EXPERIMENTAL ONCOLOGY AND MOLECULAR MEDICINE...

The Department of Experimental Oncology and Molecular Medicine(DOSMM) includes 10 Research Units dedicated to preclinical investigations.The primary goal of the Department is to serve as an important conduitthrough which new discoveries are applied to cancer diagnosis, prognosis, andtreatment. This is fostered by a collaborative environment and a strong inter-action among physician and basic scientists working in different disciplines.During 2011, DOSMM acted as an interactive platform fosteringcollaborations among preclinical investigators and with INT Clinical Depart-ments on topics of common interest, such as the identification and validationof circulating molecular markers as new tools for early detection and riskassessment, study of tumor-microenvironment related changes, and the under-lying molecular mechanisms using both animal models and patient samples,which have been awarded by national and international grant support.DOSMM supports investigators with state-of-the-art core facilities, withshared instrumentation and trained specialists.

The following resources are available. • Immunohistochemistry (technical specialists: Lorena Ventura and LuciaGioiosa): histological and cytological processing, including tissue microarrays, bya wide range of histological techniques, immunohistochemistry, in situhybridization, and autoradiography. • Cell imaging facility (Technical Specialist: Patrizia Casalini, Biol Sci D): pro-vides access to a BioRad Radiance 2000 laser confocal microscope allowingfor a wide range of fluorescent dye use, sequential and simultaneous 3 channelbright field image collection, and live cell imaging. • Flow cytometry and cell sorting (Technical Specialist: Gabriella Abolafio;Research Fellow: Andrea Vecchi, Biol Sci D): using state-of-the-art flow cyto-metric instrumentation, and software analysis. • Microbiology (Technical Specialist: Maria Teresa Radice): core servicesinclude preparation/analysis of a) competent bacteria, media, agar plates, and

UNITS

IMMUNOBIOLOGY OF HUMANTUMORS

Andrea Anichini

MOLECULAR THERAPIESSilvana Canevari

MOLECULAR IMMUNOLOGYMario P. Colombo

BIOMARKERSMaria Grazia Daidone

MOLECULAR MECHANISMS OFCELL CYCLE CONTROL

Domenico Delia

MOLECULAR MECHANISMSAngela Greco

IMMUNOTHERAPY OF HUMANTUMORS

Licia Rivoltini

TUMOR GENOMICSGabriella Sozzi

MOLECULAR TARGETINGElda Tagliabue

MOLECULAR PHARMACOLOGYNadia Zaffaroni

DIRECTOR OF DEPARTMENT

Maria Grazia Daidonephone: +39 02 2390 2238

[email protected]

EXPERIMENTAL ONCOLOGYAND MOLECULAR MEDICINE

DEPARTMENT

71 Scientific Report 2011

72Experimental Oncology and Molecular Medicine Department

other reagents; b) bacterial transformation and miniprep; c) medium/largescale plasmid DNA; d) BAC and YAC DNA; e) culture/induction of bacteria forpurification of recombinant proteins, freezing and storage of recombinant plas-mids and transformed bacteria, database management. • DNA sequencing (Technical Specialist: Donata Penso): this facility providesDOSMM Units as well as Units of other Departments with computer-readablesequences and fragment analysis of DNA templates, processing samples onABI 3100 and 3130 capillary genetic analyzers. • Functional genomics and Bioinformatics (see a detailed description onpage 146) • Proteomics/mass spectrometry laboratory (see a detailed description onpage 147) • Tissue and cell repository (see a detailed description on page 145)• Laboratory animal facility

Administrative team: Grazia Barp, Grazia Convertino, Simona Galluzzi, Ester Grande, Silvia Grassi,

Laura Mameli, Silvia Portincasa, Luisa Rivetta, Daniela Silva, Laura Zanesi, Cristina Zanini.

This team facilitates the activity of the Department by providing administrative support to research

unit leaders and core facilities, coordinating the activities of graduate students and fellows, handling

purchasing requests for laboratory consumables, and finance administration.

Laboratory Management Team: Enrico Ronchi, Domenico Di Fazio, Angelo Labori, Salvatore Venturino.

This team plays an essential role to support the research units within the Department for

maintenance of instrumentation, and management and supervision of areas for cryopreservation of

stored tissues/cells/cell extracts and reagents. In addition, the team – in association with the

administrative team - also oversees a cost-effective and efficient centralized system of ordering and

stock control for the most widely used items.

Supporting Personnel: Antonietta Calcagno, Linda Cimaglia, Antonio Illuminato, Agata Mancuso, Luisa

Mona, David Penni, Gisella Rivadossi, Pasquale Russo, Claudio Santagostini.


immunobiology of human tumors

HEAD Andrea Anichini, Biol Sci D

RESEARCH STAFF Marialuisa Sensi, Biol Sci D Roberta Mortarini, Biol Sci D

FELLOWSValentina E. Perotti Giulia Grazia

POSTDOCTORAL FELLOWElena Tassi

TECHNICIANSPaola L. Baldassari, Ilaria Bersani, AlessandraMolla, Gabriella Nicolini, Claudia Vegetti

The main goals of the research unit are: • to understand how the adaptive immune responsethat develops in neoplastic patients, or that promoted byimmunotherapy, can contribute to control of tumorgrowth; • to identify new molecular targets to overcome tumorresistance to cell death; • to define the roles in tumor biology of activated tyro-sine kinase receptors (RTK) expressed in melanoma; • to dissect the role of the tumor-host interaction inpromoting cancer development and progression. The main projects during 2011 have focused on: a) mech-anisms of immune response promoted byimmunotherapy in melanoma patients. b) Prognostic sig-nificance of immune-related markers associated withhematopoietic stem cell transplantation in pediatric solidtumors. c) Identification of new therapeutic targets inmelanoma to overcome tumor resistance toprogrammed cell death induced by pro-apoptotic drugs

and target-specific inhibitors. d) Identification ofmelanoma subsets defined by RTK expression and signal-ing pathway profiling. The main results of 2011 included: a) evidence that pro-motion of T cell infiltration and maturation, in neoplasticlesions of melanoma patients, is a main mechanism ofaction of immunotherapy based on the anti-CTLA-4monoclonal antibody ipilimumab; b) identification of anew subset of cutaneous melanomas defined by expres-sion of the RTK AXL and role of this RTK in melanomamigration and invasion; c) identification of the inhibitor ofapoptosis Apollon as a main regulator of melanomaresistance to cell death by cytotoxic drugs, MEK andBRAF-specific inhibitors, as well as to soluble ormembrane-TRAIL; d) identification of NFATc2 transcrip-tion factor as a new potential therapeutic target inmelanoma.Keywords: melanoma, tumor immunity, microenvironment

