Vav1 have become dependent upon this exchange factor, implicating Vav1 as a potent nodefor therapeutic intervention. Supported by R01 CA104125 and R03 CA155778 to M.A.M.

Tu1687

Nitric Oxide Is Essential on Regulating Tumor Initiating Properties of ColonCancer Stem CellsMaria Ausiliatrice Puglisi, Valentina Tesori, Lucia Ricci Vitiani, Giovanni Gasbarrini,Giovambattista Pani, Antonio Gasbarrini

BACKGROUND AND AIM : Several studies have indicated that continuous exposure tohigh concentrations of Nitric Oxide (NO), produced by inducibile-NO synthase (iNOS),promote neoplastic transformation within many human cancers, including colon cancer(CC). Recently, it has also been suggested that high NO synthesis is a distinctive feature of"cancer stem cells" (CSC), a tumor subpopulation with self-renewal capacity, that may beidentified by the expression of the CD133 surface marker. Aims of this study were to explorethe contribution of NO in the definition of colon CSC features and evaluate potentialstrategies to treat CC by modulating NO production. MATERIAL AND METHODS : Byimmunohistochemistry analysis we evaluated iNOS and CD133 expression in 20 samplesof human CC. Using the DAF-2DA detection system, we assayed the production of intracellu-lar NO in 5 colon CSC lines obtained from human CC tissues. By FACS sorter, we purifiedthe NO high and NO low fractions from all colon CSC lines and we compared the tumorigenicpotential of both cell fractions by in vitro and in vivo assays. To tested the potential antitumoreffects of iNOS modulation, we treated colon CSCs with the selective iNOS inhibitor 1400Wor we stably transfected these cells with two distinct iNOS-directed short-harpin RNA(shR-NAs). RESULTS : Immunohistochemistry analysis revealed that, irrespective of grade, higheriNOS expression was associated with increased CD133 levels in all CC samples. Moreover,we observed that NOhigh cells display higher expression levels of stem cell markers andincreased tumorigenic properties in vitro and in vivo than NOlow fraction. Both iNOS-directed shRNAs and iNOS inhibitor treatment decreased the rate of cell-cycle transit incolon CSCs and reduced tumorigenic capacity in vitro and in vivo, confirming an integralrole for NO and endogenous iNOS activity in the biology of colon CSCs. Interestingly, theknockdown of iNOS expression was associated with a significant down regulation of stemcell markers, such as CD133, BMI and b-Catenin. CONCLUSIONS : This study identifiesfor the first time a cell-autonomous colon CSC specific source of NO, and it highlights thebiological effects of the iNOS within colon CSCs. In the long run, these data could serveas an impetus for evaluation of iNOS-directed therapies as a component of multimodaltreatment regimens for human CC.

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Reprimo, a Novel Tumor Suppressor Gene by Inhibition of Cell Cycle ProteinsKathleen Saavedra, Alejandra M. Sandoval, Benjamin García-Bloj, Javier Cerda-Infante,Wilda Olivares, Gareth I. Owen, Juan C. Roa, Alejandro H. Corvalan

Reprimo (RPRM) is a highly glycosylated protein localized in the cytoplasmic, and mediatearrest in G2/M checkpoint. Previously, we describe the loss of protein expression of RPRMas a marker of aggressiveness in gastric cancer (GC) patients. The aim of this study is tocharacterize gene expression of RPRM in GC cell lines and explore its biologic function astumor suppressor gene (TSG). Methods: Seven GC cell lines were characterized for geneexpression of RPRM at mRNA and protein level by real-time PCR and western blot. Tostudy the role of RPRM as TSG we generated stable (RPRM-AGS) and transient (RPRM-MKN45) transfections. The effects of RPRM expression on tumorigenic characteristics inGC cells were evaluated by in vitro assays. For colony formation assay, 200 cells wascultivated for 10d, colonies were stained using violet crystal and counted under microscope.For wound healing assay, cells were cultured at subconfluent density and cell monolayerwas scratched with a plastic pipette tip. The relative migration at 0 and 20h was calculated.For cell cycle profiles, cells were synchronized by serum deprivation, recovered in completemedia, and analyzed 24h later by flow cytometry. RPRM-MKN45 cell was analyzed forsubcellular location of cyclinB1/cdc2 complex by immunofluorescence, phosphorylationlevel of cdc2-Tyr15, by western blot and Wee1 by qRT-PCR and western blot. A p<0.05was considered statistically significant. Results: RPRM levels were low to undetectable in allGC cell lines. There was no change in the percentage of cells in G2/M phase between emptyvector- and RPRM-AGS cells. RPRM-AGS cells demonstrated two-fold fewer colonies thanvector control (54,67±6,5 vs. 26.83±4.79, P=0,005). The migration potential of RPRM-AGScells was significantly lower than that of the vector control (44,52% vs 81,95%, P<0.0001).RPRM-MKN45 cells confirmed cytoplasmic location of cyclinB1/cdc2 complex, the phosphor-ylated form of cdc2-Tyr15 was significatively increased as well as levels of Wee1 in transientlytransfected cells. Conclusions: Colony formation and wound healing assays confirm thatRPRM has anticlonagenic effects and decrease migration potential in GC cells, both character-istics of TSG. We suggest that Wee1 participate in the signaling cascade of RPRM at G2/Mcheckpoint, through the inhibition of the nuclear translocation of cyclinB1/cdc2 complexand activation of cdc2 protein. Cell cycle experiments suggest that RPRM may performalternative cellular functions rather G2/M checkpoint. Grant support: Fondecyt#1111014 &Fondap#15130011 to AH Corvalan

