Su1000

Enhanced Liver Tumorigenesis in Sulfatase 1 (SULF1) Transgenic Mice IsAssociated With Activation of the Wnt-β-Catenin Signaling PathwayChunling Hu, Shaoshan Han, Catherine D. Moser, Abdul M. Oseini, Dongye Yang, LewisR. Roberts

Background and Aims: Using in vitro cell culture and in vivo xenograft mouse models, wehave previously shown that the heparan sulfate-degrading endosulfatase SULF1 functionsas a tumor suppressor in established hepatocellular carcinoma cell lines by abrogatingreceptor tyrosine kinase signaling. In contrast, whole genome gene expression analysis ofresected human HCCs showed increased expression of SULF1 in 70% of primary HCCs;and SULF1 expression in primary HCCs has been associated with activation of the oncogenicWnt/β-catenin pathway. Here we report for the first time the results of DEN-inducedhepatocarcinogenesis in a hepatocyte-specific transgenic Sulf1 mouse model in which theSulf1 gene is driven by the transthyretin promoter. The effect of Sulf1 transgenesis on Wnt/β-catenin activation was assessed. Methods: Liver carcinogenesis was induced in wild-typeor hepatocyte-specific Sulf1 transgenic mice by intraperitoneal injection of 15 mg/kg of theliver carcinogen diethylnitrosamine (DEN) at 14 days after birth. Mice were sacrificed at 8months of age and the harvested livers were examined for weight, tumor size and number.Cell proliferation was assessed using the BrdU proliferation index and Wnt/ β-catenin activa-tion was measured by assessment of β-catenin staining and cytoplasmic-nuclear translocationof β-catenin. Results: Consistent with the observation of increased SULF1 in primary humanHCCs, Sulf1 transgenic mice showed increased liver tumorigenesis as demonstrated by tumornumber, tumor volume, liver weight/body weight ratio, and BrdU proliferation index. Toinvestigate the mechanism for this, we performed immunohistochemistry for β-catenin.Wnt-β-catenin signaling was increased in Sulf1 transgenic mice compared to wild-type mice,as assessed by both increased β-catenin immunostaining and increased cytoplasmic-nucleartranslocation of β-catenin. Conclusions: In contrast to the tumor suppressor effects of SULF1in established human cell lines, which are mediated by inhibition of the co-receptor functionof heparan sulfate in receptor tyrosine kinase signaling, primary HCCs in an in vivo modelof DEN-induced HCC in Sulf1 transgenic mice show increased liver tumorigenesis associatedwith activation ofWnt-β-catenin signaling. This suggests that the effect of Sulf1 in carcinogen-esis is context-dependent, but appears to be mediated through oncogenic Wnt- β-cateninsignaling in the majority of HCCs.

Su1001

Circulating Scca-IgM Complex Is an Useful Biomarker to Monitor HCCTherapyFilomena Morisco, Giovan Giuseppe Di Costanzo, Maria Guarino, Raffaella Tortora, IlariaLoperto, Concetta Tuccillo, Carmine Ferraiuoli, Luca Beneduce, Francesco Auriemma,Nicola Caporaso

Background and aims Every year hepatocellular carcinoma (HCC) develops in about 3-4%of cirrhotic patients. The serpin squamous cell carcinoma antigen (SCCA) was found elevatedin liver cancer specimens and circulating SCCA-IgM complexes have been described inserum of patients with HCC. This study aimed to evaluate the ability of SCCA-IgM serumlevels to monitor the efficacy of HCC therapy. Methods From April 2011 to July 2012, 106patients (M/F 81/25, median age: 68 years, range 41-85) with a new diagnosis of HCC wereenrolled in two referral centres in a prospective study. The diagnosis of HCC was madeaccording to the AASLD 2010 guidelines. The patients were divided in two groups: GroupA, including 64 patients (BCLC stage A and B) treated with loco-regional therapy (RF, LTA,PEI, TACE), and Group B including 42 patients (BCLC stage B and C) treated with Sorafenib(800mg/die). Response to therapy was evaluated with imaging techniques according to themRECIST criteria. Serum SCCA-IgM levels were determined by the Hepa-IC kit (XeptagenSpA, Venezia, Italy) at basal time, after 1 and 3 months from the beginning of treatment.The quantization of the complex SCCA-IgM is expressed in Arbitrary Units(AU)/ml. ResultsAt the time of diagnosis, SCCA-IgM was reactive in 59/106 patients (56%), mean±SD:274.4±263.2 AU/ml. In particular SCCA-IgM were positive in 34/64 (53%) patients in GroupA and 25/42 (60%) patients in Group B mean±SD: 258.7±280.5 AU/ml and 295.1±242.5AU/ml, respectively. According to mRECIST criteria, 29/34 (85%) patients in Group A and16/25 (64%) in Group B showed a response to therapy at 1 and 3 months of follow-up,respectively. In responder patients of Group A the SCCA-IgM mean value decreases from263,8 AU/ml at T0 to 229.7 AU/ml at T1 (p ,0,05); in responder patients of Group B itdecreases from 276,7 AU/ml at T0 to 190,3 AU/ml at T3 (p ,0,001). In non-responderpatients SCCA-IgM levels remain stable during the follow-up. Conclusion These resultssuggest that the serial determination of SCCA-IgM complex may be helpful in monitoringthe outcome of therapy in patients with HCC.

