5 -6 Marzo 2015 | Catanzaro, Italy

ABSTRACT BOOK

MEETING VENUE Università “Magna Graecia” di Catanzaro Campus Universitario “S. Venuta” Viale Europa, loc. Germaneto 88100 Catanzaro (Italy) ORGANIZERS Enrico Iaccino, Ph.D. University of Catanzaro Magna Græcia, IT Nicola Amodio, Ph.D. University of Catanzaro Magna Græcia, IT INTERNAL SCIENTIFIC COMMITTEE Prof. Francesco Saverio Costanzo University of Catanzaro Magna Græcia, IT Prof. Giovanni Cuda ‐ Prof. Giovanni Morrone University of Catanzaro Magna Græcia, IT Prof. Pierfrancesco Tassone University of Catanzaro Magna Græcia, IT Prof. Giuseppe Viglietto University of Catanzaro Magna Græcia, IT Prof. Giuseppe Scala University of Catanzaro Magna Græcia, IT Prof. Pierosandro Tagliaferri University of Catanzaro Magna Græcia, IT SCIENTIFIC SECRETARIAT Roberta Sgrò (+39) 0961 369-4157 hemmas@unicz.it

The meeting aims to cover some of the most interesting results that

have emerged in the last years in the fields of cancer biology, tumor

microenvironment and innovative cancer therapeutics.We hope to

provide the participants with information likely to become soon

part of the fundamental scientific knowledge as well as of

therapeutic strategies.

We have been fortunate enough to have some worldwide recognized

scientific experts in their fields and we hope the scientific

community will receive the program with enthusiasm.

No registration fee is required for this meeting.

Looking forward to welcoming you in Catanzaro.

The Scientific Coordinators

Enrico Iaccino, Ph.D.

Nicola Amodio, Ph.D.

Sponsored by

- SESSION I - 5 march

Thursday, March 5, 2015

Session I Chairmen: Dr. F. Trapasso (Catanzaro); Dr. E. Iaccino (Catanzaro)

14.30 – 15.10 Prof. Soldano Ferrone – Department of Surgery, Massachusetts General Hospital,

Boston, USA Combinatorial antibody-based immunotherapy to eradicate differentiated tumor

cells and cancer initiating cells in solid tumors

15.10 – 15.25 Dr. Valeria Quarona – Department of Medical Science, University of Torino, Italy Ectoenzymes and myeloma: the bone marrow niche

15.25 – 15.40 Dr. Lavinia Raimondi – Laboratory of Tissue Engineering - Innovative Technology

Platforms for Tissue Engineering, Rizzoli Orthopedic Institute, Palermo, Italy Role of multiple myeloma cell-derived exosomes in osteoclastic differentiation

15.40 – 16.20 Dr.ssa Lorena Pochini – Department DiBEST (Biology, Ecology and Earth

Sciences), University of Calabria, Arcavacata di Rende, Glutamine transporters and cancer. New hot targets of drug discovery

16.20 – 16.50 Coffee Break

ECTOENZYMES AND MYELOMA: THE BONE MARROW NICHE

Valeria Quarona1, Alberto Horenstein1, Antonella Chillemi1, Valentina Ferri2,3, Andrea Zito1, Fabio Morandi4, Danilo Marimpietri4, Vito Pistoia4, Nicola Giuliani3, Fabio Malavasi1. 1Lab of Immunogenetics, Department of Medical Sciences, University of Torino, Italy; 2Department of Biotechnologies and Life Sciences, University of Insubria, Varese, Italy; 3Hematology and Blood and Marrow Transplantation (BMT) Center, University of Parma, Parma, Italy; 4 Istituto Giannina Gaslini, Laboratory of Oncology., Genova, Italy. Corresponding author: valeria.quarona@unito.it, +39 011 670 5995-5996 Objectives: The working hypothesis of our study is that ectoenzymes play regulatory roles in the complex interactions that take place in the myeloma microenvironment.

CD38 was reported to rule a network of ectoenzymes leading to production of adenosine (ADO), a regulator of local immunological tolerance, through a unique CD38/CD203a/CD73 pathway. The hypothesis is that ADO, a purine nucleoside with anti-inflammatory properties, plays a significant role in tumor immune escape in closed systems, as is the case for the myeloma niche.

Methods: Canonical and new pathways of ADO production were tested in cells from bone marrow aspirates and osteomedullary biopsies from myeloma patients. The pathways were analyzed for expression and function in the major components of the niche. Adenosine was measured by HPLC in the bone marrow plasma of patients and the results obtained show that the nucleoside is present at different levels. The contributions of the different components of the niche were analyzed by co-culture of myeloma cell lines with different combinations of osteoblasts, stromal cell lines and differentiated osteoclasts.

Results: In conclusion, the metabolic pathway of ADO production mediated by CD38/CD203a/CD73 works discontinuously in closed systems and is influenced by the local pH.

Conclusions: The results obtained reinforce the roles of therapeutic CD38 monoclonal antibodies in myeloma (and, possibly, myeloid leukemia): indeed ADO may support the use of anti-CD38 mAbs in myeloma therapy, where they exert cytotoxic functions and simultaneously depress the enzymatic activity of CD38, thereby reducing ADO production. Moreover, levels of ADO in the myeloma niche might be an early prognostic indicator of an aggressive form of disease.

ROLE OF MULTIPLE MYELOMA CELL-DERIVED EXOSOMES IN OSTEOCLAST DIFFERENTIATION

Lavinia Raimondi#, Angela De Luca, Nicola Amodio, Mauro Manno, Simona Taverna, Pierfrancesco Tassone, Alessandra Santoro, Gianluca Giavaresi, Riccardo Alessandro. # Laboratory of Tissue Engineering - Innovative Technology Platforms for Tissue Engineering (PON01-00829), Rizzoli Orthopedic Institute, Palermo, Italy Email: lavinia.raimondi@ior.it OBJECTIVES: Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. The pathogenesis of osteolytic bone disease is primarily correlated with aberrant osteoclasts (OCs) activation and suppressed osteoblasts (OBs) function. Within the microenvironment, MM cells stimulate OCs differentiation directly by secreting local osteoclast activating factors (OAFs) or indirectly by stimulating secretion of OAFs in others cells, such as bone marrow stromal cells and OBs. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes could be involved in OCs differentiation.

METHODS: Isolation of MM cell-derived exosomes and exosomes derived from MM patient‟s sera was

performed by differential centrifugations as described in literature (1). Characterization of exosomes was carried out by western blot analysis and Dynamic Light Scattering technique; uptake of labeled MM-exosomes on Raw264.7 cells was performed as reported in Thery et al. Evaluation of osteoclasts differentiation was assessed by TRAP staining assay, Pit formation assay, Elisa assay, western blotting and RT-PCR analysis.

RESULTS: Raw264.7 cells treated with MM cell-derived exosomes expressed specific OCs markers and differentiated into multinuclear and giant cells able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient‟s sera. Finally, exosomes derived by the metastatic

colorectal cancer cell line SW620 did not elicit the same effects.

CONCLUSIONS: The results reported in this study significantly enhance our understanding about intercellular communication in MM bone disease by demonstrating that MM cells release biologically active exosomes specifically responsible of OCs function and differentiation.

Reference:

1. Thery, C., et al., Curr Protoc Cell Biol, 2006. Chapter 3: p. Unit 3 22.

- SESSION II - 5 march

Thursday, March 5, 2015

Session II Chairmen: Prof. G. Cuda (Catanzaro); Dr. A. Morandi (Firenze)

16.50 – 17.05 Dr. Roberta Rocca – Department of Health Science, Magna Graecia University of

Catanzaro- Italy

A new anticancer target: the highly polymorphic DNA G-quadruplex

17.05 – 17.20 Dr. Duarte Mendes Oliveira – Department of Experimental and Clinical Medicine,

Magna Graecia University of Catanzaro- Italy Molecular Profiling Reveals New Potential Targets in Colorectal Cancer

17.20 – 18.00 Dr.ssa Daniela Trisciuoglio – Experimental Chemotherapy Laboratory, Regina Elena

National Cancer Institute, Rome, Italy) Enhancement of chemotherapy cytotoxicity by histone deacetylase inhibition in

preclinical models of human non-small cell lung cancer

A NEW ANTICANCER TARGET: THE HIGHLY POLYMORPHIC DNA G-QUADRUPLEX Roberta Rocca, Federica Moraca, Anna Artese, Giosuè Costa, Lucia Parrotta, Francesco Ortuso, Stefano Alcaro Dipartimento di Scienze della Salute, Università “Magna Græcia”, viale Europa, 88100, Catanzaro. e-mail: rocca@unicz.it Four-stranded G-quadruplex (G4) DNA has been subject of considerable speculation and investigation during the past decade, particularly with regard to its potential relevance to genome integrity and gene expression.1 Its main structural feature is a guanines core, obtained to superimposition of G-tetrads, planar quartets formed by four guanines that are stabilized by Hoogsteen type hydrogen bonds (Fig. 1).

Fig. 1: structure of a G4 tetrad.

They are formed especially in G-rich sequences, as in telomeres and promoters sequences of oncogenes.2 It is widely reported that ligands, able to stabilize such G4s, suppress both telomeres elongation and oncogenes transcription.3 Unfortunately, rational drug design is hampered by the high conformational polymorphism of the unimolecular telomeric G4 structures.4 Nevertheless, many synthetic and natural compounds have been reported in literature as G4 binders. In particular, our research activity is focusing on natural derivatives, such as the isoquinoline alkaloid Berberine and its derivatives, given their multiple pharmacological properties5 and the harmine scaffold, identified in our work.6 Moreover, a folding investigation about the telomeric G4 was performed to better understand the polymorphism of this target. To this aim, the use of enhanced sampling techniques such as metadynamics 7 is extremely helpful as it was demonstrated in a recent study.8 The information obtained at the end of this study is a well characterized Free Energy Surface of a local conformational search, in which three main basins were detected, corresponding to new G-quadruplex folding states. This research is supported by the Italian Ministry of Education grants (codes FIRB-IDEAS RBID082ATK_002 and 2009MFRKZ8_002). R. R. PhD grant was supported by a POR Calabria FSE 1007–2013 “HEMMAS” fellowship.

References

1. Parrotta L., Ortuso F. et al. Expert Opin Drug Discov., 2014, 11, 1-21. 2. Onyshchenko M.I., Gaynutdinov T.I. et al. Nucleic Acids Res., 2011, 39, 7114-7123. 3. Düchler M. J. Drug Target, 2012, 20(5), 389-400. 4. Petraccone L., Malafronte A. et al. J. Phys. Chem. B, 2012, 116, 2294-305. 5. Naasani, I.; Seimiya, H.; Yamori, T.; Tsuruo, T. Cancer Res. 1999, 59, 4004-4011. 6. Rocca, R.; Moraca, F. et al. Molecules, 2014, 20, 206-223. 7. Laio, A.; Parrinello, M. Proc. Nat. Acad. Sci., 2002, 99, 12562-12566 8. Limongelli, V.; De Tito, S.; Cerofolini, L. et al. Angew. Chem, 2013, 125, 2325 –2329.

MOLECULAR PROFILING REVEALS NEW POTENTIAL TARGETS IN COLORECTAL CANCER Duarte Mendes Oliveira*, Antonia Rizzuto§, Donatella Malanga*Carmelo Laudanna*, Laura Elia§, Rosario Sacco§ and Giuseppe Viglietto* * Dipartimento Medicina Sperimentale e Clinica, Università Magna Graecia di Catanzaro § Unità operativa di Chirurgia generale, Dipartimento Scienze Mediche e Chirurgiche , Università Magna Graecia di Catanzaro.

