to include mesenchymal type cell motility, transition phases motility types, and multicellular systems. The model covers a wide parameter space and will provide a deeper understanding of the conditions governing the motility of the cell at multi-scale levels.

International PhD Student Cancer Conference 85 May 20, 2010 – Lectures, poster session II and oral presentations

Spatially resolved negative regulation of B cell receptor signalling (Tim Schnyder) and Facundo D Batista Lymphocyte Interaction Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln’s Inn Fields, London, WC2A 3PX, UK

B cells constitute an important part of the immune system by producing high-affinity antibodies that target foreign antigens for destruction. Previous studies have shown that the functional role of B cells is significantly compromised when activating signals are not properly controlled leading to hyper-activation, autoimmune disease, and cancer. Therefore, we want to understand how B cell receptor (BCR)-induced signalling is negatively regulated. BCR-mediated antigen recognition on the surface of antigen- presenting cells is characterised by a two-step process of spreading and contraction. Signalling downstream of the BCR is propagated during spreading. Interestingly, signal attenuation is a requirement for accurate B cell contraction. To answer which molecules are involved in signal attenuation during the contraction phase, we have combined genetic approaches with high-resolution microscopy. We observe that the Ubiquitin ligase Casitas B-lineage lymphoma (Cbl) and the adaptor molecules Growth Factor Receptor-bound Protein 2 (Grb2) and Downstream of Kinase 3 (Dok3) promote B cell contraction. In line with this we show the dissociation of the tyrosine kinase Syk from the central antigen cluster after contraction. We consequently propose signal attenuation as a prerequisite for cellular contraction and eventual shutdown of signalling in B cells.

International PhD Student Cancer Conference 86 May 20, 2010 – Lectures, poster session II and oral presentations

Control of anti-tumor immune responses by myeloid-derived suppressor cells Eva Schlecker, Ana Stojanovic and Adelheid Cerwenka Innate Immunity Group, German Cancer Research Center, Heidelberg, Germany

Myeloid-derived suppressor cells (MDSC) represent a heterogeneous population of myeloid cells comprising immature macrophages, granulocytes, dendritic cells at different stages of differentiation. During tumor progression MDSC accumulate in blood, lymph nodes, bone marrow and at tumor sites in cancer patients and tumor-bearing mice. MDSC are potent suppressors of various T-cell functions, by means of increased p r o d u c t i o n o f r e a c t i v e o x y g e n a n d n i t r o g e n s p e c i e s a s w e l l a s e l e v a t e d l e v e l s o f a r g i n a s e - 1 . In our study we investigated whether MDSC regulate NK cell-mediated antitumor immune responses against the MHC class I deficient RMA-S lymphoma. Unexpectedly, we discovered that the mononuclear subpopulation of MDSC potently activated NK cells to produce high amounts of interferon-γ. Of note, this subpopulation suppressed proliferation of T cells via nitric oxide, confirming its T-cell suppressive phenotype. In search for the activating mechanism, we observed that a ligand for the activating NK-cell receptor NKG2D, RAE-1 was exclusively expressed on the mononuclear MDSC subpopulation. Furthermore, activated NK cells eliminated the mononuclear subpopulation in vitro and upon adoptive transfer also in vivo. In order to identify molecules and pathways that might be involved in MDSC accumulation in tumor bearing mice and their suppressive/activating function, gene expression profiling of blood and tumor-infiltrating MDSC subpopulations was performed using whole genome microarrays. Tumor-infiltrated mononuclear MDSC revealed increased expression of genes involved in inflammatory responses and chemotaxis. Our data reveal that MDSC do not suppress all aspects of anti-tumor immune responses and defines a novel, unexpected activating role of MDSC on NK cells. Understanding the reciprocal interaction of MDSC with NK cells might improve the efficiency of cancer immunotherapy.

International PhD Student Cancer Conference 87 May 20, 2010 – Lectures, poster session II and oral presentations

Finding co-occurrence and mutual exclusiveness of DNA copy number changes. (Christiaan Klijn1,2), Jan Bot2,3, David J. Adams4, Marcel Reinders2, Lodewyk Wessels1,2, Jos Jonkers1. 1 Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands 2 Information and Communication Theory Group, Delft University of Technology, Delft, The Netherlands 3 Netherlands Bioinfomatics Centre, Nijmegen, The Netherlands 4 Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1HH, UK

Cancer, a genetic disease, is only rarely caused by a single mutation. Often, multiple (epi)genetic mutations have to occur to cause oncogenic transformation. Cancer genes can be disrupted by many different processes of which DNA copy number change due to genomic instability is one. Although many studies have addressed the detection of important single copy number changes in different tumor types, no studies have thoroughly investigated the interdependence of copy number changes. Our study focuses on the detection of co-occurrent or mutually exclusive DNA copy number changes. We show that, in lymphoid tumors, there are large networks of related copy number changes over the entire genome. The genomic locations that show significant co-occurrent copy number changes also contain many more functionally related genes and cancer associated genes than expected by chance. Our observations support the notion that subtle gene dosage changes of many related genes could be an overlooked but important process in cancer. Furthermore, in a collection of mouse mammary tumors we identify mutually exclusive regions of amplification. The target genes of these amplifications can be linked to Epithelial-to-Mesenchymal transition and mitogenic signalling, possibly suggesting new roles for these known oncogenes.

International PhD Student Cancer Conference 88 May 20, 2010 – Lectures, poster session II and oral presentations

How does a protein find its mates in a crowded cell? (Xiaofan Li), Iain Moal and Paul Bates BMM Lab, Cancer Research UK London Research Institute

A living cell contains a crowded mix of heterogeneous macromolecules; yet it orchestrates highly efficient biochemical reactions with low molecular copies. This characteristic phenomenon, termed the macromolecular crowding effects (MCEs), plays important roles in cellular metabolism and signal transduction. Principles of MCEs are hypothesised as volume exclusion, with which the reduction in diffusional volume increases the effective concentration, and hence the kinetics. However, the theory of volume exclusion is insufficient to link MCEs to the enhancement of specific macromolecular interactions; quantitative investigation into mechanisms of MCEs remains a challenge. Here we reveal the possible routes through which MCEs enhance specific protein-protein binding. We show, by conducting atomistic simulations, that specific macromolecular interactions are generally not affected by crowding-induced diffusion limits, instead MCEs help to transform nonspecific collisions, proximal to the binding site, into specific encounters. This is achieved through macromolecules undergoing predominantly rotational movements towards their specific binding interface, driven by environmental electrostatic steering forces. The simulations demonstrate MCEs account for the temporal, as well as spatial, aspects of macromolecular interactions within the cell. The enhancement of specific molecular interactions, more so when protein copies are low, enables the cell to conduct efficient reactions with limited space and resource. Our simulation of heterogeneously crowded protein-protein interactions bridges the gap between structural studies of individual macromolecular complexes and systemic approaches of studying regulatory network kinetics, thereby enhancing our ability to manipulate and engineer crowded molecular interactions in signalling pathways.

International PhD Student Cancer Conference 89 May 20, 2010 – Lectures, poster session II and oral presentations

A SILAC Proteomic Approach Identified Novel Candidate Players in the Non-Clathrin Endocytic Pathway of the EGFR Gilda Nappo1, Sara Sigismund1, Simona Polo1,2, Pier Paolo di Fiore1, 2,3 1 IFOM, the FIRC Institute for Molecular Oncology, Via Adamello 16, 20139, Milan, Italy 2 Dipartimento di Medicina, Chirurgia ed Odontoiatria, Università degli Studi di Milano, Via di Rudiní 8, 20122 Milan, Italy 3 European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy

Epidermal Growth Factor Receptor (EGFR) can be endocytosed through different entry routes depending on ligand concentration. At low doses of EGF, the receptor is not ubiquitinated and is internalized exclusively through clathrin-mediated endocytosis (CME). At higher concentrations of ligand, however, a substantial fraction of the receptor is endocytosed through a non-clathrin endocytosis (NCE), as the receptor becomes ubiquitinated [1]. Importantly, the two pathways couple with distinct receptor functions. CME is mainly involved in receptor recycling and allows prolonged signalling to occur from the intracellular compartments. On the contrary, NCE targets the majority of the receptors to degradation [2]. The molecular mechanisms involved in NCE of the EGFR are mainly unknown. Essentially, NCE is defined by its insensitivity to functional ablation (KD) of clathrin and for its sensitivity to cholesterol- interfering drugs, hence its definition as a “raft-dependent pathway”. In order to identify the molecular components of NCE, we are employing a large- scale proteomic approach. To this end, pure preparation of EGFR-containing vesicles in condition of clathrin KD has been obtained by differential centrifugation technique followed by an immunopurification step using phosphospecific anti-EGFR antibodies. This method, coupled with SILAC quantitative proteomics, allowed us to select a list of candidate players, particularly enriched for specific raft-mediated endocytic proteins, which are currently being validated by RNA interference. Given the degradative function of NCE, this approach can be useful to identify negative regulators of EGFR, which might represent novel potential targets for cancer therapies.

[1] S. Sigismund et al., Proc Natl Acad Sci U S A 102, 2760 (2005). [2] S. Sigismund et al., Dev Cell, 15, 209-19 (2008).

International PhD Student Cancer Conference 90 May 20, 2010 – Lectures, poster session II and oral presentations

Using Integrative Genomics To Investigate Mechanisms of Chromosomal Instability (Rebecca A. Burrell)[1]*, Sarah E. McClelland [1]*, David Endesfelder [2], Arne Schenk [1,2], Aron C. Eklund [3], Zoltan Szallasi [3,5], Maik Kschischo [2], Charles Swanton [1,4] 1. Translational Cancer Therapeutics Laboratory Cancer Research UK London Research Institute, London WC2A 3PX 2. University of Applied Sciences Koblenz, RheinAhrCampus, Remagen, Germany 3. Center for Biological Sequence Analysis, Technical University of Denmark, DK 2800 Lyngby, Denmark. 4. Royal Marsden Hospital, Department of Medicine, Breast Unit, Sutton, SM2 5PT, UK. 5. Children’s Hospital Informatics Program at the Harvard-MIT Division of Health Sciences and Technology (CHIP@HST), Harvard Medical School, Boston, MA 02115, USA. *Equal contribution to the work presented

Chromosomal instability (CIN) occurs commonly in solid tumours, and is associated with aggressive disease and resistance to treatment. CIN describes the constant reshuffling of the genome at each cell division, generating a heterogeneously aneuploid population of tumour cells. Currently, our understanding of how aneuploidy and CIN are generated in vivo is poor. There is a significant body of evidence showing that the normal mechanics of mitosis are disrupted in CIN cancer cells. However, it is unclear whether there are common genetic aberrations underlying the abnormal cell division that characterises the CIN phenotype. We have identified genomic regions that show consistent DNA copy number change in CIN cell lines, but not in chromosomally stable cell lines. Furthermore, we have found that the same regions are aberrantly regulated in CIN tumours. Using RNA- interference we have screened 200 genes within these regions for roles in regulating mitotic fidelity, and hence chromosomal stability. We have identified a number of genes whose depletion leads to various mitotic abnormalities, including segregation errors, spindle malformation and centrosome amplification. This suggests that CIN is a complex phenotype arising from multiple cooperating defects. As these genes are commonly deregulated in CIN cancers they are likely to be important for driving CIN in vivo.

International PhD Student Cancer Conference 91 May 20, 2010 – Lectures, poster session II and oral presentations

An shRNA screen to identify novel factors involved in telomere damage induced genome instability [Marieke H. Peuscher], Jaco van der Torre, Janet van Noord and Jacqueline J.L. Jacobs Division of Molecular Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands

Telomeres help to maintain genome integrity by protecting natural chromosome ends from being recognized as damaged DNA. Although telomere dysfunction has been suggested to play a role in both cancer and aging, the precise way by which mammalian cells perceive and respond to loss of telomere function remains unclear. To increase our understanding of the cellular response to telomere damage, we are undertaking a genome wide loss-of-function genetic approach to identify novel factors involved in telomere-induced genome instability. We use inhibition of the telomere- capping protein TRF2 as a model system for telomere dysfunction and make use of a temperature sensitive (TS) TRF2 system developed in the laboratory of T. de Lange. Due to non-homologous end-joining, switching of TS TRF2 TRF2-/- p53-/- MEFs from 32 to 39°C results in the formation of telomere fusions. Using this TS setup we performed an unbiased shRNA screen, through which we aim to identify genes whose inactivation abrogates telomere damage induced genomic instability. At present we have screened half of our total shRNA library, which resulted in the outgrowth of large amounts of colonies of library-infected cells after prolonged culturing at 39°C when compared to none in the empty vector-infected cells. PCR recovery of the expressed shRNA inserts led to the indentification of a hairpin directed against NBS1, which is part of the mammalian MRN complex known to play a role in the cellular response to telomere damage. Since we could validate the observed rescue from telomere fusion formation with the NBS1 hairpin we recovered from the screen, this result serves as a proof-of-principle and demonstrates that our experimental set-up is feasible. We are currently recovering additional shRNAs from our rescued colonies and are identifying the relevant target genes.

International PhD Student Cancer Conference 92 May 20, 2010 – Lectures, poster session II and oral presentations

LOSS OF RB PROTEINS CAUSES GENOMIC INSTABILITY IN THE ABSENCE OF MITOGENIC SIGNALING [Tanja van Harn*,1], Floris Foijer*,1,2, Marcel van Vugt3, Ruby Banerjee2, Fentang Yang 2, Anneke Oostra4, Hans Joenje4 and Hein te Riele1 *: These authors contributed equally 1: Division of Molecular Biology; The Netherlands Cancer Institute; Amsterdam; The Netherlands 2: Wellcome Trust Genome Campus; Wellcome Trust Sanger Institute; Cambridge; UK 3: Department of Medical Oncology; Groningen Medical Centre; Groningen; The Netherlands 4: Department of Clinical Genetics; VU University Medical Center; Amsterdam; The Netherlands

Loss of G1/S control, an important barrier to proliferation under several growth inhibitory conditions, is a critical step in cancer development and is often caused by inactivation of the retinoblastoma pathway. However, Mouse Embryonic Fibroblasts lacking the retinoblastoma genes RB1, p107 and p130 (TKO MEFs) are still subject to cell cycle control: upon mitogen deprivation they enter and complete S phase, but then firmly arrest in G2. Another hallmark of most solid tumors is genomic instability manifested by aneuploidy. We now show that G2-arrested TKO-Bcl2 MEFs have accumulated numerous DNA Double Strand Breaks (DSBs). Upon mitogen re-addition cells resume proliferation, although only part of the damage is repaired. As a result, mitotic cells show chromatid breaks and chromatid cohesion defects. These aberrations lead to aneuploidy in the proliferative descendent cell population, manifested by losses and gains of chromosomes. Thus, unfavorable growth conditions in cells lacking proper G1/S control can cause genomic instability. In vivo this could be a mechanism for Rb-deficient cells to acquire additional mutations that further promote tumorigenesis. Interestingly, progression of retinoma to retinoblastoma, a pediatric cancer of the eye caused by loss of function of the RB1 gene, is associated with an increased level of genomic aberrations.

International PhD Student Cancer Conference 93 May 20, 2010 – Lectures, poster session II and oral presentations

Silent assassins: DNA methylation does not inhibit SINE transcription, but suppressed their recombination. Dhaval Varshney, Jana Vávrová, Robert J. White

A million copies of mammalian short interspersed nuclear elements (SINEs) comprise 11% of the human genome, but despite this abundance they are expressed at a very low level. Alu elements are estimated to harbour 33% of CpG sites in the human genome and DNA methylation of these sites was believed to silence their transcription by promoting the formation of inaccessible chromatin. However, we show the presence of RNA Polymerase III (pol III) transcription machinery on SINEs along with methylated DNA binding proteins, suggesting that DNA methylation does not affect the polymerase loading onto these elements. Loss of DNA methylation in DNA methyl transferase 1 null fibroblasts neither changes transcription nor the occupancy of the pol III machinery on SINEs. This suggests an alternative role for DNA methylation on these repetitive sequences. Homologous recombination events between Alu elements are implicated in several human diseases, including some cancers. We observe that reduction in DNA methylation causes a two-fold increase in the rate of Alu driven intra- chromosomal translocation events. Hence we propose that DNA methylation serves not to inhibit transcription of SINEs but instead it plays a role in inhibiting homologous recombination at these elements.