RELEVANT NOTES

Collaborations

ISS, Rome

Dr. Riccardo Dolcetti, C.R.O., Aviano

Dr. Ennio Carbone, Università degli Studi diCatanzaro “Magna Graecia”

Dr. R. Falcioni, Istituto Nazionale Tumori “ReginaElena”, Rome

Dr. P. Allavena, Istituto Clinico Humanitas, Milan

Prof. B. Venerando, Università degli Studi di Milano

Dr. P.F. Ferrucci, Istituto Europeo di Oncologia,Milan

Publications

Del Vecchio M, Mortarini R, Tragni G, Di Guardo L,Bersani I, Di Tolla G, Agustoni F, Colonna V, Weber

JS, Anichini A. T-cell activation and maturation attumor site associated with objective response toipilimumab in metastatic melanoma. J Clin Oncol.2011; 29: e783-8.

Sensi M, Catani M, Castellano G, Nicolini G, AlciatoF, Tragni G, De Santis G, Bersani I, Avanzi G,Tomassetti A, Canevari S, Anichini A. Humancutaneous melanomas lacking MITF andmelanocyte differentiation antigens express afunctional Axl receptor kinase. J Invest Dermatol.2011; 131: 2448-57.

Contributions

Andrea Anichini, Editorial Boards: CancerImmunology Immunotherapy, The InternationalJournal of Biological Markers, Tumori.


molecular therapies

HEAD Silvana Canevari, Biol Sci D

RESEARCH STAFFFabio Benigni, Biol Sci D (from May 2011) Marina Bagnoli, Biol Sci D Mariangela Figini, Biol Sci D Delia Mezzanzanica, Biol Sci DAntonella Tomassetti, Pharmacol Sci DAlberto Zacchetti, Vet D (until April 2011)

FELLOWSChiara Alberti, Biol Sci D Davide Bernareggi, Biotech Sci DIleana Bortolomai, Biol Sci D Barbara Frigerio, Biol Sci D Anna Granata, Biol Sci D Roberta Nicoletti, Med Biotech D (from May2011) Patrizia Pinciroli, Biol Sci D Katia Rea, Med Biotech D Alessandro Satta, Vet Biotech D

TECHNICIANSPaola Alberti, Renata Ferri, Elena Luison

Through a multidisciplinary approach and integrated func-tional/analytical methodologies, the Unit aims to: gaininsight in the molecular events involved in the tumor pro-gression; identify and validate new potential targets andprognostic/predictive markers; to develop and validatenew diagnostic/therapeutic strategies targeting ovarianand prostate carcinoma. The majority of the Unit projectsare possible thanks to fruitful interactions with otherUnits of DOSMM, Gynecological Oncology, Prostate Pro-gram, Nuclear Medicine, and Anatomic Pathology. Theresearch group is also involved in numerous national andinternational collaborations. Major results achieved in the area of characterization ofmolecular events involved in cancer progression. Wedefined the role of key enzymes of the choline metabolicpathway in cervical cancer tumor initiating cells; by meansof specific RNA interference achieved a detailed knowl-edge of the biology underlying the sustainedexpression/activity of ChoK-alpha in ovarian carcinoma;contributed to the characterization of a subset of

melanomas expressing Axl and endowed with high inva-sive potential; identified a new signaling transductionpathway, downstream to EGFR activation, characterizedby a FAK phosphorylated form concentrated at mitoticspindle. In the area of new prognostic/predictive markers forovarian carcinoma we: demonstrated that down-modula-tion of a miRNA cluster on chrXq27.3 is associated withshorter time to relapse and that forced overexpression ofthese miRNAs resensitized cells to cisplatin cytotoxicity;contributed to the identification of the prognostic role ofstathmin in p53 mutated advanced stage cases. In thearea of therapeutic approaches with antibody-basedreagents we demonstrated that, in preclinical models, ahuman anti-folate receptor fragment in dimer format wasable to strongly accrete 131I to ovarian carcinoma cellsand cure treated animals. Keywords: translational medicine, molecular mechanisms of tumor

progression, antibody-based therapy

RELEVANT NOTES

Collaborations

National Collaborations: Dr Silvano Ferrini, ISTGenoa; Prof. Marco Colombatti, University ofVerona; Dr. Franca Podo and Dr. Egidio Iorio, ISS,Rome; Dr. Sandro Pignata on behalf of the MITOgroup, Ist. Pascale, Naples; Dr. Massimo Libra,University of Catania; Dr. Gustavo Baldassarre,CRO, Aviano; Dr. Teresa Pellegrino, CNR Lecce andITT, Genoa; Dr. Laura Belvisi, University of Milan

International Collaborations: Dr. Nathalie Jacobs,CNR, Liege, Belgium; Drs. Sophia Karagiannis and

Hannah Gould, King’s College, London, UK; Dr.Zaver Bhujwalla, Johns Hopkins University,Baltimore, USA; Dr Daniel Powell, University ofPennsylvania, USA

Publications

Alberti C, Pinciroli P, Valeri B, Ferri R, Ditto A,Umezawa K, Sensi M, Canevari S, Tomassetti A.Ligand-dependent EGFR activation induces the co-expression of IL-6 and PAI-1 via the NFkB pathwayin advanced-stage epithelial ovarian cancer.Oncogene. 2011; doi: .1038/onc.2011.572.

