Article first published online: 15 OCT 2013
Copyright © 2013 American Association for the Study of Liver Diseases
Special Issue: The 64th Annual Meeting of the American Association for the Study of Liver Diseases: The Liver Meeting 2013
Volume 58, Issue S1, pages 1057A–1091A, October 2013
How to Cite
(2013), Experimental Hepatocarcinogenesis. Hepatology, 58: 1057A–1091A. doi: 10.1002/hep.26879
- Issue published online: 1 OCT 2013
- Article first published online: 15 OCT 2013
High frequency of telomerase reverse transcriptase (tert) promoter somatic mutations in hepatocellular carcinoma and pre-neoplastic lesions
Jean-Charles Nault1, Maxime Mallet1, Camilla Pilati1, Julien Calderaro2, Paulette Bioulac-Sage3, Christophe Laurent4, Alexis Laurent5, Daniel Cherqui6, Charles Balabaud7, Jessica Zucman-Rossi1;
1Inserm U674, Paris, France; 2InsermU674, Pathology department, Henri mondior, Creteil, France; 3Pathology department CHU, Bordeaux, France; 4Surgery department CHU, Bordeaux, France; 5Surgery department CHU Henri Mondor, Creteil, France; 6Surgery department Hepatobiliary center Paul Brousse, Villejuif, France; 7INSERM U1053, Bordeaux, France
Introduction: TERT is involved in the maintenance of telomere length and its reactivation is required in liver tumorigenesis. Recently, somatic mutations of the TERT promoter leading to TERT activation were identified in melanoma. We aim to search for TERT promoter mutations in hepatocellular tumors and study its role in cirrhotic (macronodules without and with dysplasia-hepatocellular carcinoma (HCC) sequence) and non-cirrhotic (hepatocellular adenoma (HCA)-HCC sequence) multistep car-cinogenesis. Methods: We sequenced the TERT promoter in 305 HCC, 20 cirrhotic macronodules, 60 classical HCA and 16 HCA with signs of malignant transformation. These tumors were characterized at clinical and histological level and for the classical genes involved in liver tumorogenesis. We assessed TERT expression using quantitative RT-PCR in 309 hepatocellular tumors and non-tumor liver tissues. Results: we identified somatic mutations of the TERT promoter in 179 (59%) among 305 human HCC. These mutations were localized at the two hot spot described in melanoma (-124G>A and -146G>A from the ATG site). TERT promoter mutations were significantly associated with CTNNB1 mutations (P<0.0001) and were more frequent in small HCC (< 5cm), with low serum AFP level and non-related to chronic HBV infection. TERT expression was significantly higher in HCC with TERT promoter mutations compared to normal liver and cirrhosis (P=0.0007). Additionally, we identified TERT promoter mutations in 25% (5/20) of cirrhotic macronodules with or without dysplasia. In contrast, we didn't found any mutations among 15 genes (CTNNB1, TP53...) classically mutated in HCC and screened in these pre-neoplastic lesions. Interestingly, cirrhotic macronodules mutated for the TERT promoter exhibited an increase TERT expression compared to macronodules without TERT promoter mutations (P=0.004). Among 60 classical HCA of different molecular subtypes we didn't identified any mutations of the TERT promoter. In contrast, 7 among 16 HCA with malignant transformation (44 %) harbored TERT promoter mutations that were systematically associated with CTNNB1 mutations. Conclusion: TERT promoter mutations are the most frequent somatic genetic alterations observed in HCC. It is also the first recurrent somatic genetic alterations identified in cirrhotic preneoplastic macronodules suggesting that TERT promoter mutations is an early genetic event in the multistep process of cirrhotic carcinogene-sis. In contrast, TERT promoter mutations is not useful to promote initially benign liver tumorogenesis on normal liver but is required at the last step of malignant transformation of HCA in association with CTNNB1 mutations.
Jessica Zucman-Rossi - Consulting: pfizer; Grant/Research Support: Integragen; Speaking and Teaching: bayer, lilly
The following people have nothing to disclose: Jean-Charles Nault, Maxime Mallet, Camilla Pilati, Julien Calderaro, Paulette Bioulac-Sage, Christophe Laurent, Alexis Laurent, Daniel Cherqui, Charles Balabaud
PDGF-BB Induces Apoptotic Priming of Cancer-Associated Fibroblasts in Cholangiocarcinoma
Sumera Rizvi1, Joachim C. Mertens1,2, Steven F. Bronk1, Nathan W. Werneburg1, Haiming Dai3, Lewis R. Roberts1, Scott H. Kauf-mann3, Gregory J. Gores1;
1Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN; 2Division of Gastroenterology and Hepatology, University Hospital of Zurich, Zurich, Switzerland; 3Division of Oncology Research, Mayo Clinic, Rochester, MN
Background and Aims: PDGF-BB secreted by cholangiocarcinoma (CCA) cells recruits cancer associated myofibroblasts (CAF) into the tumor microenvironment. These CAF have been implicated in the aggressive biology of this difficult to treat cancer. CAF display an activated phenotype that renders the cells sensitive to proapoptotic stimuli. The novel BH3 mimetic navi-toclax, which induces cell death in cells primed for apoptosis, inhibits tumorigenesis in an animal model of CCA by depleting CAF from the tumor microenvironment. However, the relationship between recruitment of CAF by PDGF-BB and their primed state is unclear. Thus, our aim was to examine the role of stro-mal PDGF-BB in cellular activation and apoptotic priming of CAF. Methods: Human cholangiocarcinoma specimens were examined by immunofluorescence, Western-Blot and qRT-PCR. Human quiescent fibroblasts (hFB), hepatic stellate cells (HSC) and CCA cell lines were used for in vitro studies. Bax oligomer-ization was examined by size exclusion chromatography using fast protein liquid chromatography. Results: We identified PDGF-BB expression by cancer cells in human CCA specimens by immunohistochemistry (IHC). CAF in human CCA specimens displayed activation of the proapoptotic Bcl-2 effector protein Bax as assessed by IHC employing an activation specific monoclonal antibody. Treatment of quiescent hFB or HSC with PDGF-BB in vitro induced an activated cellular phenotype, resulting in Bax activation, and sensitizing previously quiescent cells to navitoclax-triggered cell killing. In contrast, navitoclax did not induce apoptosis of quiescent hFB. Co-cultivation of hFB or HSC with CCA cells similarly led to apoptotic priming and increased cell death with navitoclax treatment. In addition to the observed Bax activation, profiling of PDGF-treated hFB for Bcl-2 family proteins demonstrated an increase in the proapoptotic BH3 only proteins Bid and PUMA. Bax oligomerization, which occurs after Bax activation during apoptosis, was present in PDGF treated HSC cells. In Conclusion, PDGF-BB secretion by CCA cells activates and sensitizes CAF to navitoclax-induced apoptosis. PDGF appears to mediate Bax activation and oligomerization, likely facilitated by upregulation of PUMA expression. Apoptotic priming of CAF via a receptor tyrosine kinase pathway is a novel mechanism of apoptosis regulation, and should be explored as a therapeutic approach in human CCA.
Lewis R. Roberts -Advisory Committees or Review Panels: Inova; Grant/Research Support: Bristol Myers Squibb, Bayer, Nordion; Speaking and Teaching: Nordion
Gregory J. Gores - Advisory Committees or Review Panels: Bayer, Chugia, Dai-ichi, Generon, Conatus, IntegraGen
The following people have nothing to disclose: Sumera Rizvi, Joachim C. Mertens, Steven F. Bronk, Nathan W. Werneburg, Haiming Dai, Scott H. Kaufmann
Accelerated liver cancer development in Cytoglobin-deficient mice treated with chemicals/diet via the priming of stellate cells and activation of local fibrosis, inflammation, and oxidative stress
Thuy T. Le1, Yoshinari Matsumoto1, Hoang Hai1, Katsutoshi Yoshizato1,2, Norifumi Kawada1;
1Department of Hepatology, Osaka City University, Graduate School of Medicine, Osaka, Japan; 2Academic Advisor's Office, PhoenixBio Co., Ltd., Hiroshima, Japan
Cytoglobin (Cygb) is a 21-kDa globin expressed in hepatic stellate cells (HSC) that functions as a hypoxia sensor and gas carrier. However, its pathophysiological role in vivo remains unclear. Here, we report increased liver cancer development in Cygb-deficient (KO) mice administered either diethylni-trosamine (DEN) or a choline-deficient, L-amino-acid-defined (CDAA) diet that induces hepatosteatosis. Methods: Eight-week-old Cygb KO and wild-type (WT) mice were treated with DEN in drinking water or CDAA and its control, choline-supple-mented, L-amino-acid-defined (CSAA) diet for 8–36 weeks. Macroscopic and microscopic observations were made. Gene expression and intracellular signaling pathways were analyzed. Oxidative stress, antioxidant, and macrophage deletion treatments were also performed in parallel with Cygb siRNA or CYGB overexpressing vectors transfection. Results: Model 1: 25 or 0.05 ppm DEN treatment for 25 or 36 weeks induced liver tumor formation in 1 00 or 43% of Cygb-KO mice, respectively, compared to 44 or 0% of WT mice. In KO mice, there was liver fibrosis, increased inflammatory gene expression and augmented oxidative stress. Model 2: with as little as 8 weeks of CDAA treatment, Cygb-KO mice showed marked steatohep-atitis, which resulted in advanced fibrosis at 16 weeks, compared with the WT. Surprisingly, at 32 weeks, 100% of Cygb-KO mice of both genders developed liver cancer, compared to 0% in WT mice. At all time points, there was severe inflammation associated with activated liver cancer pathways in the Cygb-KO mice. Cygb-KO HSC (HSC isolated from Cygb-KO mice) and Cygb siRNA-transfected WT-HSC were both primed, as indicated by markedly increased expression of αSma, collagen 1α1, Tnfα, Tgfβ1, Il-1β, Il-6, Ccl-2, Ccl-3, and Ccl-4 mRNA and superoxide production compared to controls. Oxidative stress and antioxidant defense PCR arrays showed altered expression of 31 genes involved in the metabolism of reactive oxygen species in Cygb-KO mice. N-acetyl cysteine treatment for 2 weeks eliminated the oxidative stress in CDAA-fed Cygb-KO mice in vivo, and in cultures of isolated Cygb-KO HSC. Macrophage deletion after 8 weeks of CDAA feeding reduced the inflammatory reaction, oxidative stress, and fibrosis in Cygb-KO mice. Moreover, Hep-G2 cells overexpressing CYGB showed decreased proliferation under hypoxia compared with plasmid controls, as well as downregulated expression of fibrosis- and angiogenesis-related genes. Conclusion: Cygb deficiency promotes liver cancer development via HSC priming and augmented inflammation, local fibrosis, and oxidative stress.
The following people have nothing to disclose: Thuy T. Le, Yoshinari Matsumoto, Hoang Hai, Katsutoshi Yoshizato, Norifumi Kawada
Inhibition of micro RNA-214 ameliorates hepatic fibrosis and development of hepatocellular carcinoma in platelet-derived growth factor-C transgenic mice
Hikari Okada1, Masao Honda1, Jean S. Campbell2, Yoshio Sakai1, Taro Yamashita1, Takayoshi Shirasaki1, Kai Takegoshi1, Takuji Tanaka3, Shuichi Kaneko1;
1Gastroenterology, Kanazawa University, Kanazawa, Japan; 2Pathology, University of Washington, Seattle, WA; 3Cancer Research and Prevention (TCI-CaRP), Tohkai Cytopathology Institute, Gifu, Japan
Objective Overexpression of platelet-derived growth factor-C (PDGF-C) in the liver of mice (Pdgf-c Tg) induces hepatic fibrosis, followed by the development of hepatocellular carcinoma. We identified differentially expressed miRNAs in Pdgf-c Tg mice and evaluated their functional relevance in the progression of hepatic fibrosis and the development of HCC. Materials and Method We used TaqMan® Array Rodent MicroRNA A Cards v2.0 containing 384 miRNA assays. miRNAs were knocked down by locked nucleic acid (LNA)-modified antisense oligonu-cleotides (LNA-antimiR) in immortalized human stellate cells, Lx-2. Pdgf-c Tg mice at 32 months of age were injected with LNA-antimiR-214 via the tail vein six times (50 μg each) with 3-week intervals by using Invivofectamine® 2.0. The expression of collagens I and IV, alpha-smooth muscle actin (α-SMA), p-SMAD3, p-AKT and p-ERK etc. was evaluated by immunohisto-chemical staining, real-time PCR, and Western blotting. Results Out of a total of 384 tested miRNAs in the liver of Pdgf-c Tg mice at 24 months of age, miR-214 was most significantly induced (4.17 fold and p=8.39E-5) with the concomitant progression of hepatic fibrosis. LNA-antimiR-214 significantly suppressed gene groups of the cytoskeleton, cell adhesion, and EGFR signaling in Lx-2 cells. In Pdgf-c Tg mice, 5'-FAM-labeled LNA-antimiR-214 was successfully introduced into hepatocytes, activated stellate cells, and macrophages, as confirmed by double staining with specific APC-labeled antibodies. Pdgf-c Tg mice treated LNA-antimiR-214 (n=5) showed markedly reduced hepatic fibrosis (40% area), liver weight (50%), tumor number (50%) and size of tumors (70% by calipers) compared with saline (n=5) or LNA-miR-scramble (n=5) injected control mice. The expression of collagens I and IV, α-SMA, p-SMAD3, p-AKT, p-ERK, EGF, p-EGF, MET, and p-MET was significantly suppressed. Moreover, serum albumin and alanine aminotrans-ferase levels were significantly improved. We found miR-214 targets the angiogenesis regulator, sema6A and the negative feedback inhibitor of EGFR, Mig-6 that were confirmed by using luciferase reporter assay. Recombinant sema6A inhibited the expression of α-SMA, collagens I and IV, and p-SMAD3 by about 40% in LX-2 and mimic-miR-214-transfected Huh-7 cells had accelerated cell growth, and greater EGF- stimulated phos-phorylation of EGFR and MET. Conclusion These results demonstrate that miR-214 participates in the development of hepatic fibrosis and tumor by targeting anti-fibrogenic gene, sema6A and EGFR/MET signal inhibitor, Mig6. LNA-antimiR-214 is therefore potentially useful in the prevention of hepatic fibrosis and HCC.
Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Aji-nomoto Co., Inc, MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, Bayer Japan
The following people have nothing to disclose: Hikari Okada, Masao Honda, Jean S. Campbell, Yoshio Sakai, Taro Yamashita, Takayoshi Shirasaki, Kai Takegoshi, Takuji Tanaka
Progress Toward a Circulating Biomarker in Biliary Cancer
Sydney D. Truong1, Heather F. Thiesset2, Michael Rizzari3, Philip Bernard1, Jason J. Schwartz4;
1Pathology, University of Utah, School of Medicine, Salt Lake City, UT; 2Surgery, Section of Transplantation, University of Utah, School of Medicine, Salt Lake City, UT; 3Surgery, University of Texas, Southwestern, Dallas, TX; 4Gen-eral Surgery, Division of Transplantation, University of Texas, Southwestern, Dallas, TX
Purpose: MicroRNAs (miRs) are small (19–25 nt), tissue-specific endogenous RNA molecules that have been suggested as potential biomarkers in human malignancies, though their diagnostic utility in biliary tract cancers remains unproven. Therefore, we sought to identify which circulating miRs are differentially expressed in patients with cholangiocarcinoma (CCA). Methods: Serum samples were collected from non-diseased control subjects (n= 19), patients with ulcerative colitis (UC) alone, primary sclerosing cholangitis arising in the setting of UC (PSC/UC, n=7), Crohn's disease (n = 10), or a known diagnosis of CCA (n=4). Total RNA was extracted, isolated, and purified following isolation of serum exosomes. Comprehensive profiling was done by miR microarray analysis and pairwise t-testing using the online software, GeneSifter. A smaller panel of 24 miRs which were significantly different between groups was analyzed further using rapid amplification of cDNA ends (RACE) -PCR. Relative quantification was performed by normalizing each miR in the samples to the same miR in a known reference. Results were further normalized to a set of endogenous miR housekeepers before conducting additional pairwise t-test comparisons of the PCR data. Results: When compared to normal controls, 9 miR sequences were enhanced in the circulation of patients with CCA (Table 1). These included sequences responsible for resisting natural apoptotic mechanisms (miR-25-3p, miR-24, miR-21), those helping to induce apoptosis (miR-451, miR-16), those suppressing growth and reducing metastasis (miR-22), and those responsible for inhibiting proliferation (miR-185). The mir-24-5pv2 sequence was depressed. CCA was distinguished from PSC in patients by decreased levels of miR-492, a sequence processed from the keratin-19 gene. Conclusion: A variety of circulating miRs are differentially expressed in the setting of CCA that may hold promise as potential biomarkers. With further validation, they may prove useful in distinguishing CCA from PSC, and allow for earlier diagnosis in equivocal settings.
Table 1. Comparison between normal controls and patients with CCA.
|MicroRNA miR-25-3p||Control (+/-SEM) 0.8 (+/- 0.13)||CCA (+/- SEM) 2.6(+/-l.l)||Ratio 3.3||P-value < 0.0028|
|miR-24-3p||1.7 (+/- 0.17)||3.7(+/-0.23)||2.2||< 0.0001|
|miR-21-Sp||2.7 (+/-O.25)||5.1 (+/-0.50)||1.9||0.0006|
|miR-451b||3.3 (+/-0.26)||5.9 (+/- 12)||1.8||0.0026|
|miR-16-Sp||5.4 (+/-0.28)||9.0 (+/-1–4)||1.7||0.0004|
|mir-22-5p||1.9 (+/- 0.12)||3.2 (+/- 0.41)||1.7||0.0006|
|miR-22-3p||4.4 (+/-0.24)||6.9 (+/-0.65)||1.6||0.0004|
|mir-25-5pv2||2.2 (+/-0.11)||1.5 (+/- 0.18)||0.7||0.0142|
|miR-451a||12.8 (+/-0.23)||15.3 (+/- 1.0)||1.2||0.0014|
Philip Bernard - Stock Shareholder: Bioclassifier LLC
The following people have nothing to disclose: Sydney D. Truong, Heather F. Thiesset, Michael Rizzari, Jason J. Schwartz
Activation of Interacting Signal Transduction Pathways in an HBV-Related Double Transgenic Murine Model of HCC
Waihong Chung1,3, Suzanne M. de la Monte1,2, Miran Kim1, Jack R. Wands1;
1Department of Medicine, Liver Research Center, RI Hospital and the Warren Alpert Medical School of Brown University, Providence, RI; 2Department of Pathology, Liver Research Center, RI Hospital and the Warren Alpert Medical School of Brown University, Providence, RI; 3Pathobiology Graduate Program, Brown University, Providence, RI
Analysis of hepatocellular carcinoma (HCC) tumors demonstrates substantial genetic heterogeneity and altered gene expression profiles. This study explores the concept that activation of interactive signal transduction pathways is necessary and sufficient to fully transform the mammalian liver to a malignant phenotype. We generated a double-transgenic mouse that overexpressed hepatitis Bx protein (HBx or ATX), as well as the insulin receptor substrate-1 (IRS-1) under liver specific promoters. The IRS-1 transgene was selected since it is upregulated in over 90% of individuals with HCC and HBx is a transcriptional transactivator expressed during active HBV replication. Methods: Approximately 1250 wild-type, single and double transgenic mice were serially studied at 3, 6, 9, 12, 15, 18, 21, and 24 month of age. We observed that 25% of the male ATX/IRS-1 mice developed HCC with a long latency of 15 months; single ATX and IRS-1 transgenic mice were tumor-free. The liver was examined for histologic changes and liver tissue and tumor lysates were prepared for mRNA and protein expression by qRT-PCR and multiplex ELISAs respectively. Expression and phosphorylation of multiple signaling molecules were analyzed in the insulin/IGF-1/IRS-1/Ras/Raf/MAPK/Erk, and PI3K/Akt/GSK3, Wnt/β-catenin, and Notch signaling cascades. These are highly conserved signaling pathways that interact and crosstalk with each other, and found active in the majority of human HCC tumors. Results: In the ATX/IRS-1 liver, early activation of the insulin/IGF pathway as evidenced by increased phosphorylation of IgF-1 R and Erk was followed by activation of the Wnt/β-catenin and Notch pathways in the form of Wnt-3, FZD-7, CCND-1, TBX-3, Notch-1, JAG-1, and Hes-1 overexpression. In the ATX/IRS-1 -derived HCC tumors, upregulation of these pathways was further accompanied by highly expressed levels of IGF-2, Wnt-3, FZD-7, cyclin D-1, and Hes-1. Conclusions: These studies demonstrate that the insulin/IGF-1, Wnt/β-catenin, and Notch signaling cascades are activated in ATX/IRS double-transgenic murine model. This was the result of the synergistic action between the HBx and IRS-1 overexpression which linked these interacting signaling pathways. The timing of pathway activation to the relationship of histopathologic changes in the liver from normal to dysplas-tic hepatocytes to HCC strongly supports the hypothesis that upregulation of the insulin/IGF-1 pathway alone results primarily in accelerated hepatocyte proliferation, but the synergistic upregulation of all three signaling pathways is necessary and sufficient to initiate HCC tumor formation.
The following people have nothing to disclose: Waihong Chung, Suzanne M. de la Monte, Miran Kim, Jack R. Wands
IL12-expressing dendritic cells enhance immunotherapy of hepatocellular carcinoma by changing the immuno-suppressive tumor-environment
Annabelle Vogt1, Elisabeth Sievers1, Veronika Lukacs-Kornek2, Georges Decker1, Esther Raskopf1, Tilman Sauerbruch1, Christian P. Strassburg1, Ingo Schmidt-Wolf1,3, Maria A. Gonzalez-Car-mona1;
1Department of Medicine I, University of Bonn, Bonn, Germany; 2Institute of Molecular Medicine and Experimental Immunology, Friedrich-Wilhelms-Universität, Bonn, Germany; 3Department of Medicine III, Center for Integrated Oncology (CIO), University of Bonn, Bonn, Germany
Background: Interleukin 12 (IL-12) as one of the most potent Th1-cytokines has been used to improve dendritic cells (DC)-based immunotherapy of cancer. However, this approach failed to achieve clinical response in patients with hepatocellular carcinoma (HCC). In this study, an improved immunotherapy with DC engineered to express IL-12 was studied in murine subcutaneous HCC. Methods: Tumor-lysate pulsed DC were transduced with IL-1 2-encoding adenoviruses or cultivated with recombinant (r)IL-12. DC were injected intratumorally (i.t.), sub-cutaneously or intravenously at different stages of tumor-development. In a further step, immunotherapy with DC was combined with a daily oral treatment of sorafenib (30mg/kg body weight). The tumor environment was characterized using flow cytometry, Elisa and immunohistochemistry regarding t-cell recruitment and cytokine expression. Results: DC overexpress-ing IL-12 through transduction by adenoviruses showed enhanced expression of co-stimulatory molecules and stronger priming of HCC-specific effector cells than DC cultured with (r)IL-12. I.t. injections of IL-12-DC induced the strongest antitu-moral effects reaching complete regressions in 75% of early-staged tumors and in 33% of advanced tumors. Interestingly, this effect showed increasing tendencies through a daily sorafenib treatment. By Ki67 flow cytometry measurement, we detect a significant decrease in tumor cells proliferation in IL-12-DC treated tumors compared to the untreated tumors. IL-12-DC increased the levels of Th1-cytokines/chemokines (IL-12, IFN-γ, GM-CSF, RANTES, MIP-2) and the recruitment of CD4+-, CD8+-T-and NK-cells in the tumor-environment. Induced immunity was tumor-specific and sustained overtime as all tumor-free animals were protected towards hepatic tumor-cell rechallenge. However, IL12-DC also enhanced immunosuppressive cytokines (IL-10, TGF-β), regulatory T cells and even myeloid derived suppressor cells within the tumors. Conclusions: IL-12 overex-pression induced by adenoviral vectors can effectively immunostimulate DC and t-cells. I.t. but not systemic injection of IL-12-DC was crucial for effective tumor regression. The effectiveness of this approach seems to be due to the induction of a Th1 tumor-environment followed by the recruitment of effector cells rather than the inhibition of tumor-immunosuppression. The combination of an i.t. IL-12-DC inoculation with sorafenib further increased the antitumoral effects, possibly through inhibition of angiogenesis. Thus, enhanced immunotherapy with IL-12-DC represents a promising approach for the treatment of HCC.