International PhD Student Cancer Conference 94 May 20, 2010 – Lectures, poster session II and oral presentations

Fancm-deficient mice reveal unique features of Fanconi anemia complementation group M Sietske T. Bakker1,2, Elly Delzenne-Goette1, Anja van der Wal1, Martin van der Valk1, Johan P. de Winter2 , Hein te Riele1 1 Division of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, NL-1066 CX Amsterdam, The Netherlands and 2 Department of Clinical Genetics, VU University Medical Center, Van der Boechorststraat 7, NL-1081 BT Amsterdam, The Netherlands

The Fanconi anemia (FA) core complex member FANCM remodels synthetic replication forks and recombination intermediates. Thus far, only one FA patient with FANCM mutations has been described, but the relevance of these mutations for the FA phenotype is uncertain. To provide further experimental access to the FA-M complementation group we have generated Fancm-deficient mice by deleting exon 2. FANCM deficiency caused hypogonadism in mice and hypersensitivity to cross-linking agents in mouse embryonic fibroblasts (MEFs), thus phenocopying other FA mouse models. However, Fancm2/2 mice also showed unique features atypical for FA mice, including underrepresentation of female Fancm2/2 mice and decreased overall and tumor-free survival. This increased cancer incidence may be correlated to the role of FANCM in the suppression of spontaneous sister chromatid exchanges as observed in MEFs. In addition, FANCM appeared to have a stimulatory rather than essential role in FANCD2 monoubiquitination. The FA-M mouse model presented here suggests that FANCM functions both inside and outside the FA core complex to maintain genome stability and to prevent tumorigenesis

International PhD Student Cancer Conference 95

May 20, 2010 – Lectures, poster session II and oral presentations

POSTER SESSION II B1 Elvan Boke B2 Eleonora Dondossola B3 Francesca Fontana B4 Willem-Jan Keune B5 Milena Mazan B6 Guillermo Menendez B7 Francesca Milanesi B8 Jennifer Miles B9 Iain Moal B10 Guenievre Moreaux B11 Sharmin Naaz B12 Noor Akmar Nam B13 Joschko Natalie B14 Samuele Notarbartolo B15 Adelaida Palla B16 Roberta Pascolutti B17 Avinash Patel B18 Chitra Pattabiraman B19 Raghunath Reddy Peesari B20 Kay Penicud B21 Alessandra Pepe B22 Gabriele Piergiovanni B23 Mariaelena Pozzebon B24 Narek Sarkisyan B25 Gian Maria Sarra Ferraris B26 Christoph Schlude B27 Chiara Segrè B28 Marta Nasila Shahbazi B29 Viktorija Sokolova B30 Monica Soldi B31 Lilly Sommer B32 Maren Stindt B33 Angelo Taglialatela B34 Gabriele Varano B35 Gianluca Varetti B36 Maria Wiezlak B37 Marcus Wilson B38 Jenifer Wood B39 Magdalena Zajac

International PhD Student Cancer Conference 97

May 20, 2010 – Lectures, poster session II and oral presentations

B1 (Elvan Boke), Marisa-Alonso Nunez, Iain Hagan Paterson Institute for Cancer Research, Manchester, UK

Errors in chromosome segragation can give rise to aneuploidy. Cells have evolved a complex regulatory network to ensure accurate duplication and division of the cellular components and chromosomes. A key transition during the life span of a cell is the commitment to mitosis which is triggered by activation of cyclin B/Cdk1, also known as mitosis promoting factor (MPF). MPF is activated by the phosphatase Cdc25 and inhibited by the kinase Wee1, and the timing of mitotic entry is controlled by the balance between these two proteins. MPF activates a number of downstream kinases including the highly conserved Polo, Aurora and NIMA related kinases. Recent studies in Xenopus egg extracts have revealed a crucial role for Greatwall kinases (Gwl) in a novel biochemical pathway that inactivates antimitotic phosphatases directed against CDK substrates. Gwl mediates the maintenance of mitosis by inhibiting PP2A. This regulation suggests that mitotic entry and maintenance is not only mediated by MPF activation but also by phosphatase regulation by Gwl. In addition to its roles in cell cycle, studies in budding yeast assigned a different role for Gwl; the budding yeast homolog, Rim15, is shown to be the convergence point of various stress pathways in S.cerevisiae. In S.pombe, three kinases are assigned to Greatwall family by homology; Cek1, Ppk18 and Ppk31, none of which is essential for survival. The ultimate aim of my project is to understand Greatwall function in fission yeast.

International PhD Student Cancer Conference 99 May 20, 2010 – Lectures, poster session II and oral presentations

B2 Effect of systemic administration of chromogranin A on tumor metastatization in animal models (Eleonora Dondossola1,2), Luca Crippa1, Barbara Colombo1 and Angelo Corti1

Chromogranin A (CgA), an acidic glycoprotein present within secretory vesicles of many normal and neoplastic endocrine and neuroendocrine cells, is abnormally released in circulation in patients with neuroendocrine tumors. We have previously shown that local production of CgA can affect tumor growth by affecting the tumor microenvironment. Furthermore, we have recently shown that circulating CgA is increased also in a subpopulation of patients with non small cell lung cancer (NSCLC), with important prognostic implications. The present study was undertaken to investigate the role of CgA in tumor metastatization. We have found that systemic administration of CgA to mice can inhibit the number of lung metastasis formed by B16F10 mouse melanoma and by TS/A mouse mammary adenocarcinoma cells, when these cells were injected into the tail vein. The dose-response curve was bell-shaped, maximal effects being obtained with a dose of 0.2 ug/mouse administered daily (i.v for four days after tumor cell injection). Remarkably, this dose produced circulating levels similar to those found in NSCLC patients. The CgA1-78 N-terminal fragment (called vasostatin-1, VS-1) was sufficient to exert anti-metastatic effects in both models, suggesting that the N-terminal region of CgA contains a bioactive site. CgA could also affect B16F10 cell migration, invasion and transmigration in vitro. These results suggest that abnormal levels of circulating CgA in cancer patients can contribute to regulate tumor metastatization, possibly by affecting different steps of the metastatic cascade.

International PhD Student Cancer Conference 100 May 20, 2010 – Lectures, poster session II and oral presentations

B3 Autophagy as a novel target in multiple myeloma therapy. Effects of 3-Methyladenine or chloroquine on myeloma cell viability and sensitivity to proteasome inhibitors (Francesca Fontana1,2,3), Elena Pasqualetto1, Riccardo Ronzoni1, Laura Oliva4, Niccolò Pengo2,4, Gianfranco Fraschini2,3, Simone Cenci2,4, Roberto Sitia1,2 1 San Raffaele Scientific Institute, Division of Genetics and Cell biology, Protein transport and secretion Unit; 2 Università Vita-Salute San Raffaele; 3 San Raffaele Scientific Institute, Department of general and specialistic surgery; 4 San Raffaele Scientific Institute, Division of Genetics and Cell biology, Age-related diseases

Proteasome Inhibitors (PI) are current cornerstones in Multiple Myeloma treatment. PI sensitivity seems to depend on the balance between proteasomal workload and proteasomal capacity (Cenci 2006; Bianchi 2009). Given the relevance of efficient protein degradation in myeloma cells, we investigated whether autophagy impacts on MM cell viability. Hallmarks of autophagy were detected by western blot and electron microscopy in myeloma cell lines (HMCL) and by immunofluorescence in HMCL and patient-derived MM cells (MMC). Inhibition of autophagy with 3-Methyladenine (3MA) or Chloroquine induced apoptosis in PI-sensitive HMCL MM.1S. In vitro specific induction of apoptosis was also observed in patient-derived MMC upon treatment with 3MA, alone or in combination with PI, at higher levels than in other bone marrow cells or PBMCs. In relatively resistant HMCL (U266 and RPMI8226), autophagy and proteasome inhibitors synergistically induced apoptosis if combined, but did not sensitize to UV rays. In conclusion, autophagy is activated in myeloma cells and it could play a role in survival and PI sensitivity. As proteocatabolism could be crucial for MM cells, inhibition of autophagy may represent a potential target to increase sensitivity to PI or to substitute them upon emergence of secondary resistance.

International PhD Student Cancer Conference 101 May 20, 2010 – Lectures, poster session II and oral presentations

B4 PIP4Kbeta and HER2 in breast cancer; co-amplified, co-regulated or just co-existent? (Willem-Jan Keune), Yvette Bultsma and Nullin Divecha Inositide Laboratory, Paterson Institute for Cancer Research, Manchester, United Kingdom

Deregulation of the cellular levels of Phosphoinositides, a minor family of second messenger phospholipids, can lead to cancer. Phosphatidylinositol (PtdIns) can be phosphorylated at the 3, 4 and 5 position of the inositol ring to generate seven distinct messengers of which PtdIns5P can be phosphorylated on the 4’-position by Phosphatidylinositol-5-phosphate-4-kinases (PIP4Ks) to generate PtdIns(4,5)P2. In 10-25% of human breast cancers, amplification of a region of chromosome 17q11-12 leads to overexpression of the HER2/Neu oncogene, which is associated with disease progression, adverse prognosis, and the development of drug resistance. The gene for PIP4Kbeta, is located within the same amplicon of HER2/Neu and appears to be co-amplified in a subset of HER2 positive cell lines. We are studying the role of PIP4Kbeta in breast cancer and in particular in HER2/ Neu positive versus HER2/Neu negative tumors. We have generated a specific antibody for PIP4Kbeta, using RNAi and overexpression we have validated its use in immunohistochemical analysis of formalin fixed tumours samples. A tissue microarray of 500 tumour samples from advanced breast cancer patients, was stained for PIP4Kbeta and for HER2. We show a strong statistical correlation between HER2 amplification and PIP4Kbeta overexpression. Interestingly, we have also identified tumours that overexpress PIP4Kbeta in the absence of HER2 upregulation. Deciphering if there is a role for PIP4Kbeta overexpression in driving or maintaining HER2 positive breast tumours may lead to novel therapeutic avenues.

International PhD Student Cancer Conference 102 May 20, 2010 – Lectures, poster session II and oral presentations

B5 Molecular and cellular characterization of the haemogenic endothelium cell population (Milena Mazan), Valerie Kouskoff & Georges Lacaud Stem Cell Biology, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK

There are different theories concerning the cellular origin of blood cells. Recent studies of our group demonstrated that the transition from haemangioblast to haematopoietic progenitors proceeds through a haemogenic endothelium intermediate stage and is correlated with changes in expression of cell surface markers such as c Kit, Tie2 and CD41. The aim of the project is to further characterize the haemogenic endothelium cell population. As a first step, the function of the Tie2 receptor will be evaluated in vitro during development of haemogenic endothelial cells and their subsequent differentiation. Tie2 signaling will be either upregulated or downregulated by expressing a functional Tie2-GFP or a dominant negative variant Tie2DIC- GFP introduced into an inducible system in embryonic stem cells. The effect of their expression will be monitored by imaging and flow cytometry analyses. The next step will be to examine the haematopoietic potential of haemogenic endothelium cell populations isolated from different location in the mouse embryo. Ex vivo experiments will be performed to evaluate the type of haematopoietic lineages generated by these cells. Finally, we will determine at the molecular level the difference between haemogenic and non-haemogenic endothelial cells by isolation of these cell populations during haemangioblast differentiation in vitro and microarray analyses.

International PhD Student Cancer Conference 103 May 20, 2010 – Lectures, poster session II and oral presentations

B6 Neurotrophin trafficking and signalling in microfluidic chambers {Guillermo Menendez}(1,2), Joao Cabral (2), Giampietro Schiavo (1) Molecular Neurobiology Lab, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX (2) Polymers & Microfluidics Lab, Department of Chemical Engineering, Imperial College, London SW7 2AZ

My project is aimed at applying microfluidics technology to the study of long-range neurotrophin signalling. Neurotrophins are target-derived growth and survival factors that, among other functions, prevent innervating neurons from undergoing apoptosis. Neurotrophins and their receptors have been shown to be retrogradely transported in axons of spinal cord neurons, following a pathway controlled by the small GTPase Rab7. The Rab7-dependent pathway is thought to trigger downstream signalling events such as the phosphorylation of the transcription factor CREB, important in promoting neuronal survival and differentiation, but direct evidence for this had not yet been provided. Time-course analysis by immunofluorescence confocal microscopy of CREB phosphorylation in DRG neurons revealed a peak of response two hours after neurotrophin stimulation of axon terminals in microfluidic cultures, where axons can be treated independently of cell bodies. I infected DRG neurons with lentivirus carrying wild type or a dominant negative mutant of Rab7 (Rab7T22N). Overexpression of Rab7T22N significantly reduced the signal generated in distal axons. CREB phosphorylation after neurotrophin stimulation of axons was also abolished using Rab7 shRNA lentivirus. My results provide a direct link between Rab7 activity and downstream effects of the signalling cascade initiated by neurotrophins at axonal networks in compartmentalised microfluidic chambers.

International PhD Student Cancer Conference 104 May 20, 2010 – Lectures, poster session II and oral presentations

B7 Molecular basis for the dual function of Eps8 on actin dynamics: bundling and capping (Francesca Milanesi) IFOM, Fondazione FIRC di Oncologia Molecolare, Milan, Italy; Maud Hertzog Institut Curie, Paris, France; Niels Volkmann Burnham Institute for Medical Research, La Jolla, USA; Doris Hanein Burnham Institute for Medical Research, La Jolla, USA; Giorgio Scita IFOM, Fondazione FIRC di Oncologia Molecolare, Milan, Italy;

Actin capping and cross-linking proteins regulate the dynamics and architectures of different cellular protrusions. Eps8 is the founding member of a unique family of capping proteins capable of side-binding and bundling actin filaments. However, the structural basis through which Eps8 exerts these functions remains elusive. Here, we combined biochemical, molecular and genetic approaches with electron microscopy image analysis to dissect the molecular mechanism responsible for the distinct activities of Eps8. We propose that Eps8 caps by wrapping around filament ends, inserting its amphipathic H1 helix into the hydrophobic pocket of the barbed end unit, acting as a clamp that prevents further addition of monomers. Single-point mutagenesis validated this mode of binding, further permitting us to dissect Eps8 capping from bundling activity in vitro and in vivo. Thus, Eps8 controls actin-based motility through its capping activity, while, as a bundler, is essential for proper intestinal morphogenesis of developing Caernorhabditis elegans.

International PhD Student Cancer Conference 105 May 20, 2010 – Lectures, poster session II and oral presentations

B8 FANCD2/I recognition and ubiquitination by FANCL in the FA DNA repair pathway (Jennifer A Miles) Ambrose R Cole, Helen Walden Protein Structure Function Laboratory, London Research Institute, Lincoln’s Inn Fields Laboratories, 44 Lincoln’s Inn Fields, London WC2A 3PX

The Fanconi Anemia DNA repair pathway removes interstrand crosslinks, which can appear during replication. 13 genes have been found to be involved in this pathway. FANCL is the Ubiquitin E3 ligase responsible for the monoubiquitination of FANCI and FANCD2 in the pathway. Without this monoubiquitination step, the interstrand crosslinks are not repaired which can lead to leukemias. Understanding this key step at a molecular level could increase knowledge on substrate recognition during ubiquitination and DNA repair. The structure of D. melanogaster FANCL has recently been solved, but a remaining question is how FANCL co-ordinates and binds its substrates. To address this question, I am overexpressing and purifying D. melanogaster FANCI and FANCD2, and orthologues from other species. Once sufficient purified protein is obtained, co-crystallisation and other structural and interaction studies will be employed to reveal the substrate recognition site on FANCL and determine whether there is a preference for either substrate. Additionally, I have generated preliminary data from pull-downs suggesting an interaction between FANCL and Ubiquitin. The role of this interaction is as yet unknown but it may be important during the monoubiquitination. This is to be further investigated with ITC for kinetic information and NMR for full structural analysis.