Bagnoli M, De Cecco L, Granata A, Nicoletti R,Marchesi E, Alberti P, Valeri B, Libra M, BarbareschiM, Raspagliesi F, Mezzanzanica D, Canevari S.Identification of a chrXq27.3 microRNA clusterassociated with early relapse in advanced stageovarian cancer patients. Oncotarget. 2011; 2: 1265-78.

Contributions

Editorial Board: Silvana Canevari, CancerImmunology Immunotherapy


molecular immunology

HEAD Mario P. Colombo, Biol Sci D

RESEARCH STAFFSilvia Miotti, Biol Sci D Claudia Chiodoni, Biol Sci D

POSTDOCTORAL FELLOWSSabina Sangaletti, PhD, Biol Sci DAgniezka Chronowska, PhD Paola Pittoni, PhD Caterina Vitali, PhD Giorgio Mauri, Med Biotech D

FELLOWAndrea Tomirotti, Med Biotech D

PHD STUDENTSAlessia Burocchi, Biol Sci D Alessandra Santangelo, Biol Sci D Alice Rigoni, Biol Sci D Ilaria Torselli, Biol Sci D

TECHNICIANSIvano Arioli, Barbara Cappetti, Ileana Facetti,Mariella Parenza, Claudia Bassani, LauraBotti, Biol Sci D, Chiara Ratti

We study the complex interplay among cells of theimmune system, the extracellular matrix, and transformingtissues. • We have studied the role of mast cells (MCs) inprostate tumor development, starting from the observa-tion that, in prostate tumors from both tumor-proneTRAMP mice and human patients, MCs are specificallyenriched and degranulated in areas of well-differentiated(WD) adenocarcinoma (AC), but not around poorly dif-ferentiated (PD) foci coexisting in the same tumors. Wederived novel cell lines, representative of WD and PDvariants, and by pharmacologically stabilizing or geneticallyablating MCs in recipient mice, demonstrated that MCpromote WD AC growth, but are dispensable for PDtumors. WD tumors rely on MCs for MMP-9 provision, asreconstitution of MC-deficient mice with wt but notMMP-9-/- MCs was sufficient to promote their growth,whereas PD tumors, consistently with epithelial-to-mes-enchymal transition, are MMP-9 self-competent. Such a

dual source of MMP-9 was confirmed in human tumors,suggesting that MCs could be a good target for earlystage prostate cancer. Nevertheless, testing whether MCtargeting could block or delay tumorigenesis in tumor-prone TRAMP mice, we observed a high incidence ofearly and aggressive tumors, characterized by a neuroen-docrine (NE) signature and c-Kit expression. These dataunderscore the contribution of MCs in tumor progres-sion and uncover a new, opposite role of MCs inprotecting against the occurrence of aggressive NE vari-ants in prostate cancer. • Fibrosis results from inflammatory tissue damage andimpaired regeneration. In the context of bleomycin-induced pulmonary fibrosis we demonstrated that thematricellular protein SPARC distinctly regulates inflamma-tion and collagen deposition depending on its cellularorigin. Keywords: mast cells, SPARC/osteonectin, tumor microenvironment

RELEVANT NOTES

Collaborations

Claudio Tripodo, Univerity of Palermo

Katia Scotlandi, Istituto Ortopedico Rizzoli, Bologna

Alessandra Carè, Istituto Superiore di Sanità, Rome

Publications

Pittoni P, Tripodo C, Piconese S, Mauri G, ParenzaM, Rigoni A, Sangaletti S, Colombo M.P. Mast celltargeting hampers prostate adenocarcinomadevelopment but promotes the occurrence ofhighly malignant neuroendocrine cancers. CancerRes. 2011; 71: 5987-97.

Sangaletii S, Tripodo C, Cappetti B, Casalini P,Chiodoni C, Piconese P, Santangelo A, Parenza M,Arioli I, Miotti S, Colombo MP. SPARC Oppositelyregulates inflammation and fibrosis in bleomycin-induced lung damage. Am J Pathol. 2011; 179:3000-10.

Contributions

Mario P. Colombo is Senior Editor of CancerResearch and Associate Editor of CancerImmunology Immunotherapy andOncoimmunology


biomarkers

HEAD Maria Grazia Daidone, Biol Sci D, PhD

RESEARCH STAFF Vera Cappelletti, Biol Sci D Silvia Veneroni, Biol Sci D Raffaella Villa, Biol Sci D

POSTDOCTORAL FELLOWSValentina Appierto, Biol Sci D, PhD Graziella Cimino Reale, Biol Sci D, PhD Daniela Sia, Med Biotech D, PhD

PHD STUDENTSMaurizio Callari, Med Biotech D Valeria Musella, Med Biotech D Lorenza Venturini, Biol Sci D

FELLOWSEleonora Di Buduo, Med Biotech D Emanuela Fina, Biol Sci D Guido Santilli, Med Biotech D Paola Tiberio, Biol Sci D Sara Toffanin, Med Biotech D

TECHNICIANSCinzia De Marco, Lucia Gioiosa, PatriziaMiodini, Biol Sci D, Rosita Motta

Research in this Unit is aimed towards identification, vali-dation and targeting of cancer-related biomarkersrelevant on cancer progression, with a focus in 2011 onbreast cancer to investigate: The subtype-dependentprognostic relevance of an interferon metagene in node-negative tumors: to identify a signature - beyond theclassical risk factors - associated with metastatization, weprofiled a comparable (for clinicopathologic features)series of ER-positive tumors from women with earlyrelapse in distant sites and from women who remaineddisease-free for a longer time, and we considered differ-entially expressed (DE) metagenes rather than single DEgenes. A metagene containing interferon-induced genes(IFN metagene) was DE (P=0.029) as a function ofmetastasis occurrence. The validation of these results onpublicly available larger datasets showed the associationof the IFN metagene with poorer prognosis in luminal(P=0.012), but with a better prognosis (P=0.0014) inERBB2+ tumors. The combined consideration of IFN and