The following people have nothing to disclose: Annabelle Vogt, Elisabeth Sievers, Veronika Lukacs-Kornek, Georges Decker, Esther Raskopf, Tilman Sauerbruch, Christian P. Strassburg, Ingo Schmidt-Wolf, Maria A. Gonzalez-Carmona
Hepatocyte gp130-mediated Signaling promotes hepatocellular carcinogenesis through activation of mTORC1 and Stat3
Yoonseok Roh, Ling Yang, Jingyi Song, Bi Zhang, Eek Joong Park, Ekihiro Seki;
Medicine, University of California, SanDiego, San Diego, CA
BACKGROUND & AIMS: Hepatocyte-specific Tak1 deletion triggers spontaneous hepatocellular carcinoma (HCC) development with liver inflammation and fibrosis. Glycoprotein 130 (gp130) activates Stat3, MAPKand mTORC1 signaling, which regulate cell survival, growth, proliferation, and carcinogenesis. However, it remains unclear whether gp130 pathway in hepatocytes promotes HCC. METHODS: Hepatocyte specific Tak1-deleted (Tak1 ΔH), Tak1/gp130ΔH and Tak1/Stat3ΔH mice were generated. Liver injury, inflammation, fibrosis, and HCC were assessed. RESULTS: gp1 30 ligands including IL-6, IL-1 1 and oncostatin M were overexpressed in HCC of Tak1 ΔH mice. The multiplicity and maximal size of spontaneous HCC in Tak1 ΔH mice at the age of 9 month was suppressed when gp1 30 or Stat3 was ablated additionally in hepatocytes. One-month-old Tak1 ΔH mice displayed spontaneous hepatocyte death and compensatory proliferation, and liver inflammation and fibrosis. These liver phenotypes of Tak1 ΔH mice were blunted in disruption of gp1 30 but not Stat3. Consistently, expressions of pro-apoptotic Bax, p21 and Puma, and pro-carcinogenic H1 9, c-Src and Mdm2 were increased in one-month-old Tak1 ΔH mice that were suppressed in Tak1/gp1 30ΔH mice but not in Tak1 /Stat3 ΔH mice. Different with results of 1 -month-old mice, additional deletion of Stat3 enhanced apoptosis of cancer cell accompanied by upregulation of p53 in tumor, which may contribute to the decreased number and size of tumor in Tak1/Stat3ΔH mice. Consistent with results of tumor development, the expression of fetal stage-specific liver genes, such as Afp, H1 9, Igf2 and Dlk1 were were suppressed in tumors of Tak1/Stat3ΔH mice compared with those of Tak1 ΔH mice. Immunoblotting analysis shows that additional ablation of gp1 30 suppressed increased activation of Stat3 and mTORC1 signaling in Tak1 ΔH mice as demonstrated by decreased phophorylation of Stat3 (Tyr705), p70S6K and eIF4E. Subsequently, we investigated whether mTORC1 activation was required for hepatocellular carcinogenesis in Tak1 ΔH mice. The mTORC1 inhibitor Rapamycin suppressed mTORC1 activation, and dramatically inhibited multiplicity and size of HCC of Tak1 ΔH mice. Accordingly, deletion of gp1 30, an upstream of Stat3 and mTORC1, resulted in more profound inhibition of spontaneous liver injury, inflammation, fibrosis and HCC development compared with those of Tak1/Stat3ΔH mice. CONCLUSIONS: gp1 30 governs parallel activation of oncogenic mTORC1 alongside Stat3 in the pathogenesis of HCC in Tak1 ΔH mice by differential regulation of hepatocyte apoptosis and compensatory proliferation in early phase, as well as cancer cell growth and apoptosis in late phase.
The following people have nothing to disclose: Yoonseok Roh, Ling Yang, Jingyi Song, Bi Zhang, Eek Joong Park, Ekihiro Seki
Tumor cell derived extracellular vesicles interact with mesenchymal stem cells to modulate the microenviron-ment and enhance cholangiocarcinoma growth
Hiroaki Haga, Irene K. Yan, Kenji Takahashi, Tushar Patel;
Mayo Clinic, Jacksonville, FL
Background: Cholangiocarcinomas are highly desmoplastic tumors that are characterized by tumor cells closely intertwined with a dense fibrous stroma. The cellular origins of the tumor stroma and contribution to cholangiocarcinoma growth remain poorly understood. Bone marrow derived mesenchymal stem cells (MSC) are a potential source of tumor stroma. We have recently shown that tumor cells can transfer genetic information through extracellular vesicles (EV). Thus, our aims were to examine the effects of tumor-cell-MSC interactions in cholangiocarcinoma growth and the role of EV signaling in these interactions. Methods: Human bone-marrow derived MSC and, KMBC malignant human cholangiocytes were used for the study. EV were isolated by differential centrifugation, verified using EM and quantitated using Nanosight nanoparticle tracking analysis. Cytokine and chemokine profiling was performed in culture supernatants. Cell phenotype was assessed by studying cell growth and migration using MTS and cell migration assays respectively. Results: Both KMBC and MSC produced EV in cell culture with a mean size of 120 nm and morphology consistent with those of exosomes. Exposure of MSC to 5 x1 05/cell KMBC-EV resulted in morphological changes, with increased migration and enhanced expression of α-SMA and FAP mRNA consistent with fibroblastic differentiation. Furthermore, KMBC-EV selectively enhanced MSC secretion of CXCL1, MCP-1, CX3CL1, PDGF and IL-6 by 10.2, 1.4, 2.2, 1.4 and 1.2-fold, respectively compared to controls. An increase in expression of CXCL1 (p=0.04), MCP-1 (p=0.03), and IL-6 (p=0.02), but not CX3CL1 mRNA was also observed in MSC incubated with KMBC-EV compared with controls. Conditioned media from MSC increased KMBC cell proliferation and migration. However, conditioned media from MSC exposed to 5 x105/cell KMBC-EV increased KMBC cell proliferation but not migration compared to conditioned media from control MSC not exposed to KMBC-EV, or to KMBC cells exposed to KMBC-EV alone. This proliferative effect was completely blocked by anti-IL-6. Summary and Conclusions: EV transfer from KMBC increases fibroblast-like activity and selectively alters mRNA expression and secretion of IL6 and other cytokines/chemokines by MSC cells that can, in turn, alter KMBC proliferation. Thus, tumor cells can "educate" MSC to modulate the microenvironment and thereby facilitate tumor growth. This is a previously unde-scribed and unique mechanism by which tumor cells can modulate the microenvironment and facilitate tumor growth. These findings offer new opportunities for therapeutic intervention in cholangiocarcinoma and possibly other cancers.
The following people have nothing to disclose: Hiroaki Haga, Irene K. Yan, Kenji Takahashi, Tushar Patel
Lack of gp130 in hepatocytes ameliorates tumor progression in a murine model of genotoxic stress
Maximilian Hatting1, Michael Spannbauer1, Gernot Sellge1, Niko-laus Gassler2, Christian Liedtke1, Christian Trautwein1;
1Medicine III, University Hospital RWTH Aachen University, Aachen, Germany; 2Pathology, University Hospital RWTH Aachen University, Aachen, Germany
Background: Hepatocellular carcinoma (HCC) is mostly triggered by chronic inflammation in the liver, as seen in Hepatitis B and C, alcoholic and non-alcoholic fatty liver disease. Earlier results indicated that the IL-6/gp130 pathway is of major relevance for hepatocarcinogenesis. After receptor binding gp1 30 dimerises and activates Janus-activated kinases (JAKs) which lead to STAT3 phosphorylation and its nuclear translocation. Here STAT3 is involved in the activation of genes controlling hepatocyte proliferation. Thus blocking gp1 30 signaling in hepatocytes could be a promising approach to treat HCCs. Aim: To investigate the role of gp1 30 in hepatocytes in a murine HCC model of genotoxic stress. Methods: Hepatocyte-specific gp1 30 knockout mice (gp130Δhepa) and littermate controls (gp130f/f) were subjected to single intraperitoneal Diethylnitrosamine (DEN) injection. The impact of gp1 30 on acute liver injury was investigated 0- 5 days after DEN administration; tumor initiation and progression were analysed 24 and 40 weeks after treatment, respectively. Results: After acute liver damage the increase in transaminases was not significantly different between gp130Δhepa animals and controls. However, inflammation was significantly reduced in gp130Δhepa livers as evidenced by decreased cytokine levels (e.g. TNFα, IL6) and less immune cell infiltration as well as changes in liver histology. Twenty-four weeks after DEN treatment both strains revealed comparable numbers of dysplastic hepatic nodules suggesting that gp1 30 is not involved in tumor initiation. Interestingly, gp130Δhepa animals developed significantly less and smaller tumors 40 weeks after DEN administration pointing to an important role of gp1 30 for tumor progression. To better understand these findings, different mechanisms and pathways (e.g. oxidative stress, apoptosis, cell proliferation, immune-cell infiltration) were investigated. Significantly higher amounts of phosphorylated Histone H2A (H2AX) were detected in gp130Δhepa liver-tumors compared to controls indicating improved repair of DNA damage in the absence of gp1 30. Conclusion: Lack of gp1 30 in hepatocytes has no effect on liver damage and tumor initiation after DEN treatment but leads to reduced tumor progression and improved DNA repair.
Christian Trautwein - Grant/Research Support: BMS, Novartis, BMS, Novartis; Speaking and Teaching: Roche, BMS, Roche, BMS
The following people have nothing to disclose: Maximilian Hatting, Michael Spannbauer, Gernot Sellge, Nikolaus Gassler, Christian Liedtke
MicroRNA-21 targets NAD+-linked 15-Hydrox-yprostaglandin dehydrogenase (15-PGDH) and promotes cholangiocarcinoma growth
Lu Lu, Chang Han, Tong Wu;
Pathology and Laboratory Medicine, Tulane Univeraity School of Medicine, New Orlenas, LA
MicroRNAs (miRNAs) are a group of small, noncoding RNAs that modulate gene expression through binding to specific target sites in messenger RNAs. This study investigated the biological function and molecular mechanism of microRNA-21 (miR-21) in human cholangiocarcinoma. In situ hybridization analysis of human cholangiocarcinoma tissues showed increased miR-21 in cholangiocarcinoma cells compared to the noncancerous biliary epithelial cells. Forced overexpression of miR-21 by lentivirus transduction enhanced human cholangiocarcinoma cell growth and clonogenic efficiency in vitro, whereas inhibition of miR-21 decreased these parameters. MiR-21 overexpression also promoted cholangiocarcinoma growth in a tumor xenograft model. The NAD+-linked 15-hydrox-yprostaglandin dehydrogenase (15-PGDH), a key enzyme that converts the pro-tumorigenic prostaglandin E2 (PGE2) to biologically inactive metabolite, was identified as a direct target of miR-21 in cholangiocarcinoma cells. In parallel, cyclooxyge-nase-2 (COX-2) overexpression and PGE2 treatment increased miR-21 expression and induces miR-21 promoter reporter activity in human cholangiocarcinoma cells. These findings reveal a novel cross-talk between COX-2/PGE2 and miR-21 signaling pathways that converges at 15-PGDH which is crucial in cholangiocarcinogenesis and tumor progression.
The following people have nothing to disclose: Lu Lu, Chang Han, Tong Wu
Hepatic myofibroblasts promote the progression of human cholangiocarcinoma through activation of epidermal growth factor receptor
Audrey Claperon1,2, Martine Mergey1,2, Lynda Aoudjehane3, Thanh Huong Nguyen Ho-Bouldoires1,2, Dominique Wendum1,2, Aurelie Prignon4,2, Fatiha Merabtene5,2, Delphine Firrincieli6,2, Christele Desbois-Mouthon1,2, Olivier Scatton1,2, Filomena Conti6,2, Chantal Housset6,2, Laura Fouassier1,2;
1Biology and Treatment of Hepatobiliary Tumours, Saint-Antoine Research Centre, Inserm UMRS_938, Paris, France; 2UPMC, Univ Paris 06, Paris, France; 3Human HepCell, Hopital Saint-Antoine, Paris, France; 4Plateforme LIMP, Bât Recherche, Hôpital Tenon, Paris, France; 5Plateforme Morphologie du petit animal, Saint-Antoine Research Centre, Inserm UMRS_938, Paris, France; 6Metabolic and biliary, fibro-inflammatory diseases of the liver, Saint-Antoine Research Centre, Inserm UMRS_938, Paris, France
Intrahepatic cholangiocarcinoma (CCA) is characterized by an abundant desmoplastic environment. Poor prognosis of CCA has been associated with the presence of α-smooth muscle actin (α-SMA)-positive-myofibroblasts in the stroma and with the sustained activation of the Epidermal Growth Factor Receptor (EGFR) in tumor cells. Among EGFR ligand, Heparin-Binding Epidermal Growth Factor (HB-EGF) has emerged as a paracrine factor that contributes to intercellular communications between myofibroblasts and tumor cells in several cancers. This study was designed to test whether hepatic myofibroblasts contributed to CCA progression through EGFR signaling. The interplay between CCA cells and hepatic myofibroblasts was examined first in vivo, using subcutaneous xenografts into immunocompromised mice. In these experiments, the cotrans-plantation of CCA cells with human liver myofibroblasts (HLMF) increased tumor incidence, size, and metastastic dissemination of tumors. These effects were abolished by gefitinib, an EGFR tyrosine kinase inhibitor. Immunohistochemical analyses of human CCA tissues showed that stromal myofibroblasts expressed HB-EGF while EGFR was detected in cancer cells. In vitro, HLMF produced HB-EGF and their conditioned media induced EGFR activation and promoted disruption of adherens junctions, migratory and invasive properties in CCA cells. These effects were abolished in the presence of gefitinib or HB-EGF neutralizing antibody. We also showed that CCA cells produced transforming growth factor-β1 (TGF-β1), which in turn, induced HB-EGF expression in HLMF. Conclusion: A reciprocal cross-talk between CCA cells and myofibroblasts through the HB-EGF/EGFR axis contributes to CCA progression.
The following people have nothing to disclose: Audrey Claperon, Martine Mergey, Lynda Aoudjehane, Thanh Huong Nguyen Ho-Bouldoires, Dominique Wendum, Aurelie Prignon, Fatiha Merabtene, Delphine Firrincieli, Christele Desbois-Mouthon, Olivier Scatton, Filomena Conti, Chantal Housset, Laura Fouassier
Modeling Hepatocarcinogenesis In Human Liver Cells
Floriane Pez1,2, Anais Lopez1,2, Lydie Lefrancois1,2, David Duran-tel1,2, Bakary Sylla3, Claude Caron de Fromentel1,2, Philippe Merle1,4;
1Immunity, Microenvironment, Virus, UMR INSERM U1052 CNRS5286, CRCL Cancer Center of Lyon, Lyon, France; 2Centre Léon Bérard, F-69008, Lyon, France; 3Molecular Oncoge-nesis, IARC, Lyon, France; 4Service d'Hépatologie et de Gastroen-térologie, Groupement Hospitalier Lyon Nord, Hospices Civils de Lyon, Lyon, France
Genetic and epigenetic abnormalities are widely heterogenous in human HCCs. The early changes leading to initiation of transformation as well as the most permissive liver cells to this process are barely known. We developed an in vitro model of transformation of liver cells by the R. Weinberg oncogene combination. We then assessed whether transformation capabilities depend on liver cells differentiation status. In addition, we assessed whether this transformation was associated with differentiation properties and pathway deregulations focusing on two main pathways : the Wnt pathway and the p53 family. We disposed of human nontransformed bipotent progenitors (HepaRG cell line), and we isolated primary human hepato-cytes (PHH). We transduced these cells with lentivirus encoding for SV40 Large T (LT) and small T (ST), a constitutive active form of HRas (HRasG12V) as well as hTERT for PHH. Cellular transformation was assessed by proliferation assay in low serum conditions, anchorage independent growth in soft agar. Differentiation and stemness markers were measured by iQRT-PCR. Wnt and p53 family pathway were explored by iQRT-PCR and WB. SV40 LT and ST expression allowed cell cycle entry of physiologically quiescent PHH, and increased HepaRG proliferation rate, but did not transform cells. By contrast, addition of the HRasG12V led to transformation of these both cell types as demonstrated by large macroscopic colony formation in soft agar, higher cell proliferation in low serum condition. Addition of hTERT increased the anchorage independent growth of PHH. Transformed PHH harbored a dedifferentiated phenotype with decreased expression of hepatocytic markers, increased expression of some stemness markers and cancer stem cell self-renewal markers. Interestingly, transformed PHH showed an increased expression of Wnt/beta-catenin target genes in association with strong overexpression of upstream activators well known as implied in hepatocarcinogenesis such as Wnt3 ligand and Frizzled-7 receptor. Regarding the p53 family, TAp73 and DNp73 were strongly upregulated. We described for the first time a unique model of in vitro transformation of human liver cells. We showed that both differentiated hepatocytes and bipotent progenitors are permissive to transformation. This process was accompanied by alteration of differentiation capabilities, and appearance of stemness markers in transformed PHH. Interestingly, some pathway deregulations previously described in human HCC tissues were also observed in our models : Wnt/beta-catenin and TP73. This could be an interesting tool for a better understanding of hepatocarcinogenesis and drug discovery.
David Durantel - Grant/Research Support: Hoffmann-La Roche
Fibrotic liver microenvironment promotes stemness of liver cancer through activation of transforming growth factor beta signaling
Mitsumasa Kondo, Taro Yamashita, Naoki Oishi, Kouki Nio, Sha Sha Zeng, Takehiro Hayashi, Yoshimoto Nomura, Mariko Yoshida, Tomoyuki Hayashi, Hikari Okada, Hajime Sunagozaka, Hajime Takatori, Honda Masao, Shuichi Kaneko;
Department of Gas-troenterology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan
[Background] Liver cirrhosis is one of the most important risk factors for the development of liver cancer. Various lineages of stromal cells including stellate cells, activated myofibroblasts, and vascular endothelial cells as well as lymphocytes are known to emerge in the cirrhotic liver. However, it is unclear how microenvironment alteration induced by these cells affects the process of liver cancer development. In this study, we evaluated the role of stromal cells on stemness of the tumor, which is closely associated with the aggressiveness of liver cancer. [Methods]Primary hepatocellular carcinoma (HCC) cells obtained from surgically resected specimens as well as Huh7 cells were co-cultured with various stromal cells in vitro using cell culture inserts. Gene and protein expression was evaluated by qRT-PCR and Western blotting. Fluorescence-activated cell sorting (FACS) was used to evaluate the frequency of cancer stem cells expressing EpCAM/CD1 33. Cancer stem cell characteristics were evaluated by spheroid formation, invasion, and tumorigenicity in immune deficient mice. Time-lapse image analysis was performed to monitor cell motility affected by stromal cells. [Results] Co-culture of HCC cells with fibroblasts resulted in the enhanced cell motility, spheroid formation, and invasion capacities of HCC cells, whereas those co-cultured with vascular endothelial cells or stellate cells did not show such phenotypes. FACS analyses demonstrated the enrichment of EpCAM/CD1 33-positive cells when co-cultured with fibroblasts. Gene expression analysis of selected cytokines and growth factors indicated the abundant expression of TGFB1 in fibroblasts but not in stellate cells or vascular endothelial cells. Supplementation of recombinant transforming growth factor beta (TGF-beta) enhanced the spheroid formation capacity of HCC cells, and this effect was abolished when cultured with neutralizing antibodies against TGF-beta. Co-injection of HCC cells with fibroblasts but not with stellate cells or endothelial cells resulted in the high motility of mice with high frequency of lung metastasis. [Conclusions] Fibrotic liver microenvironment accelerates the malignant natures of HCC with enrichment of EpCAM/CD1 33-positive cancer stem cells through activation of TGF-beta signaling. TGF-beta may be a good molecular target to reduce the risk of aggressive liver cancer development in liver cirrhosis patients.
Mariko Yoshida - Grant/Research Support: Bayer
Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Aji-nomoto Co., Inc, MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, Bayer Japan
The following people have nothing to disclose: Mitsumasa Kondo, Taro Yamashita, Naoki Oishi, Kouki Nio, Sha Sha Zeng, Takehiro Hayashi, Yoshimoto Nomura, Tomoyuki Hayashi, Hikari Okada, Hajime Sunagozaka, Hajime Takatori, Honda Masao
Role of activated hepatic stellate cells in proliferation and metastasis of hepatocellular carcinoma
Nan Lin1, Xu Linan2;
1Department of Hepatobiliary Surgery, the Third Affiliated Hospital,Sun Yat-sen University, Guangzhou, China; 2The Sixth Affiliated Hospital, SunYat-Sen University, guangzhou, China
Background::The presence of liver cirrhosis is the main risk factor for the development of hepatocellular carcinoma (HCC). Activated hepatic stellate cells (aHSC) are recognized as a central event in the development of liver fibrosis, but which role in HCC is unclear. Objective:This study investigated angiogenetic activity of aHSC in HCC and angiogenetic effect on proliferation, metastasis of HCC. Methods:In vitro, we exposured microvascular endothelial cell (MEC) to conditioned media(CM) from aHSC to observe the influence of the CM on MEC by quantifying MEC tube formation, and placed aHSC over an endothelial monolayer to assesse transendothelial migration of aHSC to hepatoma cells by examining the movement of CFSE labeled aHSC through the endothelial cell monolayer. For convenient viewing, aHSC were labeled for CFSE (one green fluorescent dye). In vivo, we established orthotopic model of HCC with nude mice receiving an intraliver injection of aHSC plus hepatoma cells. After 7 weeks, tumor size,number of metas-tases were measured. Forthermore, tumors tissue were ashah-sessed by immunostaining for expression of aHSC and microvessel. Angiogenesis activities were assessed by immunostaining tumors for CD34, an endothelial cell marker and vascular endothelial growth factor (VEGF). Results: In vitro, The CM from aHSC stimulated the tube formation by MEC, and hepatoma cells stimulated transendothelial migration of aHSC. In vivo, compared with mice receiving hepatoma cells alone, mice injected with hepatoma cells plus aHSC exhibited the most increased tumor size and regional and distant metastasis. CD34 and VEGF in primary tumors of mice injected with hepatoma cells plus aHSC were higher expression compared to hepatoma cells alone. aHSC and microvessel were both located around nests in serial sections of HCC. The CFSE labeled aHSC were found in multiple metastatic sites in each mice injected with aHSC plus hepatoma cells. Conclusion: aHSC promote proliferation, metastasis of HCC through angiogenesis, and spreaded to other parts of the body accompanying with HCC cells continuing with playing a role in metastatic tumor.