International PhD Student Cancer Conference 106 May 20, 2010 – Lectures, poster session II and oral presentations

B9 Protein-Protein Docking Using SwarmDock Iain Moal, Paul Bates

Protein-protein interactions are fundamental to almost all biological processes and their disruption is integral to many diseases including cancer. A novel docking algorithm, SwarmDock, has been developed and tested against a set of 84 complexes for which the individual protein structures, and that of the complex, is known. By taking advantage of the vibrational properties of proteins, flexibility can be modelled using a linear combination of normal modes. Swarm intelligence is used to manipulate protein structures and find the conformation and relative orientations and positions, of individual proteins in complex. SwarmDock performs well on the test cases, including difficult cases where protein undergo significant conformation change upon binding. Hence, the algorithm can give considerable insights into the structural basis of these interactions and can be used to predict and rationalise the effect of mutant proteins in the context of protein-protein interaction networks.

International PhD Student Cancer Conference 107 May 20, 2010 – Lectures, poster session II and oral presentations

B10 Dusp6 acts as a tumour suppressor negatively regulating Erk. (Guenievre Moreaux) Beatson Institute Glasgow, UK Owen Sansom Beatson Institute Glasgow, UK Steve Keyse Ninewells Hospital and Medical School, University of Dundee, UK

Dual specificity phosphatase 6 (DUSP6) is a cytoplasmic protein which negatively regulates ERK1/2. Previous in vivo studies have shown that colonic tumours expressing an oncogenic K-Ras (K-rasG12D) exhibit high levels of Dusp6 (Haigis, Kendall et al. 2008), which may help to explain why activation of K-Ras signalling in vivo rarely leads to activation of the ERK pathway. Moreover, studies have shown that Dusp6 is upregulated in early human pancreatic lesions but reduced or abolished in the ductal carcinoma (Furukawa, Fujisaki et al. 2005), suggesting that DUSP6 downregulation may be important in tumourigenesis. To investigate this, we have deleted Dusp6 in the context of K-ras activation in both the intestine and the pancreas. In both scenarios, DUSP6 genetic inactivation dramatically accelerated tumourigenesis, coincidentally with ERK activation. Taken together these findings suggest that DUSP6 is the key negative regulator of ERK1/2 in vivo and suggest it may act as a general tumour suppressor following K-Ras activating mutations.

International PhD Student Cancer Conference 108 May 20, 2010 – Lectures, poster session II and oral presentations

B11 The role of long non-coding RNAs in haematopoiesis (Sharmin Naaz), Valerie Kouskoff and Crispin Miller. All from The Paterson Institute for Cancer Research, The University of Manchester, Wilmslow Road, Withington, Manchester, M20 4BX England, United Kingdom.

Only ~2% of mammalian genomes encode mRNAs, while a vast majority are transcribed as non-protein-coding RNAs (ncRNAs). Versatile regulatory roles of ncRNAs have been demonstrated in many developmental pathways. Conversely, the dysregulation of ncRNA expression has been implicated in the aetiology of several diseases, including cancers. The aim of this project is to study the role of ncRNAs in haematopoiesis, a developmental pathway central to our understanding of leukaemia. For this study, an in vitro, mouse embryonic stem cells (mESCs) model of haematopoietic development was adopted. This involved differentiating mESCs along a Bry+ mesoderm lineage, from which the putative endothelial and haematopoietic precursor, hemangioblast, was derived. Haemangioblasts express the tyrosine receptor kinase, Flk1 and when cultured in the presence of vascular endothelial growth factor (VEGF), generate blood progenitors. The process of differentiating mESCs to blood progenitors was carried out over a 3 day time-course with cells harvested at every 12 hour interval. This was followed by high-throughput next-generation sequencing (RNA-seq) and exon microarray analysis of the time-course. The current goal is to implement bioinformatics and statistical approaches to analyse the substantial volume of data generated by these platforms in order to identify putative ncRNAs and their protein-coding partners in haematopoiesis.

International PhD Student Cancer Conference 109 May 20, 2010 – Lectures, poster session II and oral presentations

B12 RNA Polymerase III Transcription deregulation in cancer: Study of Brf1 expression in prostate cancer (Noor A. Nam)1, Imran Ahmad1, Joanne Edwards2, Hing Y. Leung1 and Robert J. White1 1 Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD UK. 2 Division of Cancer Sciences and Molecular Pathology, Department of Surgery, Glasgow Royal Infirmary, Glasgow, UK

BACKGROUND: Aberrant RNA Polymerase III (Pol III) transcription has been linked to increased cellular proliferation and progression to cancer. The importance of elevated Pol III transcripts and its associated transcription factor Brf1 in cancer has been demonstrated in cell culture and in mice. However, the significance of this deregulation has yet to be determined in patients. This study examines Brf1 expression in prostate cancer (PCa) tissues in comparison to benign prostate hyperplasia (BPH) to determine if elevated levels of Brf1 protein are detected in tumour samples and if there is any correlation with severity of disease. MATERIALS AND METHODS: Immunohistochemical staining was performed on Tissue Micro Arrays (TMAs) containing 149 cases of PCa and 21 BPH samples and the Brf1 expression was evaluated using the weighted Histoscore method. The proliferation status of these samples was assessed using Ki-67 staining. RESULTS: Brf1 expression was detected as heterogenous staining and localised predominantly in the nucleus. Brf1 expression is elevated in PCa compared to BPH (p<0.001), however there is no association between Brf1 expression with increasing Gleason Grade (p=0.545) and no significant correlation with Ki-67 expression was observed. CONCLUSIONS: Assessment of Brf1 levels in prostate cancer tissues revealed higher expression compared to benign hyperplasia and suggests that it may be a potential biomarker for cancer therapy. However a larger patient cohort that includes both hormone sensitive and hormone resistant prostate cancer patients will be of further value to determine Brf1 significance in the clinical settings.

International PhD Student Cancer Conference 110 May 20, 2010 – Lectures, poster session II and oral presentations

B13 p53 “gain-of-function” mutants inhibit apoptosis of HBV-infected hepatocytes (Joschko N.)*’, Rademacher A.’, Langhein A.’, Sun L-A.’, Steinebrunner N.’, Koch A.’, Stremmel W.’, Müller M.’ * German Cancer Research Center ‘ Department of Internal Medicine IV, Hepatology and Gastroenterology, University Hospital, Heidelberg, Germany

Chronic infection with hepatitis B virus (HBV) is one major risk factor for the development of hepatocellular carcinoma (HCC). We have previously shown in vivo that an intact p53 status and a functional CD95-signalling pathway are essential for elimination of HBV-infected cells. Whereas the hallmark of wild- type (wt) p53 is its tumor suppressor activity, tumor-associated mutant p53 proteins can exert novel anti-apoptotic “gain-of-function” (GOF) activities, which confer a selective advantage for tumor cells harboring such mutations. Combined adenoviral transfer of HBV and wt p53 led to an increased CD95 gene transactivation as well as CD95 protein expression, thus sensitizing HBV-infected hepatocytes towards CD95-mediated apoptosis. In contrast, transfection of a p53 “GOF” mutant, namely the hot-spot mutant p53R248W, inhibited viral clearance and apoptosis of HBV-infected hepatocytes. Whereas wt p53 transactivated the CD95 gene, thus leading to an increased CD95 protein expression on the surface of HBV-infected hepatocytes, p53R248W inhibited CD95 gene transactivation and therefore CD95 protein expression and CD95-dependent apoptosis. Our data show that the p53 “gain-of-function” mutant, p53R248W, contributes to viral persistence by inhibiting CD95-mediated apoptosis of HBV-infected hepatocytes. This suggests a possible synergy of alterations of the p53 pathway and HBV-induced hepatocarcinogenesis.

International PhD Student Cancer Conference 111 May 20, 2010 – Lectures, poster session II and oral presentations

B14 AN UNEXPECTED ROLE FOR THE HISTONE DEMETHYLASE JARID1B IN CHOLESTEROL METABOLISM IN MACROPHAGES (Notarbartolo, S.) and Natoli, G. Department of Experimental Oncology, European Institute of Oncology (IEO), IFOM-IEO Campus, Milan, Italy

Post-translational modifications on histones are considered important determinants of transcriptional activity. Methylation of H3K4 is highly conserved during eukaryotic evolution, and trimethylation of this residue is specifically associated with the transcription start sites of actively transcribed or poised genes. Like other histone lysines, H3K4me3 is dynamically controlled by methyltransferases (of the Set/Mll group) and demethylases (belonging to the Jarid family). We found that Jarid1b is specifically upregulated in response to LPS-triggered macrophage activation. Its depletion in bone marrow-derived macrophages resulted in the downregulation of a limited number of genes, all involved in cholesterol metabolism and all regulated by the transcription factor LXR. LXRa and LXRb are sequence-specific transcriptional activators of the nuclear receptor superfamily that function as obligate heterodimers with RXRa. LXR ligands are the oxysterols whose binding to the ligand-binding pocket of LXRs results in a conformational change that leads the transcription of target genes. LXRs play an important role in the control of reverse cholesterol transport, the process of delivering cholesterol from peripheral tissues to the liver. In macrophages LXRs regulate the cholesterol metabolism as well as the integration of lipid metabolism and inflammatory signalling. Mechanistic analyses on the link between Jarid1b and LXR function will be discussed.

International PhD Student Cancer Conference 112 May 20, 2010 – Lectures, poster session II and oral presentations

B15 Expression of Nanog in mouse and human cancer [Adelaida Palla], Daniela Piazzolla, Marta Cañamero and Manuel Serrano Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), 3 Melchor Fernandez Almagro Street, Madrid E-28029, Spain.

Nanog is an important transcription factor whose role in pluripotency and self-renewal during embryogenesis has been well studied, but its putative role during adult life or tumorigenesis remains largely unexplored. There are few studies suggesting that Nanog is expressed in human germ cell tumors. In addition, the human genome contains several Nanog pseudogenes with functional capacity that could be expressed in tumors. The aim of our study is to determine the impact and functionality of Nanog in the context of adult tissues and cancer. We are currently studying the expression of Nanog in human cancer cell lines by RT-PCR and western blot. Our data indicates that Nanog and Nanog pseudogenes are expressed in a variety of human cancer cell lines. We are now investigating their potential function. In addition we are analyzing the expression of Nanog in different primary human tumors to observe if there is a correlation between Nanog levels and tumour stage/prognosis. We also intend to perform a parallel analysis of Nanog expression in mouse tissues and tumors.

International PhD Student Cancer Conference 113 May 20, 2010 – Lectures, poster session II and oral presentations

B16 Dissecting the role of Cbl in EGFR endocytosis (Roberta Pascolutti)1, Gilda Nappo1, Veronica Algisi1, Sara Sigismund1 and Pier Paolo Di Fiore1,2,3 1 IFOM, the FIRC Institute for Molecular Oncology, Via Adamello 16, 20139, Milan, Italy 2 Dipartimento di Medicina, Chirurgia ed Odontoiatria, Universita¬í degli Studi di Milano, Via di Rudiní 8, 20122 Milan, Italy 3 European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy

Epidermal Growth Factor Receptor (EGFR) can be internalized through two different entry routes, clathrin-mediated endocytosis (CME) and non- clathrin endocytosis (NCE). EGFR ubiquitination, triggered by the E3 ligase c-Cbl, is required for this latter pathway, while it is dispensable for CME. A dual role for c-Cbl has been shown: it acts as a major E3 ligase in the ubiquitination of different plasma membrane receptors, targeting them to lysosomal degradation, and it functions also as an adaptor, by recruiting several proteins involved in the early phase of CME. In order to understand the molecular details of Cbl activity, we plan to systematically knock-down (KD) c-Cbl and its related proteins Cbl-b and Cbl-c in HeLa cells. This will be instrumental to i) characterize their effects on EGFR ubiquitination (if they act at different steps of the endocytic cascade or through different type of ubiquitin signals), ii) dissect their involvement in CME vs NCE and iii) split their roles as adaptors and E3 ligases through reconstitution of KD cells with different sets of mutants. We initially set-up both transient and stable KD of Cbl proteins in HeLa cells. Our preliminary data suggest that Cbl-b acts similarly to c-Cbl in the process of EGFR CME. Yet, Cbl-b has a minor impact on EGFR ubiquitylation compared to c-Cbl, suggesting distinct roles of the two proteins in EGFR NCE. Generation and characterization of Cbl mutants (affecting ligase vs adaptor function) is on going. Results will be presented.

International PhD Student Cancer Conference 114 May 20, 2010 – Lectures, poster session II and oral presentations

B17 Genetic Analyses of Fission Yeast Polo Kinase [Avinash Patel], A Grallert and I Hagan. Cell Division Group, Paterson Institute for Cancer Research, Manchester, United Kingdom.

Errors in the regulation of the cell division cycle can lead to cancer. The two major transitions in the cell cycle are the initiation of S phase (when the DNA is replicated) and commitment to Mitosis or M phase (when the replicated DNA are equally segregated into two daughter cells). Commitment to mitosis is universally regulated in all eukaryotes by the activation of a protein complex Mitotic Promoting Factor (MPF) consisting of the kinase CDK1 and cyclin B. Activation of MPF activates the downstream mitotic kinases (e.g. Polo, Aurora and NIMA related kinases) that lead to a number of different mitotic events. As in other systems, the fission yeast Polo kinase, Plo1 has been shown to regulate diverse mitotic events including mitotic commitment, spindle formation, the cytokinetic ring formation/contraction, and the exit from mitosis. The aim for this project is to use a genetic and biochemical approach to assess the impact of Plo1 upon the composition of the mitotic phosphoproteome. We have generated novel temperature sensitive plo1 alleles in which the Plo1 function is compromised to a sufficient degree to abolish all Plo1 known dependent events. We have also generated an analogue sensitive plo1. as allele that enables us to selectively inhibit Plo1 kinase activity by the addition of an ATP analogue. This does not lead to a complete block of Plo1 activity, rather it just blocks it’s ability to promote cytokinesis.

International PhD Student Cancer Conference 115 May 20, 2010 – Lectures, poster session II and oral presentations

B18 What are the epigenetic controls that accompany altered signalling and tumorigenic profiles in human cervical cancer sub-sets? (Chitra Pattabiraman) and Sudhir Krishna National Centre for Biological Sciences,TIFR, Bangalore, India.

Epigenetic regulators-which include DNA Methyltransferases and Histone Methyltransferases are important for normal differentiation and are often de- regulated in tumours. Work from our lab has identified a sub-set of cells in cervical cancer with enhanced tumour forming ability. We are interested in understanding the role that epigenetic processes play in this sub-set. This sub-set was identified when CaSki, a cervical cancer cell line was grown without attachment in serum free media. These cells express a cell surface marker- CD66c which functionally correlates with the increased invasiveness and enhanced tumorigenicity. Microarray analysis of CaSki spheroids revealed multiple epigenetic regulators to be differentially regulated including DNMT1 (DNA Methyltransferase) and EZH1 (Histone Methyltransferase). Combined with the effect of pharmacological inhibitors this suggests a distinct epigenetic state in the spheroids. Ongoing work examines if differential regulation of epigenetic regulators is causal to the functional phenotypes of the CD66c+ sub-set. In order to identify possible substrates on which these epigenetic regulators act, we carried out a genome wide DNA methylation analysis. We identified a small set of genes which are methylated and repressed in a state specific manner. Current work is aimed at testing their presence in patient material and their persistence over cell division.

International PhD Student Cancer Conference 116 May 20, 2010 – Lectures, poster session II and oral presentations

B19 Role of Deubiquitinase Usp9X in EGFR Endocytosis Raghunath Peesari1, Elena Cavallaro1, Simona Polo1,2 1 IFOM, the FIRC Institute for Molecular Oncology, Via Adamello 16, 20139, Milan, Italy 2 University of Milan Medical School, 20122 Milan, Italy

The role of endocytosis in EGFR signaling can be underlined by the fact that upon stimulation of cells with the EGF ligand, the growth factor receptors are activated and internalized, both processes affecting the spatial and temporal regulation of the EGFR signaling. Endocytosis is a mechanism to achieve the attenuation of signaling by committing the receptors to lysosomal degradation. This is accomplished by an interplay among various signaling proteins, such as effectors and kinases of the signaling cascade, endocytic adaptor proteins which regulate the trafficking of the receptors, ubiquitin ligases which ubiquitinate the receptors and mark them for degradation into the lysosomal compartments and other classes of proteins such as phosphatases and deubiquitinases (DUBs) that could counteract the action of the positive enzymes. We have investigated the role of deubiquitinase USP9X in EGFR endocytosis. Usp9X (ubiquitin specific protease 9, X-linked), an ortholog of Drosophila fat facets (faf) gene that plays an important role in compound eye development by regulating the levels of liquid facets (an homolog of epsin), an adaptor protein involved in endocytosis. Preliminary results suggest that Usp9X affects internalization, signaling and degradation of EGF receptor. In addition, it also affects the ubiquitination of several endocytic adaptor proteins involved in the trafficking of the EGF receptors. Among these endocytic adaptor proteins we have identified Eps15 to interact with Usp9X.

