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sMo1928

The Transient Receptor Potential Ca2+ Ion Channel TRPC1 Mediates TGFβ-Induced PTEN Downregulation and Pancreatic Cancer Cell MotilityJimmy Y. Chow, Chang-Whan Kim, Hui Dong, Tiffany A. Ornelas, Jenny M. Li, Peik L.Sia, Jeffrey Liu, John M. Carethers

Background and Aim. The pathogenesis and progression of pancreatic cancer involvesconversion of TGFβ suppressive signaling in early lesions to an unmasked, TGFβ proliferativesignaling cascade in metastatic lesions. We have previously demonstrated that TGFβ prolifer-ative signaling downregulates the tumor suppressor PTEN and induces motility of pancreaticcancer cells after ligand-receptor binding with subsequent increase in cytoplasmic free Ca2+concentration ([Ca2+]cyt) and activation of PKCα and NF-κB. [Ca2+]cyt homeostasis isimportant to maintain cell functionality - dysregulation of which contributes to tumorigenesisof pancreas. Transient receptor potential ion channels (TRPC) help regulate [Ca2+]cyt, andhere we evaluated the role of TRPC channels in regulating TGFβ proliferative signaling.Methods. BxPc3 pancreatic cancer cells were treated with TGFβ (10 ng/mL), and TRPC andPTEN expression were analyzed by RT-PCR and/or Western blots. [Ca2+]cyt was assessedusing Fura-2 fluorescence ratio digital imaging. Pharmacological reagents (TRPC inhibitors:2-APB, SKF96365, and LaCl3) and siRNAs to TRPCs were used to determine the role ofTRPCs on TGFβ-induced PTEN regulation and cell migration. Cell motility was assessedby Boyden chamber migration assay. Results. BxPc3 cells express TRPC1, 4, and 6 isoforms.TGFβ induced an increase in [Ca2+]cyt in Ca2+-free solutions and also induced an increasein [Ca2+]cyt when extracellular Ca2+ was restored. TGFβ-induced Ca2+ entry involves theactivation of TRPC, as the TRPC inhibitors 2-APB, SKF96365, and LaCl3, were able toreduce peak [Ca2+]cyt by 80%, 40%, and 29%, respectively, and reversed TGFβ-inducedPTEN downregulation. The 2-APB, SKF96365, and LaCl3 inhibitors also reduced TGFβ-induced cell motility. Using siRNA, inhibition of TRPC1 but not other isoforms, successfullyprevented TGFβ-induced cell motility. Conclusion. TRPC1 regulates TGFβ-induced downre-gulation of PTEN and pancreatic cell motility. This aspect of TGFβ regulation of PTEN andpancreatic cell motility might be exploited therapeutically to control pancreatic cancer meta-stasis.

Mo1929

CITED2 is a Novel Direct Effector of Peroxisome Proliferator-ActivatedReceptor Gamma in Suppressing Hepatocellular Carcinoma Cell GrowthKin-Fai Cheung, Junhong Zhao, Alfred S. Cheng, Joseph J. Sung, Jun Yu

Background: We previously reported that activation of peroxisome proliferator-activatedreceptor γ (PPARγ) by PPARγ agonist suppressed hepatocarcinogenesis both In Vitro and InVivo [Yu et al, Hepatology 2006, 2010]. To elucidate the molecular basis of PPARγ action,we extended to explore PPARγ downstream effectors involved in the tumor suppressiveeffect in hepatocelullar carcinoma (HCC). Aim: To identify molecular targets and potentialsignaling pathways with anti-tumor effect regulated by PPARγ in HCC. Methods: PPARγactivation was induced by PPARγ agonist (rosiglitazone) in HCC cell line (Hep3B). PrecisePPARγ binding activity was determined by enzyme-linked immunosorbent assay. The PPARγdownstream targets were identified through gene expression profiling using 44K oligo array.PPARγ-bound target was determined by chromosome immunoprecipitation (ChIP)-PCR. Theanti-tumor effect of Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminaldomain, 2 (CITED2) was evaluated by colony formation assay and cell cycle analysis byflow cytometry. CITED2 target signal pathway was identified by human cancer pathwayPCR array. Results: Optimal PPARγ binding activity was obtained with 100 μM rosiglitazonefor 3 hours. Under PPARγ activation, 329 genes were up-regulated and 245 genes down-regulated using oligo array. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathwaydatabase indicated that PPARγ-associated genes mainly involved in the pathways includingMAPK, TGF-beta, p53, and ECM receptor interaction. CITED2 was found to be the mostpredominant PPARγ-bound target by ChIP-PCR. Induced expression of CITED2 was alsoobserved after adenovirus-PPARγ transduction by Western blot. In nine primary HCCs,CITED2mRNAwas significantly down-regulated compared to the adjacent non-tumor tissues(P<0.05). The biological function of CITED2 was evaluated by loss of gene function assay.Knockdown of CITED2 was obtained in a nontumorigenic hepatocyte cell line (LO2) bysh-CITED2. Such knockdown dramatically increased the ability of LO2 to form colonies(P<0.001) and promoted S-phase DNA synthesis (22.6% vs 25.2%, P<0.01). Furthermore,knockdown CITED2 caused the upregulation of proliferative regulator (TEK), pro-invasion/metastasis genes (MMP2, SERPINB5), anti-apoptotic genes (TNF, TERT), and downregulationof proliferative inhibitor (INFA1) and pro-apoptosis genes (TNFSF1A, TNFRSF25, CASP8,GZMA, PARP). Conclusions: CITED2 is a novel direct effector of PPARγ in inhibiting HCCgrowth and proliferation. The investigation of the regulation and function of CITED2 willreveal insights into the hepatocarcinogenesis and provide novel target for treatment of HCC.