T-cell metagenes indicated a complex interaction in allthe molecular subsets except for basal tumors (whoseunfavorable outcome was independent of bothmetagenes), thus confirming the importance of analyzingprognostic variables separately within molecular subtypes.Gene expression profiling (GEP) of circulating tumor cells(CTC): CTCs might represent an easy-accessible “liquidbiopsy” whose transcriptional characterization likelyallows identification of pathways involved in metastaticdissemination and to obtain clinically-relevant information. We are currently: i) capturing CTCs by antibodies againstEpCAM and MUC-1 and by evaluating the expression ofepithelial-mesenchymal-transition and stemness-relatedmarkers, which markedly improved CTC detection rate;ii) assessing the feasibility of GEP by developing a proto-col to profile the expression of 29,000 genes by theDASL approach in as few as 50 CTCs; iii) validating theseapproaches within neo-adjuvant settings.Keywords: breast cancer, biomolecular profile, cancer stem cells

RELEVANT NOTES

Collaborations

Paolo Ciana, University of Milan, Italy; Giannino DelSal, University of Trieste, Italy; Maurizio D’Incalci(IRFMN, Milan), within the framework of theproject NEPENTE, financed by the LombardyRegion; Silvia Giordano, University of Turin andIRCC Candiolo, Italy; Josep M. Llovet, HospitalClinic (Barcelona, Spain) and Mount Sinai School ofMedicine (New York, USA), within the frame of theHCC Consortium; Angelo Paradiso, within theframework of a research project on biobankingsupported by ACC, the Italian Alliance againstCancer; Gaio Paradossi, University of Tor Vergata,Rome, Italy; Pier Giuseppe Pelicci (IEO), within theframework of a research project supported byACC, the Italian Alliance against Cancer; StefanoPiccolo, University of Padua, Italy; Daniela Pistillo,Istituto Clinico Humanitas, within the framework ofa CCM project supported by the Italian Ministry ofHealth; EATRIS Consortium, European Community

funded project to foster translational research andIATRIS Consortium, Italian initiative for translationalstudies coordinated by ISS, the Italian HealthInstitute EurocanPlatform, European Platform forTranslational Cancer Research, FP7 HEALTHEuropean Collaborative Project “SIGHT - Systemsfor in situ theranostic using of micro-particlestriggered by ultra-sound”, FP6 HEALTH EuropeanCollaborative Project “3 MICRON - Threemodality contrast imaging using multi-functionalisedmicroballoons”, FP7 HEALTH Genomnia (s.r.l.):Next generation sequencing on single cell samples

Publications

Callari M, Cappelletti V, De Cecco L, Musella V,Miodini P, Veneroni S, Gariboldi M, Pierotti MA,Daidone MG. Gene expression analysis reveals adifferent transcriptomic landscape in female andmale breast cancer. Breast Cancer Res Treat. 2011;127: 601-10.

Cordenonsi M, Zanconato F, Azzolin L, Forcato M,Rosato A, Frasson C, Inui M, Montagner M, Parenti

AR, Poletti A, Daidone MG, Dupont S, Basso G,Bicciato S, Piccolo S. The Hippo transducer TAZconfers cancer stem cell-related traits on breastcancer cells. Cell. 2011; 147: 759-72.

Contributions

Maria Grazia Daidone is Member of the Technical-Scientific Committee of AIRC, the ItalianAssociation for Cancer Research and Member ofthe Scientific Committee of the Italian School ofSenology (SIS)

Maria Grazia Daidone is Chairperson of thePathoBiology Group of the EORTC (EuropeanOrganization for Research and Treatment ofCancer) and Member of the EORTC TranslationalResearch Division M.G. Daidone is co-editor of theInternational Journal of Biological Markers andMember of the Editorial Board of BMC Cancerand Cell Proliferation


molecular mechanisms of cell cycle control

HEAD Domenico Delia, Biol Sci D

POSTDOCTORAL FELLOWSGiacomo Buscemi, PhD Benjamin Nachimuthu, PhD Laura Zannini, PhD

PHD STUDENTSAnnalisa Conti, BSc Elena Fusar-Poli, BSc Daniele Lecis, BSc

FELLOWLuigi Carlessi, BSc

TECHNICIANEnrico Fontanella

Dissection of the molecular events underlying the ATM-CHK2dependent DNA damage response and role in genomic sta-bility. One of the components of the DNA damageresponse (DDR) we have recently identified and charac-terized is DBC1 (Deleted in Breast Cancer-1), a putativetumor suppressor involved in apoptosis, transcription reg-ulation, and histone modification. DBC1 is a potentinhibitor of SIRT1 deacetylase, a regulator of p53-depen-dent apoptosis induced by genotoxic stress. We havefound that upon DNA damage, DBC1 is phosphorylatedon thr454 by ATM and ATR kinases, and that this previ-ously unknown modification of DBC1 is involved in thephysical interaction and inhibition of SIRT1. Moreover,phosphorylation of DBC1-T454 leads to the dissociationof SIRT1-p53 complex, thereby increasing p53 acetylationand p53-dependent cell death. Our findings thus furtherextend the mechanisms of DDR connected with the

ATM/ATR-dependent regulation of the SIRT1/p53 apop-totic axis. Development of pro-apoptotic SMAC-mimetic compoundswith anticancer activity.The ongoing collaboration withscientists of the University of Milan on thestructural/functional analysis and design of pro-apoptoticsmall SMAC-mimetics has lead to the development ofnew monomeric and dimeric compounds with increasedaffinity for the BIR3 and BIR3 domains of IAPs (inhibitorsof apoptosis proteins) and tumor cell killing at nanomolarconcentrations. One of these drug-like SMAC-mimetics,SMAC83, is currently being investigated for its in vivoactivity, alone or in combination with soluble TRAIL orCD34+ hemopoietic stem cells expressing membraneTRAIL, against human tumors engrafted in mice. Keywords: DNA damage, cell cycle checkpoints, apoptosis

RELEVANT NOTES

Collaborations

Profs. Martino Bolognesi and Pierfausto Seneci,University of Milan

Prof. Henning Walczak , Imperial College, London,UK

Prof. Shuki Mizutani, Tokyo Dental and MedicalUniversity, Japan

Publications

Manoukian S, Peissel B, Frigerio S, Lecis D, BartkovaJ, Roversi G, Radice P, Bartek J, Delia D. Two new

CHEK2 germ-line variants detected in breastcancer/sarcoma families negative for BRCA1,BRCA2, and TP53 gene mutations. Breast CancerRes Treat. 2011; 130: 207-15.

