Scientific Report 2012

scientific report 2012
experimental oncology and
molecular medicine department
DIRECTOR OF DEPARTMENT Maria Grazia Daidone
+39 02 2390 2238
[email protected]
UNITS
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.
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Immunobiology of Human Tumors
Andrea Anichini
Molecular Therapies
Silvana Canevari
Molecular Immunology
Mario P. Colombo
Biomarkers
Maria Grazia Daidone
Molecular Mechanisms of Cell Cycle Control
Domenico Delia
Molecular Mechanisms
Maria Angela Greco
Immunotherapy of Human Tumors
Licia Rivoltini
Tumor Genomics
Gabriella Sozzi
Molecular Targeting Unit
Elda Tagliabue
Molecular Pharmacology
Nadia Zaffaroni
AIRC Start Up Unit
Marilena V. Iorio
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scientific report 2012
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 computerreadable 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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scientific report 2012
Experimental Oncology and Melecular Medicine Department
Immunobiology of human
tumors
Head
Andrea Anichini, Biol Sci D
Research Staff
Roberta Mortarini, Biol Sci D
Marialuisa Sensi, Biol Sci D
Fellows
Giulia Grazia, Biotechnol Sci D
Ilaria Penna, Biol Sci D
Valentina Perotti, Biol Sci D
Elena Tassi, Biotechnol Sci D
Technicians
Paola L. Baldassari, Ilaria Bersani, Alessandra
Molla, Gabriella Nicolini, Claudia Vegetti
The 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 activation
Our 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 calcineurinNFATc2 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 targetspecific 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
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scientific report 2012
Experimental Oncology and Melecular Medicine Department
Molecular Therapies
Through 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
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Head
Silvana Canevari, Biol Sci D
Research Staff
Fabio Benigni, Biol Sci D
Marina Bagnoli, Biol Sci D
Mariangela Figini, Biol Sci D
Delia Mezzanzanica, Biol Sci D
Antonella Tomassetti, Pharmacol Sci D
Fellows
Chiara Alberti, Biol Sci D
Davide Bernareggi, Biol Sci D
Francesco Caroli, Technician
Barbara Frigerio, Biol Sci D
Anna Granata, Biol Sci D
Cristina Luna, Technician
Roberta Nicoletti, Med Biotech D
Patrizia Pinciroli, Biol Sci D
Katia Rea, Med Biotech D
Alessandro Satta, Vet Biotech D
Valentina Tinaglia, Sci Biotech D
Technicians
Paola Alberti, Renata Ferri, Elena Luison
scientific report 2012
Experimental Oncology and Melecular Medicine Department
Molecular immunology
HEAD
Mario P. Colombo, Biol Sci D
RESEARCH STAFF
Silvia Miotti, Biol Sci D
Claudia Chiodoni, Biol Sci D
POSTDOCTORAL FELLOWS
Sabina Sangaletti, PhD, Biol Sci D
Agniezka Chronowska, PhD
Paola Pittoni, PhD
Caterina Vitali, PhD
Giorgio Mauri, Med Biotech D
FELLOW
Andrea Tomirotti, Med Biotech D
PHD STUDENTS
Alessia Burocchi, Biol Sci D
Alessandra Santangelo, Biol Sci D
Alice Rigoni, Biol Sci D
Ilaria Torselli, Biol Sci D
TECHNICIANS
Ivano Arioli, Barbara Cappetti, Ileana Facetti,
Mariella Parenza, Claudia Bassani,
Laura Botti, Biol Sci D, Chiara Ratti
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
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scientific report 2012
Experimental Oncology and Melecular Medicine Department
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 ANCAassociated 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-ANCAassociated 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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Experimental Oncology and Melecular Medicine Department
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 FELLOWS
Valentina Appierto, Biol Sci D, PhD
Graziella Cimino Reale, Biol Sci D, PhD
PHD STUDENTS
Maurizio Callari, Med Biotech D
Valeria Musella, Med Biotech D
RESEARCH FELLOWS
Eleonora Di Buduo, Med Biotech D
Emanuela Fina, Biol Sci D
Giuseppe Merlino, MedBiotech D
Paola Tiberio, Biol Sci D
TECHNICIANS
Elena Cavadini, Cinzia De Marco, Chiara
Iacona, Patrizia Miodini, Biol Sci D, Gloria
Morandi, Rosita Motta
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, highthroughput 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
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scientific report 2012
Experimental Oncology and Melecular Medicine Department
Molecular Mechanisms of Cell Cycle Control
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
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HEAD
Domenico Delia, Biol Sci D
Postdoctoral Fellow
Benjamin Nachimuthu, PhD
Clara Ricci, PhD
Laura Zannini, PhD
Research Fellow
Luigi Carlessi, Biol Sci D
PhD Student
