experimental oncology and molecular medicine department · experimental oncology and molecular...

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scientific report 2012

DIRECTOR OF DEPARTMENT Maria Grazia Daidone+39 02 2390 2238 [email protected]

experimental oncology and molecular medicine department

The Department of Experimental Oncology and Molecular Medicine (DOSMM) includes 10 Research Units dedicated to preclinical investigations and a new start-up Unit that was awarded by AIRC to a young investigator (Marilena Iorio) to study the “Involvement of miRNA in breast cancer driving pathways: from biology to possible therapeutic application”. The primary goal of the Department is to serve as an important conduit through which new discoveries are applied to cancer diagnosis, prognosis, and treatment. This is fostered by a collaborative environment and strong interaction among physicians and basic scientists working in different disciplines.During 2012, DOSMM acted as an interactive platform to foster collaborations among preclinical investigators and INT Clinical Departments on topics of common interest, such as the identification, validation, and qualification of circulating molecular markers as new tools for early detection, risk assessment, disease monitoring; the study of tumor-microenvironment related changes, and the underlying molecular mechanisms using both animal models and patient samples, which. Allstudies have been funded by national and international grant support.DOSMM supports investigators with state-of-the-art core facilities, with shared instrumentation and trained specialists.

unitSImmunobiology of Human TumorsANDREA ANIChINI

Molecular TherapiesSIlvANA CANEvARI

Molecular ImmunologyMARIO P. COlOMbO

BiomarkersMARIA GRAzIA DAIDONE

Molecular Mechanisms of Cell Cycle ControlDOMENICO DElIA

Molecular MechanismsMARIA ANGElA GRECO

Immunotherapy of Human TumorslICIA RIvOlTINI

Tumor GenomicsGAbRIEllA SOzzI

Molecular Targeting UnitElDA TAGlIAbUE

Molecular PharmacologyNADIA zAffARONI

AIRC Start Up UnitMARIlENA v. IORIO

mailto:[email protected]

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Experimental Oncology and Melecular Medicine Department

The following resources are available.• Immunohistochemistry (Technical Specialists: lorena ventura and

lucia Gioiosa): histological and cytological processing, including tissue microarrays, a wide range of histological techniques, immunohistochemistry, in situ hybridization, and autoradiography

• Cell imaging facility (Technical Specialist: Patrizia Casalini, biol Sci D): provides access to a bioRad Radiance 2000 laser confocal microscope allowing for a wide range of fluorescent dye use, sequential, and simultaneous 3 channel bright field image collection, and live cell imaging.

• Flow cytometry and cell sorting (Technical Specialist: Gabriella Abolafio; Research fellow: Andrea vecchi, biol Sci D): state-of-the-art flow cytometric instrumentation, and software analysis.

• Microbiology (Technical Specialist: Maria Teresa Radice): core services include preparation/analysis of: a) competent bacteria, media, agar plates, and other reagents; b) bacterial transformation and minipreps; c) medium/large scale plasmid DNA purification; d) bAC and YAC DNA preparation; e) culture/induction of bacteria for purification of recombinant proteins; freezing and storage of recombinant plasmids and transformed bacteria, as well as database management.

• DNA sequencing (Technical Specialist: Donata Penso): this facility provides DOSMM Units and Units of other Departments with computer-readable sequences and fragment analysis of DNA templates, processing samples on AbI 3100 and 3130 capillary genetic analyzers.

• Functional genomics and Bioinformatics (see a detailed description on page 121)

• Proteomics/mass spectrometry laboratory (see a detailed description on page 122)

• Tissue and cell repository (see a detailed description on page 120)• Laboratory animal facility• Administrative Personnel: 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 in supporting the research units in 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, Giuseppina liguori, Agata Mancuso, luisa Mona, David Penni, Gisella Rivadossi, Pasquale Russo, Claudio Santagostini.

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ImmunobIology of human tumorsThe research activity of the Unit focuses on cutaneous melanoma. In this area, we carry out projects in collaboration with the clinical departments at our institute as well as research groups in Italy and abroad. Our main goals are: • to understand how the adaptive immune response developed by cancer

patients or promoted by immunotherapy can contribute to control tumor growth• to identify new molecular targets to overcome melanoma resistance to

target-specific therapy.• to understand the mechanisms of interaction of the tumor with its

microenvironment• to develop a functional classification of melanoma based on RTK

expression and intracellular signaling pathway activationOur main results include: a) evidence obtained by gene profiling of pre/post therapy lesions that immunotherapy targeting the CTlA-4 co-signaling molecule induces a gene program of T cell activation and differentiation to the effector stage;b) identification of Apollon, an inhibitor of apoptosis, as a crucial marker and determinant of melanoma susceptibility to cytotoxic drugs, to MEK

