rologie i - European Congress of Virology

5 th European Congress of VirologyAccumulating evidence indicates a role of miRNAs in tumorigenesis andtumor cells bear a specific and altered pattern of miRNA expression. Weperformed a high throughput screening using a TaqMan MicroRNA Assayfor the quantitation of 384 miRNAs in normal primary human keratinocytescompared to keratinocytes transformed by both E6 and E7 derivedfrom mucosal HPV 16 or cutaneous HPV 38 (K16 and K38 cells, respectively).Our analysis identified 18 miRNAs mainly deregulated in eitherK16 and/or K38 cells. Eight deregulated miRNAs (i.e. miR 10a, 18a, 19a,19b, 34a, 99a, 579 and 590) were further tested using miRNA single assayto confirm the results obtained in the screening. Since IFN may interferewith E6 and/or E7 dependent transformation, we characterized the miRNAexpression in K16 and K38 cells treated with this cytokine. Studies on thebiological significance of interesting specific targets are also in progress.REF 005The HTLV 1 encoded bZIP factor promotes cell proliferation andgenetic instability through activation of oncogenic microRNAsFranck MORTREUX 1 , Céline VERNIN 1 , Christiane PINATEL 5 ,Antoine GESSAIN 2 , Olivier GOUT 3 , Marie Hélène DELFAU LARUE 4 ,Catherine LEGRAS LACHUER 6 , Eric WATTEL 7,11 Université de Lyon 1, CNRS UMR5239, Oncovirologie et Biothérapies,Laboratoire de Biologie Moléculaire de la Cellule, Lyon, FRANCE;2 Institut Pasteur, Unité d’Epidémiologie et Physiopathologie des VirusOncogènes, Paris, FRANCE; 3 Fondation Rothschild, Service de Neurologie,Paris,France; 4 CHU Henri Mondor, Laboratoire d’Immunologie,Créteil, FRANCE; 5 Centre de Recherche sur le Cancer de Lyon, CentreLéon Bérard, Lyon, FRANCE; 6 Université Lyon I, Faculté de Médecineet de Pharmacie de Lyon, ISPBL ProfileXpert LCMT, Lyon, FRANCE;7 Université Lyon I, Service d’Hématologie, Pavillon Marcel Bérard,Centre Hospitalier Lyon Sud, Pierre Bénite, FRANCEViruses disrupt their host cells microRNAs (miRNAs) network for facilitatingtheir replication. That of HTLV 1 relies on the clonal expansionof its host CD4+ and CD8+ T cells yet the virus causes adult T cellleukemia/lymphoma (ATLL) that is regularly of the CD4+ phenotype.Infected cells express Tax and HBZ viral oncoproteins. Tax is expressedin untransformed cells where it promotes cell proliferation, genetic instabilityand miRNAs deregulation whereas in contrast, HBZ is expressedby untransformed and malignant T cells where hitherto, it is consideredto promote cell proliferation and to silence virus expression. Here weshow that an HBZ/miRNAs axis promotes cell proliferation and geneticinstability. Infected CD4+ but not CD8+ T cells were found to overexpressoncogenic miRNAs such as miR 17 and miR 21. HBZ activated these miR-NAs via a posttranscriptional mechanism while in addition to promotingcellular growth; HBZ decreased DNA stability. These effects were alleviatedby either miR 21/miR 17 knockdown or by the ectopic expression ofOBFC2A, a factor that protects genome stability and that we found targetedby miR 17 and miR 21 in HTLV 1 infected CD4+ T cells. This considerablyextends the oncogenic potential of HBZ and suggests that viral expressionmight be involved in the remarkable genetic instability of ATLL cells.REF 006Molecular signatures of tumorigenesis in penile squamous cell carcinomaand their association with high risk human papillomavirusinfectionElektra PETA 1 , Rocco CAPPELLESSO 2 , Valentina MILITELLO 1 ,Alessandro SINIGAGLIA 1,3 , Giulia MASI 1 , Matteo FASSAN 2 ,Ambrogio FASSINA 2 , Luisa BARZON 1 , Giorgio PALÙ 11 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 Department of Medicine, University of Padova, Padova, ITALY;3 IOV Istituto Oncologico Veneto, Padova, ITALYPenile squamous cell carcinoma (PSCC) is a rare tumor associated withhigh risk (HR) HPV infection in 30 60% of cases. Altered expressionof miRNAs has been reported in HPV related cervical and in head andneck cancers, but there are no studies on PSCC. The presence of HPVinfection and its relationship with p53 and p16INK4a expression and withexpression of a panel of cellular miRNAs involved in HPV related cancerwas investigated in 59 PSCCs and 8 condylomas. HR HPV DNA(HPV16 in most cases) was detected in 2/5 in situ PSCCs and in 16/54invasive PSCCs. All penile condylomas were positive for low risk HPV6 orHPV11. Immunohistochemical analysis showed that HPV positive PSCCswere typically p16INK4a positive and p53 negative. Among investigatedmiRNAs, miR 218 was significantly down regulated in PSCCs with highrisk HPV infection. In addition, miR 218 was markedly silenced in HRHPV negative PSCCs with apparently functional p53. Hypermethylationof the promoter of the SLIT2 gene, which contains miR 218 in an intronof its transcript, has been described as a mechanism of miR 218 downregulationin several cancers. Thus, methylation of SLIT2 promoter andits association with HR HPV infection and p53 status was investigatedin PSCCs. Methylation of the SLIT2 promoter was frequently observedin PSCCs and was inversely correlated with miR 218 expression levelsespecially in HR HPV negative and p53 negative PSCCs. In conclusion,this study describes different molecular signatures of tumorigenesis (i.e.,HR HPV infection, p53 inactivation, and miR 218 downregulation) inPSCCs.REF 007Tax promotes the expression of the alternative spliced isoformCD44v10 by slowing down the elongating RNA polymerase IIMorgan THENOZ 1 , Céline VERNIN 1 , Christiane PINATEL 2 , NicolasNAZARET 3 , Joel LACHUER 3 , Antoine GESSAIN 4 , DidierAUBOEUF 5 , Eric WATTEL 1 , Franck MORTREUX 11 Oncovirologie et Biotherapies, UMR5239 CNRS/ENS Lyon/UCBL/HCL,Hopital Pierre Benite, Lyon, FRANCE; 2 Oncovirologie et Biothérapies,Centre Léon Bérard, Lyon, FRANCE; 3 ProfileXpert, Neurobiotec Service,Bron, FRANCE; 4 Unit of Epidemiology and Physiopathology of OncogenicViruses, Department of Virology, Institut Pasteur, Paris, FRANCE;5 Institut National de Santé et de Recherche Médicale U590, Centre LéonBérard, Lyon, FRANCEDeregulation of gene expression is the hallmark of HTLV 1 infection,involving a complex interplay between the viral oncoprotein Tax and hostcell transcription machinery. It has been recently shown that changes inpromoter structure and occupation impinge on both transcription and alternativesplicing (AS) by modulating the RNApolII elongation rate. By usingExon microarrays, we identified here that ectopic Tax expression induceschanges in the exon expression patterns. Overall, 857 were found differentiallyspliced upon Tax expression. Of these, 309 genes were changed intheir transcriptional levels. AS events were validated by exon specific RTPCR assays. More particularly, we identified in that Tax induced NF kBdependent transcription of CD44 results in the inclusion of variable exonv10 (CD44v10) in mature transcripts. The protein expression of CD44v10at the plasma membrane of Tax+293T cells was proved by FACS analysisand CD44v10 mRNA was also detected in infected CD4+ T cells invivo. CD44 variants are described in a wide variety of cellular functionsincluding lymphocyte activation, migration, and tumor metastasis. At themechanistic level, qChIP analysis of CD44 gene occupancy showed thatTax induced splicing of CD44v10 coincides with a significant enrichmentof both RNApolII and Tax at the region encoding variable exons, indicatingthat Tax interferes with the elongation rate of RNApolII. Together, theseresults unmask new mechanisms that connect Tax, transcription and alternativesplicing with the HTLV 1 induced transcriptome reprogramming ofCD4+ T cells.Virologie, Vol 17, supplément 2, septembre 2013S119