D 1
TROP-2 IS A UNIVERSAL DETERMINANT OF CANCER METASTATIZATION AND SURVIVAL
Saverio Alberti (1) - Emanuela Guerra (2) - Romina Tripaldi (2) - Rossano
Lattanzio (2) - Giovanna Vacca (2) - Romina Zappacosta (2) - Marco
Trerotola (2) - Pasquale Simeone (2) - Valeria Relli (2) - Robert De Lange (3) -
Ulrich H. Weidle (3) - Rossana La Sorda (2) - Patrizia Querzoli (4) - Massimo
Pedriali (4) - Enzo Bianchini (5) - Domenico Angelucci (6) - Giuseppe Pizzi-
cannella (7) - Xiao-feng Sung (8) - Carla Di Loreto (9) - Carlo Alberto Bel-
trami (9) - Laura Antolini (10) - Mauro Piantelli (2)
Unit of Cancer Pathology, Center of Excellence for Research on Aging, Foundation University “G. D’ Annunzio”, Chieti, Italy & Department of Neuroscience and Imaging, University ‘G. d’Annunzio’, Via Colle dell’ Ara, 66100 Chieti Scalo, Italy (1) - Unit of Cancer Pathology, CeSI, Foun-
dation University ‘G. d’Annunzio’, Via Colle dell’ Ara, 66100 Chieti Scalo, Italy (2) - Roche Diagnostics GmbH, Pharma Research, Nonnenwald 2, D-
82372 Penzberg, Germany (3) - Institute of Pathology, University of Ferra-
ra, Via Luigi Borsari 46, 44121 Ferrara, Italy (4) - Department of Patholo-
gy, Rovigo Hospital, Viale Tre Martiri 140, 45100 Rovigo, Italy (5) - eDe-
partment of Pathology, Ortona Hospital, Contrada S.Liberata 1, 66026 Ortona, Italy (6) - Department of Pathology, Lanciano Hospital, Via per
Fossacesia 1, 66034 Lanciano, Italy (7) - Division of Oncology, Depart-
ment of Clinical and Experimental Medicine, Faculty of Health Sciences, County Council of Östergötland, University of Linköping, S-581 85 Lin- köping, Sweden (8) - Department of Pathology, University of Udine, Via
Palladio 8, 33100 Udine, Italy (9) - Biostatistics Center, Department of
Clinical Medicine, Prevention and Biotechnology, University of Milano- Bicocca, 20900 Monza, Italy (10)
Introduction: Metastases are the main cause of cancer death.
Hundreds of proteins/genes have been linked to the metastatic phenotype in different experimental systems. Yet, no unique markers of cancer aggressiveness and metastatic potential have been identified.
Materials and methods : Gene-expression profiling, metastatic
models transcriptome meta-analysis, RT-PCR and IHC analysis of training and validation patient case-series of breast, uterus, stomach, colon and ovary metastatic cancers were utilized to define global and TROP2 expression patterns. Lentiviral vector- driven down-regulation versus overexpression of Trop-2 and target downstream effectors were utilized to define the Trop-2 mechanics of metastatic induction. E-cadherin, ß-catenin and Trop-2 expression patterns were challenged against a breast cancer case series to define prognostic relevance and survival.
Results: Gene-expression profiling of murine, rat and human
metastatic cells identified TROP2 as the only globally-shared upregulated gene. Metastatic upregulation of Trop-2 was vali- dated in breast, uterus, colon, stomach and ovary cancer pa- tients. Trop-2 was then shown to induce aggressive metastatiza- tion through loss of cell-cell adhesion and increased cell migra- tion and survival. We discovered that Trop-2 binds to E- cadherin and inactivates it, by disanchoring it from the cyto- skeleton. Functional blockade of E-cadherin then triggers β- catenin activation, nuclear translocation and downstream tran-
scription. The Trop-2/E-cadherin/β-catenin module was reca- pitulated in tumor patients, with unprecedented impact on meta- static survival of aggressive triple-negative breast cancers.
Conclusions: Our findings candidate TROP2 as a universal
driver of metastatic spreading. The paradigm of a global, Trop- 2-driven pro-metastatic program paves the way for novel clini- cal approaches to patient diagnostics, prognostics and cure.