and bRAf-specific inhibitors, and to TRAIl. We found that Apollon is down-regulated by a proteasome-dependent pathway only in melanoma cells that are susceptible to drug-induced apoptosis. Moreover, knockdown of Apollon by siRNA promoted melanoma cell death in response to cytotoxic drugs and target-specific inhibitors by a caspase-2- and caspase-8-dependent mechanism and enhanced the suppression of cell-cycle–related genes such as cyclin D1, induced by MEK- or bRAfv600E-specific inhibitors. c) Identification of the calcineurin-NfATc2 pathway as a potential therapeutic target in melanoma. We found that either inhibition of calcineurin by cyclosporine A or silencing of the downstream

transcription factor NfATc2 promoted caspase-dependent melanoma apoptosis and boosted the anti-tumor activity of cytotoxic drugs, bRAfv600E- and MEK-specific inhibitors, and TRAIl. Mechanistically, upon targeting of NfATc2, melanoma cell death was associated with stabilization of p53, down-regulation of Apollon, and activation of caspase-2 -3 and -8.The Unit also collaborates with experimental and clinical Units of the INT on the following projects: a) identification of biomarkers of response to target-specific therapy in pediatric patients with diffuse intrinsic pontine glioma; b) relationship between chronic inflammation and development of lung cancer. Keywords: melanoma, microenvironment, target-specific therapy

HeadAndrea Anichini, biol Sci D

ReseaRcH staffRoberta Mortarini, biol Sci DMarialuisa Sensi, biol Sci D

fellowsGiulia Grazia, biotechnol Sci D

Ilaria Penna, biol Sci Dvalentina Perotti, biol Sci D

Elena Tassi, biotechnol Sci D

tecHniciansPaola l. baldassari, Ilaria bersani, Alessandra

Molla, Gabriella Nicolini, Claudia vegetti

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molecular therapIesThrough a multidisciplinary approach and the integration of functional and analytical methodologies, the Unit aims to: gain insight into the molecular events involved in tumor progression; identify and validate new potential targets and prognostic/predictive markers; develop and validate new diagnostic/therapeutic strategies targeting ovarian and prostate carcinoma. The majority of projects are possible thanks to fruitful interactions with other Units of DOSMM, Gynecological Oncology, Prostate Program, Nuclear Medicine, and Anatomic Pathology. The research group is also involved in numerous national and international collaborations.Characterization of molecular events involved in cancer progression: 1) we demonstrated that inhibition of PC-PlC interferes with proliferation and survival of tumor initiating cells in cervical cancer; 2) by comparing primary tumors and synchronous secondary peritoneal localizations in ovarian carcinoma, we were the first to identify a miRNA signature that might help in elucidating dissemination mechanisms; 3) using bioinformatic tools, we showed that serous ovarian carcinomas, regardless of their aggressiveness, co-regulated Il-6 together with genes associated with cell growth and early growth factor response, a result that strengthens the hypothesis that common mechanism(s) driven by EGfR ligands characterize serous ovarian carcinomas of type I and type II; 4) in various aggressive tumors, such as ovarian and thyroid carcinomas and melanomas, we found that the presence of P-fAKSer732 was crucial for the maintenance of the proliferation rate, and its absence impaired both mitotic spindle assembly and correct chromosome alignment during metaphase.New prognostic/predictive markers for ovarian carcinoma: 1) using two independent cohorts of consecutive EOC cases unselected for clinical response studied by microarray analysis, we identified a miRNA signature associated with early relapse whose high predictive value was validated by RT-qPCR; 2) we contributed to the demonstration that sAlCAM levels correlate with more aggressive type II tumors, and that ADAM17/TACE activity and sAlCAM may mediate enhanced invasiveness. Therapeutic approaches with antibody-based reagents: using preclinical models, we documented the potential of an anti-PSMA single chain to become a good theranostic reagent for early detection and therapy of prostate cancers. Keywords: molecular mechanisms of tumor progression, translational medicine,

antibody-based therapy

HeadSilvana Canevari, biol Sci D

ReseaRcH stafffabio benigni, biol Sci DMarina bagnoli, biol Sci DMariangela figini, biol Sci D Delia Mezzanzanica, biol Sci DAntonella Tomassetti, Pharmacol Sci D

fellowsChiara Alberti, biol Sci DDavide bernareggi, biol Sci Dfrancesco Caroli, Technician barbara frigerio, biol Sci DAnna Granata, biol Sci DCristina luna, Technician Roberta Nicoletti, Med biotech DPatrizia Pinciroli, biol Sci DKatia Rea, Med biotech DAlessandro Satta, vet biotech Dvalentina Tinaglia, Sci biotech D