De Amicis A, Piane M, Ferrari F, Fanciulli M, DeliaD, Chessa L. Role of senataxin in DNA damageand telomeric stability. DNA Repair (Amst). 2011;10: 199-209.

Contributions

Patent: “New homo- and heterodimeric SMACmimetic compounds as apoptosis inducers”.PCT/IB2012/000297


molecular mechanisms

HEAD Maria Angela Greco, Biol Sci D

RESEARCH STAFF Maria Grazia Borrello, Biol Sci D Claudia Miranda, Biol Sci D Debora Degl’Innocenti, Biol Sci D

PHD STUDENTSMaria Grazia Vizioli, Biotech Med D Maria Chiara Anania, Biol Sci D

POSTDOCTORAL FELLOWSMara Mazzoni, Biol Sci D Paola Romeo, Biotech Med D Emanuela Minna, Biol Sci D

TECHNICIANSSonia Pagliardini, Maria Grazia Rizzetti

The Unit is involved in studies of the molecular mecha-nisms of thyroid carcinogenesis, with particular interest inpapillary thyroid carcinoma (PTC), the most frequent thy-roid neoplasia. The final goal of ongoing studies is theidentification of markers useful for early detection, prog-nosis, and follow up, as well as novel therapeutic targets.Towards this end, the Unit employs several differentapproaches including generation of in vitro models of thy-roid carcinogenesis using tumor derived cell lines andhuman primary thyrocytes; analysis of the role of selectedpathways and molecules; mRNA and microRNA expres-sion analysis; characterization of a thyroid tumor casecollection, used both for discovery and validation studies. The results achieved during 2011 are related to: • Relevance in thyroid carcinogenesis of oncogene-induced senescence (OIS), a novel mechanism proposedas a barrier to cancer, by the demonstration OIS restrainsthyroid tumor progression, as it is present in early phase,indolent tumor papillary thyroid microcarcinoma.

• Identification of miRNAs modulated by RET/PTC1, anddemonstration that RET/PTC1 may up-regulate METexpression through miR199a. • Demonstration through functional studies that theRET-K666E mutation, detected in a MTC patient, is onco-genic, and that its transforming activity is enhanced by thein cis G691S polymorphism. • Demonstration that DUSP6/MKP3, a feedback negativeregulator of ERK1/2 pathway, is over-expressed at themRNA and protein levels in both papillary and poorlydifferentiated thyroid carcinoma and may have a pro-tumorigenic role in thyroid carcinogenesis.• Identification of genetic determinants controlling theprogression of MTC by using a mouse model (in collabo-ration with Polygenic Inheritance Unit). The Unit has also contributed to a study showing thatmutations of RAS or BRAF gene confer resistance to ima-tinib in GIST carrying imatinib-sensitive c-Kit mutations. Keywords: thyroid carcinoma, oncogenes, functional studies

RELEVANT NOTES

Collaborations

Laura Fugazzola Endocrine Unit, Fondazione IRCCSCa’ Granda, Università degli Studi di Milano

Paola Allavena Dep Immunology and Inflammation,IRCCS Humanitas Clinical Institute

Enrico Radaelli Dipartimento di PatologiaVeterinaria, Facoltà di Veterinaria, Università degliStudi di Milano

Erminio Giavini, Elena Menegola, Functional andReproductive Biology, Dipartimento di Biologia,

Università degli Studi di Milano

Clara V. Alvarez, IDIS, Neoplasia and EndocrineDifferentiation, Department of Physiology, CIMUS,School of Medicine, University of Santiago deCompostela USC, Spain

Daniel Peeper Division of Molecular Genetics, TheNetherlands Cancer Institute, Amsterdam

Jesus Gil, MRC Imperial College, London

Publications

Borrello MG, Aiello A, Peissel B, Rizzetti MG,Mondellini P, Degl’Innocenti D, Catalano V, GobboM, Collini P, Bongarzone I, Pierotti MA, Greco A,

and Seregni E. Functional characterization of theMTC-associated germ line RET-K666E mutation:evidence of oncogenic potential enhanced by theG691S polymorphism. Endocrine Related Cancer.2011; 18(4): 519-27.

Vizioli MG, Possik PA, Tarantino E, Meissl K, BorrelloMG, Miranda C, Anania MC, Pagliardini S, Seregni E,Pierotti MA, Pilotti S, Peeper DS, and Greco A.Evidence of oncogene-induced senescence inthyroid carcinogenesis. Endocr Relat Cancer. 2011;18(6): 743-57.


immunotherapy of human tumors

HEAD OF UNITLicia Rivoltini, MD

RESEARCH STAFF Chiara Castelli, Biol Sci DVeronica B. Huber, MD Monica Rodolfo, Biol Sci D

CLINICAL RESEARCH STAFFMaria Carmela Careri, MDGianluca Cutolo, MD

DATA MANAGERMarco Asioli, Paola Frati

PHD STUDENTS Chiara Camisaschi, Michela Perego, MarcellaTazzari, Elisabetta Vergani

POSTDOCTORAL FELLOWSAnnamaria De Filippo, Biol Sci DPaola Filipazzi, Biol Sci DViviana Vallacchi, Biotec Sci D

TECHNICIANSValeria Beretta, Agata Cova, Paola Deho, SimonaFrigerio, Francesca Rini, Paola Squarcina