International PhD Student Cancer Conference 117 May 20, 2010 – Lectures, poster session II and oral presentations

B20 Regulating ATM’s Response to DNA Damage Kay Penicud Cancer Research UK London Research Institute 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom

The checkpoint kinase ATM (ataxia telangiectasia mutated) transduces genomic stress signals to halt cell cycle progression and promote DNA repair in response to DNA damage. The essential ATM co-factor ATMIN (ATM INteracting protein) defines a novel NBS1-independent pathway of ATM signalling. ATMIN is required for ATM signalling in response to chloroquine and hypotonic stress, but not after induction of double strand breaks by ionising radiation (IR). ATMIN-deficient murine embryonic fibroblasts have increased spontaneous DNA damage and sensitivity to oxidative stress. An shRNA screen identified two further proteins required for regulation of ATM activity. ARP1 (ATM Regulatory Protein 1) is required for ATM substrate phosphorylation, including Smc1 and KAP-1, in response to ionising radiation. Conversely, cells deficient in ARP2 (ATM Regulatory Protein 2) exhibit increased activity of the ATM signalling pathway under basal conditions.

International PhD Student Cancer Conference 118 May 20, 2010 – Lectures, poster session II and oral presentations

B21 FUNCTIONAL ANALYSIS OF MUS81 COMPLEXES (Alessandra Pepe) and Stephen C. West Cancer Research UK, London Research Institute, Clare Hall laboratories, South Mimms, United Kingdom

The repair of DNA lesions is important for the maintenance of our genetic material. Eukaryotic cells contain a variety of mechanisms that specifically recognise different types of DNA lesions and promote efficient repair. Some of these pathways involve the nucleolytic cleavage of DNA. MUS81 interacts with EME1 to form a heterodimeric endonuclease that plays important roles in meiotic crossover formation, the resolution of recombination intermediates (e.g. Holliday junctions) and DNA crosslink repair. Studies on MUS81 knockout mice have also shown its involvement in the development of cancer. In addition to its interaction with EME1, MUS81 also interacts with a related protein known as EME2. The function of the MUS81-EME2 complex, which only exists in human cells, remains a puzzle. Recently, human MUS81-EME1 was shown to be present in a large endonuclease complex, which also includes two other heterodimeric endonucleases: SLX1-SLX4 and XPF-ERCC1. Here we provide evidence of the interaction between MUS81-EME2 and SLX1-SLX4 and XPF-ERCC1. Also, we describe the initial approaches that will be used in order to identify the functional differences between MUS81-EME1 and MUS81-EME2 complexes in human cells.

International PhD Student Cancer Conference 119 May 20, 2010 – Lectures, poster session II and oral presentations

B22 GEMC1 regulation in chromosomal replication and DNA damage checkpoint (Gabriele Piergiovanni) and Vincenzo Costanzo Genome Stability Unit, Cancer Research UK London Research Institute - Clare Hall

In order for the genome to be faithfully maintained, chromosomal DNA must be precisely replicated and segregated in each cell cycle of all organisms. Moreover, genome stability requires the coordination of DNA replication with the cell cycle progression, DNA damage sensing and repair through mechanisms called checkpoints. As a consequence, several factors involved in DNA replication are also important for the cell response to stress conditions. Most of these factors have been identified in lower eukaryote. However, the regulation of DNA replication in multi-cellular organisms is still poorly understood. Recent work in the laboratory, carried out using the Xenopus laevis egg cell-free system, identified a novel vertebrate protein required for DNA replication named GEMC1. xGEMC1 is a CDK target required for the Cdc45 initiation factor loading onto chromatin and it interacts with the checkpoint and replication factor TopBP1, which promotes its binding to chromatin during pre-replication complex formation. In this work the GEMC1 role in the DNA metabolism will be further investigated in two directions. First GEMC1 involvement in mammalian DNA replication will be evaluated. Second, using both the Xenopus system and mammalian cell lines, a possible GEMC1 role in the DNA damage response will be verified.

International PhD Student Cancer Conference 120 May 20, 2010 – Lectures, poster session II and oral presentations

B23 The adenoviral Gam1 protein affects VHL protein stability and its E3 ligase-related functions (Mariaelena Pozzebon), Agnese Collino, Susanna Chiocca. Istituto Europeo di Oncologia, Milan, Italy

Viral proteins are known to degrade target substrates by hijacking enzymes of ubiquitin pathway. Gam1 is an early protein of the avian adenovirus CELO and it possesses a SOCS-box domain that interacts with elongin C and two different cullins, Cul2 and Cul5, components of ubiquitin cullin RING ligases (CRLs). Thus, Gam1 acts as a substrate-receptor in these complexes and directly binds and ubiquitinates the SUMO-activating enzyme (E1). Moreover, Gam1 leads to SUMO- conjugating enzyme (UBC9) degradation by still unknown mechanisms. This leads to inhibition of SUMO pathway. Von Hippel-Lindau (VHL) protein associates to Cul2-based complexes and specifically targets to degradation alpha subunits of HIF (hypoxia-inducible factor) transcription factors. VHL is a tumor-suppressor protein and its loss leads to von Hippel-Lindau syndrome, characterized by the onset of renal cell carcinoma, pheochromocytoma and haemangioblastoma. Since Gam1 recruits host components of ubiquitin-E3 CRLs, it could interfere with the function of VHL. Interestingly, we observed that Gam1 leads to VHL protein decrease and to HIF-1 stabilization and activation. We verified that SUMOylation inhibition per se does not affect VHL protein stability, suggesting that Gam1 acts separately on SUMO enzymes and VHL related complexes. Since we observed by both in vitro binding and co-immunoprecipitation analysis that Gam1 and VHL directly interact, we are currently investigating whether Gam1 is an E3 ligase for VHL protein.

International PhD Student Cancer Conference 121 May 20, 2010 – Lectures, poster session II and oral presentations

B24 Investigation of Epigenetic Risk and Predictive Markers for Lung Cancer in Blood N. Sarkisyan (1), D. Scherf (1), H. Dienemann (2), K. Arab (1), R. Claus (1), C. Plass (1) and A. Risch (1) 1) Div. Epigenomics and Cancer Risk Factors, German Cancer Research Center and 2)Thoraxklinik, Heidelberg, Germany

Background: Lung cancer is a major oncological ¬ìkiller¬î worldwide and has become a global public health burden, further substantiating the need for early diagnosis, risk assessment, more effective targeted therapies, and prognosis. The key to accomplish these goals is a better understanding of genes and pathways disrupted during the initiation and progression of this disease. DNA hypermethylation is recognized as an important mechanism for tumor suppressor gene inactivation in cancer and could yield powerful biomarkers in blood for risk assessment, outcome prediction, and even early diagnosis. Here we focused on developing DNA methylation markers for adenocarcinoma and squamous cell carcinoma in lung. Methods: Using sensitive, quantitative high-throughput DNA methylation analysis (MassARRAY, Sequenom), we examined the methylation profile of 21 previously published TSGs genes in a collection of 45 lung cancer tissue samples (T) and adjacent normal lung (N) tissue from the same patients. The two-sample t-test was used to compare mean DNA methylation in tumor and normal samples. Paired tumor and normal samples were compared for differences in methylation frequency with the exact form of The Wilcoxon Matched- Pairs Signed-Ranks test. All p values reported are two sided (p≤0.05). Results: We identified 9 TSGs showing highly significant DNA methylation levels in tumor tissue vs. adjacent non-tumor lung. Of these, 7 showed highly significant hypermethylation in tumor tissue: the overall quantitative estimate of methylation level in cancer lung tissues vs. adjacent normal tissues was 35% vs. 15% for TCF21 (p≤0.001), 27% vs. 12.6% for RASSF1A (p≤0.001), 18.4% vs. 9.3% for OPCML (p≤0.001), 16.9% vs. 8.6% for DLEC (p≤0.03), 7.7% vs. 3.1% for OLIG1 (p≤0.006), 9.2% vs. 13.8% for MGMT (p≤0.004), and 7.8% vs. 3.8% for CHFR (p≤0.006). Interestingly, this is also the first report of highly significant hypomethylation of 14-3-3sigma and BLU in lung tumor tissue. The overall quantitative estimate of DNA methylation level in cancer lung tissues vs. adjacent normal tissues was 59.4% vs. 84.3% for 14-3-3sigma (p≤0.001) and 11.7% vs. 17.3% for BLU (p≤0.001). 80% of all tumor tissue samples showed hypermethylation (defined as 50% increased average methylation in T vs N) compared to adjacent normal lung in TCF21, 65.4% in OPCML, 40% in MGMT, 64.7% in DLEC, 58.1% in RASSF1A, 46.2% in OLIG, and 48.5% in CHFR. Hypomethylation (50% decreased average DNA methylation in T vs N) was shown in 59.5% of all tumor tissue samples compared to adjacent normal lung for BLU and in 53.5% for 14-3-3sigma. Conclusion and future outlook: We have identified 9 potential DNA methylation markers for lung cancer, two of which have not previously been reported to be hypomethylated in lung cancer. Further investigation of methylation status in those 9 candidate genes will be performed in cell-free DNAs of corresponding plasma samples and genomic DNAs from buffy-coat of the cancer patients as well as in cancer-free individuals to investigate the concordance and the potential of these genes to serve as biomarkers. Also, functional analysis of 14-3-3sigma and BLU will be performed to examine the expression level of those hypomethylated genes since they are not in agreement with previous reports.

International PhD Student Cancer Conference 122 May 20, 2010 – Lectures, poster session II and oral presentations

B25 Ligand-independent adhesion signaling by integrins “Gian Maria Sarra Ferraris”1, Carsten Schulte1, Chris Denis Madsen1,2, and Nicolai Sidenius1 1 Unit of Cell Matrix Signaling, IFOM-IEO-Campus, Via Adamello 16, 20139 Milan, Italy. 2 Current address: Tumour Cell Biology Laboratory, Cancer Research UK, London Research Institute, United Kingdom

Integrins are the major family of adhesion receptors responsible for the physical contact and biochemical communication between cells and the extracellular matrix (ECM). The engagement of integrins with ECM triggers “outside-in” signaling, resulting in context-dependent changes in cell morphology, migration and proliferation. We find out that most, if not all, adhesion receptors trigger integrin signaling independently of direct contacts between integrins and the ECM. The urokinase plasminogen activator receptor (uPAR) is a non-integrin vitronectin (VN) adhesion receptor linked to the outer membrane leaflet by a (GPI)-anchor. Through a structure- function analysis of uPAR, VN, b1 and b3 integrins, we document that uPAR-mediated VN adhesion triggers integrin-mediated, but ligand independent, cell spreading and signaling. This signaling is fully active on VN lacking the integrin binding site and sustained by integrin mutants deficient in ligand binding, but is dependent on an “active” conformation of the receptor as well as its binding to intracellular adaptors such as talin and kindlin. Ligand-independent integrin signaling is not restricted to uPAR as it poses no identifiable constraints to the adhesion receptor with respect to ternary-structure, ligand type, or means of membrane anchorage. Consistently, we show that cell adhesion mechanically supported by a signaling-incompetent b3 integrin is effectively translated into b1 integrin-dependent cell spreading and signaling.

International PhD Student Cancer Conference 123 May 20, 2010 – Lectures, poster session II and oral presentations

B26 Spontaneous T-cell responses against mutant forms of tumor- associated antigens in patients with colorectal carcinoma [Christoph Schlude]*, Frank Momburg*, Moritz Koch¬∞, Jürgen Weitz¬∞ and Philipp Beckhove* *Department of Translational Immunology, German Cancer Research Center, Heidelberg, Germany ¬∞Department of Surgery, University Hospital of Heidelberg, Heidelberg, Germany

Central tolerance is one of the major hurdles to successful T-cell-aimed vaccines using tumor-associated antigens (TAA). T-cells bind only with low affinity to wildtype peptides and in case of an induced immune response healthy tissue can also be attacked. One possibility to bypass central tolerance could be the use of peptides derived from mutated TAA. Therefore, we evaluated the ability of T-cells isolated from colorectal carcinoma patients to react against mutated forms of p53, KRas and B-Raf. Peripheral blood and bone marrow mononuclear cells of 26 patients with colorectal carcinoma were used to generate dendritic cells and isolate T-cells by magnetic cell sorting. IFN-γ enzyme-linked immunospot analysis (ELIspot) was then used to characterize the frequency of antigen-specific T-cells directed against synthesized polypeptides containing the most common mutations of p53, KRas and B-Raf. All tested mutant peptides of p53, KRas and B-Raf were recognized by T-cells derived from patients at response rates up to 15%. Compared to the corresponding wildtype TAA the frequency of specific T-cells was significantly higher against mutated antigens. We also found a higher frequency of antigen-specific T-cells in the peripheral blood than in the bone marrow. In this study we show that colorectal cancer patients have the ability to induce spontaneous T-cell responses against mutant forms of tumor-associated antigens.

International PhD Student Cancer Conference 124 May 20, 2010 – Lectures, poster session II and oral presentations

B27 Characterization of phosphorylation of Histone deacetylases 1 and 2 by Aurora kinases in mitosis [Chiara Segrè (1)] and Silvia Senese (1), Stefano Santaguida (1), Andrea Musacchio (1), Christian Seiser (2) and Susanna Chiocca (1) 1 SEMM (European School of Molecular Medicine), European Institute of Oncology (IEO), Milan (Italy) 2 Max F. Perutz, Medical University of Vienna, Vienna (Austria)

Histone deacetylases (HDACs) are enzymes that remove acetyl groups from lysines of proteins. HDAC1 and HDAC2 are deregulated in many cancers. HDAC1 and HDAC2 are subjected to a variety of post-translational modifications that modulate their function, but the role of this “code” in vivo is still unclear. Here we show that HDAC1 and HDAC2 are hyperphosphorylated specifically in mitosis. In vitro assays show that HDAC1 is phosphorylated by both Aurora A and Aurora B kinases, while HDAC2 only by Aurora B. Auroras are mitotic-specific kinases that orchestrate proper mitotic progression and are found deregulated in many cancers. Phospho- acceptors sites were identified by mass spectrometry and confirmed by site-directed mutagenesis. siRNA experiments confirmed that Aurora kinases are responsible for HDAC1 and HDAC2 phosphorylation in vivo. Preliminary experiments with Aurora- dependent phospho mutants seems to suggest that this phosphorylation has a very little impact over HDAC1/2 enzymatic activity, binding properties and sub-cellular localization. Since the phosphorylated forms of HDAC1 and 2 are a small fraction of the total, we developed specific antibodies against phospho HDAC1 and HDAC2. Immunofluorescence experiments showed that the bulk of HDAC1 and HDAC2 phosphorylation occur at the prophase and then it rapidly decreases at prometaphase. A second wave of phosphorylation of HDAC1 is then observed in metaphase. Our data globally suggest a mechanistic link between HDACs and Aurora kinases in mitosis. Since both HDACs and Aurora kinases inhibitors represent promising anticancer drugs, elucidating this relation could provide new insights for combinatorial approaches in cancer therapy.

International PhD Student Cancer Conference 125 May 20, 2010 – Lectures, poster session II and oral presentations

B28 CLASPing microtubules to adherens junctions (Shahbazi Marta Nasila), Epifano Carolina, Leiva-Vega Luis, Perez-Moreno, Mirna. Spanish Cancer Research Center, Madrid, Spain 28029

Adhesion receptors play key roles both in cell signaling and in the maintenance of tissue architecture, and when perturbed can lead to diseases, including cancer. We use skin as a model system to study the role of p120-catenin, a protein that interacts with the cytoplasmic domain of Cadherin adhesion receptors, in cell division, cell polarity and tissue architecture. Using a yeast-two hybrid screen, we identified a novel interaction between p120-catenin and a microtubule binding protein termed Clasp2, which specifically binds to the + ends of microtubules, and localizes in centrosomes. We observed that, in primary keratinocytes, these proteins interact with each other and colocalize at cell adhesion sites. Interestingly, absence of p120-catenin disrupts Clasp2 distribution, and leads to alterations in the organization of the microtubule network and mitotic defects. In skin, the distribution of Clasp2 is enriched in the proliferative progenitor (basal) cells of the epidermis, in a polarized apical distribution. In the absence of p120-catenin, this distribution becomes randomized. We are now characterizing which domains are important for such an interaction in vitro, and more importantly, what is the impact of this interaction in sustaining the polarized cell divisions that maintain epidermal architecture and skin physiology.