tecHniciansPaola Alberti, Renata ferri, Elena luison

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We study the complex interplay among cells of the immune system, the extracellular matrix, and transforming tissues. • Studying fibrosis, as model of impaired tissue regeneration linked to

inflammation, we have investigated its role in bone marrow (bM) failure due to neoplastic clone outgrows normal hematopoiesis. bM failure is sustained by detrimental stroma alterations, such as bM fibrosis and osteosclerosis, whose occurrence is harbinger of a dismal prognosis. We have found that the matricellular protein SPARC contributes to the stromal remodeling associated with myeloproliferation. The degree of SPARC expression in bM stromal elements, including CD146+ mesenchymal stromal cells, correlates with the degree of stromal changes, and the severity of bM failure characterizing the prototypical myeloproliferative neoplasm: primary myelofibrosis. Using Sparc KO mice and bM chimeras, we demonstrate that SPARC is required for the development of significant stroma fibrosis in a model of thrombopoietin (TPO)-induced myelofibrosis. Also, SPARC deficiency in the radioresistant bM stroma compartment impairs myelofibrosis but at the same time associates with an enhanced reactive myelopoietic response to TPO. The functional link between SPARC deficiency and favored myeloid cell expansion is confirmed by the myeloproliferative phenotype resulting from the transplantation of APCmin mutant bM cells, which are defective in hematopoietic stem-cell quiescence, into Sparc KO but not WT recipient bM stroma. Our results highlighted a complex influence of SPARC over the stromal and hematopoietic bM response in myeloproliferative conditions.

• The break of tolerance against tumor associated antigens in the long cherished arm to redirect the immune response against the tumor. The similarity between tumor- and self-antigens renders their discrimination difficult as confirmed by the possible transition from autoimmunity to

HeadMario P. Colombo, biol Sci D

ReseaRcH staffSilvia Miotti, biol Sci D

Claudia Chiodoni, biol Sci D

PostdoctoRal fellowsSabina Sangaletti, PhD, biol Sci D

Agniezka Chronowska, PhDPaola Pittoni, PhD

Caterina vitali, PhDGiorgio Mauri, Med biotech D

fellowAndrea Tomirotti, Med biotech D

PHd stUdentsAlessia burocchi, biol Sci D

Alessandra Santangelo, biol Sci DAlice Rigoni, biol Sci DIlaria Torselli, biol Sci D

tecHniciansIvano Arioli, barbara Cappetti, Ileana facetti,

Mariella Parenza, Claudia bassani, laura botti, biol Sci D, Chiara Ratti

molecular Immunology

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malignancies, at least for the hematopoietic system. This prompts to study the mechanism underlining the break of tolerance toward self antigens that is tightly related to the type of cell death and to the clearance of dying cells. A clear manifestation of autoimmunity is the development of anti-neutrophil cytoplasmic antibodies (ANCA) targeting proteins normally retained within neutrophils. Although neutrophil apoptosis and necrosis have been indicated involved in the process, ANCA pathogenesis remains obscure. In ANCA-mediated vasculitis, neutrophils are both responsible of tissue damage and target of circulating autoantibodies. ANCA activate neutrophils inducing their respiratory burst and a peculiar form of cell death, named NETosis, characterized by formation of neutrophil extracellular traps (NET), decondensed chromatin threads decorated with cytoplasmic proteins endorsed with anti-microbial activity. NET have been consistently detected in ANCA-associated small vessel vasculitis and this association suggested of studying of whether the peculiar structure of NET favors neutrophil proteins up-loading into myeloid dendritic cells (mDC) and the induction of ANCA and associated autoimmunity.

• We have shown that mDC uploaded with and activated by NET components induce ANCA and autoimmunity when injected into naive mice. DC uploading and autoimmunity induction are prevented by NET treatment with DNAse, indicating that NET structural integrity is needed to maintain the antigenicity of cytoplasmic proteins. We found NET intermingling with mDC also positive for neutrophil MPO in MPO-ANCA-associated microscopic poliangiitis providing a potential correlative picture in human pathology. These data provide the first demonstration that NET structures are highly immunogenic such to trigger adaptive immune response relevant for autoimmunity and might provide a connection to understand the transition from autoimmunity to lymphomas.