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In Vitro, Mesalazine Decreases Viability of 2D and 3D Human Colon Cancer(CC) Cell Lines Cultures by Increasing Protocadherin Mucdhl ExpressionThrough a Pathway Not Involving the CDx2 - Mucdhl AxisMathilde Lechevrel, Isabelle Gross, Céline Quesnelle, Bernard Duclos, Jean-Noël Freund,Jean-Marie Reimund

The homeotic gene Cdx2 is essential for regulating intestinal homeostasis and consideredas a tumor-suppressor gene for CC. Recently, the gene encoding the protocadherin Mucdhlhas been demonstrated to be a target of the transcription factor CDX2 [1]. In addition,mesalazine, used as a chemopreventive drug for IBD-associated CC, has been suggested to

S-819 AGA Abstracts

increase Mucdhl expression, a mechanism which may participate to its chemopreventiveeffects. Our aim was to determine if the effects of mesalazine on Mucdhl expression weredependent on Cdx2 activation as a first step, followed by Mucdhl expression activation byCDX2. Cdx2 and Mucdhl expression (Q-PCR for mRNA and immunofluorescence for pro-teins) were studied in human CC cell lines HT29, HCT116 and DLD1, 2D or 3D (colonosph-eres) cultures, after a 48 hours exposure to 20 or 40 nM of mesalazine (which correspondto the tissue concentrations obtained with a 2 or a 4 g/d therapeutic use, respectively). Inparallel, we checked cell viability and colonospheres formation efficiency (CFE) (lumines-cence assay), and used Cdx2 siRNA to appreciate the link between Cdx2 and Mucdhlincrease of expression in presence of mesalazine. Mesalazine inhibited dose-dependently CCcell lines (cultured in 2D or 3D) viability as well as CFE and increased significantly Cdx2and Mudhl mRNA expression, MUCDHL protein expression, but not CDX2 protein expres-sion. Cdx2 mRNA expression was strongly dowregulated by Cdx2 siRNA in presence ornot of mesalazine. By contrast, Cdx2 siRNA does neither affect Mudhl expression nor HT29,HCT116 or DLD1, 2D or 3D cultures viability. In conclusion, these results confirmed thatmesalazine inhibits 2D and 3D cultures of CC cell lines as well as CFE. They showed thatmesalazine increases Cdx2 mRNA expression but not protein expression (post-transcriptionalregulation?). Finally, they confirmed that mesalazine increases Mucdhl expression and dem-onstrated that this activation was not dependent on the activation of Cdx2 expression. Thiswork was ongoing in order to identify the way by which mesalazine activates Mucdhl mRNAand protein expression. [1] Hinkel et al. Gastroenterology 2012;142:875-85. Acknowledge-ments: This work was supported by a grant from the Association François Aupetit, Paris,France. Ferring Pharmaceuticals France provided us kindly mesalazine for in vitro experimen-tation.