Introduction: Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. CRC results from the accumulation of multiple genetic and epigenetic aberrations and localization of the tumor in the large intestine tract determines different surgical approaches, treatment options and survival outcome. Although major players in CRC tumor progression are known, next generation sequencing (NGS) provides tools to uncover novel or rare mutations that may unveil new targeted treatment decisions. Methods: A total of 27 CRC tumor samples from different anatomic segments with matched normal mucosa and peripheral blood were collected. The genomic DNA of each sample was extracted and NGS using the comprehensive cancer panel (life technologies) was performed on Proton Sequencer (Ion Torrent). Results: Data obtained was processed using the Ion Torrent platform-specific pipeline software Torrent Suite. We determined the frequency of expected colorectal cancer mutations (APC, ARID1A, SMAD4, KRAS, TP53 and BRAF) to confront our cohort with literature. After annotation and thorough observation of the novel variants identified we created a comprehensive list with all mutations with oncogenic potential. A somatic missense mutation on the exon 7 of the RET proto-oncogene (G533C), was found in one of the tumors, to our knowledge this is the first time this mutation is reported in colon malignancies. Another variant in the Kinase domain of RET (P1047S) was found in a different tumor, since this mutation has not been yet validated as activating, we also decided to evaluate its oncogenic potential. These mutations were validated using Sanger sequencing and ongoing functional studies will determine their role in tumor progression. Conclusions: NGS allows rapid and accurate identification of frequent mutated genes in CRC, which ultimately can determine earlier therapeutic decisions. Presence of activating RET mutations in CRC could in future provide another targeted therapy option.

- SESSION I - 6 march

Friday, March 6, 2015

Session I Chairmen: Dr. M. Rossi (Catanzaro); Dr. D. Malanga (Catanzaro)

09.30 – 10.10 Dr. Ugo Cavallaro – Department of Experimental Oncology, European Institute of

Oncology, Milano, Italy Tumor vasculature: novel mechanisms and potential targets

10.10 – 10.25 Dr. Ernestina Marianna Di Francesco – Department of Pharmacy and Health and

Nutrition Sciences, University of Calabria, Cosenza, Italy Estrogenic GPER signaling triggers HIF-1α/VEGF-mediated effects within the breast

tumor microenvironment

10.25 – 10.40 Dr. Andrea Morandi – Department of Experimental and Clinical Biomedical

Sciences, University of Florence, Florence, Italy.

Metabolic and motile reprogramming of ER positive breast cancer cells

following long-term estrogen deprivation: the role of miR155

10.40 – 11.20 Prof. Angelo Vacca – Department of Biomedical Sciences and Human Oncology,

University of Bari Medical School, Bari, Italy Bone marrow microenvironment and multiple myeloma spread

11.20 – 11.45 Coffe Break

ESTROGENIC GPER SIGNALING TRIGGERS HIF-1Α/VEGF-MEDIATED EFFECTS WITHIN THE BREAST TUMOR MICROENVIRONMENT

Ernestina Marianna De Francesco, Damiano Cosimo Rigiracciolo, Michele Pellegrino, Maria Francesca Santolla, Rosamaria Lappano, Emilia Ricchio, Sergio Abonante and Marcello Maggiolini Department of Pharmacy and Health and Nutrition Sciences, University of Calabria, 87036 Rende Contact: Ernestina Marianna De Francesco - Lab of General Pathology and Molecular Oncology Via Savinio, Edificio Polifunzionale -Phone: +39(0)984-493048 Email address: ernestinamarianna@yahoo.it Objectives: Estrogens stimulate the development of breast cancer mainly binding to and activating the estrogen receptor ER(α) and ERβ, that regulate the expression of genes involved in cell proliferation, migration and survival. The G protein estrogen receptor, namely GPER/GPR30, has been also shown to mediate estrogen action in cancer cells and cancer-associated fibroblasts (CAFs), which are main players within the tumor microenvironment toward cancer progression. We evaluated the role elicited by GPER in the estrogen-regulated expression and function of vascular endothelial growth factor (VEGF) in breast tumor both in vitro and in vivo. Methods: In ER-negative breast cancer cells and CAFs treated with 1 nM E2 and 1 μM GPER agonist G-1 we performed gene expression studies, western blotting analysis, immunofluorescence experiments and tube formation assays that evidenced the ability of estrogenic GPER/HIF-1α signaling to trigger VEGF expression and function. The experimental design included also a mouse xenograft model of breast cancer. Results: E2 and G-1 activated the GPER/EGFR/ERK/c-fos signaling pathway leading to the upregulation of VEGF through HIF-1α. In addition, endothelial tube formation occurred in a GPER-dependent manner culturing HUVECs with conditioned medium from CAFs treated with E2 and G-1. Notably, ligand-activated GPER induced in vivo tumor growth and the expression of HIF-1α, VEGF and the endothelial vessel marker CD34. Conclusions: These findings provide novel insights regarding the ability of the GPER/HIF-1α signaling to trigger VEGF expression and function. Our data contribute to shed light on the critical interaction between breast cancer cells and the surrounding microenvironment, disclosing further molecular targets toward the development of new therapeutic approaches in estrogen-sensitive tumors like breast cancer.

METABOLIC AND MOTILE REPROGRAMMING OF ER POSITIVE BREAST CANCER CELLS FOLLOWING LONG-TERM ESTROGEN DEPRIVATION: THE ROLE OF MIR155.

Andrea Morandi1, Marina Bacci2, Lesley-Ann Martin3, Gianfranco Pintus2, Maria Letizia Taddei1, Elisa Giannoni1 and Paola Chiarugi1 1Department of Experimental and Clinical Biomedical Sciences, viale Morgani 50, I-50134 Florence, University of Florence, Italy. 2Department of Biomedical Sciences, Viale San Pietro 43/B, I-07100 Sassari, University of Sassari, Italy. 3Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK Approximately 2/3 of the breast tumors are positive for estrogen receptor-α (ER) and aromatase inhibitors (AI) have become the first-line endocrine treatment choice for postmenopausal women with ER+ breast cancers. However, AI resistance still limits their benefit for many patients. Since metabolic reprogramming is now considered a hallmark of cancer we investigated whether metabolic reprogramming is linked to AI resistance. AI-sensitive MCF7-2A cells (overexpressing aromatase) treated with an AI showed a positive correlation between glycolysis and cell viability inhibition, suggesting a role for AI in impairing glycolysis. Accordingly, targeting glycolysis in combination with AI showed a synergistic effect in reducing MCF7-2A cell viability. Metabolic comparison of long-term estrogen deprived (LTED)-MCF7 cells (an established in vitro models of AI resistance) versus parental MCF7 showed an increased glycolysis dependency of the AI-resistant cells. Importantly, in silico analysis of AI-treated breast cancer patients revealed an enhanced glycolytic phenotype in patients that did not respond to AI treatment. However, when both cell lines were exposed to either 2-DG or metformin, an inhibitor of mitochondrial respiratory chain complex 1, only the parental MCF7 showed a decrease in cell viability. MCF7-LTED were able to switch to either OXPHOS or glycolysis when 2-DG or metformin were used, thus suggesting a high metabolic plasticity. Additionally, MCF7-LTED cells display higher motile and invasive abilities than parental MCF7. Crucially, single agent treatment showed that 2DG further increases MCF7-LTED motility and invasion. We showed that this metabolic and motile adaptability of MCF7-LTED cells correlates with ER-dependent miR155 enhanced expression. Indeed, targeting miR155 sensitized MCF7-LTED to metformin and impaired 2DG-induced motility. Our data showed that glycolysis is associated with breast cancer aggressiveness and tumor cell growth in the absence of estrogen. Targeting miR155 to impair the metabolic reprogramming could be a potential therapeutic tool if used in combination with current therapeutic regimes.

- SESSION II - 6 march

Friday, March 6, 2015

Session II Chairmen: Prof. G. Morrone (Catanzaro); Dr. N. Amodio (Catanzaro)

12.25 – 12.40 Dr. Eugenio Morelli –Medical Oncology Unit, Department of Experimental and

Clinical Medicine, Magna Graecia University of Catanzaro- Italy Selective targeting of IRF4 by synthetic microRNA-125b-5p mimics induces anti-

multiple myeloma activity in vitro and in vivo

12.40 – 12.55 Dr. Stefania Scicchitano – Department of Experimental and Clinical Medicine, Magna

Graecia University of Catanzaro- Italy Regulatory role of Zinc Finger Protein 521 in Medulloblastoma and co-operation with

the Sonic Hedgehog pignaling pathway

12.55 – 13.35 Dr.ssa Rita Casadonte – Proteopath GbR, Trier, Germany Tumor Classification Based on Proteomic Profiling by MALDI Imaging

13.35 – 13.50 Dr. Giulia Marvaso – Department of Experimental and Clinical Medicine, Magna

Graecia University of Catanzaro- Italy Radiation induced by-stander effect: possible RNA implication in DNA damage

transmission?

13.50 – 14.05 Dr. Andrea Resovi – Istituto di Ricerche Farmacologiche Mario Negri, Bergamo and

Milan, Italy Circulating stroma-related molecules as potential biomarkers for pancreatic ductal

adenocarcinoma.

14.05 – 14.15 Closing Remarks

14.15 – 15.00 Lunch

11.45 – 12.25 Dr.ssa Mariateresa Fulciniti – Jerome Lipper Multiple Myeloma Center, Dana-Farber

Cancer Institute, Boston, USA Multilevel control of myeloma growth and survival by transcriptional regulators

SELECTIVE TARGETING OF IRF4 BY SYNTHETIC MICRORNA-125B-5P MIMICS INDUCES ANTI-MULTIPLE MYELOMA ACTIVITY IN VITRO AND IN VIVO

Eugenio Morelli1, Emanuela Leone1, Maria Eugenia Gallo Cantafio1, Maria Teresa Di Martino1, Nicola Amodio1, Lavinia Biamonte1, Annamaria Gullà1, Umberto Foresta1, Maria Rita Pitari1, Cirino Botta1, Marco Rossi1, Pierosandro Tagliaferri1 and Pierfrancesco Tassone1, 2 1Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, T. Campanella Cancer Center, Salvatore Venuta University Campus, Catanzaro, Italy; 2Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, US. Corresponding author: Eugenio Morelli, MD, Magna Graecia University, Viale Europa, 88100 Catanzaro, Italy E-mail: gege.morelli@hotmail.it, Phone: +39-0961-3694160 Objective: We investigated the anti-multiple myeloma (MM) activity of the IRF4-targeting microRNA (miR)-125b-5p. Methods: Search for IRF4-targeting miRNAs was performed by querying microRNA Data Integration Portal. Inverse correlation between IRF4 and miR-125-5p was disclosed by an integrated analysis of 2 different MM datasets. miR-125b-5p expression was evaluated by qRT-PCR. Modulation of miR-125b-5p targets was evaluated by qRT-PCR and western blotting. Overexpression of miR-125b-5p was obtained by lentiviral vectors or synthetic mimics. Cell viabilty was evaluated by CCK-8 assay and 7-AAD flow cytometry assay. Apoptosis was investigated by Annexin V/7-AAD flow cytometry assay, TUNEL assay and western blotting of caspases expression and cleavage. Macroautophagy was explored by western blotting of LC3B, Beclin-1 and SQSTM1/p62 and by Cyto-ID autophagy detection flow cytometry assay. NOD-SCID mice bearing subcutaneous MM xenografts were used for in vivo studies. Results: We report that expression of IRF4 mRNA inversely correlates with miR-125b-5p in MM patients. Moreover, we provide evidence that miR-125b is downregulated in TC2/3 molecular MM subgroups and in established cell lines. Importantly, constitutive or transient enforced expression of miR-125b-5p impaired growth and survival of MM cells and overcame the protective role of bone marrow stromal cells. Apoptotic and autophagy-associated cell death were triggered in MM cells upon miR-125b-5p ectopic expression. Of note, we found that the anti-MM activity of miR-125b-5p was mediated via direct downregulation of IRF4 and its downstream effector BLIMP-1. Moreover, inhibition of IRF4 translated into downregulation of c-Myc, caspase-10 and cFlip, relevant IRF4-downstream effectors. Finally, in vivo intra-tumor or systemic delivery of formulated miR-125b-5p mimics against human MM xenografts in SCID/NOD mice induced significant anti-tumor activity and prolonged survival. Conclusions: Our findings provide evidence that miR-125b has tumor suppressor activity in MM. Furthermore, our data provide proof-of-concept that synthetic miR-125b-5p mimics are promising anti-MM agents to be validated in early clinical trials.