Keywords: tumor microenvironment, SPARC/osteopontin, mast cells and neutrophils

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Research in this Unit is aimed to identify and validate cancer-related and actionable biomarkers relevant for cancer progression, through multidisciplinary approaches including molecular cell biology, high-throughput techniques, and bioinformatic tools. Studies are mainly focused on breast cancer (bC) and investigate the following:• Gene expression profiles (GEPs) on “critical” samples (formalin-fixed

paraffin-embedded (ffPE) material and/or limited amount of cells, e.g. circulating tumor cells, CTC). In collaboration with the Medical Oncology Department, GEPs from ffPE samples derived from a multicenter clinical trial on operable bC were obtained with an optimized technical procedure using Affymetrix chips. Metagenes derived from a large series (n=3154) of public Affymetrix GEP were validated in this case series. The risk of recurrence was associated with proliferation and ER-related metagenes in the ER+/hER2- subtype, and with a T-cell metagene in triple negative and hER2+ cases. This enabled the identification of bC patients at a high risk of relapse despite optimal standard treatment, who could be spared useless toxicity and considered for new treatment strategies. Using blood samples we developed a protocol to reliably profile the expression of 29,000 genes by the DASl approach in CTCs, derived from bC patients subjected to primary systemic treatments.

• microRNA as blood-derived biomarkers are potentially useful for early detection and risk assessment through non-invasive approaches. To improve the reliability of circulating microRNAs (ct-miRNAs), as emerging and promising biomarkers for non invasive assessment of disease progression, much effort was spent to understand pre-analytical and analytical confounders. Isolation, stability, detection, and quantification were investigated and compared in different experimental sets, and a simple, robust, sensitive, and cost-effective system was developed to identify hemolyzed plasma/serum specimens, even in the presence of low hemoglobin concentrations and high degrees of lipemia. following proposed guidelines, we were able to identify ct-miRNAs associated with distant relapse in plasma samples of stage I bC patients enrolled in the hPR- INT study (1987-1993) and subjected only to radical or conservative surgery +RT until relapse.

Keywords: breast cancer, biomolecular profile, circulating tumor cells, blood-derived

biomarkers, microRNA

bIomarkers

HeadMaria Grazia Daidone, biol Sci D, PhD

ReseaRcH staffvera Cappelletti, biol Sci DSilvia veneroni, biol Sci DRaffaella villa, biol Sci D

PostdoctoRal fellowsvalentina Appierto, biol Sci D, PhD

Graziella Cimino Reale, biol Sci D, PhD

PHd stUdentsMaurizio Callari, Med biotech Dvaleria Musella, Med biotech D

ReseaRcH fellowsEleonora Di buduo, Med biotech D

Emanuela fina, biol Sci DGiuseppe Merlino, Medbiotech D

Paola Tiberio, biol Sci D

tecHniciansElena Cavadini, Cinzia De Marco, Chiara Iacona, Patrizia Miodini, biol Sci D, Gloria

Morandi, Rosita Motta

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Dissection of the molecular events underlying the ATM-CHK2 dependent DNA damage response and role in genomic stability. We pursued characterization of DbC1/KIAA1967, a component of the DNA damage response (DDR) signalling cascade. DbC1 is thought to act as a tumor suppressor involved in apoptosis, transcription regulation, and histone modification. We found that DbC1 is a potent inhibitor of SIRT1 deacetylase, a regulator of p53-dependent apoptosis induced by genotoxic stress, and that this inhibition requires DbC1 phosphorylation on Thr454 by ATM and ATR kinases. DbC1-Thr454 phosphorylation causes the dissociation of the SIRT1-p53 complex, thereby increasing p53 acetylation and p53-dependent cell death. We also found that in undamaged cells overexpression of DbC1-WT, but not DbC1-Thr454A, induces S-phase associated γh2AX nuclear foci, which are widely accepted markers of DSbs, suggesting a role for DbC1 in DNA replication. More recently, using TAlEN (Transcription Activator-like Effector Nuclease) we have successfully generated DbC1-knock out cells which we are currently using for ChIP-Seq analysis.Development of pro-apoptotic SMAC-mimetic (SMs) compounds with anticancer activity. In collaboration with Drs. Seneci and bolognesi at the University of Milan, we designed and generated >120 small molecule SMs. We focused our pre-clinical investigations on dimeric SM83, which possesses elevated affinity for the bIR3 domains of XIAP, cIAP1, and cIAP2, in addition to favorable ADMET features and submicromolar tumor cell killing activity. In independent experiments, we confirmed that CD34 hemopoietic stem cells expressing mTRAIl slightly inhibit the growth of primary solid tumors implanted subcutaneously in NOD/SCID mice, while the administration of SM83 as monotherapy can reduce tumor volume by about 50% in all experiments. Importantly, SM83 inhibited tumor metastases to the lungs, and this effect was further enhanced by the combination with CD34-mTRAIl. Thus, SMs appear to play a key role in preventing spontaneous tumor metastasis.Keywords: DNA damage, cell cycle checkpoints, genomic instability, IAPs, apoptosis