The following people have nothing to disclose: Nan Lin, Xu Linan
Nanoparticle based combinatorial small interfering RNA therapy against human hepatocellular carcinoma (HCC)
Thomas Decaens1, Valentina M. Factor1, Iva Kulic2, Jesper B. Andersen1, Adam Judge2, Elizabeth A. Conner1, Ian MacLachlan2, Snorri S. Thorgeirsson1;
1Laboratory of Experimental Carcinogen-esis, NCI, NIH, Bethesda, MD; 2Tekmira Pharmaceuticals Corporation, Burnaby, BC, Canada
Background: We have previously demonstrated therapeutic effects of lipid nanoparticles (LNP) loaded with single siRNA targeting CSN5 or WEE1 against human HCC cell lines in an orthotropic mouse models. Aim: To test the safety and the efficacy of a combinatorial versus single siRNA therapy in the orthotopic mouse model and to identify molecular mechanism(s) involved in therapeutic responses by global transcriptome analyses. Materials and Methods: LNP formulations of chemically modified siRNAs targeting CSN5 and WEE1 were produced by Tekmira® Pharmaceuticals. Safety was assessed in ICR mice after 9 injections. SCID-beige mice were used for intra-hepatic (Huh7-luciferase) tumor transplantation. Mice with established tumors were treated intravenously with 2 mg/kg of a single siRNA + 2 mg/kg betaGal siRNA, 2 mg/kg each of siCSN5:siWEE1 siRNA co-encapsulated in the same LNP, or 4 mg/kg of betaGal siRNA. Tumors were assayed following 1 to 9 injected doses. Tumor progression in the Huh7 orthotopic model was monitored by bioluminescence imaging and metas-tases were evaluated at endpoint. Results: Safety data show that combinatorial siRNA is well tolerated compared to single or control siRNA. We observed significant inhibition of tumor growth and metastases in mice treated with active siRNAs compared to LNP containing a non-targeting control siRNA. Significant targeted silencing was observed in tumors after single or repeat administration with no interference between the siRNAs for the CSN5:WEE1 combination. Microarray analyses of the tumors demonstrate an extensive difference of gene expression between treatment groups. Interestingly, the microarray analyses of the surrounding liver show a minimal modification of gene expression in this non-tumor tissue. Conclusion: We demonstrate that LNP-based combinatorial siRNA therapy is safe and effective in a human mouse model of HCC, with a significant decrease of tumor size associated with a massive downregulation of the targeted genes. Global gene expression of the surrounding liver is minimally affected by this therapy compared with what seen in the tumor.
Iva Kulic - Employment: Tekmira Pharmaceuticals
Adam Judge - Employment: Tekmira Pharmaceuticals, Tekmira Pharmaceuticals, Tekmira Pharmaceuticals, Tekmira Pharmaceuticals
Ian MacLachlan - Employment: Tekmira, Tekmira, Tekmira, Tekmira
The following people have nothing to disclose: Thomas Decaens, Valentina M. Factor, Jesper B. Andersen, Elizabeth A. Conner, Snorri S. Thorgeirsson
The Single-Chain Antibody Blocking Cyclooxygenase-2 Activity Inhibits the Growth of Hepatocellular Carcinoma
Yan Wu1, An Cui1, Xun Zhou1, Wenhan Wang1, Nannan Yao1, Hanwei Li1, Chang Han3, Tong Wu3, Guiying Li1,2;
1Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, School of Life Science, Jilin University, Changchun, China;2National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun, China; 3Department of Pathology, Tulane University School of Medicine, New Orleans, LA
Background/Aims: Cyclooxygenase-2 (COX-2) is a rate-limiting key enzyme catalyzing the conversion of arachidonic acid (AA) into prostaglandins (PGs). Compelling evidence has documented increased expression of COX-2 in various human cancers including hepatocellular carcinoma (HCC). Therefore, selective blockage of COX-2 activity may represent an effective strategy for anti-cancer therapy. This study was designed to construct an intrabody against COX-2 and to determine its effect on HCC cell growth (intrabodies can be directed to intra-cellular compartments to neutralize and block the function of target proteins). Methods: A single-chain fragment of antibody variable region (scFv) against COX-2 (OX-1) was isolated by antibody phage display. And then an expression plasmid pIn-tra-OX1 harboring an endoplasmic reticulum (ER)-retained scFv gene against human COX-2 (Intra-OX1) was constructed. The activity of scFv was characterized by ELISA and immunopre-cipitation. The expression and subcellular distribution of Intra-OX1 in HepG2 cells were detected by RT-PCR, Western blot and fluorescence staining. Cell cycle and apoptosis were detected by flow cytometry. The antitumor efficacy of Intra-OX1 on HCC in vivo was assessed by intratumoral injection of pIn-tra-OX1 to subcutaneous tumors in nude mice. Results: ELISA and immunoprecipitation showed that OX1 specifically bound to human recombinant COX-2 and endogenous COX-2 in HepG2 cells. OX1 inhibited the oxygenation of AA catalyzed by COX-2 enzymes. In HepG2 cells transfected with pIntra-OX1, Intra-OX1 was expressed efficiently and localized in the endoplasmic reticulum. Co-immunoprecipitation assay showed that Intra-OX1 in HepG2/pIntra-OX1 cells could recognize and bind to COX-2. Expression of Intra-OX1 inhibited PGE2 release from HepG2 cells. Compared with the control, the expression of Intra-OX1 significantly inhibited the growth of HepG2 cells, resulted in cell cycle arrest in the G0/G1 phase (P<0.01), and induced more cell apoptosis (P<0.01). Intra-OX1 also significantly suppressed the growth of subcutaneous tumors in nude mice in vivo after the intratumoral injection of pIntra-OX1. Expression of Intra-OX1 decreased the levels of EGFR, STAT3 in HepG2 cells. Conclusion: Anti-COX-2 intrabody inhibited HCC growth both in vitro and in vivo through blockage of COX-2 activity. Further studies are warranted to determine whether this approach can be utilized therapeutically for hepatocellular carcinoma.
The following people have nothing to disclose: Yan Wu, An Cui, Xun Zhou, Wenhan Wang, Nannan Yao, Hanwei Li, Chang Han, Tong Wu, Guiying Li
Transglutaminase 2-mediated epithelial-mesenchymal transition via IL-6/STAT3 signaling facilitates liver cancer progression
Wei Liu1, Guan-zhong Chen1, Kun-hua Hu2, Guo-Ying Wang1, Bin-sheng Fu1, Qi Zhang1, Gui-Hua Chen1;
1The 3rd Affiliated Hospital ofSYSU, Guangzhou, China; 2Zhongshan School of Medicine, SYSU, Guangzhou, China
Background: A number of studies have reported crucial roles of cancer-associated fibroblasts (CAFs) in providing cancer cells with proliferative, survival, invasive and metastatic propensities favouring tumourigenesis. Our recently study found that CAFs induce epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) cells through release of HGF and IL-6, and increase invasive and migratory properties of the cancer cells, but the mechanism remains obscure. In this study, we found that Transglutaminase 2 (TG2) may be involved in mediating CAFs-induced EMT in HCC cells. The signaling pathways involved in regulation of TG2 expression, as well as those underlying its tumour-promoting effect in HCC were analyzed. Methods: HCC cells were induced to EMT by separately co-cultured with CAFs (isolated from HCC) in a transwell system. EMT markers and protein expression level were assessed by western blot. TG2 were either overexpressed or silenced by lentivirus transfection in HCC cells, and in vitro cell behavior assay and in vivo metastasis assay were performed. HGF and IL-6 signaling pathways were analyzed to determine whether they were involved in regulation of TG2 expression. Additionally, to explore whether TG2 could be an important factor in determining clinical outcomes of HCC, an immunohistochemical examination of TG2 expression and a clinicopathological analysis were performed in 108 consecutive HCC patients. Results: TG2 were significantly elevated expression in HCC cells with EMT phenotype. Overexpression of TG2 promoted EMT and metastasis of HCC cells in vitro and in vivo. Knockdown of TG2 in HCC cells remarkably attenuated its EMT which induced by CAFs, as well as the migratory and invasive ability. Signaling pathway assay showed that expression of TG2 was affected by IL-6/STAT3 activation, but not HGF/Met. Further, inhibition of the phos-phorylation of STAT3 decreased the expression of TG2 in HCC cells. These results suggest that CAFs facilitates HCC metastasis by promoting EMT via IL-6/STAT3/TG2-dependent pathway. Consistently, as disclosed by immunohistochemistry results, high TG2 expression was significantly correlated to tumor size (P=0.027), clinical stage (P=0.018) and vascular invasion (P=0.022). Moreover, Kaplan-Meier analysis and multivariate analysis indicated that high TG2 expression associated with unfavorable overall survival (P<0.001), it was an independent poor prognostic marker for overall survival (P=0.043) of HCC patients. Conclusions: TG2 plays an important role in HCC invasion and metastasis and may serve as a novel prognostic biomarkerand therapeutic target. Keywords: TG2; EMT; HCC; CAFs; IL-6/STAT3 signaling
The following people have nothing to disclose: Wei Liu, Guan-zhong Chen, Kun-hua Hu, Guo-Ying Wang, Bin-sheng Fu, Qi Zhang, Gui-Hua Chen
Low-density Lipoprotein Nanoparticle Mediated Delivery of Docosahexaenoic Acid Selectively Kills Liver Cancer Cells
Ian Corbin1,2, Lacy Reynolds2, Rohit Mulik2, Xiaodong Wen2;
1Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX; 2Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX
Background: Therapeutic selectivity is highly desired property for anticancer medicines. The ideal agent should be toxic to malignant cells with minimum harm to normal cells. To date, most chemotherapeutics indiscriminately damage both cancer and healthy cells resulting in severe adverse effects. Thus, efforts to develop novel selective drugs remain a critical need in oncology, particularly for cancers like hepatocellular carcinoma (HCC) where the surrounding liver is often compromised with chronic disease. Natural products continue to be an invaluable source for anticancer drug discovery. In recent years the natural omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA) has been shown to possess promising anticancer properties. Currently, dietary consumption remains the only means of acquiring DHA. With this form of intake DHA's activity is diluted and markedly reduced as it is incorporated into plasma phospholipids or proteins. Clearly if the anticancer potential of this natural lipid is to be fully realized, novel delivery strategies must be employed. Herein, we evaluate in an in vitro cell culture system the utility of the low-density lipoprotein (LDL) as a nanoscale delivery vehicle for DHA. Methods: LDL-DHA nanoparticles were prepared and subject to extensively biophysical characterization. The therapeutic utility of LDL-DHA was evaluated in normal and malignant murine hepatocyte cell lines, TIB-73 and TIB-75 respectively, using MTT dose response, FACS, confocal microscopy and oxidative stress analyses. Results: Engineered LDL nanoparticles, uniformly loaded with unesterified DHA (LDL-DHA), closely resembled native LDL morphologically and biochemically. With regards to its biological activity, LDL-DHA nanoparticles were avidly taken up by both TIB-73 and TIB-75 cells. Dose response evaluations revealed that LDL-DHA was selectively cytotoxic to the malignant TIB-75 cells. The selectivity of LDL-DHA was further exemplified with co-cultures of these two cells, therapeutic doses of LDL-DHA that completely killed the TIB-75 proved to be innocuous to TIB-73 leaving them unharmed. FACS analysis showed that LDL-DHA activated both apoptotic and necrotic death pathways in the TIB-75 cells. Additional studies went on to show that LDL-DHA treatment selectively induced pronounced lipid peroxidation and oxidative stress in malignant TIB-75 cells. These pathways play a central role in LDL-DHA mediated cancer cell kill as supplementation with vitamin E was able to rescue the TIB-75 cells. Conclusion: These studies collectively demonstrate that LDL-DHA nanoparticles shows great promise as a selective anticancer agent against hepatocellular carcinoma.
The following people have nothing to disclose: Ian Corbin, Lacy Reynolds, Rohit Mulik, Xiaodong Wen
Role of oxidative stress and epigenetic alteration on chronic hepatitis C-related human hepatocarcinogenesis
Naoshi Nishida1,2, Masatoshi Kudo1, Tadaaki Arizumi1, Masahiro Takita1, Satoshi Kitai1, Norihisa Yada1, Tatsuo Inoue1, Satoru Hagi-wara1, Yasunori Minami1, Toshiharu Sakurai1, Kazuomi Ueshima1, Takeshi Nagasaka3, Ajay Goel4;
1Gastroenterology and Hepatol-ogy, Kinki University Faculty of Medicine, Osaka-sayama, Japan; 2Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, Kyoto, Japan; 3Gastroenterological Surgery, Okayama University, Okayama, Japan; 4Baylor Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX
Background and purpose: Chronic hepatitis C (CHC) triggers oxidative stress, which is closely associated with emergence of hepatocellular carcinoma (HCC). On the other hand, hyperme-thylation-induced transcriptional inactivation of tumor suppressor genes (TSGs) has been reported in HCC. The purpose of this study is to clarify the association between oxidative stress, epigenetic alterations and development of HCC in CHC patients. Methods: (1) We examined DNA oxidation and methylation profiles in a collection of 128 liver biopsy samples from CHC patients without any history of HCC. DNA oxidation was quantified by immunohistochemical analysis of 8-OHdG. The staining of 8-OHdG was classified as strong (2+), moderate (1+), or weak (-). HCC-free survival after biopsy was analyzed retrospectively using Kaplan-Meier method. Methylation of 10 TSGs (HIC-1, GSTP1, SOCS1, RASSF1, CDKN2A, APC, RUNX3, PRDM2, CASP8, CACNA1G) was determined by MethyLight. Significant factors contributing to increased number of methylated TSGs was determined by multivariate analysis using age, gender, fibrosis stage, amount of 8-OHdG and iron deposit as covariables. (2) HepG2 cells were treated with H2O2 and chromatin immunoprecipitation (ChIP) was performed before and after treatment using antibodies against trimethyl-H3K4, acethylated-H4K1 6 for active chromatin, trimethyl-H3K27 for repressed chromatin, 8-OHdG for damaged DNA elements, pan-histone H3 for positive ChIP control, and rabbit IgG for negative ChIP control. Quantitative PCR (qPCR) was performed for promoters of 25 different TSGs, which reportedly showed methylation in human cancer using EpiScope® Promoter qPCR Array (TaKaRa). Alterations in chromatin status on damaged DNA (DNA element with 8-OHdG) was also determined. Results: Increased 8-OHdG content was significantly associated with shorter time to HCC emergence in CHC patients (p=0.0026, log-rank test). Multivariate analysis revealed that high levels of 8-OHdG was the only variable that significantly associated with increased number of methylated TSGs (p<0.0001), and showed dose-related effect between amount of 8-OHdG and number of methylated TSGs (RR=3.53, CI=5.97∼2.15 for 2+ v.s. -; RR=2.01, CI=3.42∼1.18 for 2+ v.s 1+; RR=1.76, CI=2.83∼1.11 for 1+ v.s. -). ChIP-qPCR revealed that DNA elements carrying 8-OHdG after H2O2 treatment showed alteration of active chromatin (trimethyl-H3K4 and acethylated-H4K1 6 dominant) to repressive chromatin status (trimethyl-H3K27 dominant). Conclusion: We conclude that oxidative stress induces alteration of chromatin status, which lead to abnormal methylation of TSGs, and contribute to hepa-tocarcinogenesis in CHC patients.
The following people have nothing to disclose: Naoshi Nishida, Masatoshi Kudo, Tadaaki Arizumi, Masahiro Takita, Satoshi Kitai, Norihisa Yada, Tatsuo Inoue, Satoru Hagiwara, Yasunori Minami, Toshiharu Sakurai, Kazuomi Ueshima, Takeshi Nagasaka, Ajay Goel
Hepatitis B virus X protein and the oncogenic genes c-myc and Ras induce tumorigenic transformation and stem cell-like features in immortalized human hepatocytes
Naoki Oishi1,2, Seishi Murakami2, Wang Xuyang2, Shuichi Kaneko1,2;
1Department of Gastroenterology, Kanazawa University Hospital, Kanazawa, Japan; 2Department of Disease Control and Hoemostasis, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan
Background: Hepatocellular carcinoma (HCC) is a worldwide health issue; however, it remains poorly understood. Previously, we found that the introduction of HBV X protein (HBx) and an oncogenic allele of Ras induced the tumorigenic transformation of immortalized human fibroblasts. However, it remains unclear if these observations apply to human hepatocytes. Moreover, recent evidence suggests that HCC contains a subset of cells with stem cell features. In this study, we employed immortalized human hepatocytes and explored the requirements for transforming these cells; furthermore, we identified the relationship between HBx-induced tumorigenic transformation and stem cell-like features. Materials and Methods: We used two immortalized human hepatocyte cell lines, TTNT1 6 cells (in which hTERT was introduced) and T5B cells (in which the SV40 large T antigen (LT) was introduced). We used a retrovirus vector to introduce V12H-Ras, effector-loop mutants of oncogenic Ras, V12H-RasT35S (S35), V12H-RasE37G (G37), and V12H-RasY40C (C40), and c-myc into TTNT16 cells, HBx-expressing TTNT16 cells, LT+small T antigen (ST)-expressing TTNT16 cells, and ST+hTERT-expressing T5B cells. We then used these cells to analyze cell proliferation, senescence, transformation, and stem-like features. Results: First, we evaluated whether HBx and Ras induced the tumorigenic transformation of the immortalized human hepatocytes. TTNT1 6 cells expressing wild-type Ras or C40, but not S35 or G37, showed a profound senescence-like phenotype after transfection. In contrast, the introduction of these two genes into TTNT16-HBx or TTNT16-LT+ST cells did not induce this senescence-like phenotype. Moreover, wild-type Ras, but not S35, G37, or C40, enabled HBx- or LT+ST-express-ing human hepatocytes to form large colonies in soft agar and large tumors in nude mice. Next, we analyzed the relationship between c-myc and/or HBx introduction and the tumorigenic transformation of immortalized human hepatocytes. C-myc activated the STAT3 pathway and induced tumorigenicity in HBx-or LT+ST-expressing immortalized human hepatocytes. Furthermore, c-myc and HBx co-expression induced stem cell-like features in immortalized human hepatocytes. Conclusions: Modification of the PI3K pathway can overcome active onco-gene-induced senescence. In addition, immortalized human hepatocytes require the activation of the Raf and Ral-GEF pathways for tumorigenic transformation. Furthermore, the co-expression of HBx and c-myc introduced stem cell-like features during the tumorigenic transformation of these cells.
Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Aji-nomoto Co., Inc, MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, Bayer Japan
The following people have nothing to disclose: Naoki Oishi, Seishi Murakami, Wang Xuyang
Targeted Disruption of Fgl1 Promotes Hepatocellular Carcinogenesis
Hamed Nayeb-Hashemi1, Valeriy Demchev1, Anal Desai1, Roderick Bronson3, Jason L. Hornick2, Chinweike Ukomadu1;
1Gastroenterology, Brigham and Women's Hospital, Boston, MA; 2Pathology, Brigham and Women's Hospital, Boston, MA; 3Micro-biology and Immunobiology, Harvard Medical School, Boston, MA
Background: Fibrinogen like protein-1 (Fgl1) a liver expressed protein has been shown to be downregulated in human hepatocellular carcinoma. Reduction of Fgl1 expression in vitro also increases anchorage independent cellular proliferation of human hepatoma cell lines. Aims: To determine if targeted disruption of Fgl1 leads to enhanced carcinogenesis following exposure to diethynitrosamine (DEN) and phenobarbitol (PB). Methods: We generated a knockout mouse by the targeted disruption of Fgl1. Mice received a single intraperitoneal injection of DEN (5mg/kg) at 15 days of age followed by continuous free access to water supplemented with 0.07% PB. Mice were sacrificed at 10–12 months of age and assessed for gross evidence of cancer. Liver tissues were collected for Western immunoblot, immunohistochemistry, and quantitative poly-merase chain reaction. To identify signaling pathways activated in the tumors, tumor and non tumor tissue were also subjected to reverse phase protein array (RPPA). Results: Mice null for Fgl1 which are usually larger than wild types, were smaller at 12 months compared to wild types (22.1g +/-2.08 vs. 27.6g +/- 0.85). Macroscopic tumors were present in 75% (9/12) of Fgl1-/- mice compared to 1 7% (1/6) wild type. Tumors in Fgl1-/- were multiple (>8 per mouse) versus a solitary tumor in the wild type mice. Expression of alpha-fetoprotein mRNA was three fold higher than in non-tumor liver tissue and histologic analysis showed thickened hepatocyte cords, nuclear atypia, and a high mitotic rate. We found no changes in canonical HCC pathways that involve β-catenin, Akt and p38 by RPPA. In support, immunohistochemistry showed only cytoplasmic localization of β-catenin in tumor tissue and no changes in phos-phorylation of Akt and p38 when Fgl1 tumors were compared to non tumor tissue by Western immunoblot. However, mToR was active as multiple downstream targets including but not limited to p-4EBP1, p70 S6K, p-RPS6, fatty acid synthase and acetyl-CoA carboxylase were enhanced. Western immunoblots confirm that threonine 37 phosphorylation of 4EBP1 a downstream target of mTor is elevated in tumors from Fgl1-/- mice. Conclusions: Disruption of Fgl1 expression promotes hepatocellular cancer following administration of DEN and PB. Carcinogenesis is associated with a reversal of the larger weight phenotype of Fgl1-/- mice suggesting a cachexia effect. DEN + PB carcinogenesis does not seem to be mediated by increased nuclear localization of β-catenin, nor activation of Akt or p38 dependent pathways. Rather, mTor is active through an Akt independent mechanism.
Chinweike Ukomadu - Consulting: Gilead Sciences
The following people have nothing to disclose: Hamed Nayeb-Hashemi, Valeriy Demchev, Anal Desai, Roderick Bronson, Jason L. Hornick
The hypoxia-induced long non-coding RNA linc-RoR modulates tumor cell resistance to hypoxia by an extracellular vesicle mediated regulation of hypoxia-signal-ing pathways in hepatocellular cancer
Kenji Takahashi, Irene K. Yan, Hiroaki Haga, Tushar Patel;
Mayo Clinic, Jacksonville, FL
Background: Resistance to adverse environmental conditions such as hypoxia contribute to tumor progression. Although deregulated expression of long non-coding RNA (lncRNA) occurs in cancers, their functional contribution to tumor responses to hypoxia are unknown. We have shown that lincRNA-RoR (linc-RoR) can modulate responses to chemotherapeutic stress in human hepatocellular cancers (HCC). Thus, our aims were to examine the role and involvement of linc-RoR and other lncRNA on tumor cell survival signaling during hypoxia. Methods: HepG2, Hep3B, HepG2ST, Huh7 and PLC human HCC cells and non-malignant (HH) cells were used. lncRNA and miR-145 expression were assessed by qPCR. Hypoxic stress was induced in vitro using a hypoxia chamber and 5% CO2 / 95% N2. In vivo hypoxia was assessed by pimonidazole staining and linc-RoR expression by RNA-ISH in PLC- cell xenografts in nude mice. Extracellular vesicles (EVs) were obtained by ultra-centrifugation. EV RNA was isolated and analyzed using qPCR or digital PCR. siRNA was used to modulate lncRNA expression. Cell viability was examined by MTS assay. Expression of HIF1α was assessed using ELISA and of other signaling proteins by Western blot. Results: We identified 20 hypoxia-responsive lncRNA in HepG2 cells. Amongst these, 7 were also increased by >2-fold in HepG2 cells compared to HH cells, and including linc-RoR. In other HCC cells, linc-RoR expression was increased by 1.7–4.7 fold compared to HH. siRNA to linc-RoR decreased HCC cell viability under hypoxia. Furthermore, linc-RoR expression was increased in hypoxic areas compared to non-hypoxic areas in vivo. Linc-RoR was highly expressed in HCC-cell EVs, and EV linc-RoR was further increased during hypoxia. EVs could be taken up by other cells and transfer linc-RoR to recipient cells. EVs from hypoxic cells increased HIF-1α expression and cell survival in recipient cells during hypoxia. Compared to controls, siRNA to linc-RoR decreased p70S6K1 phosphoryla-tion, PDK1 and HIF1α protein expression, and increased expression of the linc-RoR target miR-145 in HepG2 cells and in HCC xenografts in vivo. Conclusions: These findings provide mechanistic insights into resistance to hypoxia stress by (a) identifying hypoxia-responsive lncRNA e.g. linc-RoR, (b) showing a functional link between linc-RoR and hypoxia signaling in HCC, and (c) identifying a mechanistic role of inter-cellular EV mediated transfer of linc-RoR in promoting cell survival during hypoxic stress. These observations identify previously unrecognized mechanisms by which lncRNA can modulate cellular responses to hypoxia and have biological as well as therapeutic relevance.