This work has been supported by funds of Italian Association for Cancer Research (AIRC), PI: PT. “Special Program Molecular Clinical Oncology - 5 per mille" n. 9980, 2010/15.

REGULATORY ROLE OF ZINC FINGER PROTEIN 521 IN MEDULLOBLASTOMA AND CO-OPERATION WITH THE SONIC HEDGEHOG SIGNALLING PATHWAY.

Scicchitano S*, Giordano M*#, Spoleti CB*, Lucchino V*, Di Vito A*, Nappo G*, Spina R°, Chiarella E*, Codispoti B*, Tiano F*, Marafioti MG*, Aloisio A*, De Smaele E^, Goidts V#, Mesuraca M*, Bond HM*, Morrone G*

* Laboratory of Molecular Haematopoiesis and Stem Cell Biology, University Magna Græcia, Catanzaro,Italy. # Division of Molecular Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany ^ Laboratory of Molecular Oncology, Sapienza University, Roma, Italy. ° Department of Neurological Surgery, Case Western Reserve University, Cleveland, Ohio.

The stem cell-associated transcription co-factor, zinc finger protein 521 (ZNF521) has been implicated in the control of the homeostasis of the stem cell compartment in the hematopoietic, osteo-adipogenic and neural system. ZNF521 expression is abundant in brain, particularly in the external granule layer of the developing cerebellum that contains candidate cells-of-origin of medulloblastoma, and in the majority of human medulloblastomas, with particular regard to the subset of this tumours showing constitutive activation of the sonic hedgehog (SHH) pathway. This prompted us to investigate the role of ZNF521 in human medulloblastoma cell lines and primary Ptc1+/- mouse medulloblastomas in a variety of in vitro and in vivo assays, by either inducing enforced expression with lentiviral vectors carrying the ZNF521 cDNA, or by silencing its expression using specific shRNAs.

Enforced overexpression of ZNF521 in DAOY medulloblastoma cells strongly enhanced their growth both in adherence and as spheroids, their clonogenicity in single-cell cultures and semisolid media, and their migratory ability in wound-healing assays. Importantly, ZNF521-transduced DAOY cells displayed a greatly augmented tumorigenic potential in nude mice xenotransplants. Conversely, silencing of Zfp521 in mouse Ptc1+/- medulloblastoma cells drastically reduced their growth and tumorigenic potential. We then asked whether these effects could be mediated by a functional interaction of ZNF521 with the SHH signalling pathway. Co-precipitation and chromatin immunoprecipitation assays confirmed that ZNF521 physically interacts with the prominent SHH mediators, Gli1 and Gli2, and co-localises with these factors on the promoters of several SHH target genes. In addition, overexpression of ZNF521 enhanced the transcriptional activity of Gli-responsive elements, and was associated to a significant increase in the expression of endogenous SHH target genes.

Taken together, our data indicate that zinc finger protein 521 contributes to the control of self-renewal, migration and tumorigenicity of the immature cell compartment of medulloblastoma and that these effects may be at least in part mediated by its physical and functional interaction with components of the Sonic Hedgehog signal transduction pathway.

Supported by AIRC, the HEMMAS project and the PhD Programme in Molecular Oncology.

RADIATION INDUCED BY-STANDER EFFECT: POSSIBLE RNA IMPLICATION IN DNA DAMAGE TRANSMISSION?

Giulia Marvaso1,2, Nicola Amodio1, Agnese Barone1, Cataldo Bianco1, Kathryn D Held2 1Department of Experimental and Clinical Medicine, Magna Graecia University, Catanzaro, Italy; 2Department of Radiation Oncology, Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA Objectives: Radiation-induced bystander effect (RIBE) refers to the manifestation of biological changes in cells that have not been exposed to ionizing radiation but came into direct/indirect contact with irradiated cells. In this case, RIBE is mediated by a mechanism, namely soluble factors communication. The factors that mediate such cellular communication can be released into the extracellular media following irradiation and then transferred between cells, but their nature has not been fully characterized. In this study, we tested the hypothesis that the bystander effect mediator contains RNA. The manifestation of RIBE has been commonly studied in terms of induction of micronuclei (MNI) formation, double-strand breaks reflected by –53BP1 foci formation. Recently, also microRNAs (miRNAs) has been shown to be a potential mediator of the bystander effect. Our goal was to examine miRNA expression in irradiated, bystander cells and media. In detail, let-7a and let-b expression levels where analyzed, since they are presumed to target the RAS pathway, which is known to be related to Reactive Oxygen effect and is affected by radiation.

Methods: a transwell insert culture dish was used to co-culture un-irradiated and irradiated cells. 1522 human skin fibroblasts were exposed to doses ranging 0.5-1.0 Gy of X-Ray, treated with RNAse A. Extracellular media was collected 1h after irradiation. The pattern of miRNA changes was evaluated by quantitative RT-PCR.

Results: RNase A treatment abrogated the ability of the media to induce MNI and –p53BP1 in bystander cells. A decrease in let-7a and let-b expression in directly irradiated cells as well as in the media was observed, and it occurred together with an increase of IL-6 in the media.

Conclusions: These results suggest that the bystander effect, and genomic instability, are at least in part mediated by RNA and implicate a role for it. Given the emerging role of BE in genomic instability and its possible implications in carcinogenesis, efforts to understand and modulate the bystander responses are necessary to provide new approaches to cancer therapy and prevention.

CIRCULATING STROMA-RELATED MOLECULES AS POTENTIAL BIOMARKERS FOR PANCREATIC DUCTAL ADENOCARCINOMA.

Andrea Resovi1, Dorina Belotti1, Rossana Bettolini1, Paola Allavena2, Paola Cappello3, Francesco Novelli3, Aldo Scarpa4, Giulia Taraboletti1, Raffaella Giavazzi1. 1Laboratory of Biology and Treatment of Metastasis, Department of Oncology, IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Bergamo and Milan, Italy. 2 Laboratory of immunology and Inflammation, Humanitas Clinical and Research Center, Rozzano, Italy. 3 Department of Molecular Biotechnology and Healthy Sciences, CeRMS, University of Turin, Italy. 4 Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Italy. Pancreatic ductal adenocarcinoma (PDAC) is usually diagnosed late, when treatment options are limited. Early diagnostic serological markers capable to detect the disease in its early stages might change the fate of PDAC patients. Given the relevance of the tumor stroma and extracellular matrix (ECM) in PDAC progression, this study investigates the potential value of circulating ECM-related molecules as PDAC biomarkers. Thirty-nine ECM-related candidate biomarkers were selected from published proteomic studies on PDAC. Levels of these molecules were tested by ELISA and Multiplex in a first set of plasma samples from PDAC patients (n=25), healthy subjects (n=16) and chronic pancreatitis patients (n=17). From this exploratory study, 13 biomarkers belonging to extracellular matrix, growth factors, adhesion molecules, proteases and protease inhibitors, were found to be differentially expressed in PDAC plasma compared to healthy subjects and/or chronic pancreatitis. A second analysis on an independent set of PDAC patients (n=17) and healthy controls (n=20) validated the previous findings and allowed restricting the number of selected markers to 7 molecules. Four of the selected markers appeared over-expressed in a genetically engineered mouse model of PDAC [PdxCre/LSL-KrasG12D] in association with PanIN development, hence pointing to the potential values of these markers for the early detection of PDAC. In conclusion this analysis has identified a panel of ECM-related potential biomarkers of early stage PDAC. An analysis on a larger cohort of PDAC patients is expected to identify the set of optimal biomarkers for the development of a multi-analyte diagnostic tool for the early detection of PDAC. Supported by AIRC 5 per mille, grant n.12182 A.R. is supported by “Fondazione Eugenio Morandi”

- POSTERS -

FHC SILENCING LEAD TO EPITHELIAL - MESENCHYMAL TRANSITION IN MCF7 CELL LINE Ilenia Aversa (ilenia.aversa@unicz.it), Maddalena Di Sanzo, Concetta Maria Faniello, Stefania Bulotta, Francesco Saverio Costanzo Laboratory of Molecular Oncology, Department of Clinical and Experimental Medicine, “Magna Grӕcia” University of Catanzaro Objectives: The epithelial-mesenchymal transition (EMT) is a phenotypic change of epithelial cells that is essential for embryogenesis and is also associated to migratory potential of some kind of solid tumors. This process is characterized by loss of function of E-cadherin and is associated with the altered expression of several transcriptional factors and proteins. The expression of these genes causes changes in the epithelial cellular morphology and it is associated with altered migration and invasion. Ferritin is composed by heavy (FHC) and light (FLC) subunits. In particular, FHC possesses ferroxidase activity and is involved in rapid iron uptake and release. Ferritin controls iron metabolism and plays its potential regulatory role in critical biological processes. The aim of this work was to determine the relationship between FHC subunit and EMT event in Human Breast Cancer Cell Line (MCF7). Methods: To evaluate the effect of H ferritin gene silencing on Twist, Vimentin, E-cadherin and Slug genes expression in MCF7 cells we performed real time-PCR analysis. We found that E-cadherin is down-regulated after FHC-silencing while all the other genes were found to be up-regulated. To confirm the direct correlation among intracellular FHC amount and EMT genes expression levels we restored the expression of FHC in silenced MCF7 cells and we re-evaluated Twist, Vimentin, Slug and E-cadherin expression by Real Time PCR. Results: All the genes were modulated by FHC restoring. Moreover, it has been demonstrated that microRNAs (miRNA) play a critical role in EMT in breast cancer. We explored the role of miR-125b in regulation of EMT in MCF7 cells and we found that miR-125b was significantly down-regulated after FHC-silencing and its expression were modulated by FHC restoring. Conclusions: The results so far obtained suggested that the lack of FHC, in MCF7 cells, might alter gene expression and cellular morphology, leading to a more invasive phenotype. We propose to analyze the migratory potential and the drug resistance related to FHC-silencing.