HeadDomenico Delia, biol Sci D

PostdoctoRal fellowbenjamin Nachimuthu, PhDClara Ricci, PhDlaura zannini, PhD

ReseaRcH fellowluigi Carlessi, biol Sci D

PHd stUdentAnnalisa Conti, biol Sci DElena fusar-Poli, biol Sci DDaniele lecis, biol Sci D

tecHnicianEnrico fontanella

molecular mechanIsms of cell cycle control

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HeadMaria Angela Greco, biol Sci D

ReseaRcH staffMaria Grazia borrello, biol Sci D

Italia bongarzone, biol Sci DClaudia Miranda, biol Sci D

PostdoctoRal fellowsMaria Grazia vizioli, Med biotech D

Maria Chiara Anania, biol Sci DMara Mazzoni, biol Sci D

Paola Romeo, Med biotech DDario Caccia, Med biotech D

PHd stUdentEmanuela Minna, biol Sci D

tecHniciansMaida De bortoli, Sonia Pagliardini,

Maria Grazia Rizzetti

molecular mechanIsmsThe Unit is involved in studies of the molecular mechanisms of thyroid carcinogenesis, with particular interest in papillary thyroid carcinoma (PTC), the most frequent thyroid neoplasia. The final goal of ongoing studies is the identification of markers useful for early detection, prognosis, and follow up, as well as novel therapeutic targets.• Towards this end, the Unit employs several different approaches including

generation of in vitro models of thyroid carcinogenesis using tumor-derived cell lines and human primary thyrocytes; analysis of the role of selected pathways and molecules; mRNA and microRNA expression analysis; characterization of a thyroid tumor case collection, used both for discovery and validation studies.

The results achieved during 2012 are related to:• Relevance in thyroid carcinogenesis of oncogene-induced senescence (OIS),

a novel mechanism proposed as a barrier to cancer, by demonstrating that OIS restrains thyroid tumor progression, as it is present in early phase, indolent, tumor papillary thyroid microcarcinoma. In primary thyrocytes, PTC-associated oncogenes induce senescence, with the concomitant production of inflammatory cytokines.

• Involvement of S100A11, a gene frequently up-regulated in PTC and in PTC-derived cell lines, in loss of contact inhibition, anchorage-independent growth, and resistance to anoikis, suggesting that it exerts a protumoral functional role in PTC pathogenesis.

• Identification of new miRNAs involved in thyroid carcinogenesis by integrated analysis of gene expression and miRNA profiling in PTC surgical samples and in in vitro PTC models based on RET/PTC oncogenes. Among these, miR-199a-3p was under-expressed in the PTC model and in PTC surgical samples, and was able to induce strong tumor-suppressing phenotypes in different PTC-derived cell lines.

• Demonstration through functional studies that the RET-K666E mutation, detected in a medullary thyroid cancer (MTC) patient, is oncogenic, and that

its transforming activity is enhanced by the cis G691S polymorphism.• Demonstration that DUSP6/MKP3, a feedback negative regulator of ERK1/2 pathway, is over-expressed at the mRNA and protein levels in both papillary and poorly differentiated thyroid carcinoma, and that it may have a protumorigenic role in thyroid carcinogenesis.• Identification of genetic determinants controlling the progression of MTC using a mouse model (in collaboration with Polygenic Inheritance Unit).• In collaboration with Anatomic Pathology, the Unit has also contributed to a study describing a novel double mutant KIT in GIST that responds to Imatinib.Keywords: thyroid tumors, OIS, miRNA

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Headlicia Rivoltini, MD

ReseaRcH staffChiara Castelli, biol Sci DMonica Rodolfo, biol Sci D

fellowsSara Rigoletto, biol Sci DMarcella Tazzari, PhD student

PostdoctoRal fellowsChiara Camisaschi, PhDAnnamaria De filippo, biol Sci D, PhDPaola filipazzi, biol Sci Dveronica huber MD, PhDviviana vallacchi, biotechnol Sci D, PhDElisabetta vergani, PhD

ResidentAlessandra Tuccitto, biol Sci D

tecHniciansvaleria beretta, Agata Cova, Paola Deho, Simona frigerio, francesca Rini, Paola Squarcina

ReseaRcH nURsesfelicetta Giardino, Gianluigi Rigamonti

data ManageRPaola frati

Visiting Post-doc fellow and PHd stUdentMaja burdek, PhD (fellowship from DfG, German Research Association)Olga Kuchuk, PhD student (Open University, sponsored by Digestive Surgery and liver Transplantation Unit)