The following people have nothing to disclose: Kenji Takahashi, Irene K. Yan, Hiroaki Haga, Tushar Patel
CRM197, an Inhibitor of HB-EGF, Can Suppress the Growth of Human Hepatocellular Carcinoma Cells in Vitro and in Vivo
Sumio Kawata1,2, Satoshi Ugajin1, Junji Yokozawa1, Hisayoshi Watanabe1, Takafumi Saito1, Yoshiaki Inui2, Yoshiyuki Ueno1;
1Department of Gastroenterology, Yamagata University, Faculty of Medicine, Yamagata, Japan;2Gastroenterology, Hyogo Prefectural Nishinomiya Hospital, Nishinomiya, Japan
Background: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent growth factor for hepato-cytes and is overexpressed in human hepatocellular carcinoma (HCC), suggesting an autocrine growth mechanism in the tumors as we described previously (Ref. 1,2. CRM197, a non-toxic mutant form of diphtheria toxin, is known to inhibit the action of HB-EGF (Ref.3). We demonstrate here that CRM197 can suppress the growth of human HCC in vitro and in vivo. Methods: The effects of recombinant HB-EGF, anti-human HB-EGF polyclonal antibody, and CRM197 on cell growth were examined using the human hepatoma-derived cell lines Hep3B and Huh7. The effect of CRM1 97 on EGFR phosphorylation in vitro was analyzed by Western blotting. CRM197 was also injected intraperitoneally into male nude mice that had been inoculated with Hep3B or Huh7 daily for 14 days. Results: Recombinant HB-EGF dose-dependently stimulated the growth of Hep3B and Huh7 cells. Addition of anti-HB-EGF antibody (50μg/mL) to the culture medium inhibited the growth of both cell lines (73% of control for Hep3G and 68% for Huh7) and CRM1 97 (1 0μg/mL) suppressed the growth (46% of control for Hep3B and 77% for Huh7) and HB-EGF-induced EGFR phosphorylation of the two cell lines in a dose-dependent manner. Tumor formation by Hep3B and Huh7 cells in nude mice was dose-dependently suppressed by CRM197 (1mg/kg), both when the inhibitor was administered beginning on the day of cell inoculation (39% of control for Hep3B and 42% for Huh7) or when the tumor diameter reached about 5 mm after inoculation (53% for HepB3 and 57% for Huh7). Conclusion: These data suggest that HB-EGF is a novel molecular target for treatment of human HCC. Reference 1: Inui Y, Kawata S, et al. Expression of heparin-binding epidermal growth factor in human hepatocellular carcinoma. Gastroenterology 1994; 107: 1799–804 Reference 2: Kiso S, Kawata S, et al. Liver regeneration in heparin-binding EGF-like growth factor trans-genic mice after partial hepatectomy. Gastroenterology 2003; 124: 701–7 Reference 3: Mitamura T, Higashiyama S, et al. Diphtheria toxin binds to the epidermal growth factor (EGF)-like domain of human heparin-binding EGF-like growth factor/diphtheria toxin receptor and inhibits specifically its mitogenic activity. J Biol Chem 1995; 270: 1015–9 This study was collaborated with Prof. Eisuke Mekada, Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University.
Yoshiyuki Ueno - Advisory Committees or Review Panels: Jansen
The following people have nothing to disclose: Sumio Kawata, Satoshi Ugajin, Junji Yokozawa, Hisayoshi Watanabe, Takafumi Saito, Yoshiaki Inui
15-hydroxyprostaglandin dehydrogenase expression is downregulated in hepatocellular carcinoma: role in tumor growth
Luis Castro-Sanchez1, Noelia Agra1, Cristina Llorente-Izquierdo1, Omar Motiño1, Marta Casado2,3, Lisardo Bosca1,3, Paloma Mar-tin-Sanz1,3;
1Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM, Madrid, Spain; 2Instituto de Biomedicina de Valencia (IBV-CSIC), Valencia, Spain; 3Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain
Background and aims: 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a tumor suppressor in some cancers. However, no data are available regarding 15-PGDH expression in hepatocellular carcinoma (HCC). We aimed to assess the potential role of 15-PGDH in HCC. Materials and methods: HCC cells lines were treated with EGF, HGF or different pharmacological inhibitors and vehicle as control. COX-2, mPGES-1 and 15-PGDH expression were analyzed by qPCR and Western-blot. Additionally, we induced 15-PGDH overexpres-sion or silencing in a hepatoma cell line, to test in vitro cell viability, cell cycle and apoptosis markers, so as to assess tumor growth in vivo in athymic nu/nu mice. Furthermore, this study comprised a chemical model of liver cancer induced with diethylnitrosamine, a mouse model of accelerated hepatocar-cinogenesis and human HCC biopsies where 15-PGDH expression was evaluated. Results: 15-PGDH was downregulated in human hepatoma cells with a high COX-2 and mPGES-1 expression. Moreover, EGF and HGF increased COX-2 and mPGES-1 levels and suppressed 15-PGDH expression by mainly involving ERK and p38MAPK activation. Besides, 15-PGDH expression was decreased in chemical and genetic murine models of HCC and in human HCC biopsies. Conversely, ectopic expression of 15-PGDH induced apoptosis in hepatoma cells and decreased the growth of hepatoma cells in nude mice whereas the silencing of 15-PGDH increased the tumor formation by promote increased in cell viability and S-phase cells. Conclusion: 15-PGDH expression is downregulated in HCC while the overexpression leads to apoptosis and may function as a relevant tumor suppressor and a potential therapeutic application in HCC.
The following people have nothing to disclose: Luis Castro-Sanchez, Noelia Agra, Cristina Llorente-Izquierdo, Omar Motiño, Marta Casado, Lisardo Bosca, Paloma Martin-Sanz
HDGF (hepatoma-derived growth factor)-silencing inhibits the proliferation of hepatoma cells through a sorafenib-independent pathway
Hirayuki Enomoto1, Hideji Nakamura1,2, Hiroyasu Imanishi1, Noriko Ishii1, Yukihisa Yuri1, Tomoko Aoki1, Kazunori Yoh1, Akio Ishii1, Tomoyuki Takashima1, Nobuhiro Aizawa1, Yoshiyuki Sakai1, Kazunari Iwata1, Naoto Ikeda1, Hironori Tanaka1, Yoshinori Iwata1, Masaki Saito1, Hiroko Iijima1, Shuhei Nishiguchi1;
1Division of Hepatobiliary and Pancreatic Medicine, Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya, Japan; 2Nissei Hospital, Osaka, Japan
Background/Aims: Although treatments that inhibit tumor angiogenesis (represented by sorafenib) have improved the prognosis of patients with advanced HCC (hepatocellular carcinoma), some patients do not respond to the current standard therapies. Therefore, it is important to develop a therapeutic agent that can suppress tumor progression synergistically or independently of the sorafenib treatment. HDGF (hepatoma-derived growth factor) is a unique molecule which has dual characteristics; it can act as a growth-stimulating factor for hepatoma cells and also as an angiogenic factor. The purpose of this study was to examine whether anti-HDGF treatment can provide a new therapeutic strategy for HCC. Methods: (1) We investigated whether sorafenib affected the expression level of HDGF. (2) We generated HDGF-silenced hepatoma cell lines by introducing a HDGF sh-RNA plasmid, and examined the effects of the reduced HDGF expression on the proliferation of hepatoma cells. (3) We examined whether the downregulation of HDGF can enhance the growth-inhibitory effects of sorafenib on hepatoma cells (4) We subcutaneously transplanted the control (mock-transfected) cells or the HDGF-silenced hepatoma cells into nude mice, and then examined the anti-tumor effects of oral sorafenib treatment on the mice that carried the control or HDGF-silenced hepatoma cells. All animal experiments were performed according to the criteria outlined in the "Guide for the Care and use of Laboratory Animals". Results: (1) The expression levels of HDGF in hepatoma cells (Hep3B, HepG2 and SK-Hep1) were not affected by sorafenib. (2) Two stably HDGF-silenced clones of hepatoma cell lines showed significantly lower proliferative activity compared with the control cells. (3) HDGF-silencing enhanced the growth inhibitory effects of sorafenib treatment in vitro. (4) Xenograft transplant experiments revealed that a reduction of HDGF significantly inhibited the HCC growth in vivo. Furthermore, HDGF reduction promoted the anti-tumor effects of oral sorafenib administration. Conclusions: Although HDGF is involved in HCC proliferation, the growth inhibitory mechanisms associated with the HDGF-silencing were at least partially different from those of the sorafenib treatment. Since HDGF-silencing inhibited the HCC growth through an apparently sorafenib-independent pathway, the anti-HDGF treatment is considered to represent a potentially new strategy for the treatment of HCC.
The following people have nothing to disclose: Hirayuki Enomoto, Hideji Nakamura, Hiroyasu Imanishi, Noriko Ishii, Yukihisa Yuri, Tomoko Aoki, Kazunori Yoh, Akio Ishii, Tomoyuki Takashima, Nobuhiro Aizawa, Yoshiyuki Sakai, Kazunari Iwata, Naoto Ikeda, Hironori Tanaka, Yoshinori Iwata, Masaki Saito, Hiroko Iijima, Shuhei Nishiguchi
Serum Dickkopf-1 as a biomarker for the diagnosis of hepatocellular carcinoma with stem cell features
Hajime Sunagozaka, Taro Yamashita, Naoki Oishi, Takehiro Hayashi, Hajime Takatori, Tetsuro Shimakami, Kazuya Kitamura, Kuniaki Arai, Takashi Kagaya, Yoshio Sakai, Tatsuya Yamashita, Eishiro Mizukoshi, Masao Honda, Shuichi Kaneko;
Department of Gastroenterology, Kanazawa University Hospital, Kanazawa, Japan
Background Stemness in cancer is currently of great interest as it can be used to predict prognosis of hepatocellular carcinoma (HCC). We recently proposed an HCC classification system defined by the stem cell markers epithelial cell adhesion molecule (EpCAM) and α-fetoprotein (AFP) to identify HCC subtypes closely related to certain liver lineages with distinct prognosis (Yamashita et al, Gastroenterology 2009). Here, we evaluated the utility of determining serum Dickkopf-1 (DKK-1) levels, encoded by DKK1, a gene activated by Wnt signaling and co-regulated with EPCAM, for the diagnosis of HCC with stem cell features. Material and Methods Patients diagnosed with HCC at the Liver Center, Kanazawa University Hospital, Japan from 2005 to 2012 were enrolled. We measured serum DKK-1 levels using the human DKK-1 ELISA kit (Uscn Life Science Inc.). Hepatic stem cell-like (HpSC-) and mature hepatocyte-like (MH-) HCCs were defined as previously described (Yamashita et al, Cancer Research 2008). Clinicopathological characteristics were determined and analyzed statistically in relation to serum DKK-1 concentrations using Kaplan-Meier survival analyses with log-rank tests, Cox proportional hazards models, Fisher's exact tests, and logistic regression models. Results The study included 357 HCC patients, 60 and 205 cases of whom had hepatitis B (HBV) or hepatitis C (HCV) infections, respectively. Mean serum DKK-1 levels were 209.3 pg/ml (range, 43.0–5556.3 pg/ml), and 54.4% of HCC patients showed elevated DKK-1 levels (DKK-1 high HCC) when a cut-off value of 200 pg/ml was used. Serum DKK-1 levels did not correlate with those of AFP or des-γ-carboxy prothrombin (DCP), and tended to be higher in HBV-related (mean, 248.3 pg/ml) compared with HCV-related HCCs (mean, 182.1 pg/ml). Fifty-eight percent of HCC patients who were negative for AFP and DCP were DKK-1 high. HpSC-HCCs showed poor prognosis with high serum DKK-1 levels compared with MH-HCCs who received surgery, and DKK-1 high HCCs showed a significantly high frequency of portal vein invasion (p < 0.001). Among Barcelona Clinic Liver Cancer (BCLC) stage C patients treated with sorafenib or hepatic arterial infusion chemotherapy using interferon-alpha/5-FU/cisplatin, DKK-1 high HCCs showed a significantly poor prognosis compared with DKK-1 low HCCs (median overall survival 10.6 vs. 13.2 months: p=0.031, and 3.4 vs. 26.7 months: p=0.0005, respectively). Conclusions Serum DKK-1 is elevated in HCC with stem cell features. The poor response of DKK-1 high HCCs to sorafenib or cytotoxic reagents warrants the needs for the development of a novel treatment strategy against this deadly HCC subtype.
The following people have nothing to disclose: Hajime Sunagozaka, Taro Yamashita, Naoki Oishi, Takehiro Hayashi, Hajime Takatori, Tetsuro Shimakami, Kazuya Kitamura, Kuniaki Arai, Takashi Kagaya, Yoshio Sakai, Tatsuya Yamashita, Eishiro Mizukoshi, Masao Honda
Oxidative stress induced by continuous hepatocyte apoptosis drives liver carcinogenesis independently of regeneration and DNA methylation status
Hayato Hikita, Tomohide Tatsumi, Yoshinobu Saito, Satoshi Tanaka, Satoshi Shimizu, Wei Li, Ryotaro Sakamori, Takuya Miyagi, Naoki Hiramatsu, Tetsuo Takehara;
Gastroenterology and Hepatology, Osaka University Guraduate School of Medicine, Suita, Japan
Background and Aim: Hepatocyte apoptosis is a hallmark for chronic viral hepatitis or non-alcoholic steatohepatitis, which is a high-risk condition for HCC. Although we have reported that continuous hepatocyte apoptosis led to HCC by using mice models generated by knockout (KO) of an anti-apoptotic gene, bcl-x or mcl-1, its mechanisms are still unveiled. The present study examined the mechanism of liver carcinogenesis in apoptosis prone liver. Methods: Hepatocyte-specific Mcl-1 KO mice, which develop spontaneous hepatocyte apoptosis, were examined. We detected DNA mutation by deep sequencing and quantified methylation rate by bisulfate sequencing. Results: Sixty nine% of Mcl-1 KO mice developed well-differentiated HCC in 1 year. Immunohistochemistry for 8-OHdG, Ki-67 and PCNA revealed oxidative stress accumulation and compensatory liver regeneration in Mcl-1 KO liver. Deep sequencing of whole exons of p53 (cov. 7985) and & beta-catenin (cov. 7609), of which mutations are most frequently found in human
HCC, revealed no significant difference in any base position among WT liver, KO non-cancerous liver (NC) and HCC. Ultra-deep sequencing of p53 exon7 in 1 fragment (cov. 69149) revealed no difference in mismatched base number/fragment among 3 groups. In contrast, bisulfate sequencing analysis for CpG islands of runx3, a tumor suppresser gene and reported to be hypermethylated in NC of patients with HCC, revealed that methylation rates of runx3 in NC as well as HCC was significantly higher than that in WT liver. On the other hand, apoptosis and regeneration in Mcl-1 KO mice were downregulated by further KO of pro-apoptotic genes, bak, bax or bid. The incidence of HCC at 1 year decreased from 69% (20/29) to 0% (0/9), 11% (1/9), 11% (1/9), respectively. The number of 8-OHdG-positive hepatocytes in Mcl-1 KO mice was also significantly decreased in all double KO mice. To examine the impact of oxidative stress, Mcl-1 KO mice were continuously fed with L-N-acetylcysteine (NAC; 10g/l), an antioxidant, in drinking water. NAC administration did not affect the levels of hepatocyte apoptosis, regeneration or runx3 methylation in Mcl-1 KO liver. In contrast, NAC significantly decreased not only 8-OHdG-positive hepatocytes but also incidence rate of HCC from 69% to 33%. Conclusion: Continuous hepatocyte apoptosis, which leads to HCC, produces oxidative stresses in the liver. It also generates compensatory liver regeneration and DNA hypermethylation in some genes. Anti-oxidative therapy prevents HCC without attenuating apoptosis, regeneration and methylation status.
Tetsuo Takehara - Grant/Research Support: Chugai Pharmaceutical Co., MSD K.K.
The following people have nothing to disclose: Hayato Hikita, Tomohide Tatsumi, Yoshinobu Saito, Satoshi Tanaka, Satoshi Shimizu, Wei Li, Ryotaro Sakamori, Takuya Miyagi, Naoki Hiramatsu
Generation of mesenchymal stem-like CD105+ cancer cells de novo after chemotherapy in human hepatocellu-lar carcinoma
Yoshimoto Nomura, Taro Yamashita, Naoki Oishi, Kouki Nio, Sha Sha Zeng, Takehiro Hayashi, Mariko Yoshida, Tomoyuki Hayashi, Hikari Okada, Hajime Sunagozaka, Hajime Takatori, Masao Honda, Shuichi Kaneko;
Liver Center, Kanazawa University Hospital, Kanazawa, Japan
[Background] Cancer stem cells (CSCs) are considered a pivotal target for the eradication of hepatocellular carcinoma (HCC). We recently reported that CSC markers EpCAM and CD90 are independently expressed in primary HCCs and cell lines and that CD90+ cells share features of metastatic vascular endothelial cells and express the vascular endothelial marker CD105, a co-receptor of transforming growth factor beta (Yamashita T, et al Hepatology 2013). In this study, we evaluated the effect of cytotoxic reagents on the expression of CD1 05 in human HCC. [Methods] Primary HCC cells obtained from surgically resected specimens and EpCAM+ CD90- cell lines Huh1 and Huh7 were treated with 5-FU or epirubicin in vitro. Gene and protein expression was evaluated by qRT-PCR and fluorescence-activated cell sorting (FACS). Expression of CD105 in primary HCC was evaluated by immunohistochemistry (IHC) or immunofluorescence (IF). The relation between CD105 expression status and HCC prognosis was analyzed using microarray data of 244 HCC cases and by Kaplan-Meier survival analysis. [Results] 5-FU or epirubicin treatment resulted in the generation of CD90+ and CD1 05+ cells in vitro in Huh1 and Huh7 cells originally containing no CD90+ or CD105+ cells, with enrichment of EpCAM+ cells. IF analysis validated the de novo generation of CD1 05+ cells with activation of ENG encoding CD105 and epithelial-mesenchymal transition (EMT) program regulators SNAI1 and SNAI2 evaluated by qRT-PCR analysis. IHC analysis indicated that CD105+ cells were morphologically identical to the vascular endothelial cells in untreated primary HCCs. However, surgically resected specimens after transcatheter arterial chemoembolization (TACE) clearly indicated that the CD1 05+ cancer cells survived at the periphery of the tumor. Kaplan-Meier survival analysis indicated that HCCs abundantly expressing ENG showed poor prognosis after surgery with statistical significance (P = 0.02). [Conclusions] Vascular endothelial marker CD105 is not only expressed in CD90+ mesenchymal cancer cells but is also activated after chemotherapy and TACE in EpCAM+ epithelial cancer cells accompanied by the activation of EMT regulators SNAI1 and SNAI2. CD105 may be good marker for the evaluation of EMT and could be useful for evaluating prognosis in HCC patients who receive surgery.
Mariko Yoshida - Grant/Research Support: Bayer
The following people have nothing to disclose: Yoshimoto Nomura, Taro Yamashita, Naoki Oishi, Kouki Nio, Sha Sha Zeng, Takehiro Hayashi, Tomoyuki Hayashi, Hikari Okada, Hajime Sunagozaka, Hajime Takatori, Masao Honda
Protocadherin 9 acts as a candidate tumor suppressor gene in hepatocellular carcinoma
Xiangmei Chen1, Jun Lv1,2, Pengfei Zhu1, Fengmin Lu1;
1Department of Microbiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China; 2Department of Infectious Disease, Nanfang Hospital, Southern Medical University, Guangzhou, China
Background & Aims: Our previous work has identified a frequent loss of Protocadherin 9 (PCDH9) in hepatocellular carcinoma (HCC) by using array-based comparative genomic hybridization (aCGH). However, the biochemical function of this potential tumor suppressor gene in HCC development has not been addressed. Therefore, we aimed to identify the genetic/epigenetic inactivation of PCDH9 and it's role in HCC. Methods: A total of 120 paired tumour and their corresponding non-tumour liver tissues from HCC patients with serum HBsAg positive were collected. Expression of PCDH9 and its functional targets was tested by real-time quantitative RT-PCR, western blot or immunohistochemistry anylisis. The DNA copy number variations of HCC tissues were detected by using aCGH assay. The methylation status of PCDH9 gene promoter in each paired tumor and non-tumor specimens was quantitatively analyzed, by a method composed of DNA methylation-sensitive endonu-clease digestion followed by quantitative PCR. The effect of PCDH9 on cell proliferation and tumor growth was detected by MTT, soft-agar, and xenograft tumorigenicity assays. The function of PCDH9 on cell migration was analyzed by scratch-wound healing and transwell assays. Results: Down-regulation of PCDH9 expression was detected in about 61% (73/120) of primary HCC tissues. The low expression of PCDH9 was significantly correlated with present portal vein invasion (p=0.0354). Based on aCGH data, losses of chromosome 13q21.32 where PCDH9 gene mapped was found in 6 of 25 tumor specimens (24%) and gain in 1 (4%) of cases. PCHD9 promoter hypermethylation was detected in 22% (24/109) of HCC tissues. Interestingly, PCDH9 hypermethylation was significantly correlation with larger tumor size (p=0.0139) and worse intrahepatic dissemination (p=0.0312). Demethylation treatment in PCDH9-hypermethylated HCC cells could restore its expression. Furthermore, ectopic PCDH9 expression in HCC cells significantly inhibited cell proliferation, anchorage independent growth, tumorigenicity and cell megration. PCDH9 overexpression could induce a mesenchymal-epithelial transition (MET) in HCC cell lines which was characterized by down-regulation of mesenchymal cell markers including N-cadherin, Vimentin and Fibronectin, and reactivation of epithelial cell markers such as E-cadherin and Occludin. In addition, the activation of GSK-3β signaling induced by PDCH9 was required for PCDH9-induced MET. Conclusions: PCDH9 acts as a novel tumor suppressor candidate gene in HCC. The genetic and/or epigenetic aberration mediated PCDH9 down-regulation was of clinical significance in HCC.
The following people have nothing to disclose: Xiangmei Chen, Jun Lv, Pengfei Zhu, Fengmin Lu
Lymphoid enhancer factor 1 (LEF1) Contributes to Hepatocellular Carcinoma Progression through Transcrip-tional Regulation of Epidermal-Mesenchymal Transition (EMT) Regulators and Stemness Genes
Jaw-Ching Wu1,2, Chih-Li Chen3,1, Ya-Yun Sun4,1, Chien-Wei Su1,5;
1 Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan; 2Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan; 3School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan; 4Grad-uate Institute of basic medicine, Fu-Jen Catholic University, New Taipei City, Taiwan; 5Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
Background and Aims: Lymphoid enhancer factor/T cell factor proteins (LEF/TCFs) mediate Wnt signals by recruiting beta-catenin and its co-activators to Wnt response elements of target genes. This activity of LEF 1 is important during development and its dysregulation associated with progress of several types of cancers. However, the role and mechanisms of LEF1 on the progress of hepatocellular carcinoma (HCC) remain to be investigated. Methods: Resected human HCC samples from 20 patients with postoperative recurrence and 12 without were analyzed by expression array. Immunohistochemical (IHC) staining was performed in another independent validation set of 74 HCC tissue samples. Tumor sphere formation was carried out in ultralow plates. Soft agar colony formation and trans-well invasion were performed to study the effects of downregulation of LEF1 in Mahlavu cells on tumor behaviors. Nude mice were used in xenotransplant experiments. Real time reverse transcription PCR, Western blot analysis and reporter assays were carried out to study the regulation mechanism of LEF1 on the expression of Twist, Snail, Slug, Vimentin and Oct4 genes. Chromatin immunoprecipitation (ChIP) was performed to study the binding of LEF1 on promoter regions of EMT regulators and stemness genes. Results: Microarray analysis showed that LEF 1 was associated with postoperative recurrence which was validated by IHC staining in another HCC cohort (p<0.0001). Moreover, over-expression of LEF1 was associated with Twist over-expression (p=0.018), a trend of Snail over-expression (p=0.064), multi-nodular tumors (p=0.025). In multivariate analysis, LEF 1 was one of the factors significantly associated with recurrence (p=0.002). Tumor sphere of Mahlavu cells showed upregulation of, beta-catenin, LEF1, Twist, Snail, Slug, Oct4 and increased trans-well invasion. Downregulation of LEF1 by shRNA decreased Twist, Snail, Slug, Vimentin and
Oct4 gene expression both in RNA and protein levels. Tumor sphere, soft agar colony formation, trans-well invasion were also decreased. Xenotransplant of Mahlavu cells with knockdown of LEF1 in nude mice showed smaller tumors compared to those parental Mahlavu cells. ChIP assay and reporter assays revealed that LEF1 can physically interact with and transcrip-tionally activate the promoter regions of Oct4, Snail, Slug and Twist. Conclusion: Taken together, LEF1 plays a pivotal role in the progress of HCC through transcriptional regulation of cancer stem-like cell regulator and EMT regulators.