D 2
EFFECTS OF NOREPINEPHRINE ON MIGRATION AND INVASION OF DU145 CELLS IN XENOGRAFT MOUSE MODEL OF PROSTATE CANCER.
Antonio Barbieri (1) - Sabrina Bimonte (1) - Giuseppe Palma (1) - Antonio
Luciano (1) - Domenica Rea (1) - Aldo Giudice (2) - Giosuè Scognamillo (3) -
Elvira La Mantia (3) - Renato Franco (3) - Paola Stiuso (4) - Michele Caraglia (4) - Claudio Arra (1)
Animal Facility Unit, National Institute of Tumours of Naples “Pascale” Naples, Italy, Hospital, Naples, Italy (1) - National Institute of Tumours of
Naples “Pascale” Naples, Italy, Hospital, Naples, Italy (2) - Pathology Unit,
National Institute of Tumours of Naples “Pascale”, Naples, Italy, Hospi- tal, Naples, Italy (3) - Department of Biochemistry, Biophysics and General
Pathology, Second University of Naples, Naples, Italy, University, Naples, Italy (4)
Metastasis causes most of the deaths for cancer and this process still represents an enigmatic aspect of the disease. It is note that catecholamines, such as norepinephrine, are released by the sympathetic nervous system in chronic stress conditions. Since it has also been demonstrated that norepinephrine stimulates the motility of breast and colon cells through β-adrenergic receptor, we decided to examine its possible role in the development and metastasis formation, by in vitro and in vivo studies. We de- monstrate that treatments with norepinephrine (β2 - adrenoreceptor agonist) in mice injected with human prostate cancer cells DU145 increase the metastatic potential of these cells. Our date showed that the treatment of mice with norepi- nephrine leads to a significant increase of the migratory activity of cancer cells in a concentration-dependent manner and that this process is blocked by propanolol (β-adrenergic receptors). Mice treated with norepinephrine, displayed an increased num- ber of metastatic foci of DU145 cells in inguinal lymph nodes and also showed an increased expression of MMP2 and MMP9 in tumor samples compared to controls.
All together these data show that β2-AR plays an important ro- le in the formation of metastasis in prostate cancer and suggest that the treatment with antagonist propranolol, could represents an interesting tool to control metastasis formation in cells over- expressing β2AR.
Keywords: Stress, Norepinephrine, cell migration, cell invasion, metastases, β-Adrenergic receptors.
48
D 3
PD-L1 EXPRESSION IDENTIFIES A SUBPOPULATION OF MELANOMA CELLS CHARACTERIZED BY ENHANCED INVASIVENESS AND AGGRESSIVENESS
Davide Brusa (1) - Daniela Massi (2) - Barbara Merelli (3) - Michela Ciano (1)
- Valentina Audrito (4) - Sara Serra (4) - Gianna Baroni (2) - Romina Nassini (5) - Daiana Minocci (5) - Laura Cattaneo (6) - Alessandra Carobbio (7) - Sil-
via Deaglio (4) - Mario Mandalà (3)
Human Genetics Foundation (HuGeF), University of Turin, Turin, Italy (1) -
Division of Pathological Anatomy, Department of Surgery and Transla- tional Medicine, Univerity of Florence, Florence, Italy (2) - Unit of Medical
Oncology, Department of Oncology and Hematology, Papa Giovanni XXIII Hospital, Bergamo, Italy (3) - Department of Medical Sciences, University
of Turin, Turin, Italy (4) - Unit of Clinical Pharmacology and Oncology, De-
partment of Health Science, University of Florence, Florence, Italy (5) -
Division of Pathological Anatomy, Papa Giovanni XXIII Hospital, Bergamo, Italy (6) - Research Foundation, Papa Giovanni XXIII Hospital, Bergamo,
Italy (7)
Introduction: Melanoma is one of the most aggressive malig-
nant tumors, with a high mortality in the metastatic setting. The discovery of somatic mutations in the BRAF oncogene in some patients led to the introduction of BRAF inhibitors in the clini- cal management. Clinical responses to BRAFi are dramatic, but they may be short lived. For this reason, there is intense investi- gation into alternative therapeutic strategies, including novel immunomodulatory agents such as anti-PD-1 and anti-PD-L1 antibodies. PD-L1 is a transmembrane molecule that can be ex- pressed by melanoma cells, where it mediates immunosuppres- sion by interacting with the PD-1 receptor on T cells. The clini- cal and biological significance of PD-L1 expression in metastat- ic melanoma remains to be fully uncovered.