RESEARCH NURSESFelicetta Giardino, Gianluigi Rigamonti

Clinical development of new immune-related cancertherapies: we continued to enroll melanoma andprostate carcinoma patients, in collaboration with theMelanoma Surgery, Prostate Program, and Medical Oncol-ogy, in phase II trials based on anti-CTLA4 Ab+chemotherapy (NIBIT-M1), cancer vaccines with recombi-nant tumor antigens (PRAME and NY-Eso1) or peptides(Prostavax), and BRAF/MEK inhibitors. Blood and tumorbiopsies were collected for subsequent immunomonitor-ing. Additionally, we monitored immunoregulatorypathways (myeloid-derived suppressor cells –MDSC-,Treg, cytokine/chemokine profiling) in blood, serum, salivaand tumor biopsies of sarcoma as well as head and neckcancer patients undergoing conventional or targeted ther-apies. Studies on cancer-related immune regulatorypathways: we focused on MDSC and Treg, finding amRNA/miRNA signature involving IL-6 / HIF1-α / TGF-β

related patterns in MDSC from melanoma patients. Wealso assessed the impact of metabolic microenvironmentconditions, observing that low pH induces MDSC accu-mulation and T cell anergy, while pH regulating drugsmediate recovery of tumor immunity. We further studiedtumor exosomes either as an immunosuppressive path-way or as a strategy for specific drug delivery. Molecular studies on melanoma progression markers:we analyzed gene expression and microRNA profiling insentinel lymph node biopsy (SNB), finding that positiveSNB from patients with regional node involvement andpoor prognosis at 5 years display distinct immune-relatedtranscriptional patterns. Mutated BRAF as a circulatingmolecular marker for disease progression was analyzes inmelanoma lesions and in plasma of melanoma patients,observing that 70% patients have circulating BRAFV600EDNA. Keywords: immunotherapy, myeloid-derived suppressor cells,

exosomes

RELEVANT NOTES

Collaborations

Active collaboration with members of the ItalianNetwork for Tumor Bio-Immunotherapy (NIBIT),Italian Melanoma Intergroup (IMI), InternationalMelanoma Working Group (IMWG), InternationalSociety for Biological and Cellular Therapy (ISBTC).

Collaborations with Istituto Superiore di Sanità (Dr.S. Fais), University of Siena (Dr. M.Maio), Universityof Milan (Dr. L. Guerrini), University of Genoa (Dr.G. Bianchi Scarrà), Istituto Pascale (Dr. P. Ascierto),University of Heidelberg (Dr. V. Umansky), NationalCancer Institute (Dr. F Marincola), University ofCharleston (Dr. M. Nishimura), University of Tokyo(Dr. Y. Kawakami), Karolinska Institute (Dr. S.Caramuta, A. De Milito), Faculte de Pharmacie ofChatenay-Malabry (Dr. F. Triebel), Université

Catholique de Louvain (Dr. S. Lukas) and LudwigCancer Institute Brussels (Dr. P. Coulie); Nodality(Dr. Alessandra Cesano, San Francisco, USA.

Publications

Vergani E, Vallacchi V, Frigerio S, Deho P, MondelliniP, Perego P, Cassinelli G, Lanzi C, Testi MA, RivoltiniL, Bongarzone I, Rodolfo M. Identification of METand SRC activation in melanoma cell lines showingprimary resistance to PLX4032. Neoplasia. 2011;13(12): 1132-42.

Butterfield LH, Palucka AK, Britten CM, DhodapkarMV, Håkansson L, Janetzki S, Kawakami Y, Kleen TO,Lee PP, Maccalli C, Maecker HT, Maino VC, Maio M,Malyguine A, Masucci G, Pawelec G, Potter DM,Rivoltini L, Salazar LG, Schendel DJ, Slingluff CL Jr,Song W, Stroncek DF, Tahara H, Thurin M, TrinchieriG, van Der Burg SH, Whiteside TL, Wigginton JM,

Marincola F, Khleif S, Fox BA, Disis ML.Recommendations from the iSBTc-SITC/FDA/NCIWorkshop on Immunotherapy Biomarkers. ClinCancer Res. 2011; 17(10): 3064-76.

Contributions

Patent on the Immunomodulating use of PPI

Masters on Melanoma (IMI), Masters on TumorBiotherapy (NIBIT)

Editorial Board of Journal of Immunotherapy

National guidelines on Immunomonitoring (NIBIT)

National Guidelines in Melanoma Treatment(contribution)

ACC Melanoma Guidelines

INT-Educa Courses; FAD Courses on TumorVaccines.


tumor genomics

HEAD Gabriella Sozzi, PhD

RESEARCH STAFF Luca Roz, Pharm Sci D

POSTDOCTORAL FELLOWSFrancesca Andriani, Pharm Sci DPatrizia Gasparini, Biol sci DCarla Verri, Biol Sci DMassimo Moro, PhD Giulia Bertolini, Med Biotech D

PHD STUDENTMattia Boeri, Biotech D

TECHNICIANSDavide Conte, Federica Facchinetti, RobertoCaserini, Michela Grassi

The main field of interest of the Tumor Genomics Unit isthe biological and molecular characterization of lung can-cer and the development of genetic signatures in tissuesand biological fluids useful for early detection of lung can-cer and to establish novel therapeutic approaches. Ourpast and current approaches for biomarker discovery inlung cancer are mainly focused on the identification ofmolecular changes in biological fluids collected by non-invasive procedures, such as sputum and plasma. Inparticular, we have focused on miRNAs circulating inplasma as biomarkers of invasive lung disease andvalidated those able to identify aggressive lung cancerbefore its clinical appearance in order to define molecularpredictors of high-risk disease. We investigated themiRNA profiles of lung tumors, normal tissues, andplasma samples from cases identified in two independentspiral-CT screening trials with extensive follow-up wheremultiple plasma samples, collected before radiological dis-ease detection were available. We identified specific