The following people have nothing to disclose: Jaw-Ching Wu, Chih-Li Chen, Ya-Yun Sun, Chien-Wei Su
Hepatitis C Virus strains lead to variable copy number profiles of hepatocellular carcinoma
Waleed Fateen1,4, Stefano Berri2, Henry Wood2, Judy Wyatt3, Mahmoud El-meteini4, Charles Millson5, Philip Quirke1;
1Pathology and Tumour Biology, Leeds Institite of Cancer and Pathology, Leeds, United Kingdom; 2Section of Precancer Genomics, Leeds Institite of Cancer and Pathology, Leeds, United Kingdom; 3Histopathology department, Leeds Teaching Hospitals, Leeds, United Kingdom; 4Ain Shams University Center for Organ Transplantaiton, Cairo, Egypt; 5Hepatology Department, Leeds Teaching Hospitals, Leeds, United Kingdom
Purpose: Hepatitis C Virus (HCV) is the most common cause of hepatocellular carcinoma (HCC) in the west. Many theories exist but the exact oncogenic effects of HCV remain largely unknown. We investigated whether the genotype of the HCV strain leads to differences in the DNA profile of HCC. Methods: DNA was extracted from formalin fixed paraffin embedded blocks of surgically removed HCC associated with different strains of HCV. HCV genotype 1 (group 1 n=19), 3 (group 3 n=1 1) and 4 (group 4 n=14). HCV genotype 4 samples were recruited from Ain Shams University, Egypt. DNA was tagged using home designed primer tags and multiplexed on a single flow cell of Illumina's Hiseq next generation sequencing platform. Between 5 to 8 million mapping reads were generated per genome. Each genome was divided into a series of continuous non-overlapping and equally sized windows. The number of reads per HCC windows was compared against number of reads in corresponding windows of a pool of normal genomes sequenced using the same platform and downloaded from the 1000 genome project. The data was normalized for GC content, smoothed and segmented. GISTIC 2.0 was used to identify areas of significant copy number aberrations within each of the 3 groups of HCC. Results: Variations were found between the 3 groups of HCC. Group 1 had 43 significant copy number aberrations (CNAs), 24 of which were deletions. Group 3 had 29 significant CNAs, 12 of which were deletions while group 4 had 19 significant CNAs of which 5 were deletions. Seven amplification peaks were shared between the 3 groups (1q21.2, 2p11.2, 2p11.1, 14q11.2, 14q32.33, 16p11.2, 22q11.1). Three amplification peaks were shared between groups 1 and 4 (4p11, 9p13.1, 9p11.2) and three amplification peaks were shared between groups 1 and 3 (5q13.2, 8q24.3, 15q1 1.2). A single amplification peak was shared between groups 3 and 4 (9p12). There were 6 unique amplification peaks to group 1, 6 to group 3 and 3 to group 4. No deletion peaks were shared between all 3 groups. Two deletion peaks were shared between groups 1 and 4 (8p23.1, 9p12) and five deletion peaks were shared between groups 1 and 3 (2p11.2, 16p13.3, 16q24.3, 17q25.3, 19p13.3). No deletion peaks was shared between groups 3 and 4. There were 17 unique deletion peaks to group 1, 7 to group 3 and 3 to group 4. Conclusions: Low coverage sequencing revealed differences in the DNA profiles of HCC according to the causative HCV strain. Increasing the number of cases is needed to confirm these variations. Targeted, deeper sequencing of the altered areas described above is needed understand the biology of these changes.
Stefano Berri - Employment: Illumina UK Ltd
The following people have nothing to disclose: Waleed Fateen, Henry Wood, Judy Wyatt, Mahmoud El-meteini, Charles Millson, Philip Quirke
Synergistic inhibition of Liver Cancer Stem Cells and HCC cell lines targeting Wnt/β-catenin and Ras/Raf/MAPK pathways
Roberto R. Galuppo1, Changguo Chen1, Malay Shah1, Michael F. Daily1, MarkEvers2, Brett Spear3, Paul Angulo4, Roberto Gedaly1;
1 Surgery, University of kentucky, Lexington, KY; 2Markey Cancer Center, University of Kentucky, Lexington, KY; 3Department of Microbiology and Immunology, University of Kentucky, Lexington, KY; 4Department of Internal Medicine, University of Kentucky, Lexington, KY
Background: The purpose of this study is to determine the effect of drug combination therapy in liver cancer stem cells (LCSC) and HCC cell lines targeting wtn-β-catenin and RAS/RAF/MAPK signaling pathways. Methods: Cell profiling was performed by flowcytometry to detect LCSC markers (CD133, CD44, CD24 and ALDHA1) and by xenograft tumori-genesis. In vivo model was done in NOD/SCID mice by sub-cutaneously injecting 2000 LCSC in suspension to induce tumorigenesis. Inhibition of cellular proliferation by combination of sorafenib and FH535 was assayed by 3H-Thymidine incorporation. Analysis of synergism was performed using the software CalcuSyn version 2.0 from Biosoft®. Results: We found that LCSC contain 64.4% CD133+ cells, 83.2% CD44+ cells and 96.4% CD24+ cells. Tumor growth was observed on all NOD/SCID mice 28 days after inoculation of a low LCSC suspension. FH535 and sorafenib at a 2:1 concentration ratio synergistically inhibited 3H-thymidine incorporation in LCSC (CI=0.014). FH535 and sorafenib monotherapy significantly inhibited proliferation of HCC cell lines Huh7, Hep3B and PLC. Conclusion: LCSC (CD133+, CD44+, CD24+) were able to develop poorly differentiated tumors with low cell concentrations at 4 to 6 weeks. To our knowledge, this is the first report of synergistic effect using sorafenib and FH535 on LCSC and HCC cells lines in vitro.
Paul Angulo - Grant/Research Support: NIDDK, Mochida, Genfit
The following people have nothing to disclose: Roberto R. Galuppo, Changguo Chen, Malay Shah, Michael F. Daily, Mark Evers, Brett Spear, Roberto Gedaly
Novel cancer/testis antigen FATE/BJ-HCC-2 enhances invasion and metastasis of hepatocellular carcinoma cells
Xiaoang Yang1, Lili Ge1, Junyan Piao1, Yanhui Yin2, Yu Zhang2;
1 Department of Hepatology, Henan Academy of Medical and Pharmaceutical Sciences,Zhengzhou University, Zhengzhou, China; 2Department of Immunology, Health Sciences Center, Peking University, Beijing, China
Aims: To investigate the relationship of tumor metastasis and FATE/BJ-HCC-2 expression, we analysed differential gene expression in hepatocellular carcinoma(HCC) cells induced by FATE/BJ-HCC-2,detected the tumor cell invasion function and observed tumor genesis and metastasis caused by FATE/BJ-HCC-2 in animals. Methods: The stable cell clone (HCC cell line Bel-7402) expressing FATE/BJ-HCC-2 was established by trans-fection of this gene. The total RNA was purified from FATE/BJ-HCC-2 gene-transfected Bel-7402 and mock control cells respectively. Their cDNA was amplified by reverse transcript PCR, and then labeled with fluorescence as probes. The probes were hybridized with Affymetrix Human Genome U133 plus 2.0 GeneChip array .The gene expression of above cells was analyzed. The invasive ability of FATE/BJ-HCC-2 gene-transfected Bel-7402 and mock control cells was compared by tran-swell migration assay before and after FATE/BJ-HCC-2 gene expression was silenced by siRNA technique. The FATE/BJ-HCC-2 gene-transfected Bel-7402 and mock control cells were inoculated intraperitoneally(left lower quadrant) into nude mice to observe its tumor genesis and metastasis in six weeks. Results: GeneChip analysis showed that there were 821 genes up-regulation and 873 genes down-regulation induced by FATE/BJ-HCC-2 in the tumor cells. A group of genes that might relate to tumor metastasis, such as osteopotin, fibronectin, Inte-grin Linked Kinase(ILK),α-catenin,matrix metalloproteinase-1,etc., was screened. The results of transwell migration assay showed that the number of penetrated cells in the FATE/BJ-HCC-2 gene-transfected group was much more than that in mock control group. However, the number of penetrated cells in the FATE/BJ-HCC-2 gene-transfected group was less than that in control group after FATE/BJ-HCC-2 gene expression was silenced. Statistical analysis indicated that the difference is significant (P<0.01).The animal experiments showed that tumors were formed in abdominal cavity in 70% mice of the FATE/BJ-HCC-2 gene-transfected group, but only in 10% mice of mock control group. Furthermore we also find tumor formation in livers of mice of the FATE/BJ-HCC-2 gene-transfected group with naked eyes and under light microscope. There was no tumor cell growth in liver of the mice of mock control group. Conclusion: FATE/BJ-HCC-2 could induce differential expression of a group of genes in tumor cells. It enhances invasion function of tumor cells. Tumor cells with FATE/BJ-HCC-2 expression are easier to form tumors and tumor's metastasis. This suggests that FATE/BJ-HCC-2 may be an important factor to promote metastasis of tumor cells.
The following people have nothing to disclose: Xiaoang Yang, Lili Ge, Junyan Piao, Yanhui Yin, Yu Zhang
Semaphorin7a in fibrotic liver promotes HCC development
Samuele De Minicis, Chiara Rychlicki, Laura Agostinelli, Cinzia Candelaresi, Luciano Trozzi, Stefania Saccomanno, Eleonora Min-garelli, Marco Marzioni, Antonio Benedetti, Gianluca Svegliati-Baroni;
Gastroenterology, Polytechnic University of Marche, Torrette of Ancona, Italy
Background: Semaphorin7a (sema7a) is a membrane-anchored protein involved in neuronal and immune function regulating axonal growth, immune and inflammatory response. Recent evidences have confirmed an effect of sema7a on the regulation of bleomycin-induced pulmonary fibrosis. Our group have shown the involvement of sema7a in liver fibrogenesis, but no information are currently available on hepatocarcinogene-sis. Aims: to evaluate whether SEMA7A regulate HCC development. Methods: Liver injury was induced in vivo by diethylnitrosamine (DEN) i.p. injection (25 mg/Kg) in 15 days old wild type and SEMA7A KO mice. Kupffer cells, hepatic stellate cells (HSCs) and hepatocytes were isolated with a nyco-denz-based gradient method from mice livers. Recombinant protein for SEMA7A was used for in vitro experiments in primary hepatocytes and human HepG2 cells. Results: In vivo, mice treated with DEN developed HCC formation after 6 month from the treatment. The number of tumor nodules were significantly lower in SEMA7A KO mice than in WT mice, with an average of 8,2 macroscopic nodules in WT and 3,4 in the SEMA7A KO mice. Morevoer the average of the tumour size was 4.5 mm in WT vs 2 mm in SEMA7A KO. In vitro, we observed an increased expression of SEMA7A mRNA in hepatic stellate cells and in Kupffer cells isolated from fibrotic mouse livers, while a low expression was observed in hepatocytes. Hepatocytes do express one of the main receptor of sema7a: plexin C1, demonstrated by RT PCR. Moreover, primary hepatocytes from WT mice and human HepG2 cells, incubated with sema7a recombinant protein (1nM), show an increase in Ki67 mRNA, PCNA protein expression and a modification in the mRNA levels of carcinogenetic markers: 2.1 folds reduction and 4,1 fold increase respectively in PTEN and osteopontin mRNA expression vs untreated cells. Conclusions: SEMA7A is expressed in the liver and plays a crucial role in the development of HCC. SEMA7A produced mainly by hepatic stellate cells and Kupffer cells binds to its specific receptor Plexin C1, which is expressed in hepatocytes, promoting proliferation and carcinogenetic pathways. Differential expression of SEMA7A in the liver, which occurs during fibrogenesis, may potentially explain the increased risk of HCC development in the course of cirrhosis.
The following people have nothing to disclose: Samuele De Minicis, Chiara Rychlicki, Laura Agostinelli, Cinzia Candelaresi, Luciano Trozzi, Stefania Saccomanno, Eleonora Mingarelli, Marco Marzioni, Antonio Benedetti, Gianluca Svegliati-Baroni
Identification of a MicroRNA Signature for Recurrent HCC Vascular Invasion
KuangHsiang Chuang1, Christa L. Whitney-Miller2, Mark S. Orloff1, Matthew N. McCall3, Anthony Almudevar3, Christopher T. Barry1;
1 Transplant Surgery, University of Rochester, Rochester, NY; 2Pathology and Laboratory Medicine, University of Rochester, Rochester, NY; 3Biostatistics and Biocomputing, University of Rochester, Rochester, NY
Vascular invasion has been known to be a strong predictor of hepatocellular carcinoma (HCC) recurrence after liver transplant, but clinically reliable molecular markers for vascular invasion are still not available yet. Here we report a miRNA signature that can distinguish recurrent HCC with vascular invasion from recurrent HCC without vascular invasion. We examined vascular invasion on 124 HCC tumor nodules from a cohort of 77 HCC patients, 45 of whom had recurrent HCC within 3 years of transplant. We performed miRNA expression profiling on all nodules using miRNA microarrays. High value miRNA candidates (most statistically significant and present in the HCC recurrence with macrovascular invasion) were then be validated by qPCR verification. We found that 1 3 miRNAs were differentially expressed with at least 2 fold expression change with p<0.05 (12 downregulated: miR-22, miR-29a, miR-30a, miR-34a, miR-99a, miR-100, miR-126, miR-192, miR-194, miR-195, miR-199a, and miR-497, and 1 upregulated: miR-494). Hierarchical clustering of miRNAs versus patients clearly shows that these miRNAs significantly distinguish patients with and without HCC macrovascular invasion. Further analyses of these miRNAs demonstrates that most of 12 down-regulated miRNAs can inhibit HCC cell survival, proliferation, and angiogenesis via suppressing IGF, WNT, and VEGF signaling pathways, while the up-regulated miR-494 is a convergent downstream of oncogenic transcriptional factors such as H-Ras, c-Jun, and E2F. Our study discovers a miRNA signature distinguishing between recurrent HCC with and without macrovascular invasion. This miRNA signature may serve as a prognostic biomarker and also help direct therapeutic interventions for HCC.
Christa L. Whitney-Miller - Grant/Research Support: Genentech
The following people have nothing to disclose: KuangHsiang Chuang, Mark S. Orloff, Matthew N. McCall, Anthony Almudevar, Christopher T. Barry
Sorafenib and Triptolide as Combination Therapy for Hepatocellular Carcinoma
Osama Alsaied, Veena Sangwan, Sulagna Banerjee, Rohit Chugh, Ashok Saluja, Selwyn M. Vickers, Eric Jensen;
University of Minnesota, Minneapolis, MN
Introduction Sorafenib, a multi-tyrosine kinase inhibitor, is the only FDA approved chemotherapeutic agent for metastatic hepatocellular carcinoma (HCC). We have previously shown that triptolide enhances apoptosis in HuH-7 HCC cells. In this study, we examined the effects of these agents and their combination on HCC in vitro and in vivo. Methods HuH-7 cells were treated with triptolide (T - 50 nM), sorafenib (S - 1.25 uM) or combination (C) of both. Cell viability assay (WST-8, Dojindo), caspase 3/7 activation (Promega), and proliferation assay (ECIS, Applied BioPhysics) were performed. For in vivo studies, forty mice were implanted with subcutaneous HuH7 tumors and divided into four treatment groups (n=10); saline control, sorafenib 10 mg/kg PO daily (S), Minnelide (a pro-drug of triptolide) 0.21 mg/kg IP daily (M), and combination of both (C). Tumor volumes were assessed weekly. Results The combination of triptolide and sorafenib was superior to either drug alone in inducing apoptosis, and decreasing viability (T=45%, S=38%, C = 18% at 48 hours) and proliferation (T=90%, S=90%, C=75% at 24 hours). After 2 weeks of mice treatment, tumor growth inhibition rates were S = 59%, M = 84%, and C = 95%, while control mice tumor volumes were found increased at 9-fold. When crossed over to combination treatment, control mice tumor growth volumes plateaued over the following 2 weeks. (Fig 1) Conclusion The combination of sorafenib and triptolide increased apoptosis and decreased proliferation rate in vitro. Combining sorafenib with Minnelide inhibited tumor growth in vivo with greater efficacy than single agent treatments. In vivo combination treatment allowed for using a lower dose of sorafenib (1 0 mg/kg), which is less than 10% of currently prescribed dose for HCC patients. Combination treatment could have tremendous translational potential in the management of HCC.
Rohit Chugh - Patent Held/Filed: Minneamrita
The following people have nothing to disclose: Osama Alsaied, Veena Sangwan, Sulagna Banerjee, Ashok Saluja, Selwyn M. Vickers, Eric Jensen
MicroRNA profile in chronic hepatitis B-related hepatocellular carcinoma (HCC): Overexpression of miR-224 in HCC tissue predicts recurrence after curative resection
Sun Jae Lee, Eun Jung Ko, Jong Eun Yeon, Yang Jae Yoo, Keunhee Kang, Eileen L. Yoon, Sang Jun Suh, Hyun Jung Lee, Ji Hoon Kim, Yeon Seok Seo, Hyung Joon Yim, Kwan Soo Byun;
Department of Internal Medicine, Division of Gastroenterology and Hepatology,, Korea University College of Medicine, Seoul, Republic of Korea
Background: We investigated aberrant microRNA (miRNA) expression in chronic hepatitis B (CHB) related hepatocellular carcinoma (HCC) by comparing miRNA expression of HCC tissue and non-HCC tissue to reveal which miRNAs are related to the pathogenesis of HCC development. We also investigated the association between miRNA profiles and tumor characteristics or clinical outcome. Methods: Paired tissue samples (HCC and non-HCC) from resected liver specimens were obtained from 22 patients who underwent curative resection. Microarray was performed to screen miRNAs differentiating HCC and non-HCC tissues in the relative expression. Quantitative real time polymerase chain reaction (PCR) was performed to validate the miRNA microarray data. By using miRBase, target signaling pathways of miRNA were predicted. Results: In microarray, miR-532-3p, miR-106b-5p, miR-224, miR-93-5p were up-regulated in HCC tissues, while miR-139-5p was down-regulated in HCC tissues. miR-7-5p, miR-885-3p were up-regulated in HCC with microvascular invasion compared with HCC without microvascular invasion. miR-224 were up-regulated in recurred HCC than non-recurred HCC after curative resection, while miR-194-3p and miR-192-5p were down-regulated in recurred HCC. The most common predicted target signaling pathway of aberrantly expressed miRNAs in HCC was Wnt signaling pathway. In real-time qPCR, only miR-139-5p showed aberrant expression in HCC tissues (fold change: 0.147, p=0.015), and only miR-224 was significantly up-regulated in recurred HCC after curative resection than non-recurred HCC (fold change: 3.36, p<0.001). When fold change of 3.2 (vs. non-tumor) was defined as cut-off value, sensitivity and specificity of miR-224 in predicting recurrence were 87.5% and 71.4%, respectively. miR-224 was superior in predicting recurrence after curative resection to the presence of microvascular invasion, large tumor size, multiple tumor and high serum alpha-feto protein level.
Conclusions: miR-1 39-5p was down-regulated in tissues of CHB related HCC. Overexpression of miR-224 in HCC tissues was superior in the prediction of reccurrence to other various clinical parameters.
Hyung Joon Yim - Grant/Research Support: GSK Korea, Handok Pharm, Gilead Korea; Speaking and Teaching: BMS Korea
The following people have nothing to disclose: Sun Jae Lee, Eun Jung Ko, Jong Eun Yeon, Yang Jae Yoo, Keunhee Kang, Eileen L. Yoon, Sang Jun Suh, Hyun Jung Lee, Ji Hoon Kim, Yeon Seok Seo, Kwan Soo Byun
Sublethal heat treatment induces Snail expression and ERK1 /2 to accelerate cell proliferation, migration and invasiveness of hepatocellular carcinoma cells
Shuhei Yoshida1,2, Naoki Ikenaga2, Miroslaw Kornek3,2, Masahiko Shimada1, Takayoshi Nishino1, Atsushi Mitsunaga1, Simon C. Rob-son2, Detlef Schuppan2,4;
1Division of Gastroenterology and Hepa-tology, Yachiyo Medical Center, Tokyo Women's Medical University, Yachiyo, Japan; 2Division of Gastroenterology and Liver Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; 3Department of Medicine II, Saarland University Medical Center, Homburg, Germany; 4Division of Molecular and Translational Medicine, Dept. of Medicine I, Univ. of Mainz Medical School, Mainz, Germany
BACKGROUND/AIMS. After insufficient radiofrequency ablation (RFA) local recurrent HCC has been reported to spread more aggressively than pre-RFA. We previously showed that HCC cells exposed to sublethal heat undergo EMT-like transition and a higher proliferation via ERK1/2 activation both vitro and vivo (AASLD 2012: 887). However, it remained unclear how far epithelial-mesenchymal transition (EMT) or heat shock proteins (HSPs) are involved. METHODS. The human HCC cell lines Huh7, HepG2, and Hep3B were exposed to temperatures of 37, 45, 48 and 50 degree C for 10 min. After 5 days quantitative WB was performed for Snail (EMT), MAPKs (ERK1/2, SAPK/JNK, p38MAPK) and HSP27, 70, 90. Invasiveness and migration of HCC cells were confirmed by tumor invasion assays. Inhibition of ERK1/2 was performed by 10μM of U0126 for 48 hrs. 5 x 1 06 HepG2 cells in matrigel were implanted s.c. in the right flank of nude mice (n=6 per group) at day 3 after heat treatment. Ki67 positive cells/ high power field (HPF, x400) were counted in the center and the invasion front of the tumors at day 15 after implantation. RESULTS. Compared to their untreated counterparts, heat pretreated HCC cells showed increased proliferation (as evidenced by CFSE-labeling and WB for PCNA), a high expression of EMT-related genes (Snail, COL1A1, TWIST1, CHD1L, all p<0.05), and activation of ERK1/2 (p<0.005). Snail protein was increased 5–8-fold at day 5 after heat treatment, and HCC cells showed a significantly enhanced invasiveness and cell migration in vitro at day 5. HSP27, 70 and 90 were significantly increased at day 5 post heat treatment to revert to baseline levels at day 12. Blockade of ERK1/2 by U0126 significantly attenuated all of these changes (all p<0.05). In vivo, HepG2 cells pretreated at 48 and 50 degrees C induced significantly larger tumors than control cells maintained at 37 degrees C 15 days after implantation. Ki67 in the tumor center were significantly higher in the 48 and 50 compared to the 37 degree C degree group. No difference between groups was observed in tumor necrosis, vas-cularization or invasiveness. CONCLUSIONS. HCC cells exposed to sublethal heat undergo (incomplete and reversible) EMT, enhanced tumor invasiveness and cell migration. ERK1/2 activation and enhanced HSP27, 70 and 90 expressions appear to be major driving forces of these changes, which may accelerate not only proliferation but also invasion/metastasis of HCC.