The aim of this work is to understand the prognostic role and functional significance of PD-L1 expression in metastatic mela- noma.
Materials and methods: PD-L1 expression was analyzed by
immunohistochemistry using different antibodies in primary tumors and paired metastases from 83 melanoma patients. PD- L1+ and PD-L1- subsets of the A375 cell line were stabilized in
vitro by cell sorting and compared using gene expression profil- ing and functional assays studying growth, migration and inva- sion. Results were confirmed in vivo using xenograft models.
Results: PD-L1 membrane positivity was detected in 24/83
(19%) of patients. By multivariate analysis, PD-L1 membrane expression (95% CI 1.48-4.67, P< 0.001), age >57 years [CI 95% 1.20-3.71, P< 0.009] and stage M1c (95% CI 1.01-3.38, P< 0.045) were independent predictors of poor prognosis. Fur- thermore, PD-L1 expression defined a subset of the A375 cell line characterized by a specific genetic profile. PD-L1+ A375
cells showed a highly invasive phenotype and enhanced ability to grow in vivo in immunocompromised mouse models.
Conclusions: PD-L1 may be proposed as a novel prognostic
marker in metastatic melanoma, associated to a more aggressive disease and a worse clinical prognosis. Furthermore, it defines a distinct morpho-phenotypic subset of the disease with a specific genetic signature and a different biological behavior with a more activated status in terms of invasiveness and enhanced ability to grow in vitro and in vivo. Future studies will tell
whether PD-L1 expression is also a marker of resistance to se- lected therapies and whether it may be successfully exploited alone or in combination as a target for specific subsets of mela- noma patients.
D 4
AN ANGIOPOIETIN-LIKE PROTEIN 2 AUTOCRINE SIGNALING PROMOTES EMT DURING PANCREATIC DUCTAL
CARCINOGENESIS
Carmine Carbone (1) - Geny Piro (2) - Matteo Fassan (3) - Anna Tamburrino (1) - Maria Mihaela Mina (2) - Marco Zanotto (1) - Paul J Chiao (4) - Claudio
Bassi (5) - Aldo Scarpa (3) - Giampaolo Tortora (2) - Davide Melisi (1)
Digestive Molecular Clinical Oncology Research Unit, Università degli studi di Verona, Verona, Italy (1) - Laboratory of Oncology and Molecular
Therapy, Università degli Studi di Verona, Verona, Italy (2) - ARC-Net Re-
search Centre and Department of Pathology and Diagnostics, Università degli studi di Verona, Verona, Italy (3) - Department of Molecular and Cel-
lular Oncology, MD Anderson Cancer Center, Houston, United States (4) -
Department of Surgery, Università degli studi di Verona, Verona, Italy (5)
Introduction: The identification of the earliest molecular
events responsible for the metastatic dissemination of pancreatic cancer remains critical for early detection, prevention, and treatment interventions. In this study, we hypothesized that an autocrine signaling between Angptl2 and its receptor LilrB2 might be responsible for the epithelial-to-mesenchymal transi- tion (EMT) and, in turn, the early metastatic behavior of cells in pancreatic preneoplastic lesions.
Materials and methods: We used an in vitro experimental cell
transformation model system consisting in the sequential and stable expression of activated K-Ras, HER2, and p16/p14 shRNA sequences in the human pancreatic ductal epithelial (HPDE) cell line to study the role of Angptl2 on the features of EMT in the different steps of the pancreatic ductal carcinogene- sis. The expression of Angptl2 has been analyzed in a series of 62 different human Pancreatic Intraepithelial Neoplasia (PanIN) and 15 pancreatic cancer lesions.