International PhD Student Cancer Conference 126 May 20, 2010 – Lectures, poster session II and oral presentations

B29 Mir-20a is overexpressed in colorectal cancer and contributes to subvert TGFb-driven p21WAF1 induction [Viktorija Sokolova 1], Antonio Fiorino 2, James F. Reid 1, Elisabetta Crippa 1, Manuela Gariboldi 12, Marco A. Pierotti 12 1 Molecular Genetics of Cancer, FIRC Institute of Molecular Oncology Foundation, Milan, Italy 2 National Cancer Institute Foundation IRCCS, Milan, Italy

Variations of miRNAs expression in human malignancies are associated with their localization in frequently altered chromosomal regions. By searching for miRNAs in regions showing copy number changes and concordant gene expression in 36 colorectal sporadic cancers (CRCs) compared to their normal counterpart, we identified the miR17-92 cluster on the 13q31 locus, which is gained at early stages of CRC and is highly expressed. Mir-20a, from this cluster, is a paralog of miR-106a from the miR-106b-25 cluster. Since mir-106a has been shown to abrogate the effect of TGF-beta in gastric carcinoma, we decided to investigate the relationship between enhanced miR-20a expression and TGF-beta sensitivity in CRC. To this aim, we selected two colorectal cell lines, FET and SW837, with no gains of 13q31, an intact TGF-beta/SMAD pathway, and expressing low miR-20a levels. We are studying its effect in presence of TGF-beta ligand or in combination with an inhibitor of MAPK/ERK cascade, and have found that exogenous miR-20a significantly decreases the induction of p21 by TGF-beta, both at mRNA and at protein level. Accordingly, the TGF-beta suppressive effect on cell proliferation is inhibited by miR-20a, which leads us to confirming its oncogenic function in CRC through subversion of the TGF-beta growth inhibition.

International PhD Student Cancer Conference 127 May 20, 2010 – Lectures, poster session II and oral presentations

B30 Characterization of histone PTMs and proteins associated with specific chromatin functional sub-domains (Soldi1 Monica), Bonaldi1 Tiziana 1 Department of Experimental Oncology, European Institute of Oncology, IFOM-IEO Campus, Via Adamello 16, 20139 Milano, Italy

Chromatin structural dynamics regulate diverse processes like differentiation, proliferation, and growth. One of the two main mechanisms for regulating chromatin is the covalent post translation modification (PTM). The modifications act as docking site for specific binding proteins that translate the histone PTMs into a specific biological outcome. At present, the most successful tools employed to study histone PTMs are antibodies-based strategies and mass spectrometry (MS). In order to overcome the limitations and combine the advantages of two approaches, we plan to apply ChIP to isolate specific functional chromatin domains and MS analysis to identify the PTMs co-enriched at these domains and their “chromatin interactome”. To set up the proof of principle, I have started to work on well characterized H3K9me3 modification using Hela S3 as model system. The preliminary results highlighted an enrichment of silent marks and the depletion of active marks as well as the recruitment of known binders, i.e. HP1. In the future, I intend to study how PTMs patterns and the relative interactome changes at specific chromatin loci, in a dynamic manner. The combination of biochemical and analytical approach may pave the way for elucidate the molecular mechanism underlying a histone mark and its biological outcome.

International PhD Student Cancer Conference 128 May 20, 2010 – Lectures, poster session II and oral presentations

B31 Pairing up PtdIns5P - In search for new phosphoinositide-binding proteins - Lilly Sommer, Aurelia Lewis*, Clive D’Santos#, Nullin Divecha Paterson Institute for Cancer Research, Inositide Laboratory, Manchester, UK * PROBE, University of Bergen, Bergen, Norway # CRUK Cambridge Research Institute, Li-Ka Shing Center, Cambridge, UK

Phosphoinositides are important lipid signalling molecules. Their spatio-temporal levels are tightly controlled and misregulation is linked to diseases such as cancer. Phosphoinositides are found in all cellular membranes, but also in the nucleus in a “non-membranous” form. Phosphoinositide signalling is mediated through interaction with proteins which contain specific phosphoinositide binding domains. In recent years, phosphatidylinositol 5-phosphate (PtdIns5P) has emerged as a new and important player in nuclear phosphoinositide signalling. The discovery of specific PtdIns5P binding proteins has provided insights into the significance of this lipid. It is essential to identify further downstream targets of PtdIns5P to specify its cellular function and metabolism. Our approach to find new PtdIns5P binding proteins utilises conventional protein chromatography coupled to surface plasmon resonance (SPR). Two types of SPR lipid sensor chips, differing in their mode of phosphoinositide presentation, have been developed. Both have been characterised for their ability to detect binding of nuclear and cytoplasmic proteins to phosphoinositides and for their application to detect protein-phosphoinositide interactions after chromatography. Other techniques to identify interaction partners for PtdIns5P use stable isotope labelling with amino acids in cell culture (SILAC), followed by protein pull downs with control and PtdIns5P lipid beads and mass spectrometry based proteomic screening.

International PhD Student Cancer Conference 129 May 20, 2010 – Lectures, poster session II and oral presentations

B32 Modification of p53 by SUMO-2/3 (Stindt, Maren H) [1], Carter, Stephanie [1], Vousden, Karen H [1] [1] the Beatson Institute for Cancer Research, Glasgow, UK

The p53 tumour suppressor protein is regulated by numerous post-transcriptional modifications. While most modifications occur at several sites throughout the protein, modification by the protein SUMO (small ubiquitin like modifier) is limited to a single lysine (K386) within the C-terminal domain of p53. The SUMO family consists of three members, SUMO-1 being 50% homologous to SUMO-2 and 3, which are almost identical to each other. Although several studies have investigated the role of SUMO-1 conjugation to p53, the consequences of this modification are not yet clear. However, the free pool of SUMO-2/3 is much larger than the pool of SUMO-1, suggesting a potential role SUMO-2/3 as well. We have found that the ubiquitin- and Nedd8-E3-ligase MDM2 can promote conjugation of SUMO-2/3 on wildtype and mutant p53. While p53-MDM2 binding is critical for efficient p53-SUMOylation, this activity of MDM2 is not dependent on the retention of a functional RING domain. Our initial results suggest that these effects are competitive and that inhibition of the ability of MDM2 to ubiquitinate can promote SUMOylation. Although only a small fraction of p53 is SUMOylated at a time, the modification seems to efficiently silence p53’s transcription activity.

International PhD Student Cancer Conference 130 May 20, 2010 – Lectures, poster session II and oral presentations

B33 CRKRS: a novel candidate prognostic marker and therapeutic target in breast cancer. “Angelo Taglialatela”, Manuela Vecchi, Salvatore Pece and Pier Paolo Di Fiore. IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Via Adamello 16, 20139 Milan, Italy.

Breast cancer patients outcome is difficult to predict and treatments are effective in only a minority of them while others develop resistance. Therefore, there is a great clinical need to identify new prognostic markers and therapeutic targets in order to personalize the treatment of breast cancer patients. The subversion of several kinases has been frequently implicated in malignant transformation and during last years a variety of kinase inhibitors has been developed. To provide clues on both potential new prognostic and therapeutic targets, a large-scale survey of 125 serine/threonine kinases (STK) has been recently performed by in situ analysis on tissue microarrays (TMA) (Capra et al. 2006). This study identified CRKRS, a protein putatively involved in the regulation of the splicing machinery, as overexpressed in breast cancers. Importantly, CRKRS expression significantly associates with a higher risk of disease recurrence by in situ hybridization on breast cancer TMAs. Additionally, the finding that CRKRS is amplified and overexpressed in breast cancer cell lines is particularly relevant given that gene amplification is one of the molecular mechanisms leading to oncogene activation. Therefore, these preliminary findings project a possible use of CRKRS as a prognostic marker and therapeutic target in breast cancer.

International PhD Student Cancer Conference 131 May 20, 2010 – Lectures, poster session II and oral presentations

B34 Role of B cell Receptor in Burkitt’s Lymphoma Gabriele Varano1,2*, Simon Raffel3*, Elena Vitale4, Francesco Mancuso4, Tiziana Bonaldi4, Klaus Rajewsky3 and Stefano Casola1 1 Institute of Molecular Oncology Foundation (IFOM), Milan, Italy; 2 European School of Molecular Medicine (SEMM), Milan, Italy; 3 Program of Cellular and Molecular Medicine, Children’s Hospital, and Immune Disease Institute, Harvard Medical School, Boston, USA, 4 European Institute of Oncology, Milan, Italy *contributed equally

B lymphocytes rely on the expression of a functional B cell receptor (BCR) to survive and its conditional ablation in mature B cells determines cell death. Non-Hodgkin B cell lymphomas, like Burkitt’s lymphoma, generally retain BCR expression and, therefore, we asked whether the receptor is required for survival and/or proliferation of lymphoma B cells. Using a mouse model in which deregulated expression of a human c-MYC gene is targeted to mature B cells, and a Cre/loxP-mediated strategy to induce BCR deletion in tumor cells carrying a floxed pre-rearranged variable region in the immunoglobulin heavy chain locus, I showed that BCR plays a role in controlling survival of c-MYC transformed lymphoma cells. BCR– tumor cells proliferated to the same extent as their BCR+ counterparts, but in contrast to the latter, disappeared from the culture undergoing apoptosis. When transplanted into syngeneic, recipient mice, BCR+ and BCR– tumor cells could equally home to lymphoid organs where BCR– tumor cells were dramatically counter-selected. Given the essential role of the BCR for survival of c-MYC-transformed B lymphoma cells, the antigen receptor itself and/ or components of BCR-mediated signaling cascades may serve as therapeutic targets for the treatment of mature B cell lymphomas.

International PhD Student Cancer Conference 132 May 20, 2010 – Lectures, poster session II and oral presentations

B35 Function and regulation of p31comet in the Spindle Assembly Checkpoint (Gianluca Varetti), Veronica Krenn, Claudia Guida, Anna De Antoni and Andrea Musacchio. European Institute of Oncology, Milan, Italy.

In all eukaryotes, the Spindle assembly Checkpoint (SAC) subtends to the accuracy of chromosome segregation. The molecular target of the SAC is the protein Cdc20, an activator of the Anaphase Promoting Complex (APC). The APC is an E3 ubiquitin ligase, whose activity is required for sister chromatid separation and mitotic exit. When bi-orientation is achieved, the SAC needs to be silenced to allow anaphase. The protein p31comet contributes to silencing of the SAC. p31comet binds to the SAC protein Mad2, preventing a crucial Mad2 dimerization reaction that is necessary for SAC signaling. How p31comet is regulated during SAC activation and how it acts to switch the SAC off are poorly understood issues. We show that in mitosis p31comet localizes via Mad2 to unattached kinetochores, where it cycles rapidly. p31comet becomes phosphorylated in mitotic cells in a Mad2- , Mps1- and Aurora B-dependent manner. The role of phosphorylation, however, is unclear, because a p31comet mutant that cannot be phosphorylated cycles at kinetochores with kinetics that are identical to those of the wild type protein. We also found that p31comet is necessary for APC activation. In its absence, Cdc20 becomes stabilized, the association of the Mitotic Checkpoint Complex (MCC) to the APC is increased, and mitotic exit is delayed. These observations suggest that p31 plays a role in the re-activation of the APC/C by promoting the release of the MCC, and that Cdc20 turnover is a crucial aspect of this regulatory network.

International PhD Student Cancer Conference 133 May 20, 2010 – Lectures, poster session II and oral presentations

B36 “Rho-actin dependent regulation of Phactr proteins” (Maria Wiezlak)1, Stephane Mouilleron2, Neil McDonald2 and Richard Treisman1 1 Tanscription Laboratory, 2 Structural Biology Laboratory, Cancer Research UK London Research Institute, Lincoln’s Inn Fields Laboratories, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom

Phosphatase and actin regulators (Phactr) are a family of four proteins, highly expressed in the nervous system. They have been found to bind actin and protein phosphatase 1 (PP1) but their function is not well known. A member of the family, Phactr4, was recently found to regulate neural tube and optic fissure closure by controlling PP1-Retinoblastoma cell cycle progression, but molecular basis for this regulation is not yet established. Phactr proteins bind actin through their C terminal RPEL domain. The RPEL domain was previously shown to confer Rho-regulated nuclear shuttling and activation of MAL/MKL1/myocardin –related transcription factor A. MAL is cytoplasmic in unstimulated cells and accumulates in the nucleus upon activation of Rho-actin GTPase signaling. It is F-actin assembly/G-actin depletion that regulates MAL nuclear shuttling. We have been studying regulation of Phactr proteins in NIH3T3 cells and found that localisation of Phactr is Rho-actin dependent. Further studies are aiming to describe function of Phactr family in the nervous system in the context of actin rearrangement and the molecular mechanism of Phactr1 binding to actin.

International PhD Student Cancer Conference 134 May 20, 2010 – Lectures, poster session II and oral presentations

B37 Investigating the different roles of RNA Polymerase II ubiquitylation in Saccharomyces cerevisiae (Marcus DR Wilson)1, Stefan Sigurdsson1,2, Jesper Q svjestrup1 1 Clare Hall Laboratories, Cancer Research UK London Research Institute, Blanche Lane, South Mimms, EN6 3LD, United Kingdom 2 current address Department of Biochemistry and Molecular Biology, Biomedical Center, School of Health Sciences, University of Iceland, Reykjavik, Iceland

Ubiquitylation is a common Post-translational modification that signals many cellular processes. An ubiquitin code on a protein arises from different chain topologies and different modified residues on substrates. Poly lysine-48 linked ubiquitin targets a substrate for degradation, whereas poly lysine-63 ubiquitylation does not. RNA polymerase II (RNAPII) becomes ubiquitylated when irreversibly stalled, for instance at sites of DNA damage, resulting in degradation of the largest subunit, and disassembly of the transcription machinery. The extent, nature and control of this ubiquitylation has yet to be resolved. Recently a ‘two ligase’ mechanism of ubiquitylation has been proposed, whereby the first E3 ligase, Rsp5, adds either mono-ubiquitin or poly lysine-63 linked chains onto RNAPII. In the second step, proteasomal targeting occurs by an Elc1 protein containing complex. To which substrate lysine these E3s target and in what order is unknown. I have shown that Elc1 forms a Cullin-RING type E3 ligase, which selectively polyubiquitylates monoubiquitylated substrate. Further the Elc1 complex appears to add de novo chains at a separate substrate lysine, suggesting a hierarchy of ubiquitylation. Preliminary data also suggests that there appears to be a separate role for lysine-63 linked ubiquitin chains in damage signalling on RNAPII.

International PhD Student Cancer Conference 135 May 20, 2010 – Lectures, poster session II and oral presentations

B38 Role of cofilin oxidation in cancer progression (Jenifer M. Wood) and Michael F. Olson Beatson Institute for Cancer Research, Bearsden, Glasgow, UK

Reactive oxygen species (ROS) are highly reactive molecules that are essential for normal cellular function due to their involvement in the regulation of many signalling pathways. The main way in which ROS modulate such pathways is through the oxidation of cysteine residues on key proteins. This may result in the activation or inhibition of these proteins and consequently the signalling pathways in which they are involved. However, ROS can also cause damage to DNA, proteins and lipids, and so cells have acquired defence mechanisms that allow them to maintain ROS homeostasis. The cofilin family of proteins are well known regulators of actin dynamics but recently they have also been linked to other cellular processes such as apoptosis. The best characterised mechanism of cofilin regulation is phosphorylation but there is now evidence that oxidation can also regulate its activity. The cofilin pathway has been implicated in cancer, particularly in invasion and metastasis. ROS homeostasis is also frequently altered in cancer cells and this is believed to contribute to disease development and drug resistance. We are currently investigating whether the oxidation of cofilin alters its activity in relation to actin dynamics and apoptosis, contributing to cancer progression.