Simon C. Robson - Grant/Research Support: Pfizer, NIH; Independent Contractor: eBioscience, Biolegend, EMD Millipore, Mersana; Speaking and Teaching: ACP, Elsevier, ATC; Stock Shareholder: Nanopharma, Puretech
Detlef Schuppan - Consulting: Boehringer Ingelheim, Aegerion, Gilead, Gen-zyme, GSK, Pfizer, Takeda, Sanofi Aventis, Silence
The following people have nothing to disclose: Shuhei Yoshida, Naoki Ikenaga, Miroslaw Kornek, Masahiko Shimada, Takayoshi Nishino, Atsushi Mitsunaga
Glucose transporter isoform 1 (GLUT1) expression enhances formation and growth of hepatic metastases
Andreas Koch1, Peter Wild2, Martina Müller1, Anja Bosserhoff1, Claus Hellerbrand1;
1University Hospital Regensburg, Regensburg, Germany; 2University Hospital Zurich, Zurich, Switzerland
The facilitative glucose transporter isoform 1 (GLUT1) is the key rate-limiting factor in glucose transport into cancer cells. High portal glucose levels may be one of the factors supporting tumor growth and progression in hepatic tissue, and we have previously shown that GLUT1 is a tumor-promotor in hepatocellular carcinoma, while its expression is at the detection limit in normal hepatocytes. The aim of this study was to analyze whether GLUT1 expression and a high capacity for glucose uptake, respectively, is a general pro-cancerogenic factor of the liver. For that, we used malignant melanoma as a model-tumor, which is known to preferentially metastasize to the liver. Methods and Results: Similar as observed in HCC, GLUT1 expression was enhanced in melanoma cell lines compared to primary melanocytes, as well as in melanoma compared to naevi. Furthermore, immunohistochemical analysis of a tissue microarray consisting of 1 40 human melanoma tissues showed that GLUT1 expression was significantly enhanced in metastasis compared to primary tumors. GLUT1 expression in primary tumors correlated with tumor staging, and most importantly, with progression- and overall-survival, which are known to be determined by metastasis in this tumor. To determine the role of GLUT1 in melanoma metastasis, GLUT1 expression was suppressed in the murine melanoma cell line B16 by stable trans-fection with shRNA. GLUT1 suppression inhibited anaerobic glycolysis, proliferation and migration of B16 cells. Moreover, GLUT1 suppression induced apoptosis in low glucose but not in high glucose conditions. Next, B1 6 cell clones with and without GLUT1 suppression were subjected to an established model of hepatic metastasis, in which tumor cells were injected into the spleen of syngeneic mice from where they metastasize into the liver via the portal circulation. GLUT1 suppressed cells formed significantly smaller and less invasive metastasis than mock-transfected controls. Furthermore, TUNEL staining reveled more apoptotic cells in metastases derived from GLUT1 suppressed B16 cells compared to metastases from control cells. Conclusions: Our data promote the hypothesis that high glucose levels in the portal circulation and the liver, and the capacity to utilize those, respectively, promote hepatic metastasis. GLUT1, which is almost selectively expressed in malignant cells but not in healthy liver or other non-malignant tissues, appears as attractive therapeutic target for hepatic metastasis.
Martina Müller - Grant/Research Support: Novartis
The following people have nothing to disclose: Andreas Koch, Peter Wild, Anja Bosserhoff, Claus Hellerbrand
The Roles of Ras Isoforms in Liver Cancer
Sook In Chung1,2, Hye Lim Ju1,2, Sinhwa Baek1,2, Kwang-Hyub Han1,3, Weonsang S. Ro1,3;
1Liver Cirrhosis Clinical Research Center, Yonsei University College of Medicine, seoul, Republic of Korea; 2Brain Korea 21 Project for Medical Science College of Medicine, Yonsei University College of Medicine, seoul, Republic of Korea; 3Department of Internal Medicine, Yonsei University College of Medicine, seoul, Republic of Korea
Background/Aims: Activation of Ras proteins is a key onco-genic event in human carcinogenesis. Mutations affecting the three prototype Ras oncoproteins, Hras, Nras, and Kras, show a high degree of tumor-type specificity. Kras and Nras are mutated in liver cancer, but Hras mutations are rare. In this study, we determined the liver tumorigenic potentials of the three Ras isoforms. Methods: Transgenic liver cancer mouse models expressing different Ras isoforms were developed using a hydrodynamic injection method and the Sleeping Beauty Transposon System. Transposon vectors, each encoding an oncogene (HrasG12V, KrasG12V, and NrasG12V) or down-regulating a tumor suppressor gene (shp53), were constructed. To induce liver cancer, 40 μg of the three plasmids encoding the sleeping beauty transposase and two transposons were diluted in 2.5 ml of 0.9% saline and injected into the lateral tail veins of 6-week-old C57BL/6 mice. The mice were observed at 23 days post-hydrodynamic injection or near death. Results: Co-expression of H-, K-, N-RasG12V and shp53 resulted in massive abdominal enlargement within 4 weeks after injection. Several nodular lesions emerged from the liver parenchyma and occupied most of the liver surface 23 days after injection. The ratio of liver/body weight in the KrasG12V group increased significantly compared to those in the HrasG12V (p = 0.0005) and NrasG12V groups (p = 0.0181). The ratio of the NrasG12V group showed a mild increase compared to that of the HrasG12V group, but this was not significant (p = 0.3819). The survival curve of these groups corresponded to the ratio of liver/body weight. All mice were moribund by 36 days. Conclusion: Co-expression of RasG12V and shp53 in the mouse liver promoted rapid hepatocarcinogenesis. In particular, we found that Kras was the most oncogenic among the Ras isoforms in the liver when co-expressed with shp53.
The following people have nothing to disclose: Sook In Chung, Hye Lim Ju, Sinhwa Baek, Kwang-Hyub Han, Weonsang S. Ro
Identification and validation of VWF as a potential plasma biomarker for Hepatocellular carcinoma by a Quantitative Proteomic Analysis
Xiwei Wang1,2, Min Yang1,2, Hong Li1,2, Hongmin Zhang1,2, Yix-uan Yang1,2, Peng Hu1,2, Huaidong Hu1,2, Dazhi Zhang1,2, Hong Ren1,2;
1 Department of Infectious Diseases, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China; 2Key Laboratory of Molecular Biology for Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
Background: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Although alpha-fetoprotein (AFP) is one of the most effective markers available to diagnose HCC, the sensitivity and specificity are not optimal. Therefore, new and better Biomarkers for the detection of hepatocellular carcinoma (HCC) are urgently needed. The aims of this study was to identify and validate proteins that have not been implicated in HCC . Methods: We used ITRAQ (isobaric tags for relative and absolute quantitation) combined with mass spectrometry analysis to identify the differentially secreted proteins in plasma samples from 15 HBV-related HCC, non-tumor patients of 13 liver cirrhosis (LC) and 12 chronic hepatitis B (CHB), as well as from 1 0 normal human individuals. Western blot, Enzyme-Linked Immunosorbnent Assay (ELISA) and immunohistochemical analysis were used for further validation. Results: In total, 512 unique proteins were identified and 25 proteins were found to be differentially secreted in the plasma of HCC patients compared with control subjects. Von Willebrand factor (VWF), one of the highly secreted proteins in HCC samples identified by ITRAQ and mass spectrometry analysis, was revalidated by Western blot analysis of individual plasma samples and was demonstrated significantly elevated in HCC specimens. Further validation by Immunohistochemistry revealed that the expression of VWF in tissue samples (75HCC, 70LC and 65HBV) was highly correlated with HCC. Finally, siRNA-mediated down-regulation of VWF expression significantly decreased both invasive ability and migration of BEL7402 and HepG2 cell lines in vitro. Conclusion: Through an ITRAQ based plasma proteomic approach, VWF was proved to be a potential diagnostic biomarker for HBV-related HCC. The prognostic values of VWF and its possible therapeutic applications are worth further investigation. Acknowledgements: This research was supported by the National Natural Science Foundation of China (81171560, 30930082, 81171561, 30972584), the National Science and Technology Major Project of China (2008ZX10002-006, 2012ZX1002007001, 2011ZX09302005, 2012ZX09303001-001, 2012ZX10002003), The National High Technology Research and Development Program of China(201 1AA0201 1 1), the Key Project of Chongqing Science and Technology Commission (cstc2012gg-yyjsB1 0007), the Chongqing Natural Science Foundation(cstc2011 jjA10025), the Medical Research Fund by Chongqing Municipal Health Bureau (2009–1-71).
The following people have nothing to disclose: Xiwei Wang, Min Yang, Hong Li, Hongmin Zhang, Yixuan Yang, Peng Hu, Huaidong Hu, Dazhi Zhang, Hong Ren
Suppression of hepatocellular carcinoma by inhibition of ornithine animotransferase over-expression using a non-neurotoxic GABA analogue
Ami Ben Ya'acov1, Ehud Zigmond1, Yoav Lichtenstein1, Zvi Shalev1, Lydia Zolotarov1, Hejun Lu2, Richard B. Silverman2, Yaron Ilan1;
1Hebrew University Hadassah Medical Center, Jerusalem, Israel; 2Department of Chemistry, Department of Molecular Bio-sciences, Chemistry of Life Processes Institute, and Center for Molecular Innovation and Drug Discovery,, Northwestern University, Evanston, Illinois, IL
Background & aims: Ornithine aminotransferase (OAT) is a mitochondrial enzyme that catalyses the transamination of ornithine to glutamate and is a beta-catenin target gene. OAT and GABA-aminotransferase (GABA-AT) have high sequence homology and share a common inhibitory mechanism. 5-Amino-1, 3-hexadienyl-carboxylic acid (L-gabaculine) is a natural inhibitor that irreversible inhibits GABA-AT and OAT. DNA-micro array-based gene expression profiling study performed in tumors from spontaneous HCC-developing sand rat model identified increased expression of OAT gene. In vitro, L-gabaculine significantly suppressed the proliferation of HCC cell lines. As, L-gabaculine is neurotoxic, the aim of the present study was to assess the anti-HCC effect of a non-neurotoxic L-gabaculine analogue (LHJ-II-79). Method: Screening of L-gabaculine analogues was performed for identification of the most potent anti tumor molecule, followed by assessment of the effect in vivo. Hep3B HCC-transplanted athymic Balb/c mice (n=8/group) were injected three times a week with 1 mg/kg body weight of LHJ-II-79 and were followed for their tumor growth and AFP serum levels measurements. Results: In vivo administration of LHJ-II-79 suppressed AFP secretion from HCC harboring mice. Following 14 days of treatment, serum AFP levels were suppressed and increased by 3.4 fold compared with 1 0.9 fold increase in treated vs. controls, respectively (7224 to 24857 vs. 2671 to 29155 pg/ml, respectively). In vivo administration of LHJ-II-79 also suppressed tumor size. Within 14 days of treatment tumor size was suppressed and increased by 2.45 in comparison with 8.4 folds, in treated vs. controls, respectively (0.24 to 0.49 cm3 vs. 0.034 to 0.287 cm3, respectively). Following 21 days of treatment serum AFP levels increased by 8.15 folds vs. 49.8 fold in treated vs. controls, respectively. Tumor sizes were suppressed and increased by 3.05 folds vs. 24.2, in treated vs. controls, respectively. The anti-tumor effect was associated with an increase apoptotoic effect in treated animals by 20% as compared with controls. Conclusions: OAT, a beta-catenin target gene, is highly expressed in HCC. In vivo, administration of Gabaculine and of LHJ-II-79 a GABA analogue resulted in suppression of HCC growth. The results suggest that OAT plays an important role in HCC growth and may serve as a potential therapeutic target.
Yaron Ilan - Board Membership: Exalenz, Plantylight; Consulting: Immuron, ENZO, Abbott, Taxon; Grant/Research Support: Protalix
The following people have nothing to disclose: Ami Ben Ya'acov, Ehud Zigmond, Yoav Lichtenstein, Zvi Shalev, Lydia Zolotarov, Hejun Lu, Richard B. Silverman
Repertoires of alpha-fetoprotein (AFP) specific T-cell receptors from hepatocellular carcinoma patients after AFP-derived peptides vaccine treatment
Hidetoshi Nakagawa1, Eishiro Mizukoshi1, Eiji Kobayashi2, Kazu-toshi Yamada1, Kiichiro Kaji1, Masaaki Kitahara1, Hiroshi Hamana2, Hiroyuki Kishi2, Atsushi Muraguchi2, Shuichi Kaneko1;
1Department of Disease control and Homeostasis, Kanazawa University, Kanazawa city, Japan; 2Department of Immunology, University of Toyama, Toyama city, Japan
BACKGROUND/AIM: Antigen-specific T-cells play a key role for cancer immunotherapy and gene therapy modifying T-cells toward tumor cells is a promising strategy. In this course, isolation techniques for T-cell receptor (TCR) genes are critically important. We have developed alpha-fetoprotein (AFP)-directed immunotherapy for hepatocellular carcinoma (HCC) patients. Here, we induced AFP-specific CTLs from HCC patients after AFP-derived peptides vaccine treatment and obtained the TCR genes through completely novel and rapid cloning system; hTEC10 (human TCR efficient cloning within 10 days). METHODS: We chose two patients with long-term good responses against HCCs from the participants of the clinical trial (trial registration: UMIN000003514) using HLA-A24 restricted AFP357 (EYSRRHPQL) and AFP403 (KYIQESQAL) peptide vaccines for advanced HCC patients. The patient's peripheral blood mononuclear cells (PBMCs) were cultured in vitro and AFP357-specific cytotoxic lymphocytes (CTLs) were detected by staining with AFP357-MHC tetramers and anti-CD8 antibodies and sorted as single cells. cDNAs of paired TCR chains were amplified from the single cells, cloned into expression vectors and transduced in TCR-negative cell lines or human PBMCs. Thereafter antigen-specificities were confirmed by staining with AFP357-MHC tetramers. Finally, the avidities of obtained TCRs were estimated by comparing the cytotoxicity toward C1R-A24 cells loaded with various concentrations of peptides and the EC50 values were calculated. RESULTS: Patient No.1 achieved complete remission (CR) with 27th times of vaccinations and patient No.2 had stable disease (SD) with 39th times of vaccinations in their clinical courses. In both patients, initial serum AFP levels decreased after vaccine treatments (2,503 ng/ml to 3 ng/ml and 25,410 ng/ml to 14,850 ng/ml, respectively), AFP357-specific CTLs were induced from PBMCs (1.5% and 2.6% of CD8 positive cells, respectively). 199 AFP357-specific TCRs consisting of 7 kinds of TCRs (3 TCRs from patient No.1 and 4 from patient No.2) were obtained. EC50 values for the TCRs varied from 5.8 nM to 1.31 μM and the TCR with the highest avidity was derived from patient No. 1. CONCLUSIONS: In this study, a number of TCRs were obtained and evaluated in a short period of time. By expanding this method to other patients or healthy donors, we hope that mechanisms of immune responses after AFP-derived peptides vaccine therapy will be revealed and TCR gene therapy will be implementable.
Hiroyuki Kishi - Board Membership: SC World; Consulting: Vivalis Toyama Japan; Patent Held/Filed: Valneva
Atsushi Muraguchi - Consulting: SCW
The following people have nothing to disclose: Hidetoshi Nakagawa, Eishiro Mizukoshi, Eiji Kobayashi, Kazutoshi Yamada, Kiichiro Kaji, Masaaki Kitahara, Hiroshi Hamana
Treatment of Notch signal-activated hepatoma cells with γ-secretase inhibitors results in increased survival of mouse hepatoma models
Kazunori Kawaguchi, Masao Honda, Taro Yamashita, Hikari Okada, Kouki Nio, Yoshio Sakai, Tatsuya Yamashita, Eishiro Mizukoshi, Shuichi Kaneko;
Gastroenterology, Kanazawa Univ, Kanazawa, Japan
Introduction: Molecular targeted therapy using sorafenib has been accepted for hepatocellular carcinoma (HCC); however, the anti-tumor response to sorafenib varies between patients, so more effective drugs are needed. Candidate drugs are usually identified by analyzing gene expression or proteomic profiles, but some drugs target specific genes with genomic alterations and these exhibit a more stable effect in a wide range of patients. Previously, we reported that Notch activation and Jagged1 gene amplification were observed in α-fetoprotein (AFP)-producing hepatoma cells, while clinical cases with Jagged1 amplification showed decreased survival, even after therapy. Therefore, we evaluated the effectiveness of repressing the Notch-related anti-tumor activity of HCC in vitro and in vivo. Materials and Methods: We used siRNA to knock down the expression of the Notch-related genes Jagged1 and Notch1 in AFP-producing Huh7 and HepG2 hepatoma cells or non-AFP-producing HLE or SKHep1 hepatoma cells, and we evaluated the degree of cell growth or Notch activation. Moreover, we also inhibited the Notch receptor using γ-secretase inhibitors (GSIs; L-685,458 or DAPT) and examined cell growth. We inoculated 6-week-old NOD-SCID mice with Huh7 cells and we injected the GSIs percutaneously 2 weeks later. We compared the degree of Notch inhibition, tumor growth, and survival in these mice. Results: We confirmed the suppression of the downstream Notch pathway in hepatoma cells after Jagged1 or Notch1 siRNA knockdown by assessing HES-1 gene expression. The GSIs significantly suppressed cell growth and HES-1 gene expression in AFP-producing hepatoma cells, whereas no such effects were observed in AFP-negative hepatoma cells. L-685,458 reduced the growth of Huh7 cells in NOD-SCID mice compared to controls; moreover, when the tumors were allowed to grow, the control mice died earlier. Histologically, the hepatoma cells formed spacious necrotic or apoptotic areas in the L-685,458-treated mice, along with a diminishing number of active hepatoma cells compared with control mice. Cytoker-atin 19 staining for developed tumor cells was reduced in the L-685,458-treated mice, indicating that this drug repressed the malignant characteristics of the hepatoma cells. Conclusion: We could induce effective Notch inhibition and necrosis or apop-tosis of hepatoma cells in a mouse model by GSI treatment, and the administration of L-685,458 increased survival. Therefore, GSIs could represent an effective molecular targeted therapy for HCC.
The following people have nothing to disclose: Kazunori Kawaguchi, Masao Honda, Taro Yamashita, Hikari Okada, Kouki Nio, Yoshio Sakai, Tatsuya Yamashita, Eishiro Mizukoshi
Sensitivity of human cholangiocarcinoma (CCA) cells and CCA cancer stem cells (CSCs) to chemotherapeutics and molecular targeted agents
Alice Fraveto1, Alessia Torrice1, Maria Consiglia Bragazzi1, Anas-tasia Renzi2, Guido Carpino3, Felice Giuliante4, Agostino DeRose4, Gian Luca Grazi5, Vincenzo Cardinale1, Paolo Onori2, Antonio Franchitto2, Chiara Napoletano6, Raffaele Gentile1, Cristina Napoli1, Eugenio Gaudio2, Domenico Alvaro1;
1Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Rome, Italy; 2SAIMLAL, Sapienza, University of Rome, Rome, Italy; 3Health Science, University of Rome "Foro Italico", Rome, Italy; 4Surgery, Hepatobiliary Unit, Catholic University of the Sacred Heart School of Medicine, Rome, Rome, Italy; 5Regina Elena National Cancer Institute, Hepato-Biliary Surgery, Rome, Italy; 6Experimental Medicine, Sapienza, University of Rome, Rome, Italy
CCA is currently classified by two subtypes: the pure mucin-secreting extrahepatic or intrahepatic (Muc-CCA) form and the mixed intrahepatic (Mixed-CCA) form. CCA is resistant to chemotherapeutics where resistance is conferred by CSCs which are also responsible for tumor recurrence. Aim. To evaluated the in vitro sensitivity of human CCA cells and CSC sub-populations to chemotherapeutics and molecular targeted agents. Methods. CCA samples were obtained from surgically resected patients (N= 16). CCA cells were isolated by mechanic and enzymatic digestion and CSC subpopulations (CD90+/CD13+, CD90+/CD13-, CD90-/CD13+, CD90-/CD13-) were isolated by immunomagnetic separation. Sensitivity to chemotherapeutics (cisplatin, gemcitabine) and molecular targeted agents [Hedgehog (Hg) signaling inhibitors: ciclopamine, vismodegib, LY2940680; the broad-spectrum tyrosin-kinase inhibitor, genistein and, the aminopeptidase-N inhibitor, bestatin] was tested (72 hrs incubation) by evaluating proliferation (MTS assay) and apoptosis (Caspase 3). Results: Total CCA cells isolated from Mixed-CCA were more sensitive (p<0.01) to gemcitabine (20% cell survival at 5 μM) than cisplatin (80% survival at 5 μM) while the opposite was found for Muc-CCA cells. When different subpopulations were tested, CD90+ cells, that predominated in Muc-CCA, showed the highest resistance to cisplatin and gemcitabine. Among the Hg inhibitors, Muc- and Mixed-CCA cells were completely resistant to ciclopamine (1 00% cell survival at 60 μM) and showed similar sensitivity to LY2940680 (35% survival at 100 μM) but different sensitivity to Vismodegib (Mixed- > Muc-CCA; 40 vs 60% survival at 1 00μM, p<0.01). CD13+cells, that are a predom-inat subpopulation in Mixed-CCA, showed the strongest resistance to Hg inhibitors. Mixed-CCA cells were almost completely resistant to genistein or bestatin while Muc-CCA showed a slight sensitivity (65 % survival at 120 μM genistein and 250 μM bestatin). Conclusions: Cisplatin was more active against Muc-CCA while gemcitabine was more active against Mixed-CCA; resistance being conferred by the CD90+ subpopulation. Among the Hg inhibitors, ciclopamine is not effective, LY2940680 triggers both the Mixed- and Muc-CCA subtypes, and Vismodegib is more active against Mixed-CCA; the CD13 was the CSC subpopulation showing the strongest resistance. The tyrosin kinase inhibitor, genistein, and the aminopeptidase-N inhibitor, bestatin, showed minimal effect only against Muc-CCA. In substance, cisplatin and LY2940680 should be preferentially considered for the treatment of Muc-CCA while, gemcitabine, LY2940680Y and Vismodegib should be preferred for treatment of Mixed-CCA subtype.
The following people have nothing to disclose: Alice Fraveto, Alessia Torrice, Maria Consiglia Bragazzi, Anastasia Renzi, Guido Carpino, Felice Giuliante, Agostino DeRose, Gian Luca Grazi, Vincenzo Cardinale, Paolo Onori, Antonio Franchitto, Chiara Napoletano, Raffaele Gentile, Cristina Napoli, Eugenio Gaudio, Domenico Alvaro
The Therapeutic Potential of Artesunate in Hepatocellular Carcinoma
Yves-Paul Vandeynckel1, Debby Laukens1, Anja M. Geerts1, Isabelle Colle1, Louis Libbrecht3, Eliene Bogaerts1, Lindsey Deviss-cher1, Annelies Paridaens1, Xavier Verhelst1, Christian Vanhove2, Benedicte Descamps2, Sophie Janssens4,5, Bart Lambrecht4,5, Christophe Van Steenkiste1, Hans Van Vlierberghe1;
1Department of Hepatology and Gastroenterology, Ghent University Hospital, Ghent, Belgium; 2Infinity imaging lab, Ghent University, Ghent, Belgium; 3Department of Pathology, Ghent University Hospital, Ghent, Belgium; 4Department for Molecular Biomedical Research, VIB, Ghent University, Ghent, Belgium; 5Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Introduction. Artemisinins are safe antimalarial drugs which recently have shown potent anticancer activity. Here, we evaluated the effect of artesunate, a semi-synthetic derivative of artemisinins, on tumor growth, angiogenesis, the unfolded protein response and chemoresistance in hepatocellular carcinoma (HCC). Methods. The effect of artesunate was examined in HepG2, BWTG3 cells and in a diethylnitrosamine-induced mouse model for HCC. The histology of the tumor nodules was examined by H&E and reticulin staining. The expression of chemoresistance-related transporters, angiogenic factors and the activation of the unfolded protein response was determined by quantitative RT-PCR, Western blot, ELISA and/or immuno-histochemistry. Cytotoxicity was assessed by alanine and aspar-tate aminotransferase, lactate dehydrogenase, water-soluble tetrazolium salt 1 proliferation and caspase-3 activity assays. Small animal imaging by dynamic contrast-enhanced-MRI and choline PET was performed. Results. In both cell lines, artesunate dose dependently reduced cell viability and proliferation (from 50 micromolar: p<0.05). This reduction was enhanced by hypoxia (from 12.5 micromolar: p<0.01). Moreover, artesunate downregulated the secretion of VEGF and placental growth factor in vitro (both p<0.05) and in vivo (both p<0.01).
