5β 1 integrin-emanating signals remodel nuclear architecture through the activation of ERK1/2 and p38a MAPKs during invasive cell growth

5β 1 integrin-emanating signals remodel nuclear architecture through the activation of ERK1/2 and p38a MAPKs during invasive cell growth

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  as a new lead for the development of Aurora kinase inhibitors and iscurrently under investigation in vivo. 139 Poster   5   1 integrin-emanating signals remodel nuclear architecturethrough the activation of ERK1/2 and p38a MAPKs during invasivecell growth L. Vellón 1 , L. Espinosa Hevia 1 , F. Royo 1 , L.A. Parada 1 1 CICbioGUNE, Cytogenomics, Derio, Spain  Invasive cell growth is a physiological process executed by stem andprogenitor cells during embryonic development and postnatal organregeneration. Growing evidence indicates that this program is usurped bycancer cells, resulting in metastasis. To this regard, the integrin receptorsare deeply involved in the invasive capacity of progenitor and cancer cells.Here, we assessed the role of β 1 integrins-emanating signals in thegenomic events that occur during the invasive growth of MLP29 murinehepatic progenitor and Hep16 Hepatocellular carcinoma (HCC) cells.Cytometric and immunoblot analysis of integrin expression in MLP29 cellsshowed high levels of β 1 integrins (fibronectin-FN and collagen IV-COLIVreceptors) and low levels of α v and  β 3 integrins (vitronectin-VN receptors).In concordance, MLP29 cells presented high adhesion to FN and COLIV,and low adhesion to laminin (LMN) and vitronectin (VN). By contrast, HCCcells exhibited opposite adhesive properties, which agrees with their highlevels of α v integrins and low levels of β 1 integrins. Since α 5 β 1 is the mainFN receptor in hepatocytes, we used a functional blocking antibody againstthe  α 5 β 1 integrin to investigate MLP29 cells invasion and growth. Wedetected marked cell spreading and actin cytoskeleton reorganization, andthis was associated with activation of the ERK1/2 and p38 α MAPKs cellsignalling pathways. At the nuclear level, 3D-analysis of centromeredistribution in interphase nuclei, showed that the average number ofchromocenters per nucleus increased significantly by the functionalblockade of α 5 β 1 or decreased by α 5 β 1 activation upon attachment to FN.Interestingly, these α 5 β 1 -induced alterations were abolished bypharmacological inhibition of ERK 1/2 (U0126) and p38 MAPK(SB239063). In Hep16 HCC cells, inhibition of the constitutively hyper-activated ERK 1/2 and p38 MAPKs also induced centromerereorganization. In line with these findings, gene expression analysis bymicroarray technology revealed that α 5 β 1 blocking induced the differentialexpression of a significant amount of genes involved in the nuclearstructure and nucleic acid binding. Furthermore, these α 5 β 1 -mediatedsignals drastically increased the acethylation status of Histone H3 at lys9/14. Collectivelly, these results suggest that invasive cell growth in hepaticprogenitor cells involves a remodelling of the nuclear architectureregulated, at least in part, by the α 5 β 1 integrin-mediated activation of theERK1/2 and p38 α MAPKs. This may be also applied to HCC cellspresenting a hyper-activation of major pro-survival cell transductioncascades, which may be accountable for the high invasive capacity. 140 Poster Steroidogenic factor-1 gene dose and adrenocortical tumors M. Doghman 1 , T. Karpova 2 , G. Rodrigues 1 , M. Arhatte 1 , P. Barbry 1 ,G. Zambetti 3 , B. Figueiredo 4 , C. Martinerie 5 , L. Heckert 2 , E. Lalli 1 1 Institut de Pharmacologie Moléculaire et Cellulaire Université Nice Sophia Antipolis, CNRS UMR6097, Valbonne, France; 2  University of Kansas Medical Center, Department of Molecular and Integrative Physiology, Kansas, USA; 3  St. Jude Children’s Research Hospital,Department of Biochemistry, Memphis, USA; 4  Instituto de Pesquisa Pelé Pequeno Principe, Centro de Genética Molecular e Pesquisa do Câncer em Crianças, Curitiba, Brazil; 5  INSERM U515, Hopital St Antoine UPMC,Paris, France  Adrenocortical tumor (ACT) in children is a rare form of neoplasm but itsincidence is higher in southern Brazil than in the rest of the world. In thatregion, it is almost invariably found associated with a specific germlineTP53 mutation (R337H) and loss of heterozygosity in the other allele. Wehave shown an increased copy number of the steroidogenic factor 1 (SF-1;NR5A1) gene associated with its overexpression in the majority ofchildhood ACTcompared with normal age-matched adrenal gland.Steroidogenic Factor-1 (SF-1/Ad4BP; NR5A1), a transcription factorbelonging to the nuclear receptor superfamily, has a pivotal role foradrenogonadal development in humans and mice.Using an integrated approach comprising human tumor adrenocorticalcell cultures, gene expression profiling and transgenic mice analysis, wehave defined the role for SF-1 dosage in adrenocortical tumorsdevelopment.We show that SF-1 overexpression increases human adrenocortical cellproliferation through opposing effects on cell cycle and apoptosis by usingan inducible cellular system,. This