International PhD Student Cancer Conference 136 May 20, 2010 – Lectures, poster session II and oral presentations

B39 Molecular Signature of response and potential pathways related to resistance to the HSP90 inhibitor, 17-AAG, in breast cancer. Magdalena Zajac(1), Gonzalo Gomez(2), Javier Benitez(1,3), Beatriz Martínez- Delgado(1,3). Affiliations: 1) Human Genetics Group, Spanish National Cancer Centre (CNIO), Madrid, Spain, 2) Bioinformatics Unit. CNIO, Madrid, Spain. 3) CIBERER, Centre for Biomedical Networking Research on Rare diseases

Considering the complexity of breast cancer, with its multiple genetic abnormalities, targeting a single pathway by inhibiting the activity of one component is unlikely to be effective in a long term. Identification of molecular targets that will modulate multiple components of several signalling pathways would be desired for anticancer treatment. To that end, HSP90 gained lately extreme interest and became an interesting cancer drug target. In fact, the HSP90 inhibitor, 17-AAG, currently has entered in phase II and III clinical trials as an anticancer agent in breast and other tumors. Since HSP90 inhibition leads to global depletion of oncogenic proteins involved in multiple pathways we applied global analysis using gene array technology to study new genes and pathways involved in the drug response in breast cancer. In the current microarray study we have identified a molecular signature of response to 17-AAG composed of 35 genes and described novel isoforms of HSP70 (HSPA2, HSPA1L) as good biomarkers of response in breast tumors. In addition we have found differentially expressed genes and pathways between treated resistant and sensitive cells. Both by in silico pathway analysis and by functional studies we have reported significant activation of NF-κB pathway in resistant cells upon treatment, indicating that this pathway could be potentially targeted to overcome resistance. Overall our results uncover novel genes regulating the HSP90 chaperone complex in breast cancer and describe potential biomarkers and pathways of response and resistance to 17-AAG.

International PhD Student Cancer Conference 137

May 21, 2010

SESSION Va: Signal transduction SESSION Vb: cancer genetics and drug resistence

SESSION VI: Biology of stem cells in development and cancer

International PhD Student Cancer Conference

May 21, 2010 – Keynote lecture and oral presentations

Keynote lecture Andreas Trumpp German Cancer Research Center (DKFZ), Heidelberg

Oral presentations Andrea Longatti London research Institute, London

Francesca Montani European Institute of Oncology, Milan

Lakshmi Revathi Perumalsamy National Centre for Biologicl Sciences (NCBS), Bangalore

Rinske Drost The Netherlands Cancer Institute (NKI), Amsterdam

Charlotte Ng Cambridge Research Institute, Cambridge, UK

Margherita Yayoi Turco European Institute of Oncology, Milan

Fernando Flores-Guzman German Cancer Research Center (DKFZ), Heidelberg

Stephen Goldie Cambridge Research Institute, Cambridge, UK

Cristina Elisabetta Pasi European Institute of Oncology, Milan

International PhD Student Cancer Conference 141

May 21, 2010 – Keynote lecture and oral presentations

Dormancy in normal and malignant stem cells Trumpp, Andreas1, Wilson, Anne2, Laurenti, Elisa2, Essers, Marieke1 1 Divison of Stem Cells and Cancer, Deutsches Krebsforschungszentrum (DKFZ) and Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Heidelberg, Germany 2 Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland

Abstract: Adult stem are required to maintain highly regenerative tissues such as the skin, the intestinal epithelium and the hematopoietic system. Mouse hematopoietic stem cells (HSCs) are the most well characterized somatic stem cell to date, and serve as a model for understanding other adult stem cells present in the mammalian body. Using two types of label-retaining assays we have identified a long-term dormant population within the most immature HSCs (Lin-Sca1+cKit+CD150+CD48-CD34-). Computational modeling suggests that dormant HSCs (d-HSCs) divide about every145 days, or 5 times per lifetime. d-HSCs harbor the vast majority of multi-lineage long- term self-renewal activity. While they form a silent reservoir of the most potent HSCs during homeostasis, they are efficiently activated to self-renew in response to bone marrow injury or G-CSF stimulation. After re-establishment of homeostasis activated HSCs return to dormancy, suggesting that HSCs are not stochastically entering the cell cycle, but reversibly switch from dormancy to selfrenewal under conditions of hematopoietic stress1,2. One of the reasons cancer stem cells are thought to escape anti-proliferative chemotherapy is their relative dormancy3. We now have shown that treatment of mice with Interferon-alpha family leads to the activation and proliferation of dormant HSCs in vivo, which sensitizes them to chemotherapy drugs. HSCs lacking either the interferon-a/b receptor, STAT1 or Sca-1 are insensitive to IFNa stimulation, demonstrating that STAT1 and Sca-1 mediate IFNa induced HSC proliferation4. The implications of these results for the design of strategies to target dormant CML stem cells not targetable by imatinib alone will be discussed.

1 Wilson A. et al, (2008). Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. CELL, 135: 1118-1129. 2 Laurenti E. et al., (2008). Hematopoietic stem cell function and survival depend on c-Myc and N-Myc activity. CELL, Stem Cell, Dec. 4;3(6):611-24. 3 Trumpp A. and Wiestler O.D., (2008). Targeting the evil Twin. NATURE Clinical Practice Oncology, Jun;5(6):337-47. 4 Essers M. et al. (2009). IFNa activates dormant HSCs in vivo. Nature. 2009 Apr 16;458(7240):904-8

International PhD Student Cancer Conference 143 May 21, 2010 – Keynote lecture and oral presentations

Rab-GTPases and Rab-GAPs Involved in Autophagy (Andrea Longatti) and Sharon A. Tooze London Research Institute, Cancer Research UK, London, United Kingdom

Autophagy is a bulk degradation process in which double membrane vesicles, called a u t o p h a g o s o m e s ( AVs) , f o r m a r o u n d a p o r t i o n o f c y t o p l a s m o r o r g a n e l l e s . T h e s e AVs f u s e with endosomes and lysosomes whereupon they become degradative compartments. Autophagy is a multi-step process that can be broken down into defined steps: vesicle nucleation, expansion, closure, and fusion to the endo-lysosomal system. Presumably each step involves a different set of trafficking machinery such as Rab-GTPases. These are small GTP-binding proteins that perform important functions in membrane traffic. Little is known about the membrane trafficking events required for AV nucleation and expansion and the origin of the autophagosomal membrane remains elusive. In order to address these questions, we have systematically screened the Rab-GTPase activating proteins (Rab-GAPs). We over-expressed all 38 putative human Rab-GAPs (characterised by the presence of a TBC-domain) in HEK293/GFP-LC3 cells thus inactivating their corresponding Rabs. Autophagy induced by starvation, was measured by GFP-LC3 lipidation. Through this screen 12 Rab-GAPs were identified that inhibit autophagy, including TBC1D14. We went on to investigate the candidate target Rab proteins of these inhibitory GAPs. Special focus will be on TBC1D14, a Rab-GAP that seems to bind to other autophagy-related proteins (Atg proteins) and may act on Rab11.

International PhD Student Cancer Conference 144 May 21, 2010 – Keynote lecture and oral presentations

The mitotic exit oscillator at work Romilde Manzoni*1, Francesca Montani*2, Clara Visintin2, Andrea Ciliberto*1, Rosella Visintin*2 1 IFOM, The FIRC Institute of Molecular Oncology, via Adamello, 16, Milan, 20139, Italy, 2 European Institute of Oncology, Department of Experimental Oncology, via Adamello, 16, Milan, 20139, Italy

Mitotic cyclin-dependent kinases (CDKs) promote entry in mitosis. In the budding yeast Saccharomyces cerevisiae, phosphatase Cdc14 orchestrates exit from mitosis by inactivating CDKs and reversing CDK-mediated phosphorylation events. During anaphase, Cdc14 is released from its inhibitor Cfi1/Net1 by two successive pathways: FEAR (CdcFourtheen Early Anaphase Release) and MEN (Mitotic Exit Network). Three sets of kinases play a critical role in the Cdc14-release process: the Polo-like kinase Cdc5, the mitotic CDKs themselves, and the most downstream MEN kinase Dbf2. The mechanism that coordinates these different kinases resulting in Cdc14 activation remains unclear. Here we show that Cdc14 release is a two-hit process that requires Cdc5 plus either MEN or CDKs. Once released, Cdc14 triggers a negative feedback loop composed of Cdc5-Cdc14-Cdh1-Cdc5. In the presence of stable levels of mitotic cyclins, this negative feedback generates cycles of Cdc14 release and sequestration. Interestingly this “mitotic oscillator” works also in presence of very high levels of mitotic cyclins. This observation suggests that next to the overall inactivation of mitotic cyclins, the completion of mitosis also relies on the temporally and spatially controlled phopshorylation status of different substrates determined by the mitotic-CDKs/Cdc14 ratio.

International PhD Student Cancer Conference 145 May 21, 2010 – Keynote lecture and oral presentations

A non-canonical Notch cascade converges on mitochondria for the regulation of cell survival (Lakshmi R. Perumalsamy) , Manjula Nagala and Apurva Sari National Centre for Biological Sciences, Bellary Road, Bangalore 560065, Karnataka, India

The Notch family of transmembrane receptors signal via core evolutionarily conserved elements to regulate diverse processes in metazoans. Transcriptional outputs of Notch signaling are implicated in cell fate decisions during development and the process of transformation. The regulation of apoptosis is a recently appreciated aspect of Notch function, although the molecular mechanisms underlying this activity remain to be elucidated. In this context, we have described a Notch activated cascade regulating cell survival, independent of canonical nuclear interactions. The kinases Phosphatidylinositol 3-Kinase (PI3K) and mammalian Target of Rapamycin (mTOR) - nodal elements in signaling pathways are key intermediates in the cascade, suggesting that Notch may function as a molecular sensor in the regulation of cell survival. Intriguingly, Notch activity was localized in the cytoplasm and abrogated by nuclear sequestration (Cell Death & Differentiation, 2009). Mitochondrial integration of the cellular death machinery is a core event in the apoptotic response to several stimuli. Our experiments demonstrate that Notch-mediated anti-apoptotic activity converges on molecules regulating mitochondrial architecture and integrity via the kinase Akt (Proc. Natnl. Acad. Sci. 2010). Collectively, these data suggest that non-canonical Notch signaling integrates processes regulating nutrient sensing and mitochondrial dynamics with pathways regulating cell survival.

International PhD Student Cancer Conference 146 May 21, 2010 – Keynote lecture and oral presentations

The impact of defined Brca1 mutations on tumor development, drug response and acquired resistance (Rinske Drost1), Joanna Morris2, Ute Boon1, Eva Schut-Kregel1, Hanneke van der Gulden1, Eline van der Burg1, Mark Pieterse1, Ellen Solomon2, Peter Bouwman1 and Jos Jonkers1 1 Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands 2 Department of Medical and Molecular Genetics, King’s College London, Guy’s Medical School Campus, London, United Kingdom

Women with heterozygous germline mutations in BRCA1 have a strongly increased lifetime risk of developing breast and/or ovarian cancer. BRCA-related cancers show a good response to DNA-damaging agents, like platinum compounds. But resistance to platinum agents is still a serious problem in the treatment of BRCA- related cancers. Strikingly, mammary tumors arising in the K14Cre;Brca1F/F;p53F/F mouse model (which lack Brca1 exons 5-13) do not become resistant against platinum drugs, suggesting that (partial) BRCA1 function is required for platinum resistance. Genetic reversion of BRCA1 mutant alleles may be an important mechanism underlying the induction of platinum resistance. To investigate whether genetic reversion of BRCA1 is causally related to acquired platinum resistance, mice carrying the Brca1C61G mutation were bred with K14Cre;Brca1F/F;p53F/F mice. Brca1C61G is a pathogenic missense mutation which disrupts the interaction between BRCA1 and BARD1 and thereby the E3 ubiquitin ligase activity of BRCA1. In the K14Cre;Brca1C61G/F;p53F/F model, mammary tumor development was significantly accelerated compared to the K14Cre;Brca1F/F;p53F/F model. We are currently trying to find an explanation for this difference in tumor latency. Sequencing and southern blot analysis revealed that these mammary tumors still carry the Brca1C61G mutation and have lost the floxed Brca1 and p53 alleles. Similar to Brca1del;p53del mammary tumors, Brca1C61G/del;p53del tumors display genetic instability, are ER- and vimentin-negative and cytokeratin 8-postive. Several spontaneous Brca1C61G/del;p53del mammary tumors were transplanted orthotopically into immunocompetent mice and intervention studies with platinum compounds were performed. We have observed resistance to platinum agents for various independent Brca1C61G/del;p53del tumors. Thus far, we have not observed genetic reversion of the Brca1C61G mutation as a mechanism of platinum resistance. We are currently investigating which other mechanisms can cause resistance to platinum agents in these tumors. Identical experiments are performed for tumors carrying to other BRCA1 founder mutations, respectively the Brca1185delAG and Brca15382insC mutation. This research could show if genetic reversion is one of the most important mechanisms of platinum resistance in BRCA-related cancers. These novel mouse models could offer an ideal platform to test new treatment strategies for BRCA1 mutation carriers. This research could further reveal differences in sensitivity to platinum drugs of different BRCA1 mutations. This insight could lead to various treatment strategies for carriers of different BRCA mutations and thereby hopefully to a better survival.

International PhD Student Cancer Conference 147 May 21, 2010 – Keynote lecture and oral presentations

Genomic analysis of genetic heterogeneity and evolution in high-grade serous ovarian carcinoma. Susanna L Cooke, Cancer Research UK Cambridge Research Institute, Cambridge, UK [Charlotte KY Ng], Cancer Research UK Cambridge Research Institute, Cambridge, UK Nataliya Melnyk, British Columbia Cancer Agency, Centre for Translational and Applied Genomics, Vancouver, Canada Maria J Garcia, Cancer Research UK Cambridge Research Institute, Cambridge, UK Tom Hardcastle, Cancer Research UK Cambridge Research Institute, Cambridge, UK Jillian Temple, Cancer Research UK Cambridge Research Institute, Cambridge, UK Simon Langdon, Division of Pathology and Edinburgh Breakthrough Research Unit, University of Edinburgh, Edinburgh, UK David Huntsman, British Columbia Cancer Agency, Centre for Translational and Applied Genomics, Vancouver, Canada James D Brenton, Cancer Research UK Cambridge Research Institute, Cambridge, UK

Drug-resistant relapse is a major reason of treatment failure in ovarian cancer, despite a high initial response rate. Intra-tumour genetic heterogeneity has been put forward as a possible cause, where initial treatment fails to clear a subpopulation of intrinsically resistant cells, which then grow out to cause relapse. An alternative hypothesis suggests that secondary resistance evolves in tumours that evaded treatment because of extrinsic reasons, such as drug delivery. To investigate the genetic relationship of diseases at presentation and at relapse, we performed 24-colour FISH and genome- wide copy number and loss of heterozygosity analyses on three pairs of ovarian cancer cell lines, each pair established from tumours sampled at the two disease stages from a single patient. We showed that the disease taken at different timepoints was clonal but very divergent. As relapsed diseases had rearrangements that were mutually exclusive with changes present in disease at presentation, this suggests that the clones at relapse diverged from the dominant clone at presentation prior to diagnosis and evolved independently. This implies that resistant clone could have existed as a minor subclone at presentation and that intra-tumour genetic heterogeneity could explain high rates of relapse in ovarian cancer.