Su1002

Hepatocellular Carcinoma Derived Colony Stimulating Factor-1 PromotesArginase Expression and Activity in Tumor Associated MacrophagesAbdul Rehman, Priyanka Thakur, Michael Duncan

Tumor-associated macrophages are an important component of the tumor microenvironmentand play a crucial role in hepatocellular carcinoma (HCC) development and prognosis. Pro-inflammatory (M1-type) macrophages enhance antitumor immunity while M2-type macro-phages are immunosuppressive and promote cancer through tissue remodeling and angiogen-esis. These cells demonstrate a high degree of plasticity and can revert from one phenotypeto another under appropriate stimuli. Tumor-derived factors including colony stimulatingfactor-1 (CSF-1) promote macrophages towards an M2 phenotype and may potentiatetumor growth and metastasis. It is well established that CSF-1 signaling is critical in thedifferentiation, survival and function of macrophages. The enzyme arginase is highlyexpressed in tumor-associated macrophages and plays a significant role in the immunosup-pressive activity of these cells by catalyzing the conversion of the amino acid L-arginine toL-ornithine and urea. We examined whether CSF-1 signaling influences arginase activityand hypothesized that CSF-1 regulates arginase expression and activity in tumor associated

S-991 AASLD Abstracts

macrophages. In order to test our hypothesis we developed an in vitro model of paracrine-mediated differentiation of murine bone marrow cells to macrophages. We treated murinebone marrow derived cells with condition media derived from murine hepa 1-6 HCC cellline and converted a significant fraction of these cells to f4/80 positive macrophages. Thesecells highly expressed M2-type markers and were largely negative for M1-type markers.Importantly, we observed a significant increase in arginase gene expression in our differen-tiated macrophage population. In order to determine the role of CSF-1 and its receptor-mediated pathways in arginase gene expression and activity, we selectively blocked CSF-1receptor as well as the phosphatidylinositol 3-kinases (PI3K) and p38 mitogen activatedkinase (p38MAPK) pathways using selective small molecule inhibitors. We observed signifi-cant decrease in arginase gene expression by quantitative real time PCR when CSF-1 receptorand the PI3K and p38MAPK pathways were inhibited. Also, an arginase enzymatic assayshowed reduced activity of arginase with the inhibition of CSF-1 as well as downstreamPI3K and p38MAPK signaling pathways. This data clearly demonstrates a critical role forCSF-1 signaling in arginase gene expression and enzymatic activity and provides a potentiallink between the CSF-1 pathway and immunosuppressive activity of tumor-associated macro-phages.

Su1003

Role of Activated Hepatic Stellate Cells in Metastasis of HepatocellularCarcinomaNan Lin, Xu Linan