Immunotherapy of human tumorsClinical and immunological effects of immune-based cancer therapies. We continued enrolling patients in Phase I-II studies of cancer vaccines (PRAME and NY-Eso1) in melanoma patients (in collaboration with the Melanoma Surgery Unit). We also continued the vaccine trial of survivin-derived peptides in prostate carcinoma patients with biochemical recurrence (in collaboration with the Prostate Program). We completed enrollment in the NIbIT-M1 trial (anti-CTlA-4 mAb and fotemustine in melanoma patients, in collaboration with the Medical Oncology Unit). Clinical and immunological evaluations are presently ongoing. finally, we collected blood samples and tumor biopsies from melanoma patients treated with bRAf and MEK inhibitors to test the involvement of the immune system in clinical response (in collaboration with the Medical Oncology Unit).Studies on cancer-related immune regulatory pathways. We monitored immunoregulatory pathways (including myeloid-derived suppressor cell (MDSC), Treg, cyto/chemokine profiles) in blood, serum, saliva, and tumor biopsies from sarcoma, melanoma, hepatocellular carcinoma (in collaboration with the Gastrointestinal, hepatopancreatobiliary Surgery, and liver Transplantation Units) and head and neck (h&N) cancer patients. We found evidence that the level of MDSC correlates with poor prognosis in melanoma patients, and that these cells seem to express a mRNA/miRNA signature including Il-6, hIf1-alpha and TGf-beta. We also obtained data showing the association between a decrease in MDSC and clinical response in sarcoma patients. In h&N cancer, salivary cytokines were found to predict the severity of mucositis after chemo/radiotherapy (in collaboration with head and Neck Cancer Medical Oncology Unit). We assessed the impact of the metabolic microenvironment, observing that low ph contributes to MDSC activation and T cell anergy, while drugs buffering local acidity mediate the restoration of effective tumor immunity. We studied tumor exosomes as an immunosuppressive pathway in cancer, discovering a novel decoy effect of these organelles on T cell activity, and as a tool for specific drug delivery, i.e. TRAIl-induced tumor apoptosis. finally, we analyzed the phenotypic and functional behavior of pDC, a novel subset of antigen-presenting cells, in cancer patients. Molecular studies on melanoma progression markers. Gene expression studies identified a lymphocyte subpopulation expressing the activation marker CD30 with a potential role in suppressing immune response in melanoma-draining sentinel lymph nodes. Profiling studies identified circulating miRNA in melanoma patients by comparison with healthy donors. We assessed the immunological properties of melanoma cells with acquired resistance to bRAf inhibitors (bRAfi), collecting evidence that bRAfi-resistant tumors up-regulate chemokine secretion, potentiate MDSC accrual, and express higher levels of ph regulators compared to bRAfi-sensitive counterparts. Keywords: myeloid-derived suppressor cells, exosomes, regulatory T cells,

cancer vaccines, melanoma genetics

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Our research activity covers all aspects of lung cancer with the final aim of making an impact on a disease that is a major health-care burden in terms of morbidity and mortality. We use an integrated approach that combines cellular and molecular biology, biochemistry, and pharmacology to gain new insights in the pathogenesis of lung cancer and to find novel ways to provide early diagnosis and new treatment options. The goal of our translational studies is the implementation of highly sensitive molecular tests that can be included in screening programs to improve both detection and clinical management of lung cancers.Major research projects involve the biologic and molecular characterization of lung cancer• miRNAs: sensors and players. The implementation of molecular markers

for risk stratification is a priority, and microRNAs (miRNAs) are an extremely promising new class of blood-based biomarkers for cancer detection and definition of prognosis. We recently reported that specific miRNA signatures can be identified in plasma samples of patients up to two years before detection of disease by spiral-CT, and can also predict tumor aggressiveness. We have now performed an extended validation of these signatures in cases and controls enrolled in the MIlD trial. An investigation of the functional potential of miRNAs in different aspects of lung cancer biology is underway to provide novel tools for early detection and therapy.

• Development of novel experimental models. Mouse models are still the most valuable tool for preclinical evaluation of novel therapeutic strategies in cancer and, among these, patient-derived xenografts (PDX) provide a faithful representation of the patient’s original tumor both immunohistochemically and genetically. We have recently developed in vivo lung cancer PDX models by directly implanting fragments of the primary tumors in the flank of immunocompromised mice. To date, starting from 74 human lung primary tumors, we successfully grew 30