International PhD Student Cancer Conference 148 May 21, 2010 – Keynote lecture and oral presentations

THE RALP ADAPTOR PROTEIN MODULATES MOUSE EMBRYONIC STEM CELL DIFFERENTIATION (Margherita Yayoi Turco), Allegra Pianaroli, Ernesta Fagiani, Stefano Casola, Giuseppe Testa, Antonio Simeone*, Luisa Lanfrancone European Institute of Oncology, Department of Experimental Oncology, Campus IFOM-IEO, Milano, Italy; *CEINGE, Napoli, Italy

Development requires the strict coordination of signalling pathways of proliferation, differentiation and migration. The molecular mechanisms that regulate cell behaviour during embryogenesis are often dysregulated or reactivated during tumourigenesis. In melanomas their highly metastatic behavior has been hypothesized to reflect the reactivation of molecular pathways typical of their migratory and plastic embryonic progenitors. Adaptor proteins play an important role in the regulation of specific signalling pathways by linking activated cell-surface receptors to their downstream intracellular targets. RaLP, the latest identified member of the Shc family of adaptor proteins, might represent another example of a developmental modulator reactivated in tumour cells. RaLP is expressed early during embryogenesis in the pluripotent epiblast and then in tissues deriving from the neuroectoderm and neural crest. In the adult, it is expressed in the olfactory lobe, cerebral hemisphere and cerebellum. In the pathological context, RaLP is expressed in melanomas and its expression level correlates with disease progression. To understand the role of RaLP in melanoma pathogenesis, it is necessary to elucidate its function during embryonic development. To this end, RaLP knockout embryonic stem cells were differentiated towards epiblast cells and neural lineages. RaLP expression is tightly regulated during these differentiation programs and the genetic deletion of RaLP confers a differentiation impairment towards both these lineages. In particular, during the differentiation to neural fate KO cells showed severe defects in neuronal formation. The data seems to suggest a role of RaLP in the commitment of ES cells to differentiation. The signaling pathway in which RaLP is implicated will be further elucidated.

International PhD Student Cancer Conference 149 May 21, 2010 – Keynote lecture and oral presentations

Potential cancer stem cells in ret transgenic mouse model of spontaneous melanoma (Fernando Flores-Guzman), Dirk Schadendorf, Viktor Umansky. German Cancer Research Center, DKFZ. Heidelberg, Germany

The cancer stem cell (CSC) hypothesis suggests that neoplastic clones are maintained exclusively by a rare fraction of tumor cells with stem cells properties. CSC could represent disseminated dormant tumor cells without clinical signs of progression. We used a transgenic mouse spontaneous melanoma model, in which after a short latency around 25% of all transgenic mice developed skin tumors with metastases in distant organs like liver and lungs. We found that CD133+ melanoma cells represent a small subset in primary skin tumors and lymph node metastases (< 1.5%). The amount of CD133+ melanoma cells both in primary skin tumors and lymph node metastases was depended on the primary tumor weight and stage of tumor progression. We found CD133+ single melanoma cells in the bone marrow of tumor bearing mice suggesting thereby that these cells could be potential melanoma stem cells. We also found CD20, CD24, CD44, CD166 and Nestin expression in primary tumors, which correlated with the tumor progression. In conclusion, our data demonstrate an existence of the subpopulation of CD133+ melanoma cells in primary skin tumors, metastatic lymph nodes and in the bone marrow of tumor bearing transgenic mice and suggest that these subsets could be potential CSC.

International PhD Student Cancer Conference 150 May 21, 2010 – Keynote lecture and oral presentations

FRMD4A is a potential marker of cancer stem cells in human head and neck squamous cell carcinoma (HNSCC) (Stephen J. Goldie) - 1 Richard Price - 2 Fiona M. Watt - 1 1 - Cambridge Research Institute, Cambridge, UK 2 - Dept of Plastic Surgery, Addenbrooke’s Hospital, Cambridge, UK

Head and neck squamous cell carcinoma (HNSCC) often recurs despite radical treatment. Cancer stem cells may evade current therapies and allow a cancer to re-grow. By targeting the tumour stem cell population, we may control the disease and cause less harm to the patient’s normal tissues. FRMD4A has been shown to be more abundant in human keratinocytes with a stem-like phenotype, and highly over expressed in a panel of human SCCs. FRMD4A in human skin was studied using in-situ hybridization and immunofluorescence staining. Laser capture microscopy (LCM) was used to collect samples of the basal and granular layers of human epidermis in order to compare levels of FRMD4A by Q-PCR. In cell cultures derived from human HNSCCs FRMD4A was stably knocked down using lentivirus shRNAs. The effect on function was tested in vitro and by xenografting to show changes in tumorigenicity. Results of these studies revealed much higher levels of expression of FRMD4A in the basal layer compared to the granular layer. Knockdown of FRMD4A disrupted normal cell-cell adhesion in HNSCCs. Growth of the HNSCC lines in vitro and in vivo was reduced in the FRMD4A knockdowns, making it a potential target for future therapies.

International PhD Student Cancer Conference 151 May 21, 2010 – Keynote lecture and oral presentations

Regulation of self-renewal in mammary stem cells (Pasi Cristina Elisabetta), author, IEO-Milan Bonizzi Giuseppina, IEO-Milan Cicalese Angelo, IEO-Milan Pier Giuseppe Pelicci, IEO-Milan

It has been well established that tumours are hierarchically organized tissues composed by different cell subsets. Particularly, only a small fraction of these cells has the ability to self-renew and form tumours if transplanted in recipient mice; in light of these properties, these cells have been named cancer stem cells (CSCs). In recent years, the hypothesis of the existence of CSCs has gained consent, as they could be isolated in different tumours both in humans and in mice. Nevertheless, the origin of CSCs and the mechanisms that account for the generation of these cells are yet unknown. We have recently demonstrated that the control of self-renewing SC divisions may be involved in tumorigenic processes in mammals and that p53 plays a key role in this regulation. To better understand the mechanisms that account for this control, we have investigated the role of the Myc oncogene on stem or progenitor cells, and have been able to show a role for this protein on the regulation of self-renewal and symmetric or asymmetric division in mammary SCs. The results of this study will help in broadening the available targets for a CSC-mediated anti-tumour therapy.

International PhD Student Cancer Conference 152 KEYNOTE SPEAKERS

International PhD Student Cancer Conference

Keynote Speakers

PETER FRIEDL

Education Univ. Bochum, Germany M.D. 1992 Infectious immunology Mc Gill Univ., Montreal, Canada Ph.D. 1996 Immunology and cell biology Univ. Würzburg, specialist accreditation dermatologist (2002) and clinical allergologist (2003) A. Positions and Honors 1991-1996 Research fellow Inst. Immunology, Univ. Witten, Germany 1996-2003 Resident, Department of Dermatology, Univ. Würzburg, Germany 2003-2007 Associate Professorship, Molecular Cell Dynamics, Rudolf Virchow Center, Univ. Würzburg since 2007 Chair, Microscopical Imaging of the Cell, Radboud University Nijmegen Medical Centre, Nijmegen,The Netherlands Editorial Board Member Histochem. Cell Biol., Clin. Exp. Metast., Cell Adhesion Migration, J. Clin. Exp. Cancer Res. Felix-Wankel Animal Protection Award (1994); German Skin Cancer Award (2004); Oscar-Gans Award (2005); German Cancer Award 2008 B. Selected peer-reviewed publications (in chronological order) (Publications selected from 52 peer-reviewed publications) Friedl P, Maaser K, Klein CE, Niggemann B, Krohne G, Zänker KS (1997) Migration of highly aggressive MV3 melanoma cells in 3-D collagen lattices results in local matrix reorganization and shedding of a2 and b1 integrins and CD44. Cancer Res. 57, 2061-2070. Gunzer M, Schäfer A, Borgmann S, Grabbe S, Zänker KS, Bröcker EB, Kämpgen E, Friedl P (2000) Antigen presentation in three-dimensional extracellular matrix: interactions of T cells with dendritic cells are dynamic, short lived, and sequential. Immunity 13, 323-332. Wolf K, Mazo I, Leung H, Engelke K, von Andrian UH, Deryugina EI, Strongin AY, Bröcker EB, Friedl P (2003) Compensation mechanism in tumor cell migration: Mesenchymal-amoeboid transition after blocking of pericellular proteolysis. J. Cell Biol. 160, 267-277. Friedl P, Wolf K (2003) Tumour cell invasion and migration: Diversity and escape mechanisms. Nat. Rev. Cancer 3, 362-374. Wolf K, Muller R, Borgmann S, Bröcker EB, Friedl P (2003) Amoeboid shape change and contact guidance: T cell crawling through fibrillar collagen is independent of matrix remodeling by MMPs and other proteases. Blood 160, 267-277. Friedl P, den Boer AT, Gunzer M (2005) Tuning immune responses: diversity and adaptation of the immunological synapse. Nat. Rev. Immunol. 5, 532-545. Wolf, K., Wu, Y.I., Liu, Y., Tam, E., Geiger, J., Overall, C., Stack, M.S., Friedl, P. 2007. Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion. Nat. Cell Biol. 9:893-904. Friedl P, Wolf K. 2008. Tube travel: protease functions in individual and collective cancer invasion. Cancer Res. 68:7247-7249. Friedl P, Weigelin B (2008) Interstitial leukocyte trafficking and immune function. Nat. Immunol. 9:839-848. Andresen V, Alexander S, Heupel WM, Hirschberg M, Hofmann RM, Friedl P (2009) Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging. Curr. Opin. Biotechnol. 20:54-62. Friedl P, Gilmour D (2009) Collective cell migration in development, regeneration and cancer. Nat. Rev. Mol. Cell Biol. 10:445-457. Friedl P, Wolf K (2010) Plasticity of cell migration – a multiscale tuning model. J. Cell Biol. 188:49-57.

C. Research Support DFG, Deutsche Forschungsgemeinschaft 01/04 – 09/10 Klinische Forschergruppe: “Tumormikromillieu” TP5 DFG, Deutsche Forschungsgemeinschaft 02/06 – 02/11

International PhD Student Cancer Conference 155 Keynote Speakers

The Tumor – Vessel Interface EU: 201842 06/08 – 06/12 ENCITE: European Network for Cell Imaging and Tracking Expertise Dutch Cancer Foundation: KUN 2008-4031 ---- 12/08 – 12/12 ---- Serial Killers EU people program: 237946 01/10 – 01/13 T3net: Tissue Transmigration Training Network Government of Economical Affairs and the Province of Gelderland: 06/09 – 06/13 PID082022: Pieken in de Delta Oost Nederland: Healthy Ageing

ANDREAS TRUMPP Andreas Trumpp is Professor and head of the Division of “Stem Cells and Cancer” at the German Cancer Research Center (DKFZ), and the Founding Director of the Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM) in Heidelberg, Germany. Andreas received his PhD at EMBL-Heidelberg in 1992 for his studies on molecular embryology. As a postdoctoral fellow he worked with J. Michael Bishop and Gail R. Martin at UC San Francisco on conditional knockouts of the Fgf8 and c-Myc genes. In 2000 he started the Genetics and Stem Cell Laboratory at the Swiss Institute for Experimental Research (ISREC) in Epalinges/Lausanne, Switzerland and was Professor at the Swiss Federal Institute of Technology Lausanne (EPFL) 2005-2008. His Laboratory focuses on understanding the molecular and cellular control of stem cell fate and function. Of particular interest are the complex interactions between hematopoietic stem cells and their bone marrow niches as well as their switch from a dormant to an activated state in response to injury cues. In addition, his group addresses the biology of malignant stem cells in cancer and metastasis as well as developing strategies to overcome their resistance to classical chemotherapies.

International PhD Student Cancer Conference 156 SPECIAL SESSIONS

International PhD Student Cancer Conference

Special Sessions

SILVIA CAMPORESI AND ALESSANDRO BLASIMME Two senior Folsatec PhD students will present the current debate on the controversial field of human embryonic stem cells (widely understood, ie. iPS cells, hybrids and chimeras included). The arguments used to support and to oppose such research will be discussed, together with a comparative analysis of the world different regulatory frameworks and funding policies. A debate with a panel of discussants and the public will be encouraged.

Folsatec - Foundations of the life sciences, ethics and epistemology. This PhD program is open to students having either a scientific or a philosophical degree as well as to medical doctors. The course aims to create internationally recognized scholars in the fields of the foundations of the Life Sciences, ethical analysis and the relationships between biomedicine and society. Please visit: http://www.ifom-ieo-campus.it/research/boniolo.php

PIER PAOLO DI FIORE Pier Paolo Di Fiore is the founder of IFOM, an internationally competitive research centre, where he carries out most of his research. He is Full Professor of General Pathology at Milan University and he co-founded the European School of Medicine, which provides Ph.D. programs in Molecular Medicine, Medical Nanotechnology and Bioethics. He was made an EMBO member in 1998 and has received numerous international awards. He has recently been nominated Director of the Molecular Medicine Programme at the European Institute of Oncology. Di Fiore is a scientist of international renown whose work in the field of cancer is widely recognized, as witnessed by more than 180 published papers. He is particularly interested in the mechanisms of signal transduction by growth factor receptors in tumours and the role of endocytosis in this process. In recent years, he has developed translational research lines that aim to provide practical applications to cancer treatment. His innovative work has led him to link endocytosis to cancer by demonstrating that loss of expression of the endocytic protein and cell fate determinant, Numb, is causal in breast tumorigenesis and is predictive of aggressive disease. He has also identified a signature for metastatic risk in breast cancer, a predictor of survival for early-stage lung cancer, and a stem-cell specific signature for breast cancer that has led to the identification of numerous breast stem cell-specific markers, a first step for the development of new tools for stem cell-based diagnosis, prognosis and therapeutic intervention in breast cancer.

GIUSEPPE TESTA Giuseppe Testa is Principal Investigator at the European Institute of Oncology in Milan where he heads since 2006 the laboratory of stem cell epigenetics. His academic training was in Medicine (MD at the University of Perugia), Molecular Biology (PhD at the EMBL), Bioethics and Science and Technology Studies (MA at Manchester University, visiting fellowship in the program on Science, Technology and Society at the Harvard Kennedy School). He completed his postdoctoral training in Dresden where he pioneered new stem cell genome engineering technologies and was awarded the European Doctorate for excellence of research in Biotechnology by the European Association for Higher Education in Biotechnology. His lab investigates the epigenetic mechanisms that enable the differentiation of stem cells and that mediate the reprogramming of somatic cells into pluripotent stem cells. He was recently selected to join as Associate Principal Investigator the EuroSyStem Project, aimed at interlinking biological and computation expertise in stem cell biology. In 2007 he was appointed on the Ethics and Public Policy Committee of the International Society for Stem Cell Research (ISSCR) and serves on the ethics advisory panel of the European stem cell networks ESTools and NeuroStemCell.

International PhD Student Cancer Conference Special Sessions

He authored publications both in molecular biology and bioethics in leading journals including Nature Biotechnology, Cell, Cell Stem Cell, Science, EMBO Journal and Bioethics, and is the recipient of several awards, including the Roche Prize for Leading Bioscientist of the Next Decade (2003) and the National Prize of the Accademia dei Lincei for the best MD thesis (1997). His first book ‘Die Gläsernen Gene. Die Erfindung des Individuums im molekularen Zeitalter (Naked genes. Reinventing the Human in the Molecular Age – translation forthcoming, MIT Press)’, coauthored with Helga Nowotny, was published in 2009 by Suhrkamp in the Edition Unseld.