Objective: The presence of liver cirrhosis is the main risk factor for the development ofhepatocellular carcinoma (HCC). Activated hepatic stellate cells are recognized as a centralevent in the development of liver fibrosis, but which role in HCC is unclear. This studyinvestigated angiogenetic activity of activated hepatic stellate cells in HCC and angiogeneticeffect on proliferation, metastasis of HCC. Methods: In vitro, we exposured microvascularendothelial cells to conditioned media(CM) from activated hepatic stellate cells to observethe influence of the CM on microvascular endothelial cells by quantifying microvascularendothelial cell tube formation, and placed aHSC over an endothelial monolayer to assessestransendothelial migration of activated hepatic stellate cells to hepatoma cells by examiningthe movement of CFSE labeled activated hepatic stellate cells through the endothelial cellmonolayer. For convenient viewing, microvascular endothelial cell and activated hepaticstellate cells were labeled for CFSE (one green fluorescent dye). In vivo, we establishedorthotopic model of HCC with nude mice receiving an intraliver injection of activated hepaticstellate cells plus hepatoma cells. After 7 weeks, tumor size, number of metastases weremeasured. Furthermore, tumors tissue were ashahsessed by immunostaining for expressionof activated hepatic stellate cells and microvessel. Angiogenesis activities were assessed byimmunostaining tumors for CD34, an endothelial cell marker and vascular endothelialgrowth factor (VEGF). Result: In vitro, The CM from activated hepatic stellate cells stimulatedthe tube formation by microvascular endothelial cell , and hepatoma cells stimulated transen-dothelial migration of activated hepatic stellate cells . In vivo, compared with mice receivinghepatoma cells alone, mice injected with hepatoma cells plus aHSC exhibited the mostincreased tumor size and regional and distant metastasis. CD34 and VEGF in primary tumorsofmice injectedwith hepatoma cells plus activated hepatic stellate cells were higher expressioncompared to hepatoma cells alone. Activated hepatic stellate cells and microvessel were bothlocated around nests in serial sections of HCC. The CFSE labeled activated hepatic stellatecells were found in multiple metastatic sites in each mice injected with activated hepaticstellate cells plus hepatoma cells. Conclusion: Activated hepatic stellate cells promote prolifer-ation, metastasis of HCC through angiogenesis, and spreaded to other parts of the bodyaccompanying with HCC cells continuing with playing a role in metastatic tumor.

Su1004

High Mobility Group Box-1 Can Increase miR-21 Expression in HepatocellularCarcinoma Through STAT3 SignalingMan Chen, Ying Chang, Hai Huang, Jusheng Lin, Allan Tsung

Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwideand is associated with a very poor prognosis. The discovery of microRNA (miRNA) hassignificantly altered traditional concepts of gene regulation and miRNAs have emerged asnovel modulators of gene expression in cancer. As an oncomir, the over-expression of miR-21 has been reported in many cancers including HCC. However, the role and regulationof miR-21 in HCC are not elucidated. High mobility group box 1 (HMGB1) is a prototypicalDamage-Associated Molecular Pattern molecule (DAMP) with multiple extracellular func-tions, including modulation of inflammation and immunity. We hypothesize that HMGB1induces the expression of miR-21 in HCC to promote tumorigenesis. Methods: Human HCCcell lines (Huh7, HepG2, Hep3B) and normal human hepatocytes were stimulated withvarying doses of recombinant HMGB1 (rHMGB1) or treated with neutralizing antibody toHMGB1. Signal transducer and activator of transcription 3 (STAT3) signaling was inhibitedusing the STAT3 inhibitor wp1066 to examine its role inHMGB1-mediatedmiR-21 induction.miR-21 expression was quantitated by Reverse transcription and TaqMan real-time polymer-ase chain reaction (Taqman qRT-PCR). Protein expressions of HMGB1, total STAT3, andSTAT3 phosphorylation status at Tyr 705 (pSTAT3) were assessed by Western blot analysis.Results: miR-21, HMGB1 and pSTAT3 were all significantly up-regulated in HCC cell lines(Huh7, HepG2 and Hep3B) compared with normal hepatocytes. We also detected thesignificant over-expression of miR-21, HMGB1 and pSTAT3 in 20 pairs of HCC tumorscompared to their matched adjacent non-tumor tissues (P.0.05). To determine if HMGB1regulated miR-21 expression, rHMGB1 was used to stimulate the various HCC cell lines.Interestingly, miR-21 was up-regulated more than 3-fold of basal expression at 24 hourswith 1ug/ml rHMGB1 stimulation. This increase of miR-21 expression was inhibited whenneutralizing antibody to HMGB1 was used to treat the same cells. To determine the mecha-nism of HMGB1-mediated miR-21 regulation, we examined STAT3 signaling. Western blotanalysis for pSTAT3 demonstrated that with rHMGB1 treatment, pSTAT3 expression wasunregulated about 1.5-2.5 fold compared to total STAT3, while treatment with neutralizingantibody to HMGB1 inhibited the induction of pSTAT3. In addition, using the STAT3

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sinhibitor wp1066, miR-21 expression was suppressed in HMGB1 treated cell lines. Conclu-sions: As a damage-associated molecular pattern molecule, extracellular HMGB1 can activatemiR-21 in HCC through STAT3 signaling. This study provides new insights into the interac-tion of inflammatory mediators andmiRNA regulation of oncogenic pathways in HCC tumori-genesis.