Mo1930

Decreased Liver Tumorigenesis in Sulfatase 2 (SULF2) Knockout Mice May BeMediated Through Inhibition of the Epithelial-Mesenchymal Transition (EMT)Ikuo Nakamura, Michael K. Asiedu, Keith Knutson, Kadra H. Ahmed, Catherine D.Moser, Chunling Hu, Lewis R. Roberts

Background and Aims: Sulfatase 2 (Sulf2) plays a critical role in tumorigenesis of hepatocellu-lar carcinoma In Vivo. Analysis of genes expressed in association with SULF2 in humanHCCs suggests that SULF2 is associated with the epithelial to mesenchymal transition(EMT) during liver carcinogenesis. We determined the effect of knockout of Sulf2 on livercarcinogenesis in mice. Methods: Liver carcinogenesis was induced in wild-type (WT) orSulf2 knockout (Sulf2-KO) mice using the liver carcinogen diethylnitrosamine (DEN). Weexamined tumor size and number, DNA synthesis, the expression of cytokines and growthfactors, BrdU staining as a measure of cell proliferation, E-cadherin staining as a marker ofthe epithelial cell phenotype, and Slug and Vimentin as markers of the mesenchymal cellphenotype. We also stained liver sections with β-catenin to study the relationship betweenSULF2 and Wnt signaling. Results: Knockout of Sulf2 decreased the size and volume of

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DEN-induced HCC tumors at 8 months after DEN injection. The number of BrdU-positivenuclei was significantly decreased in Sulf2-KO mice compared with WT mice. E-cadherinstaining was stronger in tumors of Sulf2-KO mice than in tumors induced in WT mice. Onthe other hand, staining of the EMT markers Slug and Vimentin was stronger in tumorsoccurring in WT mice than in Sulf2-KO mice. In addition β-catenin immunostaining wasstronger in nuclei of tumors occurring in WT mice than in Sulf2-KO mice. Conclusions:Knockout of Sulf2 reduced DEN-induced liver tumorigenesis in mice. The decrease in livertumorigenesis in Sulf2-KO mice was associated with a more epithelial phenotype, suggestingthat Sulf2 plays a role in the EMT during liver carcinogenesis.

Mo1931

Clinical Outcome of Polypoid Lesion of Gallbladder 10 mm or LargerWon Jae Yoon, Yong-Tae Kim, Jaihwan Kim, Dong-Won Ahn, Ji Kon Ryu, Yong BumYoon

