EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 S31

linteus, Agaricus bisporus, Hericium erinaceus, Auricularia auricula-judae andGrifola frondosa. Generally methanol extracts showed significantly strongereffect than water extracts. In fact only hot alkali extracted polysaccharides fromFomes fomentarius showed moderate cytotoxicity toward tumor cells (IC50 =0.564mg/ml for HeLa, 0.312mg/ml for K562 and 0.879mg/ml for MDA-MB-453cells).Conclusion: Results obtained showed that some compounds of methanolextracts from Piptoporus betulinus, Ganoderma lucidum and Ganodermaapplanatum, could be promising agents for the treatment of human tumors andare candidates for further analyses on experimental animals, in vivo. Our studyprovides a first comprehensive evaluation of the cytotoxicity of various Serbianfungi species and forms an important basis for the isolation and structuralelucidation of cytotoxic compounds from these species in the future.No conflict of interest.

138 Induction of cellular senescence and hair follicle stem celldysfunction upon p14ARF and p16Ink4a expression in the skin

N. Azazmeh1, R. Tokarsky-Amiel1, I. Ben-Porath1. 1Institute for MedicalResearch − Israel − Canada Hadassah School of Medicine The HebrewUniversity of Jerusalem, Department of Developmental Biology and CancerResearch, Jerusalem 91120, Israel

Introduction: Cellular senescence is a physiologic stress response program,in which cells cease to proliferate and undergo dramatic alterations inmorphology, metabolic activity and protein secretion. Despite its importancein aging and tumor suppression, many fundamental aspects of cellularsenescence remain poorly elucidated. Senescence is executed most oftenby the p19ARF/p53 and p16Ink4a/Rb pathways, which are interconnected andmediate partially redundant effects, yet their distinct contributions to thesenescence program are not fully understood.Materials and Methods: We developed mice that allow inducible activation ofthe two central mediators of senescence, p14ARF and p16Ink4a, in the epidermisof the skin. We set out to test the comparative ability of the two genes to inducesenescence, and their effects on tissue structure and stem cell function.Results and Discussion: We found that both p14ARF and p16Ink4a acutelyreduced proliferation in the epidermis and induced the formation of senescentcells. However, activation of p14ARF gave rise to substantially more senescentcells than p16Ink4a activation. The endogenous Cdkn2a products − p19ARF

and p16Ink4a − were induced upon the activation of both transgenes, revealinga senescence-promoting feed-forward loop. Activation of p14ARF, but notof p16Ink4a, also caused a rapid, but transient, wave of apoptosis, prior tothe generation of senescent cells. The influence of p14ARF and p16Ink4a onhair follicle stem cell function was also distinct: while the proliferation ofthe stem cells and the entry of the hair follicle to anagen were completelyprevented upon p14ARF activation, leading to a severe hair loss (alopecia),a partial arrest was detected upon p16Ink4a activation. We found that hairfollicle bulge stem cells were resistant to p53-induced apoptosis, and insteadunderwent senescence, causing an irreversible blockage of proliferationpotential. Interestingly, induction of skin hyperplasia prevented the appearanceof senescent cells in the epidermis, yet was not sufficient to overcome bulgestem cell dysfunction.Conclusion: Our findings indicate that the p19ARF/p53 pathway is a morepowerful inducer of senescence in the epidermis than the p16Ink4a/Rb pathway,and can execute complete blockage of stem cell function. This, despite theinterconnectivity of the two pathways. These data shed light on one of themost important cellular tumor suppression mechanisms.No conflict of interest.

140 MET signaling in colorectal cancer-initiating cells blunts responseto EGFR inhibition: Implications for targeted therapy

P. Luraghi1, G. Reato1, E. Cipriano1, E. Menietti1, F. Sassi1, V. Bigatto1,F. Orzan1, A. Bertotti1, L. Trusolino1, C. Boccaccio1. 1Candiolo CancerInstitute − FPO (IRCCS) University of Turin, Experimental Clinical MolecularOncology, Candiolo (TO), Italy

Metastatic colorectal cancer remains largely incurable, although in a subsetof patients survival is prolonged by new targeting agents such as anti-EGFR antibodies. Reportedly, colorectal cancer contains a tumorigenic cellhierarchy sustained by a subpopulation of colorectal cancer-initiating cells(CC-ICs), which may confer therapeutic resistance. However, how CC-ICsrespond to EGFR inhibition has not been fully characterized. From apreviously set-up platform of molecularly annotated patient-derived xenograftsof metastatic colorectal cancer (xenopatients), we derived sphere culturesendowed with properties of CC-ICs (xenospheres), which faithfully retain thesame genetic alterations as the tumor of origin, and generate secondarytumors (spheropatients), highly resembling those of both xenopatient andpatient of origin. Xenospheres and spheropatients offered the unprecedentedopportunity to investigate the molecular and biological response to targetedtherapies at cancer-initiating cell level. Interestingly, we found that xenospheresretained the genetic determinants of response to anti-EGFR therapy: in line