Su1005

Type XVIII Collagen Promotes Hepatocellular Carcinoma Cell AdhesionTrey Forehand, Priyanka Thakur, Michael Duncan

It is estimated that 80 to 90% of HCC results from cirrhotic liver disease characterized, inpart, by the accumulation and extensive remodeling of the hepatic extracellular matrix.While these stromal changes correlate strongly with HCC progression and hamper therapeuticoutcomes, few studies provide insights on the role that components of the extracellularmatrix play in promoting HCC progression. Type XVIII collagen (ColXVIII) is a prominentliver extracellular matrix (ECM) component. Recent analysis of transgenic de novo mousemodels as well as other studies in human specimens suggests divergent expression of themajor variants of type XVIII collagen. The longer variants are decreased in HCC while theshort variant is increased. The functional consequences of increased short variant type XVIIIcollagen remain largely unknown. Because of its known role as an extracellular matrixprotein, we hypothesized that HCC tumor derived type XVIII collagen is increased and playsa critical role in HCC cell adhesion. Utilizing an orthotopic murine model of hepatocellularcarcinoma we validated that the short variant of type XVIII collagen is significantly overex-pressed in tumors. This tumor derived type XVIII collagen showed strong co-localizationwith other extracellular matrix proteins including laminin and fibronectin.We over-expressedthe short chain type XVIII collagen in HEK293T cells plated on various ECM substrataincluding laminin, type IV collagen, and fibronectin. This over-expression resulted in deposi-tion of type XVIII collagen in these substrata and on the cell surface. Collectively, theseresults suggest that the short variant of type XVIII collagen is an ECM associated protein.To determine a functional role for type XVII collagen, we utilized an RNA interferenceapproach in an HCC cell line, Hepa1-6. Lentiviral mediated overexpression of a short hairpinRNA sequence resulted in a significant decrease in type XVIII collagen gene expressiondetermined by quantitative real time PCR and protein levels as determined by Western blot.We observed significant morphological differences in Hepa1-6 with reduced type XVIIIcollagen. These cells appeared unable to establish firm adhesion in culture. Using phalloidinfluorescence staining, we also observed significant defects in the ability of these cells toassemble F-actin. We next assessed the loss of type XVIII collagen on cell adhesion. Knock-down of type XVIII collagen resulted in a significant loss of cell adhesion by the hepa1-6to various ECM substrates including type I collagen and type IV collagen. These resultspoint to a critical role for type XVIII in HCC adhesion to the ECM and suggest that thismolecule may be an important component of the HCC tumor microenvironment.

Su1006

Nitrosative Stress Marker (iNOS) Is Positively Correlated With HepatocellularCarcinoma Tumor Grade, Oxidative Damage Marker (O8HdG) and HighMetabolic Marker (HK2) LevelsBryan Rea, Rohini Chennuri, Ada Quintana, Nissim Hay, Scott Cotler, Grace Guzman

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths and likelydevelops on a continuum from cirrhosis to dysplasia to cancer. We have previously reportedincreased expression of HK2, a marker for increased metabolic activity, and 8-OHdG, amarker for reactive oxygen species, across the progression of HCC. iNOS, a marker fornitrosative stress, has also been implicated in carcinogenesis. This study aims to correlateexpression of iNOS with expression of HK2 and to examine the relationship of iNOS, HK2,and 8-OHdG with grade, stage, and type of HCC. Method: A liver tissue array containing45 well characterized subjects with cirrhosis plus HCC and 8 normal controls was analyzed.Areas of cirrhosis, dysplasia, and HCC were examined by standard immunohistochemistryto determine levels of iNOS, HK2, and 8-OHdG; staining was analyzed by Aperio ImageAnalysis. Regression analysis, Kruskal-Wallis, Mann-Whitney, and Friedman tests wereemployed using SPSS 20. Results: Mean levels of iNOS expression was significantly increasedin areas of HCC compared to areas of cirrhosis (p ,.05). Strong positive correlation wasfound between iNOS and HK2 in areas of cirrhosis (R=.812;p ,.001), dysplasia (R=.797;p,.001), and HCC (R=.674;p,.001). In areas of HCC, high grade tumors had higherlevels of expression of HK2 and 8-OHdG compared to medium and low grade tumors (HK2,p,.05;8-OHdG, p,.001). In areas of dysplasia, Stage 3 and 4HCCs had increased expressionof HK2 and iNOS compared to Stage 1 and 2 HCC (HK2, p ,.01; iNOS, p,.05). Poorlydifferentiated pleomorphic type HCC had higher levels of iNOS and HK2 expression thannon-pleomorphic type HCC (HK2, p,.01; iNOS, p,.05). Conclusion: These results showthat iNOS expression is significantly increased in HCC lesions compared to pre-cancerouslesions. iNOS is also strongly correlated with HK2 expression, which provides evidence thatnitrosative stress is associated with areas of increased metabolic demand. Furthermore, thesebiomarkers are also correlated with higher grade tumors, higher stage, and the poorlydifferentiated pleomorphic type of HCC, indicating the possibility that these markers mayhave prognostic and clinical significance in the pathology of HCC. This pilot study has asmall sample size. A larger cohort will be necessary to determine whether anti-ROS and/oranti-nitrosative stress therapeutics could play a role influencing outcomes of liver carcinogen-esis.