MULTIPLE MYELOMA CELLS DOWNREGULATE MIR-29B IN DENDRITIC CELLS TO PROMOTE A TUMOR PERMISSIVE INFLAMMATORY MICROENVIRONMENT AND IMMUNE-ESCAPE

Cirino Botta1, Maria Cucè1, Maria Rita Pitari1, Daniele Caracciolo1, Lucia Fiorillo1, Annamaria Gullà1, Eugenio Morelli1, Teresa Del Giudice1, Maria Eugenia Gallo Cantafio1, Nicola Amodio1, Maria Teresa Di Martino1, Pierpaolo Correale2, Marco Rossi1, Pierosandro Tagliaferri1 and Pierfrancesco Tassone1,3

1Medical Oncology, Department of Experimental and Clinical Medicine,”Magna Graecia” University, Catanzaro, Italy; 2Unit of Radiotherapy, Siena University School of Medicine, Siena, Italy; 3Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USA First author: Cirino Botta Medical Oncology Unit - Department of Experimental and Clinical Medicine “Magna Graecia” University of Catanzaro e-mail: cirino.botta@gmail.com - Phone number: +39 3280369043 Objectives: Multiple myeloma (MM) is an incurable plasma-cells malignancy in which the bone-marrow microenvironment (BM) plays a pivotal role in tumor growth, survival, drug resistance and immune-escape. Due to the central role of dendritic cells (DCs) in orchestrating immune response against cancer, we hypothesized that MM-cells can recruit DCs and reprogram them at the microRNA (miRNA) level to sustain a tumor-permissive microenvironment.

Methods: miRNA profiling of immature DCs (iDCs), mature DCs (mDCs) and tumor-associated DCs (taDCs), were retrieved from GEO datasets and analyzed to find differentially expressed miRNAs. A subsequent in silico target prediction analysis was performed to identify pathways potentially perturbed by miR-29b. All results were then confirmed through western blot, qRT-PCR, flow cytometry and luciferase assays.

Results: In our datasets analysis we found miR-29b to be upregulated in mDCs and downregulated in taDCs compared to iDCs. We confirmed these results in DCs cocultured with different MM-cells. We then transiently transfected iDCs with miR-29b and co-cultured them with MM-cells. We observed a significant reduction in CD86 and B7H3 expression accompanied by a downregulation of VEGFA and IL-23 (both IL12B and IL23A) production at mRNA and cytokine level, thus reducing the capability of DCs to induce a pro-inflammatory response.

On these bases we performed an in silico gene ontology analysis to identify immune/inflammatory pathways predicted to be affected by miR-29b. We found that miR-29b transfection in DCs impaired pro-inflammatory signaling at different levels by reducing STAT3, JUN and NFKB signaling and by inducing SOCS1. Additionally, we observed that miR-29b transfected DCs, reduced the proliferation of co-cultured MM-cells compared to control, thus evidencing an important role for this miRNA in microenvironment modulation.

Conclusion: Our results showed that replacement of miR-29b in DCs reduces their inflammatory potential and their capability to sustain MM growth thus rendering miR-29b an attractive candidate for miRNA-based immune therapy.

NOREPINEPHRINE ESTABLISHES IMMUNOSUPPRESSIVE NETWORK IN HUMAN MELANOMA MICROENVIRONMENT.

Maura Calvani, Arianna Casini, Matteo Becatti, Elisa Giannon , Chiara Azzari, Luca Filippi and Paola Chiarugi

Introduction Stress related factors, such as catecholamines, can impact on immunological response and cancer progression through β-adrenoreceptors (βARs) activation. The exact role of the immune system on stress-induced cancer progression has not been well elucidated. In many cancers, malignant progression is accompanied by profound immune suppression that arises from the ability of tumors to subvert normal immune regulation to their advantage. The tumor microenvironment can prevent cytotoxic T cells expansion by promoting production of proinflammatory cytokines and growth factors, leading to the accumulation of suppressive cell populations (e.g. regulatory T cells -Treg) that inhibit instead of promoting immunity.

Material and methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors (Meyer Hospital) by centrifugation using a Ficoll Histopaque solution; these cells were exposed to hypoxia and/or Norepinephrine (NE) and then labeled with fluorescent antibodies. Analysis of natural killer (NK) citotoxicity was assessed by FACS analysis through the identification of CD107a in CD16/CD56 positive cells. Treg cells were identified by positivity of CD4 and CD25.

Results and discussion Our results show that that stress related factors like NE create an immunosuppressive environment by coordinating a network of events both in cancer and immune cells, such as NK and Treg. Particularly, NE reduces citotoxicity of NK cells both by direct stimulation of PBMCs and by cocolturing NE -prestimulated A375-Melanoma cells with lymphocytes, but that this effect is reverted by administration of specific β-blockers. Moreover, NE and hypoxic conditions increase expansion of Treg cells both in tumor microenvironment and in peripheral blood cells. Furthermore, NE increases immunosuppressive environment by reducing the action of interleukin 15 (IL-15), an established inductor of NK activity. However, lymphocytes combined stimulation with IL-15 and β3-blockers restores and increases NK activity, as demonstrated by dramatic reduction of A375-Melanoma cells viability.

Conclusion. Our work provides a new concept that indicates stress induction by NEas strong down-regulator of the immune response against tumor.

SYNTHETIC MIR-22 IMPAIRS NHEJ REPAIR SENSITIZING MULTIPLE MYELOMA CELLS TO BORTEZOMIB-INDUCED BRCANESS

Daniele Caracciolo, MD1, Teresa Calimeri, MD, PhD, Nicola Amodio, PhD1, Ciro Botta, MD1, Eugenio Morelli, MD1, Emanuela Altomare, PhD1, Cinzia Federico, PhD1 , Enrica Romeo, Lavinia Biamonte, PhD student1, Maria Angelica Stamato, PhD student1, Maria Rita Pitari, PhD1, Maria Eugenia Gallo Cantafio, PhD student1, Antonino Neri2, Maria Teresa Di Martino, PhD1, Pierosandro Tagliaferri1, MD1 and Pierfrancesco Tassone1, MD1,3 1Department of Experimental and Clinical Medicine,Magna Graecia University, S. Venuta University Campus, Catanzaro, Italy 2 Department Medical Sciences, Hematology 1 CTMO, University of Milan, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy 3 Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USA First author: Daniele Caracciolo Medical Oncology Unit - Department of Experimental and Clinical Medicine “Magna Graecia” University of Catanzaro e-mail: mercury86p@gmail.com Phone number: +39 3206898427

Objectives: Multiple Myeloma (MM) is an incurable hematologic malignancy characterized by deep genomic instability. c-MYC promotes error-prone Alternative pathway of Non-Homologous End Joining (A-NHEJ), by repression of microRNAs that regulates LIG3 and PARP1. Based on this rationale, we aimed at elucidate the role of miRNAs in the regulation of MM genomic instability.

Methods: Cell proliferation and apoptosis were evaluated respectively with CCK-8 assay and Annexin V-7ADD staining. y-H2AX, LIG3,PARP1 and c-MYC levels were analyzed by western blot of whole and nuclear protein extracts. NHEJ repair was evaluated with a dual gene plasmid assay utilizing Luciferase (LUC) as a test gene to measures end joining, and Alkaline Phosphatase (SEAP) as a reporter gene. Transient transfection of MM cells was carried out by electroporation. miRNA expression profiling was carried out using Total RNA and Affymetrix GeneChip miRNA 1.0 and qRT-PCR.

Results: Among MYC-repressed miRs that have predicted binding sites in the 3‟-UTR of LIG3 and PARP1, we investigated miR-22 given the significative anti-correlation with c-MYC in our MM and PCL microarray dataset. First, we confirmed this inverse correlation evaluating miR-22 and c-MYC expression in basal conditions and after treatment with two validated c-MYC inhibitor (10058-F4 and JQ1) in a panel of 10 MM cell lines. Transfection of synthetic miR-22 mimics in MM cells (R8226, H929, AMO-1, MM-1S) induced growth inhibition and triggered apoptosis which were accompanied by an increase of the DNA damage marker yH2AX, a reduction of LIG3 and PARP-1 mRNA and protein levels and a strong inhibition of NHEJ repair. Finally, we found that miR-22 mimics potentiate effects of Bortezomib-induced Homologous-Recombination (HR) impairment, thus sensitizing MM cells to DNA damaging agents such as melphalan.

Conclusions: Taken together, our findings indicate that miR-22 regulates DNA repair and chemosensitivity likely by repressing the highly error-prone A-NHEJ in MM cells.

ABUNDANT EXPRESSION OF ZINC FINGER PROTEIN 521 (ZNF521) CONTRIBUTES TO THE LEUKAEMIC PHENOTYPE IN AMLS BEARING MLL REARRANGEMENTS.

Chiarella E*, Mesuraca M*, Aloisio A*, Codispoti B*, Tiano F*, Lupia M@, Scicchitano S*, Lucchino V*, Giordano M*, Nappo G*, Spoleti CB*, Marafioti MG*, Horton SJ§, Schuringa JJ^, Bond HM*, Bullinger L°, Morrone G*

* Laboratory of Molecular Haematopoiesis and Stem Cell Biology , University Magna Græcia, Catanzaro, Italy. @ Molecular Medicine Program, European Institute of Oncology, Milano, Italy. ^ Dept. of Hematology, Groningen University Medical Center, Groningen, Netherlands. § Dept. of Haematology, Cambridge Institute for Medical Research, Cambridge, United Kingdom. ° Department of Internal Medicine III, University of Ulm, Ulm, Germany.

ZNF521 is a transcription co-factor containing 30 zinc finger motifs, first identified for its abundance in primitive haematopoietic progenitors. This protein displays features of a transcriptional co-repressor, and has been implicated in the regulation of the homeostasis of haematopoietic, osteo-adipogenic and neural stem cells. These effects are, at least in part, mediated by the inhibition of transcription factors whose activity is critical for cell fate choice of multipotent progenitors, among which EBF1, GATA1 and Runx2. ZNF521 silencing in CD34+ cells depletes the immature compartment and enhances B-lymphopoiesis.

Inappropriate expression of regulatory proteins, associated to genetic aberrations, is often critical for the development and/or maintenance of the leukaemic phenotype. To establish whether ZNF521 does play a role in this process we have investigated its expression in gene profiling datasets of acute leukaemias. Our results show that the ZNF521 transcript is relatively abundant in most AMLs and T-ALLs, but not B-ALLs (where, instead, the expression of the cognate protein ZNF423 is consistently high). In AMLs, ZNF521 expression is higher in stem cells-enriched subsets compared to the stem cells-depleted population. Discrete ZNF521 mRNA levels correlate with specific genetic aberrations: low expression is detected in AMLs with FLT3 internal tandem duplications and in acute promyelocytic leukaemias, whereas consistently high expression is associated to MLL rearrangements in both paediatric and adult AMLs. ZNF521 appears to be a direct target of MLL fusion oncoproteins, since its expression is consistently upregulated in CD34+ cells transduced with MLL-AF9 or MLL-ENL, and down-regulated by MLL-AF9 knock-down in the THP-1 MLL-AF9+ cell line. Silencing of ZNF521 in THP-1 cells, that produce high amounts of the protein, inhibits their growth and clonogenicity; conversely, its enforced expression in ZNF521-negative MV4;11 cells strongly enhances their clonogenicity. Finally, ZNF521 silencing in MLL-AF9-transformed primary CD34+ cells inhibits their proliferation and leads to their extinction whereas its enforced co-expression significantly enhances growth and clonogenicity of the transformed cells. Thus, ZNF521 is implicated in the control of the homeostasis of the immature compartment in AMLs, and its expression appears critical to sustain MLL-mediated leukaemogenesis.