Background and Aim: Polypoid lesions of the gallbladder (PLGs) are detected more frequentlydue to increased use of abdominal ultrasonography (US). The significance and appropriatemanagement of PLGs are not well understood. Most reports are based on surgical series.The aims of this study was to evaluate the clinical outcome of PLGs with diameter ≥ 10mm. Materials and Methods: We analyzed the data of patients who were diagnosed withPLGs with diameter ≥ 10 mm by US, endoscopic ultrasonography (EUS), and/or computedtomography between 2000 and 2008 at Seoul National University Hospital and were followedup for ≥ 6 months or underwent surgery. Size increase was defined as diameter increase> 3 mm. Data was collected until December 31st, 2009. Results: Two hundred sixty-ninepatients (131 men, median age at PLG diagnosis 49 years) were collected. Mean diameterof the PLG was 13 mm. Solitary PLG was diagnosed in 153 patients; sessile PLGs werediagnosed in 76 patients. Coexisting gallstone was detected in 36 patients. One hundredeighty-one patients underwent surgery. In the 88 patients who did not undergo surgery(median follow-up period 23.8 months), only 3 patients demonstrated size increase of thePLG, with 2 patients developing malignancy; these patients showed size increase at 1 year.Of 188 patients who underwent surgery, 59 (31.4%) had neoplastic polyps (42 adenoma,16 adenocarcinoma, and 1 lymphoma) In univariate analysis of factors associated withmalignant PLG, age at diagnosis ≥ 50 years (P=0.035), initial PLG diameter ≥ 12 mm (P=0.003), sessile PLG (P=0.016) were associated with malignancy; hyperechoic spots on USor endoscopic ultrasonography (EUS) was negatively associated with malignant PLG(P<0.001). In multivariate analysis, age at diagnosis ≥ 50 years (OR 3.72, 95% CI 1.09-12.69; P=0.036) and initial PLG diameter ≥ 12 mm were associated with malignancy;hyperechoic spots on US or EUS was negatively associated with malignant PLG. In the 38patients with initial diameter of PLG < 12 mm and hyperechoic spots on PLG, no malignantPLG was found. Conclusion: In patients with PLG diameter ≥ 10 mm, malignancy shouldbe expected when PLG size increases (> 3 mm) at 1 year. Even in patients with PLG diameter≥ 10 mm, the risk of malignancy seems low in patients with initial diameter of PLG < 12mm and hyperechoic spots on PLG.

Mo1932

Different Expression of E-Cadherin and β-Catenin in Intestinal Type andPancreatobiliary Type of Ampulla Vater CancerSeon Mee Park, Joung-Ho Han, Soon Man Yoon, Hee Bok Chae, Sei jin Youn, Eui KeunSeo, Young Shim Cho, Rohyun Sung

Background and aims: Ampulla vater cancers(AVC) are classified histologically as an intestinaltype and a pancreatobiliary type. The molecular pathogenesis of both histologic types arenot well characterized. We aimed to investigate the expression patterns of E-cadherin andβ-catenin in both histologic types and the relations with clinicopathologic features of AVC.Methos: Twenty-six resected AVC specimens were stained with antibodies to E-cadherin andβ-catenin. Normal expression was defined as exclusive membraneous staining. Dysregulatedexpression was defined as cytoplasmic staining in more than 50% of tumor cells and/ornuclear staining. Demographics and histopathological data were collected by retrospectivechart review. Results: Intestinal type was noted in 12(46%) cases and pancreatobiliary typein 14(54%) cases. Preserved membranous staining of E-cadherin was frequent in intestinaltype than in pancreatobiliary type(83% vs. 29%; p=0.008). E-cadherin expression was wellpreserved in early stages(p=0.039), well differentiated type(p=0.034) and histologic featuresof intestinal type-pseudostratification(p=0.007) and carcinomas with adenoma compon-ent(p=0.014). Preserved membranous staining of β-cadherin was frequent in pancreatobiliarytype than in intestinal type(75% vs. 25%, p=0.039). Aberrant cytoplasmic and/or nuclearstaining of β-cadherin was prominent in the histopathologic features of intestinal type,such as pseudostratification(p=0.004) and carcinomas with adenoma component(p=0.027).Conclusions: Dysregulation of E-cadherin and β-cadherin expression may play a role differ-ently in the carcinogenesis of AVC, E-cadherin in pancreatobiliary type and β-cadherin inintestinal type.

Mo1933

Neoadjuvant Chemoradiation Therapy for Pancreatic Cancer is Enhanced byEarly Placement of Uncovered Self-Expanding Biliary Metal Stents (SEMS)John Y. Nasr, David Y. Lo, A. James Moser, Michael Sanders, Adam Slivka, Herbert Zeh,Georgios I. Papachristou, Jennifer Lewis, Eric Wright, Gennadiy Bakis, RameshSrinivasan, Abby Crume, Lisa A. Rutstein, Douglas A. Howell

Background: Neoadjuvant chemoradiation (NeoRx) for pancreatic cancer obviates surgeryfor patients with unrecognized micrometastases and may improve 5 year survival (Evans,JCO 2008). Reports of frequent plastic biliary stent occlusion during NeoRx suggests a rolefor initial SEMS placement. Methods: We evaluated outcomes of pancreatic cancer patientstreated with uncovered biliary SEMS and NeoRx at two referral centers since 2006. NeoRxconsisted of 6-8 weeks of Gemcitabine-based RT and 4 weeks of recovery prior to plannedsurgical resection. Results: 77 patients (38 males: 39 females) with age range 37-86 were