miRNA signatures in plasma samples collected up to twoyears before cancer detection by spiral-CT. We alsodefined a signature that discriminates subjects accordingto aggressiveness of their future tumors, and in particularthe occurrence of early metastatic but spiral-CT invisiblelung tumors or small spiral-CT detected lesions withaggressive potential. Interestingly, the plasma signatureassociated with tumor aggressiveness seemed relatedwith miRNA expression not only in tumor, but also innormal lung tissue from patients (Boeri M. et al., ProcNatl Acad Sci USA. 2011; Sozzi G. et al., Cell Cycle. 2011).Very recently, we validated the performance of plasmamiRNA signatures in an extended series of 51 patientsand 100 individual controls belonging to the MILDscreening trial at the INT, proving the strength of our sig-natures in the prediction of lung cancer development andin the classification of the disease aggressiveness.Keywords: lung cancer, microRNA, cancer stem cells

RELEVANT NOTES

Collaborations

PET Unit, HSR, Milan (Dr. F. Fazio, Dr. R. Moresco);CERMS, A.O.U. San Giovanni Battista, Turin (Prof. R.Ferracini)

Dr. F. Pezzella, Oxford University, UK; Prof. C.M.Croce, OSUMC, Columbus (USA)

Chemores consortium (FP6, European Commu-nity); Curelung consortium (FP7, European Com-munity)

Publications

Boeri M, Verri C, Conte D, Roz L, Modena P,Facchinetti F, Calabrò E, Croce CM, Pastorino U,Sozzi G. MicroRNA signatures in tissues andplasma predict development and prognosis ofcomputed tomography detected lung cancer. ProcNatl Acad Sci USA. 2011; 108(9): 3713-8.

Sozzi G, Pastorino U, Croce CM. MicroRNAs andlung cancer: from markers to targets. Cell Cycle.2011; 10(13): 2045-6.

Contributions

CBS member (Comitato Borse Studio) AIRC-FIRC

Scientific Committee Societa’ Italiana diCancerologia (SIC)

Scientific Committee “Fondazione Edo Tempia”,Biella, Italy

Scientific Advisory Board American-Italian CancerFoundation (AICF)

Selection Committee Pezcoller-AACR award 2011

Editorial Board: Lung Cancer Steering Committeeof the European project “Chemores”( VI shared-cost RTD action Patent: Italian and US provisional“MICRO-RNA BIOMARKERS AND METHODSOF USING SAME” (Inventors G. Sozzi, M. Boeri, U.Pastorino)


molecular targeting

HEAD Elda Tagliabue, Biol Sci D

RESEARCH STAFF Manuela Campiglio, Biol Sci D Rosaria Orlandi, Biol Sci D (membership ofDOSMM Functional Genomics Facility)Serenella M. Pupa, Biol Sci D

FELLOWS Valentina Ciravolo, Biotech Sci D Gaia C. Ghedini, Biotech Sci D Alessandra Meini, Biol Sci D Ilaria Plantamura, Biol Sci D Manuela Ratti, Biol Sci D Francesca Ripamonti, Biotech Sci D Tiziana Triulzi, Biotech Sci D (AIRC)

POSTDOCTORAL FELLOWSMarilena V. Iorio, Biotech Sci D Francesca Bianchi, Biotech Sci D (AIRC)

PHD STUDENTS Claudia Piovan, Biol Sci D Marianna Sasso, Biol Sci D

TECHNICIANSPierangela Aiello, Patrizia Casalini(responsible for DOSMM imaging facility),Cristina A. Ghirelli

CONSULTANTSSylvie Ménard, Biol Sci D Marco Sandri, Stat Sci D

We aim to gain insight into the molecular pathways rele-vant for progression and response to therapy of breastcarcinomas, especially those with HER2 overexpressionand triple-negative (TN) features. During 2011, we com-pleted two observational studies carried out, respectively,in HER2-positive breast carcinoma patients treated withtrastuzumab-based therapy in a metastatic and an adju-vant setting. In the metastatic study, 272 patients undertrastuzumab treatment during 2000-2001 whoprogressed after first-line trastuzumab treatment showedclinically and statistically significant benefits from contin-ued trastuzumab treatment in both RECIST responderand non-responder patients. Analysis in the adjuvant set-ting, which included 1012 patients treated withtrastuzumab between 2005 and 2009 in 42 Italian hospi-tals, showed that trastuzumab is differentially-protective

depending on the site of metastasis; in fact, in addition tothe well-known CNS relapses, bone metastases were alsoresistant to the antibody treatment in our retrospectiveseries. Excluding these two sites, protection bytrastuzumab was more than 70% in all other distantmetastases, suggesting that the overall 50% protectionrate observed in trials is not equally distributed amongdifferent anatomical sites. Our analysis of TN breast carci-nomas demonstrated that these tumor cells contributedirectly to the tumor vasculature through endothelial celldifferentiation and that the wound-healing process pro-motes tumor cell progression through specific activationof PDGFR and VEGFR and their downstream pathways.Accordingly, targeting of PDGFR and VEGFR significantlyimpaired growth of TN breast carcinoma xenografts. Keywords: breast carcinoma, targeted therapy, HER2

RELEVANT NOTES

Collaborations

Augusto Amici, Genetic Immunization Lab., Dept ofMolecular, Cellular and Animal Biology, Univ. ofCamerino; Manuela Iezzi, Lab. di Immuno-Oncologia, CeSi, Chieti Scalo; Claudio Tripodo,Sezione di Anatomia Patologica, PoliclinicoUniversitario P. Giaccone, Palermo; RossellaCanese, Dept. of Cell Biology and Neurosciences,Istituto Superiore di Sanità, Rome; Egidio Iorio,Dept. of Cell Biology and Neurosciences, Istituto

Superiore di Sanità, Rome; Jiong Wu, CancerHospital, Fudan University, Shanghai

Publications

Campiglio M, Bufalino R, Sandri M, Ferri E, AielloRA, De Matteis A, Mottolese M, De Placido S,Querzoli P, Jirillo A, Bottini A, Fantini M, Bonetti A,Pedani F, Mauri M, Molino A, Ferro A, Pupa SM,Sasso M, Ménard S, Balsari A, Tagliabue E, on behalfof the Demetra Group. Increased overall survivalindependent of RECIST response in metastaticbreast cancer patients continuing trastuzumab

treatment: evidence from a retrospective study.Breast Cancer ResTreat. 2011; 128: 147-54.