effect is dependent on an intact SF-1transcriptional activity. Gene expression profiling showed that an increasedSF-1 dosage regulates transcripts involved in steroid metabolism, cellcycle, apoptosis, and cell adhesion to the extracellular matrix. Consistentwith these results, increased SF-1 levels selectively modulate the steroidsecretion profile of adrenocortical cells, reducing cortisol and aldosteroneproduction and maintaining DHEA-S secretion. We identified a novel pro-apoptotic factor for adrenocortical cells, NOV/CCN3, whose levels aresignificantly reduced by SF-1 overexpression in human adrenocortical cellsand are also reduced in primary adrenal tumors. In mice, increased Sf-1dosage produces adrenocortical hyperplasia and formation of tumors whichsrcinate from the subcapsular region of the adrenal cortex. These tumorsexpress gonadal markers and activated Stat3.Our studies reveal the critical role of SF-1 gene dosage for adrenocorticaltumsrcenesis and constitute a rationale for the development of drugstargeting SF-1 transcriptional activity for ACTtherapy. 141 Poster NPM-ALK modulates the p53 tumour suppressor pathway in a JNKand PI 3-Kinase dependent manner: MDM-2 is a potential therapeutictarget for the treatment of ALK-expressing malignancies Y. Cui 1 , A. Kerby 1 , F.K.E. McDuff 1 , S.D. Turner 1 1 University of Cambridge, Department of Pathology, Cambridge, United Kingdom  Anaplastic large cell lymphoma (ALCL) is in the majority of cases apaediatric disease of a T- or null-cell phenotype and is characterised by thepresence of the t(2;5)(p23;q35) or variant translocations involving the ALKgene on chromosome 2. This chromosomal translocation generates theNucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK) fusion protein, ahyperactive kinase with transforming properties. The p53 tumoursuppressor gene is rarely mutated in ALK-expressing ALCL, perhaps onereason why this disease has a good prognosis. However, the mechanismcontrolling p53 activity in ALCLhas not been fully elucidated. We show inpatient-derived ALCLcell lines and NPM-ALK transformed BaF3 cells thatNPM-ALK induces post-translational modification of the p53 antagonistMDM2, leading to inactivation of the p53 tumour suppressor pathway.Furthermore, we demonstrate that the PI 3-Kinase-Akt pathwaydownstream of NPM-ALK is responsible for this activity. It therefore followsthat inactivation of MDM2 with the specific inhibitor nutlin-3 results in adecrease in proliferation and subsequently apoptosis of NPM-ALK-expressing ALCLcells, a response that is enhanced when cells areexposed to nutlin-3 in conjunction with the PI 3-Kinase inhibitor LY294003.We also demonstrate that NPM-ALK activates JNK by phosphorylation inturn leading to JNK-mediated sequestration and degradation of p53. Thisactivity can be attenuated following administration of a specific JNKinhibitor. We conclude that NPM-ALK regulates the activity of the p53tumour suppressor pathway via sequestration by JNK and MDM2 leadingto its degradation. MDM2 antagonists in combination with JNK/PI 3-Kinaseinhibitors may therefore be potential targets for the treatment of ALK-expressing malignancies such as ALCL. 142 Poster PKC theta increases phosphorylation and stability of the Fra-1protein in invasive breast cancer cell lines K. Belguise 1 , S. Milord 1 , F. Galtier 1 , R. Hipskind 2 , M. Piechaczyk 3 ,D. Chalbos 1 1 Institut de Recherche en Cancérologie de Montpellier, Control of Hormono-dependent Cancer Progression, Montpellier, France; 2  Institut de Génétique Moléculaire de Montpellier, Intracellular Signalling and Gene Regulation, Montpellier, France; 3  Institut de Génétique Moléculaire de Montpellier, Oncogenesis and Immunotherapy, Montpellier, France  In contrast to cells expressing estrogen receptor α (ER+), the most invasiveER- breast cancer cell lines express high constitutive AP-1 binding activitymainly due to high concentration of Fra-1, a member of the FOS family. Fra-1, which induces the expression of genes implicated in breast cancerprogression, enhances in vitro proliferation and invasiveness of these cells.These results led us to investigate the molecular mechanisms responsiblefor high expression of Fra-1 in the most invasive cells.The effect of PKC θ on Fra-1 expression and phosphorylation wasevaluated by transient transfection of constitutively active and dominantnegative PKC θ mutants in ER+ MCF7 cells and ER- Hs578Thuman breastcancer cells, respectively. Implication of ERK1/2 and/or ERK5 in thisregulation was determined by the use of the MAPK inhibitors UO126 andPD98059 and of Fra-1 proteins mutated on S252 and S265 whosephosphorylation by ERK1/2 prevents Fra-1 degradation by the proteasome.Results show that PKC θ , whose expression can be detected in ER- butnot ER+ cells, increases Fra-1 expression. Ectopic expression of aconstitutively active PKC θ mutant in MCF7 cells increases Fra-1 level andFra-1 phosphorylation as observed by the appearance of low migratingbands. Moreover, introduction of a dominant negative PKC θ mutant in   3606 July 2008Poster Session
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