with data in original bulk tumors, xenospheres harboring a mutated KRASgene were resistant, while those harboring wild-type RAS pathway genes(RASwt) were sensitive. However, EGFR inhibition in sensitive CC-ICs could becounteracted by cytokines secreted by cancer-associated fibroblasts. Amongsuch cytokines, HGF, the ligand of the MET receptor, was particularly activein supporting in vitro CC-IC proliferation and resistance to EGFR inhibition.Consistently, in spheropatients genetically engineered to produce humanHGF, so as to achieve endocrine and paracrine activation of human MET inxenografted CC-ICs, MET inhibition enhanced and sustained tumor regressioninduced by anti-EGFR antibodies, leading to a virtually complete and durableresponse. These data show that RASwt CC-ICs rely on both EGFR and METsignaling, and provide a strong proof of concept for concurrent targeting of thetwo pathways in the clinical setting.No conflict of interest.

141 RET/PTC1 in vitro models unveil a novel tumor suppressor miRNAin papillary thyroid carcinoma

E. Minna1, P. Romeo1, L. De Cecco2, M. Dugo2, G. Cassinelli3, C. Lanzi3,S. Pilotti4, M.A. Pierotti5, A. Greco1, M.G. Borrello1. 1Istituto NazionaleTumori, Molecular Mechanisms Unit Department of Experimental Oncologyand Molecular Medicine, Milan, Italy, 2 Istituto Nazionale Tumori, FunctionalGenomics and Bioinformatics Unit Department of Experimental Oncologyand Molecular Medicine, Milan, Italy, 3Istituto Nazionale Tumori, MolecularPharmacology Unit Department of Experimental Oncology and MolecularMedicine, Milan, Italy, 4Istituto Nazionale Tumori, Department of Pathologyand Laboratory Medicine, Milan, Italy, 5 Istituto Nazionale Tumori, ScientificDirectorate, Milan, Italy

Background: Thyroid cancer (TC) is the most common endocrine malignancy,with increasing incidence. The majority of TCs are classified as papillarythyroid carcinomas (PTCs) and RET/PTC oncogene is the second mostfrequently identified driving genetic lesion in this tumor histotype. Inaddition, several studies have reported aberrant miRNA expression in PTC.Nevertheless, the mechanisms leading to miRNA deregulation in this neoplasiaas well as the consequences of their altered expression are still poorlyunderstood.Methods: MiRNA/gene microarray expression profiles were defined in twoPTC cell models: thyrocytes infected with RET/PTC1 oncogene and TPC1cells, harbouring endogenous RET/PTC1, treated with the inhibitor RPI. Theexpression of miR-199a-3p was evaluated by meta-analysis of miRNA profilesof a large cohort of PTCs/non-neoplastic thyroids publicly available on TheCancer Genome Atlas and by qRT-PCR in PTC surgical specimens and inthyroid derived cell lines. The functional role of this miRNA was then assessedby transfecting a miR-199a-3p mimic in thyroid derived cell lines and analysingproliferation, migration and cell death.Results:We identified a total of 30 miRNAs and 301 coding genes significantlyand concordantly de-regulated in the two cell models accordingly with thepresence of an active RET/PTC1 oncoprotein. Among de-regulated miRNAwe focused on miR-199a-3p. We showed that this miRNA is under-expressedin PTC surgical samples and in PTC-derived cell lines, and its restorationin PTC cells causes MET and mTOR protein levels reduction. Moreover,we demonstrated that miR-199a-3p can exert oncosuppressor functions inPTC cell lines by impairing migration and proliferation and, more interesting,inducing lethality through an unusual form of cell death similar to methuosis,caused by macropinocytosis dysregulation. However, neither MET nor mTORspecific silencing recapitulate miR-199a-3p-induced cell death, suggesting theinvolvement of additional target genes.Conclusions: Cancer cell modeling and functional approaches lead to theidentification of miRNAs relevant in thyroid carcinogenesis as suggested byour results that indicate miR-199a-3p as a novel tumor suppressor miRNA inPTC.No conflict of interest.

142 Proteolytic regulation of the EphB4 receptor in prostate cancer

J. Lisle1, I. Mertens-Walker2, C. Stephens2, S. Stansfield2, A. Herington2,J. Clements2, S. Stephenson2. 1Princess Alexandra Hospital, TranslationalResearch Institute, Woolloongabba, Australia, 2Translational ResearchInstitute, Cancer Program, Woolloongabba, Australia

Introduction: EphB4, a receptor tyrosine kinase, is over-expressed in66% of prostate cancers (PCa) where it promotes tumour angiogenesis,increases cancer cell survival and facilitates cell invasion and migration;however, the mechanism of action is still unclear. Recently, we developedan overexpression model to study EphB4 in PCa using the cell line 22Rv1(22Rv1-B4) and identified the presence of novel EphB4 fragments. UsingEphB4-specific antibodies directed to either the C- or the N-terminus of theprotein we predicted these were the result of sequential specific proteolyticcleavage events releasing both extracellular (ECF − 70kDa) and intracellular(ICF − 47kDa) fragments. This study focused on the possible mediators offragment generation and the function of the ICF in PCa.