Tagliabue E, Campiglio M, Pupa SM, Balsari A,Ménard S. The HER2 world: better treatmentselection for better outcome. J Natl Cancer InstMonogr. 2011; 2011: 82-5.

Contributions

Elda Tagliabue is on the Editorial Boards of TheBreast and the Chinese Journal of Clinicians

molecular pharmacology

HEAD Nadia Zaffaroni, Biol Sci D, PhD

RESEARCH STAFFMarco Folini, Biol Sci D, PhD Cinzia Lanzi, Biol Sci D Paola M.C. Perego, Biol Sci D, PhD Rosanna Supino, Biol Sci D

POSTDOCTORAL FELLOWSGiovanni L. Beretta, Biol Sci D, PhD Giuliana Cassinelli, Pharm D, PhD Michelandrea De Cesare, Vet D Paolo Gandellini, Biotech Sci D, PhD Laura Gatti, Biol Sci D, PhD Chiara Giommarelli, Pharm D, PhD Marzia Pennati, Biol Sci D, PhD

FELLOWSErika Borghini, Biol Sci D Anna Casamichele, Biol Sci D Denis Cominetti, Biol Sci D Valentina Profumo, Biotech Sci D Valentina M. Zuco, Biol Sci D

PHD STUDENTSGiacomo Cossa, Biotech Sci D Alessia Lopergolo, Biotech Sci D

TECHNICIANSNives Carenini, Elisa Campi, ElisabettaCorna, Laura Dal Bo, Enrica M. Favini, MariaStella Tinelli, Monica Tortoreto

CONSULTANTFranco Zunino, Biol Sci D

Novel therapeutic targets. The confirmation that notonly telomeric G-quadruplexes (G4) but also G4 presentin gene promoters represent valuable therapeutic targetswas obtained exposing telomerase-negative and -positivetumor cells to hybrid G4 ligand-alkylating agents. Cyto-toxic activity was mainly ascribable to the induction oftelomeric dysfunction (TRF2 and POT1 displacement) intelomerase-negative cells, and to the inhibition of telom-erase activity, caused by interference with G4 structurespresent in the promoters of c-myc and hTERT genes intelomerase-positive cells. Mechanisms of drug resistance.The characterization ofcellular changes associated with resistance totopoisomerase I inhibitors indicated that tyrosyl-DNAphosphodiesterase 1, whose expression is increased inresistant variants, can account per se for mild levels ofresistance to the new camptothecin gimatecan. However,the enzyme strongly cooperates with BRCA1 andXRCC1 to repair drug-mediated DNA damage.New drug combinations. In a human model of RET-dri-

ven medullary thyroid carcinoma, the combination of cis-platin and sunitinib resulted in in vitro synergisticinteraction and increased in vivo antitumor activity due tothe enhancement of both intrinsic and extrinsic apoptoticpathway activation mediated by Ret targeting. Preclinical development of new anticancer agents.Thenew camptothecin namitecan showed a promising thera-peutic profile in a panel of pediatric sarcoma models,including bone and soft-tissue tumors. Favorable drugpharmacokinetics and ability to inhibit angiogenesisappear as major determinants of antitumor activity. miRNA-based therapeutic approaches. Wedemonstrated that, in the context of a network involvingp63, miR-205 is able to control the deposition of laminin-332 in normal prostate cells, and that therapeuticreplacement of this miRNA in prostate cancer cells canrestore basement membrane deposition and 3D organi-zation into normal-like acini.Keywords: therapeutic targets, drug resistance, preclinical drug

development

RELEVANT NOTES

Collaborations

Prof. P. Chiarugi, University of Florence; Dr. L.Fugazzola, Fondazione Policlinico IRCCS, Milan;Prof. M. Magnani, University of Urbino; Prof. M.Palumbo, University of Padua; Prof. E. Scanziani,University of Milan. Prof. D.A. Altieri, The WistarInstitute Cancer Centre, Philidelphia (PA); Prof. F.Caruso, University of Melbourne (Australia); Prof.S.B. Howell, University of San Diego (CA); Prof.N.W. Keith, University of Glasgow (UK); Dr. S.Linder, Karolinska Institute, Stockholm, (Sweden);Prof. R.R. Reddel, Children’s Medical ResearchInstitute, Sydney (Australia); Dr I. Vlodavsky, TheBruce Rappaport Faculty of Medicine, Haifa(Israel).

Publications

Nicolini V, Cassinelli G, Cuccuru G, Bongarzone I,Petrangolini G, Tortoreto M, Mondellini P, Casalini P,

Favini E, Zaffaroni N, Zunino F, Lanzi C. Interplaybetween Ret and Fap-1 regulates CD95-mediatedapoptosis in medullary thyroid cancer cells.Biochem Pharmacol. 2011; 82: 778-88.

Becker AL, Orlotti NI, Folini M, Cavalieri F, ZelikinAN, Johnston AP, Zaffaroni N, Caruso F. Redox-active polymer microcapsules for the delivery of asurvivin-specific siRNA in prostate cancer cells.ACS Nano. 2011; 5: 1335-44.

Contributions

Editorial board: Critical Reviews inOncology/Hematology, European Journal ofCancer, BMC Cancer, Journal of Chemotherapy,Apoptosis, Critical Reviews in Oncogenesis (NadiaZaffaroni)

International Journal of Oncology, ISRNBiochemistry (Paola Perego)

Anti-Cancer Agents in Medicinal Chemistry, ISRNOncology (Marco Folini)


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