Supported by AIRC, the HEMMAS project and the PhD Programme in Molecular Oncology.

ZOLEDRONIC ACID IMPAIRS STROMAL REACTIVITY BY INHIBITING M2-MACROPHAGES POLARIZATION AND PROSTATE CANCER-ASSOCIATED FIBROBLASTS.

Comito G, Pons Segura C , Sobierajska K, Ippolito L, Taddei ML, Giannoni E and Chiarugi P.

Department of Experimental and Clinical Biomedical Sciences, University of Florence

INTRODUCTION: Cancer associated fibroblasts (CAFs), the major cellular components of tumor microenvironment, promote epithelial mesenchymal transition (EMT) and acquistion of stemness traits in prostate cancer cells. Of note, prostate CAFs are active players in promoting monocyte recruitment to tumor site. CAFs secretion of stromal derived growth factor-1 (SDF-1) delivery promote macrophage trans-differentiation to the M2- macrophages phenotype. Moreover, M2 macrophages are able to induce mesenchymal- mesenchymal transition of fibroblasts, leading to their enhanced reactivity and highlighting the mutual relationship between these cell compartments. This complex interplay among cancer cells, CAFs and M2-macrophages, lead to i) an increase in tumor cell motility, that promote cancer cells escape from primary tumor and metastatic spread, and ii) an activation of both endothelial cells and their bone-marrow-derived precursors to drive de novo angiogenesis. Zoledronic acid (ZA), an amino-bisphosphonate compound in use for the treatment of symptomatic skeletal events, has recently been shown to have immunomodulatory properties that need to be exploited in cancer immunotherapy. There is in vivo evidence showing that ZA can reduce the tumorigenic phenotype of M2- polarized macrophages. METHODS: Human Monocytes were obtained from normal donor buffy coat, fibroblasts were isolated from aggressive carcinoma (CAFs) or from benign prostate hyperplasia (HPFs). RESULTS: Here we show the key effects of ZA on M1/M2 macrophages and CAFs in prostate carcinoma progression. First, we analyzed the phenotype of the differentiated macrophages treated with ZA evaluating their M1 and M2 phenotype, by looking at the expression of IL-12 or IL-10, respectively. Our data revealed that ZA treatment impairs M2- macrophages polarization, while it is ineffective on M1-macrophages polarization. As a consequence, ZA-treated M2-polarized macrophages lose their ability to foster the motility of prostate cancer cells. We also investigated the effect of ZA on fibroblasts activation and found that this molecule is able to reduce stromal fibroblasts reactivity, as well as their ability to elicit EMT in prostate carcinoma cells. Finally, we demonstrated that CAFs treated with ZA lack their ability to protect prostate cancer cells to docetaxel toxicity. CONCLUSION: Our data suggest that the benefit of ZA in the therapy of prostate carcinoma patients, potentially goes beyond the simple skeletal/bone symptoms treatment, but is enlarged to regulation of stromal inflammatory events.

MULTIPLE MYELOMA CELL-DERIVED EXOSOMES SUPPORT MIGRATION AND VIABILITY OF PRE-OSTEOCLAST CELLS

Angela De Luca#, Lavinia Raimondi, Daniele Bellavia, Riccardo Alessandro and Gianluca Giavaresi. #Angela De Luca: Laboratory of Tissue Engineering - Innovative Technology Platforms for Tissue Engineering (PON01-00829), Rizzoli Orthopedic Institute, Palermo, Italy.

Email: angela.deluca@ior.it

OBJECTIVES: In multiple myeloma (MM) bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in pathogenesis of skeletal disorders. Bone disease is sustained by a supportive microenvironment based on complex cross-talk involving stromal, immune, endothelial and bone cells, as well as extracellular matrix components. Exosomes are nanovesicles (40-150 nm) secreted by most cells and recognized as a mode of cell-cell communication during tumor growth and progression. Several studies have showed that exosomes released by cancer cells may affect survival, apoptosis, migration, angiogenesis and resistance to chemotherapy as well as prepare the metastatic niche. On these premises, we investigated if MM cell-derived exosomes may affect migration of pre-osteoclast cells as well as viability and apoptotic mechanism. METHODS: The ability of cells to migrate through 8-mm pore size of filter was determined using Transwell migration plates, according to the manufacturer's instructions. Migration-related signaling molecule expression was evaluated by flow-cytometry and RT-PCR. Viability assay was evaluated by MTT-assay at 72 hrs and 6 days. Apoptotic cells were analyzed by citofluorimetric assay using Annexin V-FITC conjugated. Activity of Caspase3/7 was assessed by Caspase-Glo® 3/7 Assay System. The anti-apoptotic and survival factors expression was investigated by western blotting and qRT-PCR analysis. RESULTS: MM-exosomes supported migration of pre-OCs cells and induced migration-related signaling molecule expression. MM cell-derived exosomes increased pre-osteoclast cells viability within 72hrs; a significative reduction in cell viability was evident in cultures treated with MM-exosomes after 6 days of exposure when induction of mature osteoclasts differentiation occurred. Exosomes treatment triggered anti-apoptotic mechanisms increasing Raw264.7 cell survival. CONCLUSIONS: Taken together, these findings provide evidence that MM cell-derived exosomes influence pre-OCs migration as well as cell viability by inhibiting apoptotic mechanisms in favour of cell survival.

P27KIP1 ROLE IN CANCER THERAPY: NEW INSIGHTS FROM A KNOCK-IN MOUSE MODEL

Carmela De Marco

Department of Experimental and Clinical Medicine University “Magna Graecia” of Catanzaro Tel: 0961 369 4165 Mail address: cdemarco@unicz.it

Objective. Since the cyclin-dependent kinase inhibitor, p27kip1, is frequently inactivated in cancer and its expression levels have both prognostic and therapeutic implications, we generated a mouse model to investigate the effects, in cancer onset and progression, of restoring p27kip1 expression,

Methods. Throughout the cell cycle, the expression levels of p27kip1 are controlled by transcriptional, translational and post-translational mechanisms. In particular the post-translational modification of C-terminal Threonine 198 (Thr197 in mouse) is involved in the regulation of protein stability. We generated a knock-in mouse model in which p27kip1 gene was replaced, by homologus recombination, with a mutant p27T197A, in which Thr197 is mutated to Ala: T197A substitution makes p27 protein less stable. In order to restore p27kip1 protein expression, we treated mice and embryonic fibroblasts (MEFs) with a proteasome inhibitor, Bortezomib (Velcade), already used in clinical practice.

Results. The homozygous p27T197A/T197A mice were considerably heavier than controls but they were not obese. Essentially, p27 knock-in mice showed the enlargment of almost all organs, due to an increase in cell number, rather than an increase in cell size or of the amount of extracellular material. Indeed, we observed hyperplasia in multiple organs: i.e. pituitary and mammary glands, thymus, uterus, intestine. In agreement with these results, MEFs from p27T197A/T197A mice had a less stable p27kip1 protein and an increased proliferation rate. We found that the treatment with Bortezomib, in vitro and in vivo, restores p27 protein expression causing a reduction of cell proliferation rate and a rescue of hyperplasia.

Conclusions. All togheter, these data make the p27T197A/T197A mouse model a promising preclinical system to study the mechanism and efficay of new anticancer drugs that inhibit p27kip1 degradation.

THE HAT INHIBITOR CPTH6 PREFERENTIALLY INHIBITS IN VITRO AND IN VIVO PROLIFERATION ON

PATIENT-DERIVED LUNG CANCER STEM-LIKE CELLS Di Martile Marta et al. Laboratory of Experimental Chemotherapy,Regina Elena National Cancer Institute, Rome, Italy. dimartile@ifo.it. INTRODUCTION: Epigenetic enzymes targeting is emerging as promising approach for cancer therapy. In this context, we recently identified the thiazole derivative 3-methylcyclopentylidene-[4-(4‟-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6) as a novel pCAF and GCN5 histone acetyltransferases inhibitor (HATi). CPTH6 exerts in vitro anti-tumoral effect in a panel of human tumor cell lines derived from different histotypes. Here, we evaluated the efficacy of CPTH6 using in vitro and in vivo models of Non Small Cell Lung Cancer (NSCLC), including patient-derived Lung Cancer Stem-like Cells (LCSC). METHODS: The biological effect of CPTH6 was analyzed in a panel of NSCLC cell lines and in patient-derived Lung Cancer Stem Cell (LCSC) models. Cell viability was tested by MTT and CellTiterGlo. Apoptosis induction and cell cycle perturbation were assessed by flow cytometry. DNA damage induction was revealed by Western Blot and Immunofluorescence analyses. Nude and NOD/SCID mice were used for in vivo experiments.

RESULTS AND CONCLUSIONS: The HATi CPTH6 reduced cell viability of both NSCLC and LCSC lines in vitro. Interestingly, it was more efficient in LCSC (IC50 values ranging from 10 to 2μ5M) than NSCLC (IC50 values ranging from 60 to 300μM) lines. Furthermore, the growth inhibitory effect seemed to be related to baseline expression of acetylated alpha-tubulin, which was particularly prominent in sensitive LCSC cells. Unlike LCSC models, in which CPTH6 treatment induced cell cycle perturbation and apoptosis even at low doses, in NSCLC lines CPTH6 triggered cell cycle perturbation associated to DNA damage. In vivo experiments confirmed the antitumor efficacy of CPTH6 particularly in LCSC-derived models. In fact, CPTH6 induced a pronounced growth inhibitory effect and apoptosis on LCSC136 derived tumors, while it did not significant affect H1299 tumor growth. Overall, CPTH6 could be a valuable agent for the treatment of NSCLC and it should be further studied as a possible antineoplastic agent.

PROTEOMIC ANALYSIS OF KIDNEY IN DICER CONDITIONAL KNOCKOUT MOUSE

Claudia Vincenza Fiumara

Laboratory of Proteomics, Dpt. of Experimental and Clinical Medicine Magna Græcia University of Catanzaro phone: +39 0961 3694107 fax: +39 0961 3694090 e-mail: claudia.fiumara@live.it

In this study we have analyzed the protein profile of a murine model of polycystic kidney: the dicer conditional knockout mouse. Dicer, an RNase III type endonuclease, is the key enzyme involved in miRNA and RNAi pathways. The lack of functional Dicer alleles causes the block of the maturation of pre-miRNAs to its mature form and recent studies demonstrate that Dicer is essential for several aspects of postnatal kidney function and homeostasis.

Proteomic analysis was performed using kidney tissue extracts of Dicer KO mice one and two months old, which show, by immunohistochemistry, differential renal cysts development and morphological alterations. Kidney tissue extracts were analyzed by 2-D Gel Electrophoresis coupled with mass spectrometry. Using nanoscale LC-MS/MS analysis, several differentially expressed proteins were identified.

Fifty-four protein spots resulted to be differentially expressed in Dicer KO tissue compared with control tissue; among these same were up-regulate such as Tropomyosin-1 and Ezrin/Moesin/Radixin, while same were down-regulate such as Prohibitin, Selenium binding protein 1, Hsp60, HspA8. The most interesting among those were validated by western blot.

Ingenuity Pathway analysis was performed to examine functional correlations within differentially expressed proteins. Proteins differentially expressed were overlaid onto global molecular networks developed from information contained in the knowledge base. Networks were “named” on the most common functional group(s) present. Ingenuity pathway analysis lead to the identification of five interesting network; of these the most significant were Cellular Assembly and Organization, Immunological Disease, Hematological Disease; Post-Translational Modification, Protein Folding, Cellular Assembly and Organization. Moreover an in-silico network, was created to study and elucidate the interactions between dicer, miRNA and deregulated proteins.