International PhD Student Cancer Conference AUTHORS’ INDEX

International PhD Student Cancer Conference

Authors’ index

AUTHORS’ INDEX

Federica Alberghini, Poster session I [email protected] pag. 35 Seema Alexander, Poster session I [email protected] pag. 36 Veronica Algisi, Poster session I [email protected] pag. 37 Sietske Bakker, Oral Presentation [email protected] pag. 95 Roheet Bantval Rao, Oral Presentation [email protected] pag. 84 Ajoeb Baridi, Poster session I [email protected] pag. 38 Julian Blaser, Poster session I [email protected] pag. 39 Elvan Boke, Poster session II [email protected] pag. 99 Helene Bon, Poster session I [email protected] pag. 40 Vanessa Borges, Poster session I [email protected] pag. 41 Paola Brescia, Poster session I [email protected] pag. 42 Rebecca Burrell, Oral Presentation [email protected] pag. 91 Katja Butterbach, Poster session I [email protected] pag. 43 Marieta Caganova, Poster session I [email protected] pag. 44 Marìa Carretero, Poster session I [email protected] pag. 45 Federica Castellucci, Poster session I [email protected] pag. 46 Adrian Charbin, Poster session I [email protected] pag. 47 Avradip Chatterjee, Poster session I [email protected] pag. 48 Filippo Ciceri. Poster session I [email protected] pag. 49 Catherine Cowell, Poster session I [email protected] pag. 50 Matteo D’Antonio, Poster session I [email protected] pag. 51 Amrita Dasgupta, Poster session I [email protected] pag. 52

International PhD Student Cancer Conference 163 Authors’ index

Andrew Davidson, Poster session I [email protected] pag. 53 Sahra Derkits, Poster session I [email protected] pag. 54 Ivana Dokic, Poster session I [email protected] pag. 55 Eleonora Dondossola, Poster session II [email protected] pag. 100 Sara Donnelly, Poster session I [email protected] pag. 56 Sarah Dowding, Poster session I [email protected] pag. 57 Rinske Drost, Oral Presentation [email protected] pag. 147 Charlotte Durkin, Poster session I [email protected] pag. 58 Mike Fletcher, Poster session I [email protected] pag. 59 Fernando Flores-Guzman, Oral Presentation [email protected] pag. 150 Francesca Fontana, Poster session II [email protected] pag. 101 Giulia Fragola, Poster session I [email protected] pag. 60 Daniele Frangioni, Poster session I [email protected] pag. 61 Meiling Gao, Poster session I [email protected] pag. 62 Ernst Geutjes, Poster session I [email protected] pag. 63 Stephen Goldie, Oral Presentation [email protected] pag. 151 Gordana Halec, Poster session I [email protected] pag. 64 Willaim Harris, Poster session I [email protected] pag. 65 David Hobson, Poster session I [email protected] pag. 66 Charlotte Hodson, Poster session I [email protected] pag. 67 Joerg Hoeck, Oral Presentation [email protected] pag. 30 Hung Yi Kristal Kaan, Poster session I [email protected] pag. 68 Mirjam Ketema, Poster session I [email protected] pag. 69

International PhD Student Cancer Conference 164 Authors’ index

Willem-Jan Keune, Poster session II [email protected] pag. 102 Guy Kingham, Poster session I [email protected] pag. 70 Christiaan Klijn, Oral Presentation [email protected] pag. 88 Ulrike Krebs, Poster session I [email protected] pag. 71 Anne Lempens, Oral Presentation [email protected] pag. 83 Xiaofan Li, Oral Presentation [email protected] pag. 89 Andrea Longatti, Oral Presentation [email protected] pag. 144 Serena Lunardi, Poster session I [email protected] pag. 72 Cerys Manning, Poster session I [email protected] pag. 73 Stefano Marchesi, Poster session I [email protected] pag. 74 Sara Mari, Poster session I [email protected] pag. 75 Matteo Marzi, Oral Presentation [email protected] pag. 29 Andrzej Mazan, Poster session I [email protected] pag. 76 Milena Mazan, Poster session II [email protected] pag. 103 Guillermo Menendez, Poster session II [email protected] pag. 104 Francesca Milanesi, Poster session II [email protected] pag. 105 Jennifer Miles, Poster session II [email protected] pag. 106 Iain Moal, Poster session II [email protected] pag. 107 Francesca Montani, Oral Presentation [email protected] pag. 145 Guenievre Moreaux, Poster session II [email protected] pag. 108 Sharmin Naaz, Poster session II [email protected] pag. 109 Noor Akmar Nam, Poster session II [email protected] pag. 110 Gilda Nappo, Oral Presentation [email protected] pag. 90

International PhD Student Cancer Conference 165 Joschko Natalie, Poster session II [email protected] pag. 111 Charlotte Ng, Oral Presentation [email protected] pag. 148 Samuele Notarbartolo, Poster session II [email protected] pag. 112 Adelaida Palla, Poster session II [email protected] pag. 113 Roberta Pascolutti, Poster session II [email protected] pag. 114 Cristina Elisabetta Pasi, Oral Presentation [email protected] pag. 152 Avinash Patel, Poster session II [email protected] pag. 115 Chitra Pattabiraman, Poster session II [email protected] pag. 116 Raghunath Reddy Peesari, Poster session II [email protected] pag. 117 Kay Penicud, Poster session II [email protected] pag. 118 Alessandra Pepe, Poster session II [email protected] pag. 119 Lakshmi Revathi Perumalsamy, Oral Presentation [email protected] pag. 146 Marieke Peuscher, Oral Presentation [email protected] pag. 92 Gabriele Piergiovanni, Poster session II [email protected] pag. 120 Mariaelena Pozzebon, Poster session II [email protected] pag. 121 Narek Sarkisyan, Poster session II [email protected] pag. 122 Gian Maria Sarra Ferraris, Poster session II [email protected] pag. 123 Eva Schlecker, Oral Presentation [email protected] pag. 87 Christoph Schlude, Poster session II [email protected] pag. 124 Tim Schnyder, Oral Presentation [email protected] pag. 86 Chiara Segrè, Poster session II [email protected] pag. 125 Marta Nasila Shahbazi, Poster session II [email protected] pag. 126 Viktorija Sokolova, Poster session II [email protected] pag. 127

International PhD Student Cancer Conference 166 Monica Soldi, Poster session II [email protected] pag. 128 Lilly Sommer, Poster session II [email protected] pag. 129 Maren Stindt, Poster session II [email protected] pag. 130 Angelo Taglialatela, Poster session II [email protected] pag. 131 Shefali Talwar, Oral Presentation [email protected] pag. 31 Melda Tozluoglu, Oral Presentation [email protected] pag. 85 Margherita Yayoi Turco, Oral Presentation [email protected] pag. 149 Tanja van Harn, Oral Presentation [email protected] pag. 93 Gabriele Varano, Poster session II [email protected] pag. 132 Gianluca Varetti, Poster session II [email protected] pag. 133 Dhaval Varshney, Oral Presentation [email protected] pag. 94 Feng Wang, Oral Presentation [email protected] pag. 32 Maria Wiezlak, Poster session II [email protected] pag. 134 Marcus Wilson, Poster session II [email protected] pag. 135 Jenifer Wood, Poster session II [email protected] pag. 136 Magdalena Zajac, Poster session II [email protected] pag. 137

International PhD Student Cancer Conference 167 IMPORTANT ADDRESSES

CONFERENCE VENUE IFOM-IEO CAMPUS Via Adamello, 16 20139 Milan, Italy Phone +39.02.574303200 Fax +39.02.574303231 www.ifom-ieo-campus.it

ORGANIZING COMMITTEE Federica Castellucci - +39.02.574 303 312 Francesca Milanesi - +39.328.6482358 Gian Maria Sarra Ferraris - +39.02.574 303 328 Chiara Segré – phone +39.02.9437 5053 Gianluca Varetti – phone +39.02.9437 5042 [email protected]

EVENT COORDINATOR Sabrina Frata SEMM Foundation c/o Campus IFOM-IEO Via Adamello, 16 20139 Milan, Italy Phone +39.02.574 303 296 Fax +39.02.94 375 991 [email protected] www.semm.it

APARTMENT HOTEL RIPAMONTI RESIDENCE (4 star) Via dei Pini, 3 20090 Pieve Emanuele MI Phone +39 02 90763018 Fax +39 02 90782945 [email protected] http://www.atahotels.it/struttura.asp?id=ripamontiresidence&ln=eng

International PhD Student Cancer Conference 168 Know before you go - Milan

LOCAL TRAVEL

GENERAL INFORMATION: All public transportation in Milan is managed by ATM (website www.atm-mi.it). There are three major underground lines (MM1 red - MM2 green - MM3 yellow, see map no. 1) and several buses and tram lines. Please remember that the service starts at 6 am and the underground stops at midnight (1.00 am on Saturday night) while trams and buses stop at 2 am. TICKETS: You can buy ATM tickets at any of the authorized outlets (bars, tobacconists, stationers, newspaper stands) found throughout the Milan area and on intercity routes. The closest outlet to IFOM-IEO Campus is the Bar Devoti, located in Via Ripamonti (see attached map no. 2). You can also buy tickets in all underground stations at the dedicated ticket machines; these machines accept both coins and bank notes, credits cards and bank cards (please use preferably coins and bank notes). The closest underground stations to the IFOM-IEO Campus are Lodi and Brenta on the yellow line (M3). You can also reach Crocetta station (M3) using the tram 24 (direction Duomo) or on foot (20 minutes walking).

Several kinds of tickets are available:

For your stay in Milan we suggest you to buy the “Abbonamento bigiornaliero” (2-day pass) that is valid 48 hours after the validation without travel limits within Milan. The cost is 5,50 € per ticket. For a single trip you can buy instead the urban ticket (“Biglietto urbano”). The cost is 1 € and it is valid for 75 minutes. During this window of time, you can take all the trips you wish on buses and trams, but only one access to the underground is allowed (but you can make one trip in the underground and then take buses or trams with the same ticket within 75 min). Please be informed that if you reach Ripamonti residence by public transportation, you need an extra-urban ticket that costs € 2,00 and it is valid for 75 minutes.

International PhD Student Cancer Conference 169 TRANSPORTATION BETWEEN IFOM-IEO CAMPUS AND RIPAMONTI RESIDENCE Private shuttle services are organized for transportation between campus/residence and for the social dinner on Thursday 20th (see the table below).

Wednesday 19th Thursday 20th Friday 21th From Ripamonti residence 8.30 am 9.30 am to IFOM-IEO Campus From IFOM- IEO campus 9.00 pm 6.10 pm to Ripamonti residence From Ripamonti residence 8.00 pm to Skyline restaurant From Skyline restaurant 12.00 am to Ripamonti residence

Ripamonti residence also provides guests with a free shuttle bus to/from some stops scattered in the center of Milan and to/from Abbiategrasso underground station (M2). For further details please see the attached timetable (no. 3) and the map (no. 4) with the major Ripamonti shuttle bus stops. Taxis are also available but please note that they can be quite expensive. During the night they impose an extra charge of about 7 € to the normal fee. Taxi stations are present in all the main tourist points in Milan. For calling or booking: ph. 02/8585 - 02/4040 - 02/6969 - 02/4000).

SOCIAL EVENTS

On Wednesday night there will be an informal Pizza and Beer party at the bar located inside the Campus. A private bus will then leave around 9.00 pm to bring you to the Ripamonti Residence. If you want to drink something later on, you will find some bars inside the Ripamonti Residence. If instead you want to enjoy some nightlife in Milan you can easily reach some nice places by public transport from the institute. 1. You can reach the “Navigli” area by taking the tram 24, direction Duomo, for 4 stops getting off at the stop “Via Ripamonti- Viale Sabotino”. Then take the tram 9 (direction Porta Genova) or 29/30 (direction Via Messina) for 5 stops, until the stop “Piazza 24 Maggio”. To come back you will have to go by taxi or by public transport (underground M2 from Porta Genova station until S. Ambrogio station) until the shuttle stops (the nearest one to the Navigli area is in Piazza S. Ambrogio, buses will leave from there at 0.40 am and 1.50 am). Remember that the M2 line stops around 00:15 am. 2. You can go to Colonne di San Lorenzo, by taking the tram 24, direction Duomo, for 7 stops until the stop “Corso di Porta Romana- Via Santa Sofia”. Then go to Via Francesco Sforza and take the bus 94 (direction Cadorna) for 5 stops, until the stop “Colonne di San Lorenzo”. To come back, go on foot to the shuttle stop in Piazza S. Ambrogio (about 10 minutes walking).

International PhD Student Cancer Conference 170 3. If you want to go to the very centre of the city, take the tram 24, direction Duomo. Get off at the last stop: you will be in front of the most astonishing example of gothic architecture in Italy: the Duomo. To come back to the residence, walk to Piazza Diaz, where you can find a shuttle stop (departure at 10.00 pm, 0.15 am).

On Thursday night we will have a social dinner at the Skyline Restaurant located in Corso Como. A DJ and music will be provided to dance afterward. A private bus will bring you back to the Ripamonti Residence. If you want to stay outside later on, several bars and discos are located in the Corso Como area. In this case you will have to go back to the residence on your own. Please remember that the underground stops at midnight. The easiest way is to walk to Via Fatebenefratelli (20 minutes walking) where a shuttle stop will be present (departure at 0.30 am, 1.55 am and 4.35 am). In alternative you can take a taxi to reach Sant’Ambrogio station (M2), departure at 0.40 am, 1.50 am and 4.30 am.

TOURISTIC INFORMATION:

Main museums:

• Museo della Scienza e della Tecnica/Museum of Science and Technology (Via S. Vittore 21 - Metro M2 stop S. Ambrogio) • Pinacoteca di Brera (Via Brera, 28 – Metro M2 stop Lanza or Metro M3 stop Montenapoleone) • Triennale (Viale Alemagna, 6 – Metro M1-M2 stop Cadorna) • Museo di Storia Naturale/Museum of Natural History (Giardini Indro Montanelli, Corso Venezia 55- Metro M1 stop Palestro) • PAC Padiglione di Arte Contemporanea/Pavilion of Contemporary Art (Via Palestro 14- Metro M1 stop Palestro)

Main Monuments:

• The Duomo cathedral (piazza del Duomo – Metro M1-M3 stop Duomo or 24 tram from IFOM-IEO Campus). We recommend you in particular to visit the Cathedral rooftop. It’s open from 9.00 am to 10.00 pm and it represents one of the most fascinating locations in the city of Milan, allowing you to see a superb panorama of the whole city and, when the weather is fine, you can also enjoy the view of the Alps mountains (more informations are available on the website www.duomomilano. it). Close to the Duomo cathedral you will find also the Teatro La Scala (5min on foot). Of particular interest is a wonderful museum inside the theatre that retrace its history (for further information: www.teatroallascala.org). • The Castello Sforzesco (piazza Castello – Metro M1 stop Cairoli). You can visit either the castle and the park around it, and also the numerous art museums located inside the castle (for further informations : www.milanocastello.it). The castle also hosts one of the Pieta’ sculptures by Michelangelo. • Santa Maria delle Grazie Church (piazza Santa Maria delle Grazie, 2 - Metro M1 stop Conciliazione or Metro M2 stop Cadorna). In the “refettorio” close to the church

International PhD Student Cancer Conference 171 there is the famous wall paint “The Last Supper” by Leonardo da Vinci. Reservation is strictly required, and it is recommended to reserve in advance due to the long waiting list. Reservation can be done at 0039 02 92 800 360 or on the website http://www.vivaticket.it/?op=cenacoloVinciano

Nightlife Milan is famous for its exciting nightlife. In particular, we recommend you to visit the Navigli area (underground M2, stop Porta Genova) and Brera area (Metro M2, stop Lanza), pedestrian precincts where it is possible to have a drink in really nice bars and pubs. If you like enjoying open air, you should also go to Corso di Porta Ticinese and the area in front of San Lorenzo Church (called Colonne di San Lorenzo) and close to Giardini di Piazza Vetra. This area is particularly recommended in spring and summer, with many people enjoying drinks in the streets.

For further information: Tourist Offices A.P.T., Azienda di Promozione Turistica del Milanese Via Marconi, 1 – Ph. +39.02.725241 – email: [email protected] I.A.T, Informazione e Accoglienza Turistica Via Marconi, 1 – Ph. +39.02.72524300 Stazione Centrale, Departure Gallery – Ph. +39.02.72524360/370 TCI, Touring Club Italiano C.so Italia, 10 – Ph. +39.02.5359971 – email: info-touring@ touringclub.it

City Sightseeing Sightseeing of the city Servizio Autostradale: a city sightseeing tour by bus and by walking. Departure is from Piazza del Duomo at h. 9.30 a.m. The tickets are available at the APT office or in the major hotels in Milan. For info: +39.02.72524300 Tourist Tram, Ciao Milano. The Azienda Trasporti Municipali (ATM) organizes city sightseeing tours by antique tram. Departures from Piazza Castello. Tickets sold on the tram itself. For info: +39.02.72002584

ACKNOWLEDGEMENTS

We would like to thank all the people that helped us in the organization of this conference. First of all Sabrina Frata, who guided us through this new and exciting adventure. Then Deborah Agostini for making such an appealing website and all the IT staff for their help in setting up all the informatic devices we have been used during this meeting.

International PhD Student Cancer Conference 172 International PhD Student Cancer Conference International PhD Student Cancer Conference International PhD Student Cancer Conference International PhD Student Cancer Conference NOTES

International PhD Student Cancer Conference NOTES

International PhD Student Cancer Conference NOTES

International PhD Student Cancer Conference NOTES

International PhD Student Cancer Conference NOTES

International PhD Student Cancer Conference