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MiR-130b Plays an Oncogenic Role by Repressing PTEN Expression inEsophageal Squamous Cell CarcinomaRisheng Cao, Tingting Yu, Shuo Li, Mingen Fu, Lihua Ren, Weixu Chen, Xiqiao Zhou,Ruihua Shi

Esophageal carcinoma is one of the most common malignancies with high cancer- relatedmorbidity and mortality worldwide. MicroRNAs (miRNAs) are a class of small non-codingRNAs that regulate a wide variety of cellular processes. MiRNAs also play an important rolein the development and progression of cancers. In previous microarray study, we demon-strated that miR-130b was up-regulated in esophageal squamous cell carcinoma (ESCC)tissues. However, biological function and molecular mechanism of miR-130b in ESCC remainto be elucidated. Methods: qRT-PCR assays were used to quantify miR-130b expressionlevels in ESCC samples. ESCC cells overexpressing or down-expressing miR-130b wereanalyzed for in vitro biological functions. Novel target of miR-130b was identified by bioinfor-matics searching and dual-luciferase reporter. Western blotting and qRT-PCR assays wereused to detect the expressions of target gene PTEN (phosphatase and tensin homolog) anddownstream Akt. Results: The high levels of miR-130b were identified in 20 ESCC samplescomparing with adjacent non-neoplastic tissues. The over-expression of miR-130b increasedthe proliferation ability of ESCC cells and also enhanced their ability to migrate and invade.In contrast, cellular proliferation, migration and invasion of ESCC cells were weakened whenmiR-130b expression was suppressed. We confirmed that miR-130b interacted with the 3'-untranslated region (3'-UTR) of PTEN, and that an increase in the expression level of miR-130b negatively affected the protein level of PTEN. However, the dysregulation of miR-130b had no obvious impact on the mRNA level of PTEN. As Akt is a downstream effectorof PTEN, we explored if miR-130b affected Akt expression, and found that miR-130bindirectly regulated the level of phosphorylated Akt protein. Interestingly, the total Aktprotein levels remained unchanged. Conclusion: The above findings indicate that miR-130bplays an oncogenic role by repressing PTEN expression and Akt phosphorylation, whichcould be helpful in developing microRNA-based treatment for ESCC in the future.

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Differential Mechanisms Utilized by Pancreatic Tumor Cells and AssociatedFibroblasts to Remodel Stroma During Metastatic InvasionHong Cao, Robbin Eppinga, Eugene Krueger, Jing Chen, Gina L. Razidlo, Mark A.McNiven

Metastatic invasion of tumors into peripheral tissues is known to rely upon protease-mediateddegradation of the surrounding stroma. This remodeling process is dependent upon complex,actin-based specializations of the plasma membrane termed "invadopodia" that act to bothsequester and release matrix metalloproteases. Here we report that cells of mesenchymalorigin, including fibroblasts, pancreatic stellate cells, and tumor-associated fibroblasts,degrade substantial amounts of surrounding matrix by a mechanism independent of conven-tional invadopodia. First, as tumor cell-derived invadopodia are dependent upon the actionof the GTPase Dyn2, loss of Dyn2 function inhibits invadopodia-based matrix degradationby pancreatic tumor cells. In surprising contrast, we have found that loss of Dyn2 functionin mesenchymal cells does not inhibit degradation, but actually leads to a marked increase.Second, these degradative sites lack the punctate shape of conventional invadopodia butare instead spread along the cell base and are reticular and/or fibrous in character. Third,inhibition of either Src or Cdc42 function, known to drive invadopodia formation, had noeffect on the mesenchymal matrix degradation induced by loss of Dyn2. Taken together, thesedata indicate that this mechanism of degradation is independent of classical invadopodialdegradation. To define the mechanistic processes that differ between tumor and stromal celltypes, we screened for matrix metalloprotease activity using zymography. An unexpectedand significant upregulation of MMP2 activity in mesenchymal-derived cells upon loss ofDyn2 was observed, whereas MMP2 was nearly completely absent in the tumor cells. Further,loss of Dyn2 resulted in an upregulation of surface-bound MT1-MMP, known to activateMMP2. In contrast, loss of Dyn2 in the pancreatic tumor cells reduced surface levels ofMT1-MMP. Thus, in mesenchymal cells, loss of Dyn2 induces retention of MT1-MMP at thesurface, leading to large scale matrix degradation through an MT1-MMP/MMP2-dependentmechanism. Excitingly, co-culture invasion experiments demonstrate that the matrix degrada-tion induced in these stromal fibroblasts is sufficient to promote invasion by pancreatic

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