In the mouse model for HCC, artesunate decreased vessel density and tumor burden (both p<0.05). No apparent hepatotox-icity or mortality was observed. In vitro and in vivo activation of the PKR-like endoplasmic reticulum kinase pathway (resp. p<0.05 and p<0.01) together with decreased ATP-binding cassette transporter G2 expression (resp. p<0.01 and p<0.05) by artesunate was demonstrated. Conclusions. These observations exhibit the potential of artesunate as a safe treatment of HCC optionally combined with current hypoxia-inducing therapies such as transarterial embolization or sorafenib. As a low-cost drug, artesunate might be of particular interest for the developing countries with high incidence of HCC.
Isabelle Colle - Advisory Committees or Review Panels: MSD, Janssen, MSD, Gilead; Patent Held/Filed: Trombogenics; Speaking and Teaching: BMS, Janssen
The following people have nothing to disclose: Yves-Paul Vandewynckel, Debby Laukens, Anja M. Geerts, Louis Libbrecht, Eliene Bogaerts, Lindsey Devisscher, Annelies Paridaens, Xavier Verhelst, Christian Vanhove, Benedicte Descamps, Sophie Janssens, Bart Lambrecht, Christophe Van Steenkiste, Hans Van Vlier-berghe
Aptamer as a potential novel targeted therapy for hepatocellular carcinoma
Yun Bin Lee1, Jung-Hwan Yoon1, Jung Hwan Lee2, Eun Ju Cho3, Dong Hyeon Lee1, Yuri Cho1, Su Jong Yu1, Jeong-Hoon Lee1, Yoon Jun Kim1, Hyo-Suk Lee1, Chung Yong Kim1;
1 Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea; 2Aptamer Initiative, POSTECH Biotech Center, Pohang University of Science and Technology, Pohang, Republic of Korea; 33Department of Internal Medicine, Kangwon National University Hospital, Chuncheon, Republic of Korea
Background/Aim: Aptamers are small synthetic oligonu-cleotides that bind to protein targets with high specificity and affinity. AS1411 is the first nucleic acid aptamer in clinical development for cancers. AS1411 binds to the protein nucle-olin, which is over-expressed in the cytoplasm and on the surface of cancer cells. Glypican-3 (GPC3), a cell surface protein that is highly expressed in hepatocellular carcinoma (HCC), has emerged as a candidate therapeutic target. We aimed to investigate the therapeutic potential of aptamers for HCC by evaluating AS1411 effect on HCC cell growth and by confirming the affinity and specificity of GPC3 aptamer in HCC cells. Methods: Human HCC cell lines (Huh-7, SNU 761 cells) and human cholangiocarcinoma (CCA) cell lines (KMBC, Witt cells) were used. Cell growth was assessed using MTS assay and cell death signaling was explored by immunoblot analysis. Fluorescence-activated cell sorting was performed to evaluate the affinity and specificity of GPC3 aptamers in HCC cells. Results: Treatment of AS1411 aptamer decreased HCC cell proliferation, and modified AS1411 aptamer showed additional decrease in cell proliferation. However, AS1411 or modified AS1411 did not induce caspase 9 and 7 activation. Moreover, decrease of cell growth by AS1411 or modified AS1411 was neither prevented by caspase inhibitor nor necrosis inhibitor. Out of many GPC3 aptamers newly synthesized, we found a specific one showing high affinity and specificity in HCC cells as compared to CCA cells. Conclusions: We found that AS 1411 and modified AS1411 can suppress HCC cell growth without inducing cell death. Additionally, we confirmed that GPC3 aptamer may selectively bind to HCC cells with high affinity, implicating the therapeutic potential of aptamer as a novel targeted therapy for HCC.
The following people have nothing to disclose: Yun Bin Lee, Jung-Hwan Yoon, Jung Hwan Lee, Eun Ju Cho, Dong Hyeon Lee, Yuri Cho, Su Jong Yu, Jeong-Hoon Lee, Yoon Jun Kim, Hyo-Suk Lee, Chung Yong Kim
Histological characterization of biliary intraepithelial neoplasia with respect to pancreatic intraepithelial neoplasia
Yasunori Sato, Kenichi Harada, Motoko Sasaki, Yasuni Nakanuma;
Department of Human Pathology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
Background: Biliary intraepithelial neoplasia (BilIN) is a precursor lesion of cholangiocarcinoma arising in the hilar region of the liver and the extrahepatic bile duct. BilIN represents the process of multistep cholangiocarcinogenesis, and is a concept of biliary counterpart of pancreatic intraepithelial neoplasia (PanIN). Previous studies on histopathological characteristics of BilIN and PanIN have been performed individually. Aim: This study was performed to compare the histological characteristics of BilIN to those of PanIN. Methods: Paraffin-embedded tissue sections of surgically resected specimens of cholangiocarcinoma with hepatolithiasis (n = 25) associated with the foci of BilIN and pancreatic ductal adenocarcinoma (n = 22) associated with the foci of PanIN were used. Immunohistochemical staining was performed using the primary antibodies against MUC1, MUC2, MUC5AC, CEA, S1 00P, p53, cyclin D1 and p21. For mucin staining, alcian blue pH2.5 was used. The results were semi-quantitatively graded in consideration of the signal intensity or the percentage of positive cells in each lesion, and were compared for the foci of BilIN-1, BilIN-2/3, PanIN-1A/1B and PanIN-2/3. Results: Cytoplasmic mucin expression tended to be abundant in PanIN rather than BilIN, and PanIN-1A/1B showed significantly increased mucin expression compared to that of BilIN-1. Approximately 20% of the foci of BilIN-1 and BilIN-2/3 showed MUC2 expression, while it was almost negative in PanIN. The nuclear and cysto-plasmic expression of S100P was frequently observed in BilIN and PanIN, and its expression was significantly high in PanIN-1 A/1 B compared to that of BilIN-1. The expression of p53 was negative in BilIN-1 and PanIN-1A/1B. Twenty % of the foci of BilIN-2/3 and 64% of PanIN-2/3 foci showed positive immunohistochemical expression of p53, in which a significant difference was observed between them. The expression of MUC1, MUC5AC, CEA, cyclin D1, and p21 was similarly increased along with the increase in the epithelial atypia in both BilIN and PanIN. Conclusion: In addition to similarities in the histopathological features, several differences were observed between BilIN and PanIN. These results suggest that the carcinogenesis of cholangiocarcinoma and pancreatic ductal adenocarcinoma done not necessarily undergo the same process.
The following people have nothing to disclose: Yasunori Sato, Kenichi Harada, Motoko Sasaki, Yasuni Nakanuma
MAPK-activated protein kinase 2, a new binding partner of EBP50, promotes survival in liver cancer cells exposed to oxydative stress
Thanh Huong Nguyen Ho-Bouldoires1,3, Audrey Claperon1,3, Mar-tine Mergey1,3, Dominique Wendum1,5, Olivier Scatton1,4, Chantal Housset2,3, Laura Fouassier1,3;
1Biology and Treatment of Hepato-biliary Tumours, Saint-Antoine Research Centre, Inserm UMRS_938, Paris, France; 2Metabolic and biliary, fibro-inflamma-tory diseases of the liver, Saint-Antoine Research Centre, Inserm UMRS_938, Paris, France; 3UPMC, Univ Paris 06, Paris, France; 4Service de Chirurgie Hépato-Biliaire et Transplantation Hépatique, AP-HP, Hôpital Saint-Antoine, Paris, France; 5Service d'Anatomie et Cytologie Pathologiques, AP-HP, Hôpital Saint-Antoine, Paris, France
Introduction: Liver tumors develop in a context of oxidative stress and inflammation. Oxidative stress stimulates intracellular signaling such as the MK2 (mitogen-activated protein kinase-activated protein kinase 2) pathway. Activation of MK2, a target of p38 MAPK, promotes cell survival upon stress conditions via the phosphorylation of AKT and the heat shock protein HSP27. MK2 is also involved in the inflammatory response through the regulation of cytokines synthesis. Recently, we have identified the scaffolding protein EBP50 (Ezrin-radixin-moesin Phosphoprotein 50) as a new partner of MK2 in liver by a two-hybrid screening. The objective of the study was to investigate the role of MK2/EBP50 in tumor liver cells exposed to oxidative stress. Methods: Expression of MK2, HSP27 and EBP50 mRNA was analyzed by RT-qPCR in 40 human hepatocellular carcinoma and adjacent non-tumor liver tissue. Validation of the MK2-EBP50 interaction was addressed by co-immunoprecipita-tion and GST-pull down. In vitro, oxidative stress was induced by hydrogen peroxide. MK2-dependent signaling was analyzed by western blot in human hepatocellular (PLC/PRF/5) and biliary (Mz-ChA-1, EGI-1) carcinoma cell lines. Cell survival and apoptosis were assessed by MTT, flow cytometry (sub G1 fraction) and western blot (cleavage of caspase 3/PARP). Expression of interleukins was determined by RT-qPCR. The role of MK2 was investigated using a pharmacological inhibitor (MK2iIII) and that of EBP50 by siRNA. Results: In vivo, the expression of MK2, HSP27 and EBP50 mRNA was increased in HCC compared with the matched non tumor liver tissue. We showed that MK2 interacts with the PDZ domains of EBP50. In liver tumor cells, oxidative stress decreased cell viability and increased apoptosis. These effects were amplified in the presence of MK2iIII. MK2 was activated and induced the phosphorylation of AKT and HSP27 in response to oxidative stress. Oxidative stress increased the expression of IL-1 b/IL-8 mRNA that was also inhibited by MK2iIII. This response was not abolished by actinomycin D, an inhibitor of mRNA transcription indicating that MK2 regulates the expression of interleukins by a post-transcriptional mechanism. Upon oxidative stress, loss of EBP50 by siRNA in liver tumor cells caused a decreased phosphorylation of AKT/HSP27 and of IL-1 b/IL-8 mRNA levels. Conclusion: MK2 pathway is activated in liver tumor cells exposed to oxidative stress and contributes to cell survival. EBP50 is also involved in the oxidative stress response, potentially through its interaction with MK2. These data suggest a contribution of the MK2/EBP50 pathway in the resistance of liver tumor cells to oxidative stress.
The following people have nothing to disclose: Thanh Huong Nguyen Ho-Bouldoires, Audrey Claperon, Martine Mergey, Dominique Wendum, Olivier Scatton, Chantal Housset, Laura Fouassier
Overexpression of the insulin-like growth factor 2 (IGF2) mRNA binding protein (IMP) p62 as a marker of poor outcome in gallbladder cancer
Sonja M. Kessler1,2, Eva Lederer2, Robert Reihs2, Alexandra K. Kiemer1, Johannes Haybäck2;
1 Pharmaceutical Biology, Saarland University, Saarbruecken, Germany; 2Institute of Pathology, Medical University, Graz, Austria
Background: The oncofetal protein IMP2–2/p62 has been described to be overexpressed only in a few types of cancer. Gallbladder cancer is a rare but highly malignant, aggressive cancer entity with poor prognosis. Aim of this study was to investigate the implication of p62 on human gallbladder cancer. Methods: Tissue microarrays (TMAs) of 457 gallbladder cancer patients were analysed by immunohistochemistry. Eight different bile duct cancer cell lines were used to develop mouse xenografts. p62 was knocked down by siRNA in different bile duct cancer cell lines. Cell viability was measured by MTT assay. mRNA expression was investigated using real-time RT-PCR, protein expression was determined by Western Blot analysis. Results: Investigation of TMAs stained for p62 revealed overexpression in 66.6% of the tumor samples. Among the positive samples 8.6% highly expressed p62 whereas 91.4% showed mild to moderate expression of p62. Kaplan-Meier curves demonstrated a poor survival linked to high p62 expression (p=0.041; Figure 1). Cell lines, which caused the fastest increase in tumor volumes in a xenograft model, highly expressed p62. Upon knockdown of p62 in different bile duct cancer cell lines in vitro, the cytotoxic effect of the chemothera-peutics was increased suggesting a protective role of p62 in cell survival of gallbladder cancer cells. Conclusion: p62 is overexpressed in gallbladder cancer and is directly associated with poor survival. p62 might therefore display a new bio-marker or therapeutic target for the treatment of gallbladder cancer.
Figure 1. Kaplan-Meier survival analysis of gallbladder cancer patients with different expression levels of IMP2–2/p62.
The following people have nothing to disclose: Sonja M. Kessler, Eva Lederer, Robert Reihs, Alexandra K. Kiemer, Johannes Haybäck
Maid is a specific guardian gene to regulate DNA damage in liver fibrosis and hepatocarcinogenesis
Shuji Terai1, Naoki Yamamoto1, Taro Takami1, Issei Saeki1, Koichi Fujisawa1, Tomoaki Murata3, Hiroshi Nishina2, Isao Sakaida1;
1 Department of Gastroenterology & Hepatology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan; 2Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University,, Tokyo, Japan; 3Center for animal, Yamaguchi University, Ube, Yamaguchi, Japan
(Objective) Generation of hepatocellular carcinoma (HCC) is related with the progression of liver fibrosis. Maid (maternal inhibitor of differentiation) gene, which is HLH transcriptional regulator, binds to Cyclin D1, Rad51, E12, Jab1 and oligo1 and regulates cell cycle and differentiation. From the aspect of TGF-beta signaling and the structure, Maid was stress response regulator and regulated cell inhibition and ECM. In human hepatocarcinogenesis process, we found that HHM (Human homologue of Maid) is specific marker of dysplastic nodule and well-differentiated HCC. Previously we showed that Borte-zomib, a proteasome inhibitor, improved liver fibrosis and generation of HCC. To clarify the mechanism of Maid, we analyzed liver fibrosis and hepatocarcinogenesis in persistent injured Maid KO livers. (Material and Method) We generated Maid KO mice. The phenotype of Maid KO mice is normal. We therefore added 3 month repeated CCl4 injection (0.5mg/kg body) into Maid KO mice and wild type mice, and then analyzed liver fibrosis and hepatocarcinogenesis. We did DNA chip analysis and Ingenuity pathway analysis using 3 month CCl4 injected Maid KO and wild type livers and control no damage. We used 20 nM Bortezomib on HepG2 cells and human hepatocytes, and analyzed cell proliferation and HHM expression. (Results) CCl4 injected Maid KO mice accelerated to induce liver fibrosis compared with wild type mice (p<0.05), and had AFP positive-HCC (25%). DNA chip analysis revealed that "Cell cycle" related genes such as Cyclin A2, Cyclin E1, Cyclin B and CDC25c were increased. We also found that these genes related with "double strand DNA break repair" such as RAD51, RAD54L and RAD50 were also increased. DNA repair genes such as BLM, MSH2, MHH1, RAD50 and MRE1 1 were also activated in Maid KO livers. Moreover, we found that TGF-beta 3, Collagen 1A and Collagen 3A were significantly increased. In vitro analysis, Bortezomib specifically inhibited HepG2-proliferation with up-regulated HHM expression. (Discussion) We found that Maid KO mice itself activated DNA damage related and cell cycle related genes. Concerning with TGF-beta signaling, Maid disruption does not stop TGF-beta related cell inhibition and ECM production. From these systems, Maid KO mice induced both liver fibrosis and generation of HCC. Bortezomib induced HHM expression with inhibition of cell proliferation in HepG2 but not hepatocyte. These results indicated that Maid specifically regulate DNA damage and progression of liver fibrosis and hepatocarcinogenesis. (Conclusion) Maid is a specific guardian gene to regulate DNA damage in liver fibrosis and hepatocarcinogenesis.
Tomoaki Murata - Board Membership: Naoki Yamamoto, Taro Takami, Issei Saeki, Koichi Fujisawa, Hiroshi Nishina, Isao Sakaida; Speaking and Teaching: Shuji Terai
The following people have nothing to disclose: Shuji Terai, Naoki Yamamoto, Taro Takami, Issei Saeki, Koichi Fujisawa, Hiroshi Nishina, Isao Sakaida
Anti-proliferative mechanisms downstream of c-MET of the kinase inhibitor tivantinib (ARQ 197)
Shuai Lu1, Antonia Rizzani1, Frank T. Kolligs1, Eike Gallmeier1, Sabrina Arena2, Alberto Bardelli2,3, Burkhard Göke1, Alexander L. Gerbes1, Enrico N. De Toni1;
1Internal Medicine 2, University of Munich, Munich, Germany; 2Department of Oncology, IRCC, Institute for Cancer Research and Treatment and University of Torino, Candiolo, Italy; 3FIRC Institute of Molecular Oncology, Milan, Italy
Tivantinib has been tested in clinical trials as c-MET inhibitor and demonstrated clinical benefit as a second line treatment of hepatocellular carcinoma (HCC). Since its efficacy was predicted by elevated expression of c-MET in previous phase-2 and -3 randomized trials, a phase-3 trial in HCC patients selected according to c-MET expression has been initiated. Unexpectedly, recent in vitro studies challenged the notion of tivantinib as a selective c-MET inhibitor. To provide a possible explanation for these conflicting data, we investigated the molecular mechanisms of action of tivantinib and whether their regulation is influenced by c-MET by using a panel of 8 cell lines showing different c-MET expression status. We found that tivantinib exerted a strong antiproliferative effect in all the tested cell lines by inducing apoptosis and a G2/M cell cycle arrest. These changes were accompanied by cleavage of Cas-pase-8 and Bid, decreased p-Akt, Bcl-xl and Mcl-1, and increased Cyclin-B1. No obvious correlation between efficacy of tivantinib and c-MET expression in individual cell lines could be found. Nevertheless, although c-MET activation by HGF did not affect Cyclin B1, administration of HGF enhanced Akt-phos-phorylation, Mcl-1 and Bcl-xl showing that tivantinib impinges on targets downstream of c-MET. These findings might help explaining the mechanisms of action of this promising compound and account for the conflicting results between recent in vitro studies and the predictive significance of c-MET expression in clinical studies.
The following people have nothing to disclose: Shuai Lu, Antonia Rizzani, Frank T. Kolligs, Eike Gallmeier, Sabrina Arena, Alberto Bardelli, Burkhard Göke, Alexander L. Gerbes, Enrico N. De Toni
Vitamin D for prevention of liver cancer in the setting of disrupted TGF-β signaling pathway
Lior H. Katz1, Nina M. Muñoz1, Vivek Shukla2, Andrea C. Cortes1, Sara Peleg1, Kirti Shetty3, Jian Chen1, Randa El-Zein1, Lopa Mishra1;
1 University of Texas, MD Anderson Cancer Center, Houston, TX; 2NIH, Bethesda, MD; 3MedStar Washington Hospital Center, Washington, DC
Hepatocellular carcinoma (HCC) is the third most common cause of cancer related death worldwide. Vitamin D (VD) has been implicated in the prevention of multiple cancers- some with inactivation of TGF-β signaling. Recently, inactivation of TGF-β signaling has been associated with HCC. We analyzed whole genome data forTGF-β signaling and examined the role of VD in the context of TGF-β inactivation in HCC. Methods: Databases of HCC Genomics (COSMIC) and transcriptomics (TCGA) were analyzed. The effect of calcitriol on cell proliferation was measured in HCC cell lines; Hep-G2 cells, knocked down to β2-spectrin (β2SP); and in MEF cells, produced from mice knocked out to Sptbn1. Wild-type (WT), Sptbn1 +/- and Smad3+/- mice were fed with diets containing 200 IU VD/kg or 1 0,000 IU VD/kg for 9 weeks. Hepatocyte proliferation was analyzed through Ki67 staining. The expression of cyclin D1 was evaluated by Western blotting. A whole genome gene expression profiling array was performed from liver samples of those mice using Illumina MouseWG-6 v2.0 gene expression arrays®. Lastly, liver samples from patients with HCC who had received VD supplementation were evaluated by immunohisto-chemistry. Results: Whole genome data revealed aberrant TGF-β signaling in ∼70% of HCCs. Treatment with calcitriol suppressed the growth of HCC cell lines, regardless of their TGF-β pathway status. Inhibition of proliferation was noted in WT MEFs as well as in the Sptbn 1 +/- and Sptbn 1 -/- cells. High dose VD reduced proliferation in livers from normal mice and in from the Sptbn 1+/-and Smad3+/- mutants, and lowered their cyclin D1 levels. The microarray data showed that the most significant changes in gene expression were associated with acute phase inflammatory response (p<0.005). Validation studies showed a significant fold change differences of P-PARA, OAZ1, TLR7 and TLR 9 after high dose VD diet between wild type mice and the mutants (p<0.05). VD supplementation to patients with HCC was associated with higher expression of β2SP (p<.0001) and TβRII (p=.01 3) compared with that of patients who did not receive the supplement. Conclusions: Whole genome data revealed aberrant TGF-β signaling in ∼ 70% of HCCs. In contrast to other types of cancer, VD suppresses the proliferation of HCC cell lines as well as normal hepatocytes regardless their TGF-β signaling status. This might happen due to restoring of TGF-β signaling by VD. However, genes related to acute phase inflammation react differently to VD treatment in the setting of TGF-β signaling inactivation. Taken together, these results suggest that VD treatment strategies could potentially be pivotal in prevention and treatment of HCC.
Kirti Shetty - Grant/Research Support: Ikaria, Novartis, Onyx-Bayer, Hyperion; Speaking and Teaching: Merck-Schering Plough, Salix, Gilead, Onyx
The following people have nothing to disclose: Lior H. Katz, Nina M. Muñoz, Vivek Shukla, Andrea C. Cortes, Sara Peleg, Jian Chen, Randa El-Zein, Lopa Mishra
A Highly Invasive Subpopulation of Hepatoma Cells Is Associated with Enhanced Hedgehog Signaling and Epithelial Mesenchymal Transition
Ya-Han Fan1,2, Samantha Nguyen1, Jia Ding3, Jian Wu1,3;
1Internal Medicine/GI & Hepatology, UC Davis Medical Center, Sacramento, CA; 2Dept. of Gastroenterology, Xinqiao Hospital, Third Military University of Medical Sciences, Chongqing, China; 3Fudan University Shanghai Medical College, Shanghai, China
Background: Hepatoma consists of heterogeneous subpopula-tions in terms of their cell surface markers, tumorigenicity, invasion and metastatic capability. In our previous study, we indicated that the CD133-/EpCAM- hepatoma subpopulation was more metastatic than its counterpart; however the controlling mechanisms are unexplored (J Hepatol 2011 ;55:838–845). The present study aims to delineate the significance of hedgehog signaling in the development of metastases. Methods: Huh-7 cells were FACS-enriched into CD133+/EpCAM+ (double positive, DP) and CD1 33-/EpCAM- (double negative, DN) subpopulations. The double negative cells further underwent Transwell-selection for metastatic cells (Transwell-selected, TS). The metastatic rate, matrix metalloproteinase (MMP) gene expression, epithelial mesenchymal transition (EMT) markers, and hedgehog signaling activities were determined in these subpopulations. Results: TS cells displayed much greater metastatic activity as evidenced by an increased Transwell invasion rate (60 vs. 37 and 32%, p<0.05), extremely elevated expression of MMP1, 2 and 9 genes (36–3000-fold, p<0.05–0.001) compared to DN and DP subpopulations. There was a nearly 2-fold increase in TGF-β1 gene expression in TS cells compared to DN and DP subpopulations. TS cells lost E-cad-herin and were all vimentin-positive as shown by immunocyto-chemistry in contrast to DP cells. There was a transitional increase in Gli-1 gene expression levels from DP, DN to TS subpopulations, which is consistent with elevated Zeb1 and Gli-2 levels in the nuclear fraction as detected by Western blot analysis. Flow cytometric analysis verified that these TS cells maintained a negative cell surface marker profile in their sub-passages. The freshly-sorted DN Huh-7 cell-derived xenografts were all metastatic through either local invasion or distal metastases in contrast to no metastases from DP-derived xenografts in immunodeficient NSG mice as demonstrated by bioluminescent imaging, autopsy and histopathology. Conclusions: The highly metastatic capability of unique Transwell-selected cells is associated with negative expression of CD133 and EpCAM surface molecules, significant EMT and enhanced hedgehog activity. Further exploration of molecular mechanisms of hedgehog signaling in modulating metastases will establish molecular targets for the therapeutic intervention of hepatoma metastases.