Annalisa Conti, Biol Sci D
Elena Fusar-Poli, Biol Sci D
Daniele Lecis, Biol Sci D
Technician
Enrico Fontanella
scientific report 2012
Experimental Oncology and Melecular Medicine Department
Molecular mechanisms
Head
Maria Angela Greco, Biol Sci D
RESEARCH STAFF
Maria Grazia Borrello, Biol Sci D
Italia Bongarzone, Biol Sci D
Claudia Miranda, Biol Sci D
POSTDOCTORAL FELLOWS
Maria Grazia Vizioli, Med Biotech D
Maria Chiara Anania, Biol Sci D
Mara Mazzoni, Biol Sci D
Paola Romeo, Med Biotech D
Dario Caccia, Med Biotech D
PHD STUDENT
Emanuela Minna, Biol Sci D
TECHNICIANS
Maida De Bortoli, Sonia Pagliardini,
Maria Grazia Rizzetti
The 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, PTCassociated 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 overexpressed 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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scientific report 2012
Experimental Oncology and Melecular Medicine Department
Immunotherapy of Human Tumors
Clinical 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 upregulate 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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Head
Licia Rivoltini, MD
Research Staff
Chiara Castelli, Biol Sci D
Monica Rodolfo, Biol Sci D
Fellows
Sara Rigoletto, Biol Sci D
Marcella Tazzari, PhD student
Postdoctoral Fellows
Chiara Camisaschi, PhD
Annamaria De Filippo, Biol Sci D, PhD
Paola Filipazzi, Biol Sci D
Veronica Huber MD, PhD
Viviana Vallacchi, Biotechnol Sci D, PhD
Elisabetta Vergani, PhD
Resident
Alessandra Tuccitto, Biol Sci D
Technicians
Valeria Beretta, Agata Cova, Paola Deho,
Simona Frigerio, Francesca Rini, Paola
Squarcina
Research Nurses
Felicetta Giardino, Gianluigi Rigamonti
Data Manager
Paola Frati
Visiting post-doc fellow and
PhD student
Maja Burdek, PhD (Fellowship from DFG,
German Research Association)
Olga Kuchuk, PhD student (Open University,
sponsored by Digestive Surgery and Liver
Transplantation Unit)
scientific report 2012
Experimental Oncology and Melecular Medicine Department
TUMOR GENOMICS
HEAD
Gabriella Sozzi, PhD
Research Staff
Luca Roz, Pharm Sci D
Postdoctoral Fellow
Francesca Andriani, Pharm Sci D
Giulia Bertolini, Med Biotech D
Patrizia Gasparini, Biol Sci D
Massimo Moro, PhD
Carla Verri, Biol Sci D
PhD student
Mattia Boeri, Biotech D
Technicians
Roberto Caserini, Davide Conte
Federica Facchinetti, Mavis Mensah
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
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scientific report 2012
Experimental Oncology and Melecular Medicine Department
Molecular Targeting
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
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Head
Elda Tagliabue, Biol Sci D
Research Staff
Manuela Campiglio, Biol Sci D,
Rosaria Orlandi, Biol Sci D
Serenella Pupa, Biol Sci D
Fellows
Lorenzo Castagnoli, Biotech Sci D
Valentina Ciravolo, Biotech Sci D
Gaia 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 level
Anna Rossini, Biol Sci D
Marianna Sasso, Biol Sci D (PhD Student)
Federica Turdo, Biol Sci D
Postdoctoral Fellow
Tiziana Triulzi, Biotech Sci D
Consultants
Sylvie Ménard, Biol Sci D
Marco Sandri, Stat Sci D
Technicians
Pierangela Aiello, Patrizia Casalini
Cristina Ghirelli
scientific report 2012
Experimental Oncology and Melecular Medicine Department
Molecular Pharmacology
Head
Nadia Zaffaroni, Biol Sci D, PhD
Research Staff
Marco Folini, Biol Sci D, PhD
Cinzia Lanzi, Biol Sci D
Paola Perego, Biol Sci D, PhD
Postdoctoral Fellows
Giovanni L. Beretta, Biol Sci D, PhD
Joanna Bidzinska, Biotech 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
Alessia Lopergolo, Biotech Sci D, PhD
Marzia Pennati, Biol Sci D, PhD
Valentina Zuco, Biol Sci D
Fellows
Denis Cominetti, Biol Sci D
Nicola Fenderico, Biotech Sci D
Valentina Profumo, Biol Sci D, PhD Student
Francesca Santambrogio, Biol Sci D
Stefania Sbarra, Biol Sci D
Technicians
Elisa Campi, Nives Carenini, Elisabetta
Corna, Laura Dal Bo, Enrica Favini,
Maria Stella Tinelli, Monica Tortoreto
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 multitargeting 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
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scientific report 2012
Experimental Oncology and Melecular Medicine Department
AIRC Start Up Unit
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 HER2mediated 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 miR205 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
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Head
Marilena V. Iorio, Biotech Sci D
Research Staff
Ilaria Plantamura, PhD, Fellow
Elvira D’Ippolito, PhD student, Fellow
Claudia Piovan, PhD, collaborator
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