Overall, our data confirm that the proteomic approach is a powerful tool to shed light in the complexity of biological systems, such us the kidney.

LOCKED NUCLEIC ACID (LNA)-MIR-221 INHIBITOR AS PROMISING NEW ANTI-MYELOMA AGENT

Maria Eugenia Gallo Cantafio1, Annamaria Gullà1, Nicola Amodio1, Emanuela Leone1, Mariamena Arbitrio1, Pierosandro Tagliaferri1,2, Pierfrancesco Tassone1,2,3 and Maria Teresa Di Martino1,2 1Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, 2T. Campanella Cancer Center, Salvatore Venuta University Campus, Catanzaro, Italy; 3Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USA Corresponding author: Maria Eugenia Gallo Cantafio, eugy_86@libero.it; Maria Teresa Di Martino, teresadm@unicz.it- Università Magna Graecia, Catanzaro, viale Europa, 88100

Objective: miR-221/222 cluster is upregulated in several type of cancer and promote cell proliferation. Previously we proved that silencing of both miRNAs induces significant anti-MM activity and triggers canonical targets in vitro and in vivo. Then, in the aim to progress to clinical translation, we selected an originally inhibitor, specifically designed for systemic delivery, named LNA-i-miR-221, and we evaluated its specificity on endogenous miR-221 and its properties of tissue distribution and pharmacokinetics.

Methods: We evaluated miR-221/222 silencing by qRT-PCR and targets modulation by q-RT-PCR and western blotting. In vivo, male SCID/NOD mice were inoculated(sc) with MM cells. Animals was treated i.p. with 25mg/kg of LNA inhibitors. Detection of LNA-i-miR-221 in paraffin murine organs, for pharmacokinetics report, was carried using in situ hybridization (ISH). Pilot toxicity study was performed in Cynomolgus Monkey.

Results: LNA-i-miR-221 inhibited growth of MM cells that overexpress endogenous miR-221; miR-221 downregulation and increase of p27Kip1 mRNA and protein, a miR-221 canonical target, were detected. In vivo, LNA-i-miR-221 systemic delivery exerted significant anti-tumor activity in MM xenografted SCID/NOD mice (about35%). After single i.p. dose of 25mg/kg, we detected the presence of LNA-i-miR-221 from 2 up to 7 days in tumors and mouse tissues by ISH. Interestingly, any toxicity was detected with long lasting presence of the inhibitors in vital organs. We also evaluate the LNA-i-miR-221 half-life in mouse plasma using HPLC-MS/MS that confirmed the rapid LNA-i-miR-221 uptake by cell tissue. To evaluate the maximum tolerated doses, in vivo dose escalation treatments was performed (10-100mg/kg). All animal treatments were well tolerated.

Conclusions: These preliminary observations need further investigations to better explore dynamic and kinetics of our LNA inhibitor. The antitumor activity observed in MM and the safety profile in mouse and in monkey support the rationale for development of this novel LNA-miR-221 inhibitor as a promising anti-MM agent.

Supported by the Italian Association for Cancer Research (AIRC), PI: PT. “Special Program Molecular Clinical Oncology - 5 per mille" n. 9980, 2010/15.

NATURAL KILLER CELLS TARGETING OF HUMAN AND MOUSE CANCER INITIATING CELLS

R. Tallerico*, L. Conti, S. Lanzardo, C. Garofalo, R. Sottile, M. Todaro, G. Stassi, G. Parmiani, F. Dieli, F. Cavallo and E. Carbone

*University Magna Graecia of Catanzaro, Laboratory of Tumor Immunology and immunopathology (L.I.T.I.P.), Department of Experimental Medicine and Clinical Germaneto, Catanzaro.

1) Tallerico R et al. J Immunol 2013 Mar 1;190(5):2381-90.

Objectives: Tumor cell populations is composed by two compartments: cancer-initiating cells (CICs) and differentiated cancer cells. The resistance of CICs to drugs and irradiation often allows them to survive traditional therapy. Natural Killer (NK) cells are potent cytotoxic lymphocytes that can recognize tumor cells in human and mice. In a human setting we already demonstrate in vitro that freshly purified allogeneic NK cells can recognize and kill Colorectal Carcinoma CICs while the tumors either autologous or allogeneic is less susceptible to NK cells [1]. To validate in vivo the in vitro data we used a mouse model that recapitulate the breast cancer natural history. Mammospheres expressing markers associated with CSC phenotype have been generated from the tumor lesion.

Methods: Epithelial murine ErbB-2+ tumor cell line, named TUBO. Mammospheres (P1, P2, P3 generations), derived from TUBO cells, express markers associated with CIC phenotype, form tumors in syngeneic mice. Cytotoxicity assay were performed using the fluorescent CFDA. Samples were analysed by FACS analysis. Tumourigenesis capacity of CICs: BALB/c mice were used. Freshly dissociated CIC cells were subcutaneously injected. Statistical analysis. Significance levels were determined by a pairwise Student‟s T-Test two tails Analysis: P value ≤ 0,05 was considered significant. ANOVA Analysis was performed with GraphPad Prisme 5.0 Sofware. In vivo experiments were performed in three groups (PBS, tilorone, Tmb-1) of BALB/c mice to test the role of NK cells in the recognition of cancer stem cells.

Results: We demonstrate that NK cells purified from C57/BL6 and Balb/c mice, selectively recognize the CICs (named mammospheres P1, P2, P3) derived from an epithelial murine ErbB-2+ positive tumor cell line, named TUBO. The in vivo data showed that NK cells selectively recognize the CICs (named P3) and control lung metastatization.

Conclusions: This study strengthens the idea that biology based therapy harnessing NK cells could be an attractive opportunity in solid tumors.

SILENCING OF HEAVY CHAIN FERRITIN (FHC) IN A CANCER CELL LINE OF OVARIAN ORIGIN (SKOV3) INDUCES AN ENRICHMENT OF CELLS WITH STEM-LIKE PROPERTIES

Authors: Nadia Lobello (nadialobello@hotmail.com), Alessia Noto, Giuseppe Roscilli, Rita Mancini, Gennaro Ciliberto, Francesco Saverio Costanzo

Laboratory of Molecular Oncology, Department of Clinical and Experimental Medicine, “Magna Graecia” University of Catanzaro

Objectives: Cancer is sustained by a population of cells with stem cell-like properties whose distinctive feature is their extensive capacity to self-renew, and to produce progenitor cells and terminally differentiated progeny [1]. The putative CSCs are themselves mostly quiescent for proliferation, are resistant to chemotherapeutic agents and therefore are thought to be responsible for disease relapse and for the emergence of resistance to therapies [2-4]. Many studies have indicated that abrogation of iron metabolism, elevation of ROS, or modification of redox regulatory mechanisms in cancer cells, could be considered as therapeutic approaches for cancer [5]. To evaluate the role of iron in CSCs, we silenced SKOV3 cells for ferritin heavy chain gene (SKOV3 shFHC) and we compared the effect on spheres obtained from SKOV3 WT, SKOV3 shRNA and SKOV3 shFHC.

Methods: We stably silenced SKOV3 cells for ferritin heavy chain gene (SKOV3 shFHC) and plated silenced and un-silenced cells in non-adherent culture in order to form spheres. Then analysed FHC silencing by comparing the mRNA and the protein expression levels of SKOV3 WT, SKOV3 shRNA and SKOV3 shFHC spheres through Real-time PCR and Western Blotting analyses, respectively. We studied SKOV3 migration and glucose consumption by wound healing and glucose assay and analysed different levels of stemness and EMT markers between WT, shRNA and shFHC SKOV3 spheres.

Results: Spheres obtained from SKOV3 shFHC were larger than those obtained by SKOV3 shRNA and WT analysed in the same generation. Moreover we noted that silenced cells not only formed the new generations of spheres faster than un-silenced cells but also continued to form spheres until ten generations while WT and shRNA cells arrested to the third and fourth generation, respectively. We also observed that shFHC spheres showed higher level of stemness and EMT markers compared to WT and shRNA SKOV3 spheres. Finally we evaluated the cytokines levels in cellular supernatant and observed higher IL-2, IL-6 and EGF levels in shFHC spheres compared to WT and shRNA SKOV3 spheres.

Conclusions: These preliminary data suggest that silencing of heavy chain ferritin in a cancer cell line of ovarian origin induces an enrichment of cells with stem-like properties and a more aggressive behaviour.

References

1. A Noto, S Raffa, C De Vitis, G Roscilli, D Malpicci, P Coluccia, A Di Napoli, A Ricci, M R Giovagnoli, L Aurisicchio, M R Torrisi, G Ciliberto and R Mancini.

Stearoyl-CoA desaturase-1 is a key factor for lung cancer-initiating cell. Cell Death Dis. 2013

2. Akunuru S, James Zhai Q, Zheng Y. Non-small cell lung cancer stem/progenitor cells are enriched in multiple distinct phenotypic subpopulations and exhibit plasticity. Cell Death Dis 2012; 3: e352.

3. Bartucci M, Svensson S, Romania P, Dattilo R, Patrizii M, Signore M et al. Therapeutic targeting of Chk1 in NSCLC stem cells during chemotherapy. Cell Death Differ 2012; 19: 768–778.

4. Abdullah LN, Chow EK. Mechanisms of chemoresistance in cancer stem cells. Clin Transl Med 2013; 2: 1326–2-3

5. Bystrom LM1, Guzman ML, Rivella S. Iron and reactive oxygen species: friends or foes of cancer cells? Antioxid Redox Signal. 2014 Apr 20;20(12):1917-24. doi: 10.1089/ars.2012.5014. Epub 2013 Jan 15.

PEPTIDE-GUIDED TARGETING OF GPR55 FOR NEW THERAPEUTIC STRATEGIES OF CANCER

Maria Mangini*, Enrico Iaccino§, Selena Mimmi§, Ileana Quinto§, Giuseppe Scala§ and Stefania Mariggiò*

* Institute of Protein Biochemistry, National Research Council, Naples, Italy § Department of Clinical and Experimental Medicine, University “Magna Graecia” of Catanzaro, Italy

Objectives

G-protein-coupled receptors (GPCRs) are „druggable' targets in cancer treatment. GPR55 was recently identified as the lysophosphatidylinositol receptor, and its expression correlates with the invasive potential of metastatic cells and bone metastasis formation1. This study aims to interfere the GPR55 function in cancer cells with peptidic binders.

Methods

Whole-cell-based screening of M13-phage-displayed random library2 was performed using as bait the GPR55 receptor heterologously expressed in HEK293 cells. The 7-mer insert peptides in M13-phage library were flanked by a pair of cysteine residues resulting in cyclized peptides. Binding specificity to GPR55-positive cells was verified using synthetic FITC-labelled peptides by FACS and confocal microscopy. The effect of peptide binding to GPR55 was analysed in lysphosphatidyinositol-triggered GPR55 signalling, including intracellular calcium, AKT phosphorylation, modulation of actin cytoskeleton, and receptor internalization.

Anticipated data

By screening M13-phage-displayed random library, we selected a peptide that binds specifically to HeLa cells expressing GPR55 and not to cells interfered for GPR55. Binding specificity of the FITC-labelled peptide was further investigated in competition assays with unlabelled peptide, and confirmed using scramble analogues. The identified peptide did not affect significantly GPR55 signalling; however, it showed a significant modulation of both basal and agonist-induced GPR55 internalization. This analysis has been extended to lymphoblastoid leukemic cells.