Results: We demonstrated that the sequential activation of K-
Ras, expression of Her2 and silencing of p16/p14 are sufficient to progressively and significantly reduce the expression of the epithelial marker E-cadherin and to induce migratory properties in HPDE cells. In this multistep model, we measured the pro- gressively increased secretion of Angptl2, and the overexpres- sion of LilrB2. Exogenous Angptl2 significantly increased pro- liferation rate and migratory properties of LilrB2-expressing HPDE/K-Ras cells, and HPDE/K-Ras/Her2/p16p14shRNA cell lines. Conversely, silencing the expression of ANGPTL2 re- verted EMT and reduced migration in HPDE/K-Ras, and HPDE/K-Ras/Her2/p16p14shRNA cell lines. An increasingly significant overexpression of Angptl2 was observed in human PanIN and neoplastic lesions if compared with normal pancreat- ic parenchyma.
Conclusions: These findings showed that the autocrine signal-
ing of Angptl2 and its receptor LilrB2 plays key roles in sus- taining EMT and the early metastatic behavior of cells in pan- creatic preneoplastic lesions supporting the potential role of Angptl2 for early detection, metastasis prevention, and treat- ment in pancreatic cancer.
49
D 5
ENDOTHELIAL PODOSOME ROSETTES REGULATE VASCULAR BRANCHING IN TUMOR ANGIOGENESIS
Giulia Chiaverina (1) - Giorgio Seano (2) - Paolo Armando Gagliardi (1) -
Laura Di Blasio (1) - Alberto Puliafito (1) - Roberto Sessa (3) - Claire Bou-
vard (4) - Guido Tarone (5) - Dominique Helley (6) - Lydia Sorokin (7) - Guido
Serini (1) - Federico Bussolino (1) - Luca Primo (1)
Institute for Cancer Research and Treatment at Candiolo, Turin, Italy, Department of Oncology, University of Torino, Turin, Italy, Candiolo, Italy
(1) - Harvard Medical School, Massachusetts General Hospital, Boston,
MA, USA, Harvard Medical School, Massachusetts General Hospital, Boston, United States (2) - Cardiovascular Research Institute, University of
California, San Francisco, CA, USA, University of California, San Francis- co, CA, USA, San Francisco, United States (3) - UMR-S 765, Université
Paris Descartes, Sorbonne Paris Cité, Paris, France, The Scripps Re- search Institute, La Jolla, CA, USA., Paris, France (4) - Department of Mo-
lecular Biotechnology and Health Sciences, University of Torino, Molecu- lar Biotechnology Center, Turin, Italy, University of Torino, Turin, Italy, Turin, Italy (5) - UMR-S 765, Université Paris Descartes, Sorbonne Paris
Cité, Paris, France, Université Paris Descartes, Sorbonne Paris Cité, Par- is, France, Paris, France (6) - Institute of Physiological Chemistry and
Pathobiochemistry, Muenster University, Muenster, Germany, Muenster University, Muenster, Germany, Muenster, Italy (7)
Introduction: The mechanism by which angiogenic endothelial
cells break the physical barrier of vascular basement membrane and consequently sprout, forming new vessels in mature tissues, is unclear. Here we show how the angiogenic endothelium is characterized by the presence of podosome rosettes. Podosomes and invadopodia, collectively called invadosomes, are special- ized cell-matrix contacts with an inherent ability to degrade ECM components, thus they are considered key structures of cells that are able to cross anatomical boundaries.
Materials and methods : We studied the formation of podo-
some rosettes in HUVECs, in mouse aortic ring (mAR) assay and in genetic mouse models.
Results: We found that VEGF-A stimulation induces the for-
mation of endothelial podosome rosettes by up-regulating integ- rin α6β1; in contrast, the binding of α6β1 integrin to vascular basement membrane laminin impairs the formation of podo- some rosettes by restricting α6β1 integrin to focal adhesions and hampering its translocation to podosomes. By analyzing genetic mouse models of cancer and by exploiting a function blocking antibody, we demonstrate how the interaction between α6β1 integrin and laminin controls tumor blood vessel branching by influencing the dynamics of podosome rosettes.