S-992AASLD Abstracts

Su1007

High Mobility Group Box 1 Mediates Tumor Growth Through Toll-LikeReceptor 9Wei Yan, Yu Fu, Dean Tian

High-mobility group box 1 (HMGB1) is an evolutionarily conserved, chromatin-bindingprotein that has been implicated in several disease states, including sepsis, arthritis, ischemia-reperfusion injury, and cancer. Overexpression of nuclear Damage Associated MolecularPattern (DAMP) molecule HMGB1 is associated with tumor progression. HMGB1 is anevolutionarily conserved and critical regulator of cell death and survival; however, theexact mechanisms related to HMGB1-induced tumor growth remains unclear. Our findingselucidate how HMGB1, when released under hypoxic stress, can promote tumor growth inhepatocellular carcinoma (HCC) cells. Overexpression of HMGB1 enhanced cell proliferationwhile stable knockdown of HMGB1 remarkably reduced proliferation in HCC cells. Inaddition, during hypoxia, HMGB1 translocated to the cytosol and bound to mitochondrialDNA (mtDNA) released from mitochondria. This translocation subsequently activated Tolllike Receptor 9 (TLR9) signaling pathways. We further demonstrate that treatment with TLR9agonists increased HCC cell proliferation while treatment of TLR9 antagonist significantlydiminished cell proliferation. Moreover, TLR9 antagonists suppressed HCC tumor growthin vivo. In all, these results suggest that in hypoxic HCC cells, HMGB1 binds mtDNA andsubsequently activates TLR9 signaling pathways to induce tumor growth.(This work issupported by Grants from National Science Foundation of China,No. 81000928 andNo.81000159)

HMGB1 promotes tumor growth in vivo

HMGB1 binds TLR9 in cytoplasm of hypoxic HCC cells

Su1008

Netrin-1 Induces Epithelial-Mesenchymal Transition and PromotesHepatocellular Carcinoma InvasivenessWei Yan, Yu Fu

Hypoxia is often found in solid tumors and is associated with tumor progression and poorclinical outcomes. The exact mechanisms related to hypoxia-induced invasion and metastasisremain unclear. We elucidated the mechanism by which the neuronal guidance molecule,Netrin-1, released under hypoxic stress can induce epithelial-mesenchymal transition (EMT)to promote invasion and metastasis in hepatocellular carcinoma (HCC) cells. EMT inductionwas found to occur in hypoxic HCC cells in a process that was dependent on the extracellularrelease of Netrin-1 and subsequent activation of DCC signaling pathways. We further demon-strate that overexpression of Netrin-1 or treatment with recombinant Netrin-1 enhancedinvasiveness of HCC cells while stable knockdown of Netrin-1 remarkably reduced HCCinvasion. Conclusion: These results suggest that in hypoxic HCC cells, Netrin-1 activatesDCC signaling pathways to induce EMT activation with subsequent production of multipleinflammatory mediators which in turn promote cancer invasion.(This work is supported byGrants from National Science Foundation of China,No. 81000928 and No.81000159)

Su1009

Virtual Palpation Model - Combining Spiral CT and Elastography Data: AProof-of-Concept StudyCostin T. Streba, Ioana Andreea Gheonea, Larisa D. Sandulescu, Adrian Saftoiu, DanIonut Gheonea

Introduction and aims Palpation traditionally involves the examination of a patient throughdirect contact, by using the sense of touch thus attempting a diagnosis through the feel ofthe area examined. Elasticity imaging methods combine some forms of tissue excitation withmethods for detection of tissue response. Medical simulations are now widely viewed as apotential solution to many of the problems associated with medical training. Our aim was