PDXs (take rate 40.5%) in SCID mice and additional PDXs are continuously established. To use these models for testing novel treatments, we set up metabolic imaging in vivo of PDXs using weekly [18f]fDG-PET together with coronal and 3D-reconstruction at different days. We observed a good correlation of metabolic activity between the primary tumor and PDXs which support the use of these “human in mouse” models for functional studies.Keywords: lung cancer, tumor genetics,

biomarkers, microenvironment

HeadGabriella Sozzi, PhD

ReseaRcH staff luca Roz, Pharm Sci D

PostdoctoRal fellowfrancesca Andriani, Pharm Sci D

Giulia bertolini, Med biotech DPatrizia Gasparini, biol Sci D

Massimo Moro, PhDCarla verri, biol Sci D

PHd stUdentMattia boeri, biotech D

tecHniciansRoberto Caserini, Davide Conte

federica facchinetti, Mavis Mensah

tumor genomIcs

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Extracellular matrix components as breast cancer prognostic markers. Our unsupervised clustering analyses of more than 600 breast carcinoma samples identified a subgroup of tumors (extracellular matrix 3 - ECM3) as an independent group in all the tested datasets (26-41% of cases). ECM3 tumors showed a homogeneous ECM gene pattern encoding mainly structural ECM proteins. Multivariate analysis of distant metastasis-free survival cases in untreated breast tumor patients revealed a significant interaction between ECM3 and histological grade. In Cox models, estimated separately in grade I-II and grade III tumors, ECM3 status and worse survival probability were significantly associated only in grade III tumors (hR=3.0, 95% CI=1.3-7.0), whereas no significant differences between ECM3 and non-ECM3/grade III tumors were found considering clinicopathological features and intrinsic molecular subtypes. Our data suggest that ECM3 classification can provide information on tumor biology and progression.Mechanisms of resistance to chemotherapy. Our analysis of the possible role of maspin, a member of the serpin protease inhibitor family, in breast cancer response to chemotherapy showed that tumors overexpressing maspin are more likely to be resistant to doxorubicin (DXR)-based chemotherapy than those not overexpressing it, as demonstrated both in human breast carcinomas and in mice xenotransplanted with tumor cells genetically manipulated to overexpress maspin. Maspin-associated resistance to DXR was due mainly to low diffusion of DXR in the maspin-induced collagen-enriched microenvironment. The neutralizing monoclonal antibody we prepared to control maspin reversed the collagen-dependent DXR resistance. Activity and resistance mechanisms to HER2-targeted therapies. by analyzing data from clinical studies, we identified antibobody-dependent cell-mediated cytotoxicity (ADCC) as the main mechanism through which trastuzumab exerts its antitumor effects in neoadjuvant monotherapy. In treatment settings involving concomitant use of trastuzumab and chemotherapy, the antibody appears to act primarily through inhibition of DNA repair, while, in sequential protocols, the antibody acts mostly by exerting cytostatic activity through inhibition of hER2-mediated tumor cell proliferation. Since trastuzumab resistance is likely to depend directly on the mechanisms responsible for its antitumor activity, resistance mechanisms must also be considered in different clinical settings. Keywords: breast cancer, targeted

therapy, HER2

molecular targetIng

HeadElda Tagliabue, biol Sci D

ReseaRcH staffManuela Campiglio, biol Sci D, Rosaria Orlandi, biol Sci D Serenella Pupa, biol Sci D

fellowslorenzo Castagnoli, biotech Sci Dvalentina Ciravolo, biotech Sci DGaia C. Ghedini, biotech Sci D (PhD Student/fIRC)Marta Giussani, biotech Sci D (PhD Student)Alessandra Meini, biol Sci D (PhD Student)viola Regondi, biotech Sci D 1st levelAnna Rossini, biol Sci DMarianna Sasso, biol Sci D (PhD Student)federica Turdo, biol Sci D

PostdoctoRal fellowTiziana Triulzi, biotech Sci D

consUltantsSylvie Ménard, biol Sci D Marco Sandri, Stat Sci D

tecHniciansPierangela Aiello, Patrizia Casalini Cristina Ghirelli

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Novel therapeutic targets, with special reference to microRNAs. We demonstrated that miR-205 functions as a brake against prostate cancer metastasis by blocking both the afferent and efferent arms of the circuit between tumor cells and associated fibroblasts. In fact, miR-205 not only counteracts the epithelial–mesenchymal transition (EMT) in prostate cancer cells upon stimulation with cancer-associated fibroblasts, thus impairing cell invasion, tumorigenicity, and metastatic dissemination, but also blocks tumor-driven activation of surrounding fibroblasts by reducing pro-inflammatory cytokine secretion. In the search for telomerase-related