We propose the use of GPR55-peptidic binders for in-vivo diagnosis of GPR55-positive tumor cells, specific delivery of chemotherapeutic agents to cancer cells, and direct modulation of receptor activities for therapeutic applications.

1- Ross RA (2011) Trends Pharmacol Sci 32:265-9.

2- Palmieri C et al. (2010) Blood 116:226-38.

CANCER IN ITS MICROENVIRONMENT: NEW INSIGHTS FROM GENETIC ALTERATION STUDY IN COLON CANCER

Cinzia Marinaro*, Duarte Mendes Oliveira*, Antonia Rizzuto§, Chiara Mignogna*, Lia Congiusta§, Emilia Dora Giovannone*, Rosario Sacco§, Giuseppe Viglietto* .

* Laboratorio di Oncologia Molecolare, Dipartimento di Medicina Sperimentale e Clinica, Università Magna Graecia Di Catanzaro § Unità Operativa di Chirurgia Generale, Dipartimento di Scienze Mediche e Chirurgiche, Università Magna Graecia Di Catanzaro

Colorectal carcinoma (CRC) is frequently found in industrialized countries and lead to a high incidence of cancer-related mortality. Defined by histomorphological features, CRC and its pre-invasive lesions are quite heterogeneous. In particular intra-tumor heterogeneity is strictly linked to tumor progression and aggressiveness, acquiring a critical importance for response to therapeutic intervention. Unfortunately molecular characterization aimed to obtain an accurate assessment of the mutational status on tumor tissue biopsy is often compromised due to specimen size and presence of normal cells.

Methods: In order to investigate the molecular heterogeneity in colon cancer we isolate tumor cells from archival FFPE samples using DEPArrayTM microchip sorting technology. To evaluate the prevalence of various mutations, tumor cells were separated from stromal cells by using two different antigen labels, cytokeratin for epithelial tumor cells and vimentin for mesenchymal stromal cells. DNA extraction, amplicon library preparation and sequencing of "hot spot" regions frequently mutated in human cancer genes were then applied (ION Torrent platform).

Results: A highly pure collection of tumor cells from FFPE tissue samples allowed accurate and sensitive molecular characterization. Compared to bulk mixed tissue, tumor cells exhibited a higher frequency of specific mutations involved in CRC carcinogenesis and small subpopulations sharing rare private mutations appeared. Furthermore stromal cells presented a small percentage of somatic mutations not identified in the tumor fraction, highlighting the possibility of the existence of an altered genetic state of the microenvironment surrounding the tumor.

Conclusions: DEPArrayTM sorting of cytokeratin-positive tumor cell fractions and vimentin-positive stromal cells gave us the possibility to identify somatic mutation that are present exclusively in tumor or stromal cells. Knowledge of specific alterations for each subpopulations is crucial to further elucidate the intra-tumor heterogeneity and specific contribution of microenvironment to target therapy response.

ID-PEPTIDES AS NEW TOOLS FOR DIAGNOSIS AND TREATMENT OF B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA (B-CLL)

Selena Mimmi, Eleonora Vecchio, Iaccino Enrico, Marco Rossi, Cristina Falcone, Marilena Pontoriero, Annamaria deLaurentiis, Antonio Pisano, Simona Ceglia, Giuseppe Fiume, Camillo Palmieri, Ileana Quinto and Giuseppe Scala

Department of Clinical and Experimental Medicine, University “Magna Graecia” of Catanzaro, Viale Europa-Germaneto, 88100 Catanzaro, Italy.

Introduction: Several features of the B-cell receptor (BCR) complex have emerged as major markers for prognostic classification of B-cell chronic lymphocytic leukemia (B-CLL). In particular, characterizing BCR signaling profiles may help to identify signaling markers useful for patient stratification, disease monitoring and therapeutic targeting in B-CLL. In this regard, we used random peptides libraries (RPLs) to identify peptide ligands of the Ig-BCR of neoplastic B-cells, and to determine the epitope diversity recognized by the Ig-BCR.

Material and method: The full-length Ig gene transcripts (VDJ) from a B-CLL human sample were amplified by two nested PCRs using a combination of forward primers specific for the respective VH, Vκ or Vλ leader regions and a reverse primer specific for the respective constant region. The PCR products were sequenced and analysed by IgBlast to identify the IgV mutation status. The IgVH and IgVL PCR products were cloned into eukaryotic expression vectors and coexpressed in 293T cells in order to produce the corresponding IgBCR. The quality of the recombinant IgBCR was assessed by SDS-PAGE under non-reducing and reducing conditions. A flow cytometry analysis showed that the Id-peptides specifically recognized the respective target cells.

Results and discussion: We developed a methodology that combines the IgBCR variable genes repertoire analysis and the IgBCR production. We identified small bioactive peptides, "Id-peptides", that bind to the idiotypic determinants of Ig-BCR of the to B-CLL. This approach will allow us to:

1) identify of a pool of Id-peptides for B-CLL shared by a substantial number of B-CLL patients;

2) understand the general differences in antigenic reactivity between mutated and unmutated IgBCR,

3) understand the correlation between IgBCR signal transduction in leukemic cells and the clinical course of the disease.

Conclusion: The described strategy is able to provide us of (i) patient-specific and tumor-specific Ig-BCR that will be used for screening RPLs (ii) monitoring the CLL clinical progression by recognizing the circulating B-CLL in lymph nodes and bone marrow and (ii) prognostic information based on the IgVH genetic status of each patient.

References

-SmithGP and Petrenko VA (Chem Rev 1997) Phage Display

-Palmieri et al. (Blood 2010) - In vivo targeting and growth inhibition of the A20 murine B-cell lymphoma by an idiotype-specific peptide binder

-Croce CM et al (Semin Cancer Biol 2010) Molecular basis of CLL

BISPHOSPHOGLYCERATEMUTASE: A KEY METABOLIC SWITCH FOR CELL PROLIFERATION INDUCTION

Camilla Mugnaioni

Dep. Scienze Biomediche, Sperimentali e Cliniche Università degli Studi di Firenze, Firenze, Italy. Tel.: +39 0552751248; cell.: +39 3491059743; E-mail address: camillamugnaioni@alice.it

INTRODUCTION. Many kind of cancer cells exploit glycolysis rather than oxidative phosphorylation for energy production even in the presence of oxygen. This kind of metabolism, although less efficient in terms of ATP production, generates high levels of glycolytic intermediates necessary to support the high biosynthetic flux of rapidly proliferating cells. In this work we investigate the role of Bisphosphogliceratemutase (BPGM) an enzyme involved in the metabolism of highly proliferating cells which promote a low efficiency glycolysis (in terms of ATP production). BPGM acts both as a mutase, converting the glycolytic intermediate 1,3- bisphosphoglycerate (1,3-BPG) to 2,3-bisphosphoglycerate (2,3-BPG) and as a phosphatase, converting the 1,3-bisphosphoglycerate to 3-phosphoglycerate.

METHODS. Different kind of cell lines are used: Human dermic fibroblast (HDF); prostatic cancer cell line (DU145 and PC3); melanoma cancer cell line (A375).

BPGM is silenced by siRNA interfering techniques and proliferation is evaluated with cell counting.

Metabolism of each cell line is evaluated using [U-14C] labeled radioactive glucose and analyzing at the scintillator. The 2,3-BPG levels is analyzed by GC-MS analysis.

RESULTS. Cells knocked down for BPGM proliferate less than control and shifting to a more respiratory metabolism, decreasing glucose up-take and increasing CO2 production. The PP pathway results increased in cells with BPGM expression silenced, assuming a block of flux glycolytic for the abrogation of BPGM shunt which generates high level of glycolytic intermediates undergoing to the PP pathway. In cells not expressing BPGM, 2,3-BPG is decreased, demonstrating that the physiologic function of BPGM is directly related to its mutase activity.

CONCLUSIONS. Here we show that BPGM is expressed in many primary and trasformed cell lines and, shunting glycolytic pathway, is involved in the metabolic reprogramming of proliferating cells.

TITLE: OXIDATIVE STRESS INDUCES MIR-21 OVER-EXPRESSION IN K562 CELLS SILENCED FOR FERRITIN

HEAVY CHAIN (FHC). Authors: Mariafranca Panebianco (mpanebianco@unicz.it), Flavia Biamonte, Fabiana Zolea, Francesco Saverio Costanzo. Laboratory of Molecular Oncology, Department of Clinical and Experimental Medicine, “Magna Græcia”

University of Catanzaro. Objectives: MiR-21 is involved in the control of cell proliferation, apoptosis, invasion and metastasis. Moreover, its expression is elicited in response to oxidative stress. The iron-binding protein ferritin, and particularly its heavy subunit (FHC), plays a key role in iron and redox homeostasis. In this study we analyzed the expression of miR-21 in K562 cells silenced and unsilenced for FHC. Methods: We used Real-time PCR to measure miR-21 levels in shRNA and shFHC K562 cells. The analysis of ROS amount in both cell types was performed using the DCF assay. The effects of miR-21 modulation on several target proteins were analyzed using Western Blot; the analysis of the cellular damage was performed using either DNA laddering assay or Comet assay. Results: FHC silencing induced an increase of about 2-fold in ROS levels and this was accompanied by a significant up-regulation (10-fold) in miR-21 expression. Treatment of shFHC K562 cells with the ROS inhibitor NAC (N-acetyl-L-cysteine) brought miR-21 levels back to that of shRNA cells. To investigate the effect of miR-21 over-expression, we determined the protein expression of three validated miR-21 targets (p53, MLH1 and MSH2) and we found that the up-regulation of miR-21 was strongly associated with a decreased expression of the predicted targeted gene. The analysis of the cellular damage of silenced and unsilenced cells, via DNA laddering and Comet Assay, showed a more sfavorable baseline condition of the silenced cells demonstrated by a higher DNA fragmentation and an increased Comet Tail Length and Tail DNA%. This phenomenon seemed to reverse when treating both cell types with H2O2. ShFHC K562 cells, in fact, appeared to be more resistant after a further stress stimuli and this was associated with a further increment of miR-21. Conclusions: These findings suggest that induction of miR-21, mediated by oxidative stress, might promote tumorigenicity in shFHC K562 cells.