Conclusions: We provide the first evidence that endothelial
podosome rosettes are critical regulators of sprouting angiogen- esis and promote tumor blood vessels branching. The vascular normalizing effects of podosome rosette inhibition thus open new avenues to study their role in tumor angiogenesis and blood vessel branching in general.
D 6
PROSTAGLANDIN E2 TRANS-ACTIVATES THE COLONY-
STIMULATING FACTOR-1 (CSF-1) RECEPTOR AND SYNERGIZES WITH CSF-1 IN THE INDUCTION OF MACROPHAGE CHEMOTAXIS VIA THE MITOGEN-ACTIVATED PROTEIN KINASE ERK1/2
Graziana Digiacomo (1) - Marina Ziche (2) - Persio Dello Sbarba (1) - San-
dra Donnini (2) - Elisabetta Rovida (1)
Università, Università degli studi di Firenze, Firenze, Italy (1) - Università,
Università degli Studi di Siena, Siena, Italy (2)
Introduction: Prostaglandin E2 (PGE2) is a key mediator of
immunity, inflammation and cancer. Besides the existence of four G-protein-coupled E-prostanoid receptors (EP1-4), the complexity of PGE2 signaling is further increased by the existence of a cross-talk between EP receptors and tyrosine kinase receptors (TKR) such as Fibroblast Growth Factor Receptor 1 (FGFR1) or Epidermal Growth Factor Receptor (EGFR). The Colony-Stimulating Factor-1 Receptor (CSF-1R) is a TKR that sustains the survival, proliferation, and motility of monocytes/macrophages, which are essential components of innate immunity and cancer development.
Materials and Methods: The experiments were performed in
two murine macrophage cell lines, BAC-1.2F5 and RAW264.7, that physiologically express high amounts of CSF-1R protein. BAC-1.2F5 cells were incubated in the absence of CSF-1 for 18 h, whereas RAW264.7 cells in the absence of FBS for 24 h, respectively, before being stimulated with the appropriate stimulus. Cells were then lysed and protein lysates subjected to SDS-PAGE and immunoblotting with the indicated antibodies. To determine whether CSF-1R is necessary for PGE2-induced ERK1/2 activation and for the macrophage chemotaxis, we in- hibited CSF-1R genetically (by specific siRNA) or pharmaco- logically (by a CSF-1R specific drug, GW2580). Migration as- say was performed with modified Boyden chambers.
Results: In either BAC-1.2F5 or RAW264.7 1 µM PGE2
induced rapid CSF-1R phosphorylation, wich was dependent on Src family kinases activation. Indeed, SFK pharmacologic inhibition prevented PGE-induced CSF-1R hosphorylation. Genetic inhibition of CSF-1R reduced PGE2-elicited ERK1/2 phosphorylation and macrophage chemotaxis, indicating that CSF-1R plays a role in PGE2-mediated immuno-regulation. Furthermore, at low concentrations PGE2 synergized with CSF- 1 in inducing macrophage chemotaxis, as well as ERK1/2 phosphorylation. Accordingly, ERK1/2 inhibition completely blocked chemotaxis induced by the combination CSF-1/PGE2.
Conclusions: Our results indicate that PGE2 trans-activates
CSF-1R and synergizes with CSF-1R –dependent signaling at the level of ERK1/2 in promoting macrophage chemotaxis. This synergistic interaction is likely to play an important role in the initiation and progression of inflammatory diseases as well as cancer.
50
D 7
MELANOMA-DERIVED EXOSOMES AND MIR-222 PROMETASTATIC FUNCTIONS.
Alessandra De Feo (1) - Carolina Coscia (1) - Maria Bellenghi (1) - Maria
Cristina Errico (1) - Nadia Felli (1) - Gianfranco Mattia (1) - Alessandra Carè* (1) - Federica Felicetti* (1)
Istituto Superiore Sanità, Dipartimento Ematologia, Oncologia e Medici- na Molecolare, Rome, Italy (1)
Introduction: Our previous results indicated that miR-
221&222 and miR-126&126* take part in a complex balance able to induce or inhibit melanoma progression. In particular, miR-221&222 are key factors for melanoma development and dissemination, whereas miR-126&126* play tumor suppression functions. Growing evidence is showing that miRNAs are not strictly cellular, but are secreted through the release of small vesicles, called "exosomes", and therefore present in extracellu- lar compartments, including blood and other body fluids, and in cell culture media. Tumor exosomes contain miRNAs as well as intact and functional mRNAs and proteins that can alter the cel- lular environment to favor tumor growth. Increasing our under- standing of exosomes and extracellular miRNAs should clarify this new communication network.