miRNAs, we found that miR-380* was the most down-modulated miRNA in telomerase-positive versus telomerase-negative peritoneal mesotheliomas. Restoring miR-380* expression in peritoneal mesothelioma cells resulted in significant impairment of cell growth, as a consequence of the interference with the expression of telomerase subunit hTERT and the enzyme’s catalytic activity. Mechanisms of drug resistance. The study of the alterations of ovarian carcinoma cells displaying reduced sensitivity to platinum drugs and receptor tyrosine kinase inhibitors highlighted a major role for deregulated pro-survival signaling pathways. In fact, increased phosphorylation of ERK1/2 was found in resistant cells, in which genome-wide expression analysis revealed down-regulation of apoptosis-related factors (PUMA) and inhibitory phosphatases targeting ERK1/2 (DUSP5, DUSP6). New drug combinations. The combination of histone deacetylase and proteasome inhibitors was found to induce marked synergistic anti-proliferative and pro-apoptotic effects in ovarian carcinoma cells, which were particularly pronounced in p53 mutant variants displaying a platinum drug-resistant phenotype. A novel therapeutic approach based on heparanase/heparan sulfate system targeting was tested in preclinical models of pediatric sarcomas. The non anti-coagulant heparin derivative SST0001 induced a significant tumor growth inhibition in all models. The absence of toxicity upon prolonged treatments, together with the multi-targeting nature of the drug involving tumor-microenvironment interactions, suggested its use in combination with anti-angiogenic agents (bevacizumab, sunitinib) to prevent compensatory mechanisms of resistance. Strong antitumor activity was documented by the high rate of complete responses. Our results point towards cooperative inhibition of angiogenesis and tumor growth promoting signaling pathways.Keywords: therapeutic targets, drug resistance, preclinical drug development, miRNAs

HeadNadia zaffaroni, biol Sci D, PhD

ReseaRcH staffMarco folini, biol Sci D, PhD

Cinzia lanzi, biol Sci DPaola Perego, biol Sci D, PhD

PostdoctoRal fellowsGiovanni l. beretta, biol Sci D, PhD

Joanna bidzinska, biotech Sci D, PhDGiuliana Cassinelli, Pharm D, PhD

Michelandrea De Cesare, vet DPaolo Gandellini, biotech Sci D, PhD

laura Gatti, biol Sci D, PhDAlessia lopergolo, biotech Sci D, PhD

Marzia Pennati, biol Sci D, PhDvalentina zuco, biol Sci D

fellowsDenis Cominetti, biol Sci D

Nicola fenderico, biotech Sci Dvalentina Profumo, biol Sci D, PhD Student

francesca Santambrogio, biol Sci DStefania Sbarra, biol Sci D

tecHniciansElisa Campi, Nives Carenini, Elisabetta

Corna, laura Dal bo, Enrica favini, Maria Stella Tinelli, Monica Tortoreto

molecular pharmacology

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The goal of the Start Up Unit is the identification and study of microRNAs involved in the most important pathways activated in human breast cancer, with the aim to better define the role of these small but powerful molecules in this neoplasia, and to provide the experimental bases for their possible use as targets or tools in specific therapies.To achieve this challenging but promising purpose, the strategy is described in the following tasks:• Investigation of microRNAs

involved in the ER- and hER2-mediated pathways;

• Investigation of the microRNAs involved in triple negative (TN) breast cancer biology;

• Evaluation of the biological effects of the microRNAs of interest identified in the previous tasks and validation of putative targets;

• Evaluation of the prognostic/predictive significance of selected microRNAs on series of primary breast carcinomas;

• Identification of the mechanisms regulating the expression of microRNAs of interest.

In 2012, we reported (Piovan C et al., 2012) that miR-205 is directly activated by P53 and plays an oncosuppressive role in TNbC, inhibiting tumor growth both in vitro and in vivo, at least partially through direct targeting of E2f1 and lAMC1.We are now exploring the possible therapeutic application of miR-205, either using an inducible (Tet-ON) system to turn on miR-205 expression, and through intratumoral or systemic delivery of miR-205 in liposome carriers. Moreover, in collaboration with Dr. Croce (OSU,Oh, USA) we developed a KO mouse and are now analyzing the effect of miR-205 knocking down on both mammary gland development and tumor occurrence.Concerning miRs and TNbC, we have obtained interesting preliminary data on two miRs involved in the regulation of vasculogenic mimicry properties of this breast cancer subgroup.Regarding miRs and hER2, we have preliminary data on miR-205 and response to trastuzumab.for miR and ER, we have contributed to a manuscript (Di leva G et al., 2012) describing a regulatory loop between ER and miR-191.Keywords: microRNA, breast cancer, signalling pathways, therapy

aIrc start up unIt

HeadMarilena v. Iorio, biotech Sci D

ReseaRcH staffIlaria Plantamura, PhD, fellowElvira D’Ippolito, PhD student, fellow Claudia Piovan, PhD, collaborator

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