CRL3IBTK REGULATES THE TUMOR SUPPRESSOR PDCD4 THROUGH UBIQUITYLATION COUPLED TO

PROTEASOMAL DEGRADATION Antonio Pisano, Simona Ceglia, Camillo Palmieri1, Eleonora Vecchio2, Giuseppe Fiume, Annamaria deLaurentiis, Selena Mimmi, Cristina Falcone, Enrico Iaccino2, Annarita Scialdone, Marilena Pontoriero, Francesca Fasanella Masci, Rosanna Valea, Shibu Krishnan, Marco Gaspari, Giovanni Cuda, GiuseppeScala1 and Ileana Quinto1 From the Department of Experimental and Clinical Medicine, University “Magna Graecia” of Catanzaro, 88100 Catanzaro, Italy

Background: The human Inhibitor of Bruton's Tyrosine Kinase gene (IBTK) encodes a 150 kDa protein, named IBtkD, which has not yet functionally characterized1. IBtkD is a BTB/POZ domain-containing protein, and represents the human orthologue of S. pombe btb1, which is a substrate receptor for Cullin 3 RING-finger ubiquitin Ligases (CRL3)2. We investigated the possibility that this protein could be a component of a new human CRL3 complex, promoting self degradation and that of the tumor suppressor Programmed cell death protein 4 (Pdcd4). Results: By in vivo coimmunoprecipitation, we demonstrated that either exogenously expressed IBtkD or

endogenous IBtkD associated with Cul3 and Rbx1, generating in vivo a CRL3-like complex. We analyzed the

stability of a few partners of IBtkD that are involved in protein translation. We observed a consistent effect of

IBtkD expression on the protein levels of the Pdcd4, a negative regulator of protein synthesis and a well-

known tumor suppressor3. We analized interaction domain of IBtkD and Pdcd4, localized respectively in the RBD domain of Pdcd4 and C-terminal amino acid portion of IBtkD. By an in vivo assay of Pdcd4

ubiquitination, we observed that the overexpression of IBtkD significantly enhanced Pdcd4 ubiquitination,

promoting the relative proteasome-mediated degradation. Depletion of IBtkD through RNAi system induced a

consistent Pdcd4 accumulation. By regulating Pdcd4 stability IBtkD modulated the suppressive effect of Pdcd4 on translation of reporter luciferase mRNA constructs, with stem-loop structured and unstructured 5‟UTR. Conclusion: IBtkD is a substrate receptor of a Cul3 dependent ubiquitin ligase promoting ubiquitination and proteasomal degradation of Pdcd4. Thus, CRL3IBTK could modulate different signaling pathway involved in cell survival, such as K-Ras dependent pathway and mTOR pathway, representing a potential target for cancer therapy4,5.

BIBLIOGRAFIA

1. Spatuzza, C., Schiavone, M., Di Salle, E., Janda, E., Sardiello, M., Fiume, G., Fierro, O., Simonetta, M.,Argiriou, N., Faraonio, R., Capparelli, R., Quinto, I., and Scala, G. (2008) Physical and functional characterization of the genetic locus of IBtk, an inhibitor of Bruton's tyrosine kinase: evidence for threeprotein isoforms of IBtk. Nucleic Acids Res 36, 4402-4416

2. Petroski, M. D., and Deshaies, R. J. (2005) Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol 6, 9-20

3. Biyanee, A., Ohnheiser, J., Singh, P., and Klempnauer, K. H. (2014) A novel mechanism for the control oftranslation of specific mRNAs by tumor suppressor protein Pdcd4: inhibition of translation elongation. Oncogene

4. Dorrello, N. V., Peschiaroli, A., Guardavaccaro, D., Colburn, N. H., Sherman, N. E., and Pagano, M. (2006) S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth. Science 314, 467-471

5. Luo, J., Emanuele, M. J., Li, D., Creighton, C. J., Schlabach, M. R., Westbrook, T. F., Wong, K. K., andElledge, S. J. (2009) A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell 137, 835-848

TARGETING MIR-21 IN BONE MARROW STROMAL CELLS RESTORES RANKL/OPG RATIO WITHIN

MULTIPLE MYELOMA MICROENVIROMENT Maria Rita Pitari1, Marco Rossi1, Nicola Amodio1, Cirino Botta1, Eugenio Morelli1, Cinzia Federico1, Annamaria Gullà1, Daniele Caracciolo1, Maria Teresa Di Martino1, Pierosandro Tagliaferri1, and Pierfrancesco Tassone1,2 1Department of Experimental and Clinical Medicine and T. Campanella Cancer Center, Magna Graecia University, S. Venuta University Campus, Catanzaro, Italy; 2Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, US. Corresponding author: Maria Rita Pitari, PhD, Magna Graecia University, Viale Europa, 88100 Catanzaro, Italy; E-mail: mariaritapitari@yahoo., Phone: +39-0961-3694160 Objective: the present study was designed to investigate whether modulation of miR-21 expression, an oncogenic microRNA (miRNA) with an emerging role as therapeutic target in human malignancies, including multiple myeloma (MM), might be involved in MM-related BD by interfering on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) production within bone marrow stromal cells (BMSCs) adherent to MM cells. Methods: to evaluate miR-21 expression level, primary BMSCs or HS-5, a human BMSC cell line, in a co-culture system, were cultured together with either RPMI 8226 or U266 MM cell lines or primary CD138+ PCs isolated from MM patients. To evaluate whether miR-21 inhibition restored RANKL/OPG ratio, primary BMSCs or HS-5 were transduced with lentiviral vectors carrying miR-21 inhibitory sequences or GFP reporter and cultured together with MM cells, as described above. Mir-21, OPG and RANKL mRNA expression levels were evaluated by q-RT-PCR; RANKL and OPG production were evaluated by western blotting and ELISA assays. Results: miR-21 expression is dramatically enhanced, while OPG is strongly reduced in BMSCs adherent to MM cells. On this basis, we validated the 3‟UTR of OPG mRNA as miR-21 target. Antagonism of miR-21 in BMSCs adherent to MM cells restored OPG expression and secretion. Interestingly, miR-21 inhibition also reduced RANKL production and secretion by BMSCs. High levels of protein inhibitor of activated STAT3 (PIAS3), which is a validated target of miR-21, reduced STAT3-mediated RANKL gene activation. Finally, we demonstrate that restored RANKL/OPG ratio in cell co-culture medium of BMSCs stably expressing miR-21 inhibitors dramatically impairs the resorbing activity of mature osteoclasts.

Conclusions: our data provide proof-of-concept that miR-21 expression within MM-microenviroment plays a crucial role in bone resorption/apposition balance and that its targeting overcomes OPG down-regulation and RANKL up-regulation MM-cell induced, supporting the design of innovative miR-21 inhibition-based strategies for MM-related BD.

CANCER-ASSOCIATED FIBROBLASTS TRANSFER PROTEINS AND LIPIDS TO CANCER CELLS THROUGH CARGO VESICLES SUPPORTING TUMOR GROWTH

Alice Santi1,*, Anna Caselli1, Paolo Paoli1, Camilla Mugnaioni1, Elena Michelucci2, Paolo Cirri1.

1 Dipartimento di Scienze Biomediche Sperimentali e Cliniche, Università degli Studi di Firenze, Italy 2 Centro di Spettrometria di Massa, Università degli studi di Firenze, Italy.

INTRODUCTION. In pluricellular organism, the intercellular communication can occur through the secretion of membrane-delimited extracellular vesicles (EVs) containing both genetic material and proteins. EVs are secreted by numerous cell types and, once uploaded by recipient cells, can induce the modification of their phenotypic behavior. Studies on EVs have a great impact on cancer biology field and, in particular, on tumor microenvironment studies. Tumor microenvironment is composed both by cancer cells and by non-transformed stromal cells. The main stromal cellular component of tumor microenviornment is an “activated” form of fibroblasts known as Cancer- Associated Fibroblasts (CAFs). CAFs are able to influence many aspects of tumor biology by promoting cancer cells proliferation and motility. Our aim is to study the crosstalk between tumor cells and CAFs by analyzing the transfer of material (i.e. proteins and lipids) between these cell types mediated by the secretion of EVs.

METHODS. The transfer of material between CAFs and tumor cells was evaluated by labeling proteins and lipids with fluorescent probes. Donor cells were labeled and then plated in coculture with recipient cells. Proteins and lipids transfer was quantified by cytofluorimetric analysis. The transferred proteins were identified by mass spectrometry analysis.

RESULTS. Our data show that CAFs transfer proteins and lipids to tumor cells, while the passage of material from tumor cells to CAFs is notably less. We have identified the proteins transferred from CAFs to tumor cells, they include structural proteins, glycolytic enzymes and a group of heterogeneous proteins. Protein transfer is essentially mediated by fibroblasts released EVs and sustains cancer cells growth.

CONCLUSIONS. The material received by tumor cells could reduce the time to reach the lower limit of cell mass necessary for duplication, but we can also hypothesize that some of the subset of proteins could have a particular role in promoting tumor cells proliferation.

* Corresponding author: Dipartimento di Scienze Biomediche Sperimentali e Cliniche, Sezione di Scienze Biochimiche, Università degli Studi di Firenze, viale Morgagni 50, 50134, Firenze (Italy). Tel.: +39 0552751248; fax: +39 0552751201. E-mail address: alice_santi@hotmail.it

ROLE OF IBTK IN B LYMPHOMAGENESIS

Eleonora Vecchio, Camillo Palmieri, Cristina Falcone, Simona Ceglia, Iaccino Enrico, Selena Mimmi, Marilena Pontoriero, Antonio Pisano, Annamaria de Laurentiis, Giuseppe Fiume, Ileana Quinto and Giuseppe Scala.

Department of Clinical and Experimental Medicine, University “Magna Graecia” of Catanzaro, Viale Europa- loc. Germaneto, 88100 Catanzaro, Italy.

Introduction: Aberrant expression of genes involved in B-cell development has been implicated in pathogenesis of B-lymphoproliferative diseases. Aim of this study is to investigate the role of IBtk in B cell tumorigenesis. Taking advantage of the availability of the Ibtk -/- mice, we have analyzed the the impact of

-Myc transgenic mice.

Methods: -Generation of mice: Em-myc mice (C57BL/6 strain) and Ibtk+/- mice were crossed. The F1 offspring were crossed to Ibtk+/- mice to generate Ibtk+/+, Ibtk+/-, Ibtk-/-Em-myc transgenic littermates. Myc tumor initiation was scored by peripheral lymph node palpation. Mice with obvious tumors were sacrificed. -Real Time (RT) PCR: Total RNA was extracted from single-cells suspensions obtained from part of tumors of wild-type Ibtk+/+ EP--Myc and Ibtk+/- EP--Myc lymphoma. Reverse-transcribed cDNAs were subjected to an angiogenesis RT2 Profiler RT-PCR array. Flow cytometry: Single-cell suspensions of spleen, bone marrow, lymph node were stained with a combination of fluorescence-conjugated antibodies to evaluate the expression of cell surface antigens as markers of differentiation stages of B cells.

Results: Due to the limited number of Ibtk-/- EP--Myc mice, the following data are referred to Ibtk+/+ EP--Myc and Ibtk+/- EP--Myc mice. Macroscopic observations of internal organs from tumor–bearing Ibtk+/- EP--Myc mice showed more vascularised lymph nodes than wild-type Ibtk+/+ EP--Myc. Based on this observation, we sought to evaluate the effect of heterozygosity of IBtk on the expression of angiogenic factors in tumor cells by RT-PCR. Our results showed that Ibtk single allele knock-down led to an increased expression of a panel of proangiogenic and lymphangiogenic factors (e.g., FIGF, FLT, NOS3, TGFA). We proceed to perform an immunophenotypic analysis of primary lymphoma cells. Tumors arising in Ibtk+/+ EP--Myc mice were approximately evenly distributed between B-cell (B220pos CD43neg IgMpos IgDlow) and early pre-B cell (B220pos CD43pos BP-1pos CD24neg SL156pos) tumors as expected from previous studies [1, 2].

Differently, Ibtk+/- EP--Myc mice mainly harbored lymphomas with phenotypic markers of late pre-B cell stage (B220low CD43neg BP1pos CD24neg SL156neg).

Conclusions: These preliminary data indicate that the loss of one allele of Ibtk gene could enhance lymphoma aggressivity.

1. Adams, J.M., et al., The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature, 1985. 318(6046): p. 533-8.

2. Iritani, B.M. and R.N. Eisenman, c-Myc enhances protein synthesis and cell size during B lymphocyte development. Proc Natl Acad Sci U S A, 1999. 96(23): p. 13180-5.