Materials and methods: Exosomes were isolated by ultracen-
trifugation or Exoquick-TC methods. The expression of miR- NAs, mRNAs and/or proteins in exosomes was evaluated by Real Time PCR or Western Blot analysis. Biological assays were performed according to standard procedures.
Results: Our data show that miR-222 is carried by exosomes
secreted from melanoma and that its expression show a direct correlation with melanoma malignancy. In addition a significant level of miR-222 seems to represent a key factor in increasing exosome release, suggesting that this miRNA may drive traffic of microvesicles within tissues and tumor mass. We performed a series of biological assays looking for the capability of miR- 222-containing exosomes (exo-miR-222) to convey the same effects obtained by directly overexpressing miR-222 in mela- noma cells. Interestingly, in exomiR-222 fused melanoma cell line we observed a significant induction of the invasion and chemotactic capacities compared to exo-control cells. In order to gain insight in the molecular mechanisms underlying this exo-based increased malignancy, we investigated (by TaqMan Array Plate) genes differentially expressed in exo-miR-222 and exo-control. Some tumor promoting genes, like VEGF, FGF2, MGAT5, PNN and FXYD5 were upregulated in exosomes se- creted by miR-222 overexpressing cell line.
Conclusion: These data support the conclusion that at least a
fraction of miR-222 is secreted by exosomes. In turn, miR-222 was identified as a component required for exosomal secretion in melanoma, further confirming the role played by this miRNA in the genetic changes underlying tumor progression.
D 8
NOTCH3 DEREGULATES CXCR4 EXPRESSION IN IMMATURE THYMOCYTES
Francesca Ferrandino (1) - Giovanni Bernardini (2) - Ambra Ciuffetta (2) -
Antonello Campanese (2) - Paola Grazioli (2) - Diana Bellavia (2) - Alberto
Gulino (2) - Isabella Screpanti (2) - Maria Pia Felli (3)
Department of Experimental Medicine, "Sapienza" University of Roma, Roma, Italy (1) - Department of Molecular Medicine, “Sapienza” Universi-
ty, Viale Regina Elena 291, 00161 Roma (2) - Department of Experi-
mental Medicine, “Sapienza” University, Viale Regina Elena 324, 00161 Roma (3)
Constitutive activation of Notch signaling is one of the major cause of acute T cell lymphoblastic-leukemia (T-ALL) in hu- mans and mice. The oncogenic function of Notch3 in T-ALL was demonstrated by a murine model of our laboratory, charac- terized by enforced expression of the Notch3 active form (N3- IC) in immature thymocytes (N3-ICtg). Deregulated prolifera- tion and maturation at the preT/T transition phase and constitu- tive activation of preTCR were observed in N3-ICtg mice . T cell differentiation depends on multiple signals furnished by the stroma, which elaborates cytokines, chemokines and ligands and sustains thymocyte maturative process within the thymic micro-environment. Cooperative signaling among the preTCR, CXCR4 and Notch are required at β selection for the continued differentiation from Double Negative (DN) to Double Positive (DP) T cells. The stromal cell derived factor SDF-1(CXCL12) and its receptor CXCR4 promote survival of DN thymocytes and regulate the migration during the DN/DP transition. The CXCR4/SDF-1 axis has been suggested to play a role in the pathogenesis of T-ALL.
FACS experiments demonstrated decreased percentages of CXCR4 positive cells in DN-gated thymocytes of N3-ICtg mice with respect to wt, not attributable to any reduction of DN cell numbers in N3-ICtg mice. Furthemore, mRNA from selected DN thymocytes evidenced a reduced transcription of CXCR4 gene in N3-ICtg with respect to wt mice. Thus it may suggest that Notch3 disrupts early event in preT-cell progressive matu- ration accompanied by altered migration through the thymus, as