The following people have nothing to disclose: Ya-Han Fan, Samantha Nguyen, Jia Ding, Jian Wu
Antiproliferative and proapoptotic effect of the MLK/JNK inhibitor CEP-1347 in hepatocellular carcinoma
Shuai Lu1, Johanna Liebl2, Antonia Rizzani1, Burkhard Göke1, Eike Gallmeier1, Alexander L. Gerbes1, Angelika Vollmar2, Enrico N. DeToni1;
1 Internal Medicine 2, University of Munich, Munich, Germany; 2Department of Pharmacy, Pharmaceutical Biology, University of Munich, Munich, Germany
BACKGROUND: Sorafenib is the only systemic agent approved for the therapy of hepatocellular carcinoma (HCC). However, in spite of its efficacy, the investigation of alternative therapeutic targets is urgently required. JNK is overexpressed in the majority of HCC and chemical induction of HCC is prevented byJNK inhibition. Notably, expression of JNK was recently shown to predict a poor response to sorafenib. Nevertheless, since no JNK inhibitor is currently undergoing investigation as therapy of HCC, JNK remains a largely unexploited therapeutic target. CEP-1347 is a MLK/JNK inhibitor originally designed to prevent the progression of Parkinson's disease, and underwent extensive Phase 3 clinical investigation proving save and well tolerated. We aimed at assessing the potential efficacy of this compound as therapy of HCC. METHODS: the effect of CEP-1 347 was assessed in liver cancer cell lines by viability assays, FACS-based analysis of cell cycle and apoptosis and by western blot. In vivo, its effect was assessed in a model of xenograft tumor induction by daily intraperitoneal injections of CEP-1347 or vehicle. RESULTS: Administration of CEP-1 347 in nanomolar range led to a dramatic loss of cell viability and enhanced the antiproliferative effect of sorafenib by causing G2/M cell cycle arrest and apoptosis. Concomitantly, caspase cleavage and the downregulation of apoptosis regulators Mcl-1 and Bcl-2 were observed. In vivo CEP-1347 strongly inhibited tumor growth of Huh7 cells. CONCLUSIONS: We provide preclinical in vitro and in vivo data showing the antitumor activity of CEP-1 347 and propose the utilization of this compound as experimental therapy of hepatocellular carcinoma.
The following people have nothing to disclose: Shuai Lu, Johanna Liebl, Antonia Rizzani, Burkhard Göke, Eike Gallmeier, Alexander L. Gerbes, Angelika Vollmar, Enrico N. De Toni
Protein kinase R modulates c-Fos and c-Jun signaling to promote proliferation of hepatocellular carcinoma with hepatitis C virus infection
Takao Watanabe1, Yoshio Tokumoto1, Masashi Hirooka1, Masanori Abe1, Morikazu Onji1, Raymond T. Chung2, Yoichi Hiasa1;
1Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Toon, Japan; 2Gastrointestinal Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA
Background and aims: Double-stranded RNA-activated protein kinase R (PKR) is upregulated by hepatitis C virus (HCV) and overexpressed in hepatocellular carcinoma (HCC). We previously reported that PKR is overexpressed and activated in HCC with HCV infection, as compared with surrounding non-HCC tissues. However, the precise roles of PKR in HCC with HCV infection remain unclear. The present study aimed to identify the roles of PKR in HCC with HCV infection, and to determine whether overexpressed PKR in HCC has beneficial or malignant effects in patients with this disease. Methods: We modulated PKR expression in the HCV-replicating cell lines JFH-1 and H77 (generated by Huh7.5.1 cell transfection) using siRNA and a PKR expression plasmid, and then assessed cancer-related genes by real-time PCR and Western blotting. Cell lines were further analyzed using wound healing and MTS (proliferation) assays. The modulation of genes by PKR was also assessed in 34 specimens of human HCC. Results: The expression of c-Fos and c-Jun genes was altered in parallel by PKR. An increase in PKR resulted in the upregulation of phosphorylated c-Fos and c-Jun that was related to levels of phosphorylated Erk1/2 and JNK1, namely the MAPK pathway, which is associated with cell proliferation. We therefore assessed cell proliferation in mono-layer wound-healing experiments. We found that JFH1 and H77s cells recovered more slowly after PKR knockdown than controls. Cell proliferation determined by MTS assays significantly decreased and increased after PKR knockdown and PKR upregulation, respectively, and cell proliferation was dependent on PKR-modulated c-Fos and c-Jun expression. We also confirmed the coordinate expression of c-Fos and c-Jun with PKR in human HCC specimens with HCV infection. The amounts of c-Fos and c-Jun and their phosphorylation levels were increased in specimens with high levels of PKR expression. Conclusions: The activities of c-Fos and c-Jun were upregulated by PKR through the activation of Erk1/2 and JNK1, respectively. Such modulations resulted in HCC cell proliferation with HCV infection. These findings suggest that PKR-related proliferation pathways could serve as an attractive therapeutic target.
Raymond T. Chung - Advisory Committees or Review Panels: Idenix; Consulting: Enanta; Grant/Research Support: Gilead, Merck, Mass Biologic, Gilead
The following people have nothing to disclose: Takao Watanabe, Yoshio Tokumoto, Masashi Hirooka, Masanori Abe, Morikazu Onji, Yoichi Hiasa
Dysregulation of Purinergic Signaling in Hepatocellular Carcinoma
Janielle P. Maynard1, Randy L. Johnson2, Dolores Lopez-Terrada3, John A. Goss4, Sundararajah Thevananther1;
1Pediatrics - Gastroenterology, Hepatology & Nutrition, Baylor College of Medicine, Houston, TX; 2Biochemistry and Molecular Biology, UT MD Anderson Cancer Center, Houston, TX; 3Pathology and Immunology, Baylor College of Medicine, Houston, TX; 4Surgery, Baylor College of Medicine, Houston, TX
Hepatocellular carcinoma (HCC) is the third most lethal cancer worldwide, but molecular mechanisms of its pathogenesis are not well understood. Recent studies suggest that extracellular ATP-mediated activation of P2Y2 purinergic receptor induces hepatocyte proliferation in response to partial hepatectomy and ATP treatment alone was sufficient to induce hepatocyte proliferation in vitro. The purpose of this study was to characterize extracellular nucleotide effects on HCC cell proliferation and to examine the role of P2 purinergic signaling in the pathogenesis of HCC in patients and Mst1/2-/-, a mouse model of HCC. Hypothesis: Dysregulation of purinergic signaling facilitates aberrant cell proliferation underlying hepatocellular carcino-genesis. Methods. HCC human-derived Huh7 cells, maintained in serum free media for 24h, were treated with ATPγS, or ADP (100μM) for different time intervals. SP600125 pretreatment was used to inhibit c-Jun N-terminal Kinase (JNK) signaling. Western blotting, qRT-PCR and 5-Bromo-2'-deoxy-uridine (BrdU) incorporation analysis were done. Mst1/2-/- and WT mouse livers (1, 3, & 6 months) and HCC patient livers (n=27) were analyzed by qRT-PCR for all 15 P2 purinergic receptor isoforms. Results. Extracellular nucleotide treatment alone was sufficient to induce cell cycle progression in Huh7 cells, evidenced by increased BrdU incorporation and increased cyclin D3, E, and A mRNA and protein expression. We observed downregulation of cyclin D1 mRNA, however, as previously reported in a subset of HCC with high tumor grade. JNK inhibition attenuated nucleotide-induced cyclin D3, E and A protein expression, but enhanced downregulation of cyclin D1. Mst1/2-/- mouse tumors (at 3–6 months) exhibit dysregulated expression of multiple P2 purinergic receptor isoforms as compared to WT. In HCC patients, multiple P2 purinergic receptor isoforms were elevated >2-fold in liver tumors as compared to uninvolved areas in up to 52% of patients. P2 purinergic receptor upregulation was more prevalent among HCC patients infected with hepatitis C virus (HCV) (75%) as compared to non-viral groups (20%) identifying a unique subset of viral-induced HCC over-expressing P2 receptors. Conclusions. Our results suggest that extracellular nucleotides are potent mitogens in Huh7 cells, inducing downregulation of cyclin D1 and upregulation of cyclin E, which are associated with poor prognosis in HCC patients. Our analysis of HCC patient and Mst1/2-/- mice livers has uncovered a likely role for purinergic signaling in the pathogenesis of HCC, highlighting P2 purinergic receptors as potential biomarkers and novel therapeutic targets for HCC.
The following people have nothing to disclose: Janielle P. Maynard, Randy L. Johnson, Dolores Lopez-Terrada, John A. Goss, Sundararajah Thevananther
Visualization of stem cell features in human hepatocellular carcinoma; tumor-host interaction and clinical imapct
Shinji Tanaka1, Shunsuke Muramatsu1, Arihiro Aihara1, Rama Adikrisna1, Kaoru Mogushi2, Satoshi Matsumura1, Daisuke Ban1, Takanori Ochiai1, Takumi Irie1, Atsushi Kudo1, Noriaki Naka-mura1, Koh Nakayama2, Hiroshi Tanaka2, Shoji Yamaoka3, Minoru Tanabe1, Shigeki Arii1;
1Department of Hepato-Biliary-Pan-creatic Surgery, Tokyo Medical and Dental University, Tokyo, Japan; 2Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; 3Department of Molecular Virology, Tokyo Medical and Dental University, Tokyo, Japan
Hepatocellular carcinoma (HCC) is one of the aggressive malignancies mainly due to the recurrence and/or metastasis even after curative resection. There is emerging evidence that tumor metastasis and recurrence might be driven by a small subpop-ulation of stemness cells, so-called cancer stem cells (CSCs). Previous investigations revealed that breast CSCs were found to exhibit intrinsically low proteasome activity, and lower level of reactive oxygen species (ROS). In this study, two stem cell features, low proteasome activity and low intracellular ROS were visualized in human HCC cells. By use of two-color FACS sorting to isolate the cells with stem cell features, we analyzed the cell division behavior in normoxia and hypoxia, expression of stem cell markers, tumorigenicity, metastatic potential, specific expressing gene signatures and their clinical impact. A visualized small subpopulation of HCC cells demonstrated asymmetric divisions in normoxia, but symmetric divisions in hypoxia. Their remarkable tumorigenicity suggested the cancer initiation potential of the HCC CSCs. Comprehensive gene expression analysis revealed that chemokine-related genes were upregu-lated in the CSCs subpopulation. Our identified HCC CSCs facilitated the migration of macrophages in vitro, and also demonstrated the metastatic potentials with recruitment of macrophages in vivo. The analysis of clinical samples revealed the CSC-specific gene signature has a significant correlation to the recurrence in the patients after curative operation for HCC. Our monitoring system of the stem cell features is a promising tool to analyze the in vivo significance of CSCs microenviron-ments in human HCC.
The following people have nothing to disclose: Shinji Tanaka, Shunsuke Muramatsu, Arihiro Aihara, Rama Adikrisna, Kaoru Mogushi, Satoshi Matsumura, Daisuke Ban, Takanori Ochiai, Takumi Irie, Atsushi Kudo, Noriaki Nakamura, Koh Nakayama, Hiroshi Tanaka, Shoji Yamaoka, Minoru Tanabe, Shigeki Arii
Tolerability and Safety Data in Patients with Hepatocellular Carcinoma who received multiple intratumoral and/or intravenous treatments of Pexa-Vec (JX-594) in two Phase 2 Studies
Caroline Breitbach1, Jeong Heo2, Mong Cho3, Riccardo Lencioni4, Tae-Ho Hwang2, James Burke1;
1Jennerex Biotherapeutics, San Francisco, CA; 2Pusan National University Hospital, Busan, Republic of Korea; 3Pusan National University Yangsan Hospital, Busan, Republic of Korea; 4University of Pisa, Pisa, Italy
Pexa-Vec (pexastimogene devacirepvec; JX-594) is a targeted oncolytic & immunotherapeutic vaccinia virus engineered to express human granulocyte-macrophage colony stimulating factor (GM-CSF). To date two Phase 2 studies of Pexa-Vec have been completed in patients with advanced hepatocellular carcinoma (HCC): (1) in a randomized dose-finding study, 30 patients received 3 IT Pexa-Vec injections into liver tumors every 2 weeks at 1e8 plaque forming units [pfu] versus 1e9 pfu and (2) in a single-arm Phase 2 study, 25 patients received an IV Pexa-Vec infusion (day 1) followed by 2 IT injections (days 8 & 22) (each dose at 1 x 1 09 pfu) prior to initiation of sorafenib therapy. A subset of patients received an optional IT boost treatment at Week 12. The extent of Pexa-Vec exposure amongst these patients, and the most common AEs and frequency of > Grade 3 treatment-related adverse events (AEs) are summarized. In the randomized dose-finding study, 97% (29/30) of patients received the complete regimen, 3 scheduled IT injections; one patient treated with low-dose Pexa-Vec received 2 IT doses. The most common AEs on the Phase 2 randomized study were fever (97%), chills (80%), injection site pain (50%), vomiting (50%), nausea (47%), and headache (37%). The most common treatment-related Grade 3 AEs for patients treated at a dose of 1e8 pfu were: lymphopenia (14%) and aspartate aminotransferase increased (14%). For patients treated at 1e9 pfu, pyrexia (1 9%) was the only Grade 3 AE recorded as treatment-related in more than one patient. No Grade 4 or 5 AEs related to treatment were reported on this trial. In the single-arm study all patients (25) received the IV Pexa-Vec infusion as per protocol; 22 patients (88%) received two subsequent IT injections and 10 patients (40%) received the optional IT boost at Week 12. The most common AEs were fever (96%), chills (76%), headache (48%), abdominal pain (40%), lymphopenia (40%) and nausea (40%). Prior to sorafenib treatment, the most common treatment-related Grade 3 AEs were: lymphopenia (16%), neutropenia (12%), leukopenia (12%), and hemoglobin decreased (12%). Treatment-related Grade 4 AEs included: lymphopenia (20%), neutropenia (4%) and leukopenia (4%). Notably, the reduction in white blood cell count subsets was mostly transient; clinically-significant infection or febrile neutropenia was not commonly reported. Treatment with Pexa-Vec was generally well-tolerated in patients with advanced HCC when administered by IV infusion and/or IT injection. The majority of patients generally adhere to the schedule of multiple injections. Further studies with Pexa-Vec IT and IV are warranted in this indication.
Caroline Breitbach - Employment: Jennerex Biotherapeutics
Jeong Heo - Grant/Research Support: Jennerex, Green Cross
Riccardo Lencioni - Consulting: Jennerex Inc.; Speaking and Teaching: Bayer Healthcare
Tae-Ho Hwang - Grant/Research Support: Jennerex
James Burke - Employment: Jennerex
The following people have nothing to disclose: Mong Cho
A novel mechanism of PXR mediated Sorafenib drug resistance in HCC through MDR and CYP3A4
Yin Ying Lu1, Fan Feng1, Fan Zhang2, Xudong Gao1, Chunping Wang1, Xiujuan Chang1, Jianhui Qu1, Hong Wang1, Zhen Zeng1, Mingliang Cheng3, Chunzhang Yang4, Yongping Yang1;
11Center of Therapeutic Research for Hepatocarcinoma, Beijing 302 hospital, Beijing, China; 2Tumor center, PLA General Hospital, Beijing, China; 3Infectious Department, Guiyang Medical college, Guiyang, China; 4National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda,, MD
Hepatocellular carcinoma (HCC) is the sixth most common cancer with high fatality and mortality worldwide. The rapid development of drug resistance to current chemotherapies and targeted therapies has hindered the effectiveness of HCC treatments, leading to a poor prognosis for HCC patients. Recently, we identified a potential mechanism that how HCC obtains drug resistance against Sorafenib through Pregnane X receptor (PXR) pathway. PXR is a nuclear receptor that senses the presence of foreign toxic substances including drugs and up-regu-lates the expression of proteins involved in drug metabolism, detoxification and clearance process. Surface plasmon resonance (SPR) has demonstrated that Sorafenib exhibits high affinity to PXR, functions as PXR's ligand, and triggers gene transcriptions/translations that are necessary for drug detoxification and clearance, leading to tolerance. Our results showed that Sorafenib promoted accumulation of PXR in nucleus and recruitment of PXR to the promoter region of multi drug resistance (MDR). Further results from Luciferase and Western blot assays showed that Sorafenib induced the transcription and translation of PXR downstream genes including MDR and CYP3A4 in a dose dependent manner. In addition, down-regulation of PXR expression by siRNA blocked Sorafenib-associated up-regulation of MDR and CYP3A4. On the other hand, up-regulation of PXR activity induced by Anisomycin significantly reduced the inhibitory effect of Sorafenib on HepG2 cell proliferation. In conclusion, our study indicates a novel molecular mechanism that drug resistance against Sorafenib in HCC is mediated by PXR activation and PXR downstream gene transcription/translation, offering a potential to target the drug resistance pathway and improve future HCC's treatment outcome.
The following people have nothing to disclose: Yin Ying Lu, Fan Feng, Fan Zhang, Xudong Gao, Chunping Wang, Xiujuan Chang, Jianhui Qu, Hong Wang, Zhen Zeng, Mingliang Cheng, Chunzhang Yang, Yongping Yang
The new oral iron chelator, Deferasirox, is the new drug to prevent Liver Fibrosis and Hepatocarcinogenesis
Naoki Yamamoto1,2, Takahiro Yamasaki1, Koichi Uchida1, Norikazu Tanabe1, Taro Takami1, Issei Saeki1, Koichi Fujisawa1, Masaki Maeda1, Shuji Terai1, Isao Sakaida1;
1Gastroenterology & Hepatology, Yamaguchi University Graduate School of Medicine, Ube Yamaguchi, Japan; 2Health Administration Center, Yamaguchi University, Ube Yamaguchi, Japan
<Background/Aim>Hepatitis B,C and non-alcoholic steatohep-atitis (NASH) can progress to advanced liver fibrosis and to hepatocellular carcinoma(HCC).Deferasirox(DFX) is a new oral iron chelator which inhibits proliferation of some cancer cells.The aim of this study is to investigate whether DFX has any effects on the development of liver fibrosis and preneoplastic lesions in animal model. <Methods>In vitro)We examined cell growth by MTS assay and apoptosis by Caspase 3 activity using human hepatoma cell(HepG2,HuH7,Hep3B).In vivo) 1 )The effects of DFX were examined using the choline-defi-cient L-amino acid-defined (CDAA) diet-induced rat liver fibrosis model.The total study periods were 16,and20weeks.One group received CDAA diet with DFX(20mg/kg/adult),The other was CDAA diet only.Liver fibrosis was analyzed by Azan,Sirius-red,aSMAexpression and hydroxyproline level.The preneoplastic lesion was assessed by glutathione S-transferase placental form(GST-P) expression.The change of laboratory data was analyzed.Type I procollagen,TIMP1 ,2,TGFb mRNA were analyzed using both Real time-PCR and DNA array.2)We examined the effects of DFX using N-nitrosodiethylni-trosamine(DEN)-induced liver cancer mouse model. One group received with DFX(20mg/kg/adult) from 5 months to 8 months after DEN injection of 1 mg/kg at 14 days,The other was DEN injection only.Liver cancer was analyzed by HE,AFP,PCNA,CD44 expression.The oxidative stress was analyzed by 4HNE,8OHdG expression.We compared many gene expressions of cancer and non cancer tissues between DFX group and control. <Results> The cell growth of hepatoma cells was inhibited with DFX in a dose-dependent manner(P<0.01).The caspase3 activity was increased with DFX in a dose-dependent manner(P<0.01).In CDAA model,DFX prevented liver fibrosis by Azan,Sirius-red,aSMA expression(p<0.05)and hepatic hydroxyproline level was decreased.DFX reduced both the area and numbers of GST-P positive preneoplastic lesions(p<0.01).Administration of DFX reduced levels of 4HNE (DFX 3.3,CDAA only 8.0,p<0.01), 8OHdG (DFX 1.3,CDAA only 2.0 ng/ugDNA,p<0.05).DFX inhibited Type 1 procollagen,TIMP1 ,2 mRNA expression (all of p<0.01).In DEN model, DFX reduced both the area and numbers of tumor lesions (p<0.01).DFX prevented AFP,CD44 expression (p<0.05)and significantly reduced levels of 4HNE,8OHdG. <Conclusion>Our results indicated that DFX inhibited liver fibrosis and preneoplastic lesions. Deferasirox may be the new drug for Liver Fibrosis and Hepatocellular Carcinoma.
The following people have nothing to disclose: Naoki Yamamoto, Takahiro Yamasaki, Koichi Uchida, Norikazu Tanabe, Taro Takami, Issei Saeki, Koichi Fuji-sawa, Masaki Maeda, Shuji Terai, Isao Sakaida
FOXM1 Expression Correlates with both in vivo and in vitro AFP production and Therapeutic Effect of Thiostrep-ton on Human Hepatocellular Carcinoma
Huang S. Feng, Ai J. Gang, Wu Yan, Chen J. Juan, Liping Zhang;
The First Affiliated Hospital of Chongqing Medical University, Chongqing, China
Background: Forkhead box M1 (FOXM1) transcription factor plays an important role in hepatocarcinogenensis. Given the space and time consistency of the expression patterns between AFP and FOXM1 during normal embryonic liver development, liver regeneration and liver carcinogenesis, we sought to explore the possible correlation between FOXM1 expression and AFP production and to evaluate the cellular effects of a FOXM1 inhibitor, Thiostrepton, in hepatocellular carcinoma (HCC) cells and its potential as a novel therapeutic for HCC. Methods: FOXM1 expression in 52 clinical HCC tissues was examined both by immunohistochemistry and Western blot; Chromatin Immunoprecipitation (ChIP) was performed to examine the possible occupancy of the AFP promoter by FOXM1; Cytotoxicity of Thiostrepton were tested in HepG2 and HepG2.2.15 cell lines; cell cycle profiles were assessed by flow cytometry and the possible molecular targets were explored. Results: Up-regulation of FOXM1 was confirmed in 69.2% (36/52) of HCC tissues. Clinicopathologically, FOXM1 up-regulation was associated with metastasis (P=0.039). More importantly, a significant positive correlation was observed between either tissue AFP mRNA or plasma AFP concentration and tissue FOXM1 expression levels before surgery (r=0.3, p=0.03; r=0.332, p=0.016; respectively). Meanwhile, a significant positive correlation between tissue AFP mRNA and pre-operative serum AFP level was demonstrated by the spearman rank correlation test (r=0.574, P<0.01). Both FoxM1 interference and Thiostrepton treatment led to significant reduction of AFP secretion in cell culture media. Moreover, CHIP assay confirmed that FOXM1 can bind to eight Forkhead response elements (FHREs) located at the proximal region of the AFP promoter. In addition, Thiostrepton dramatically reduced FOXM1 expression in HCC cells, leading to cell cycle blockade at G1/S transition, concomitant with down-regulated CyclinD1, CDK2, CDK4 and SKP2 expression, and significantly induced FOXO3A expression. Conclusions: These data suggested that FOXM1 expression is closely correlated with both in vitro and in vivo AFP production possibly through its transcriptional regulation of the AFP promoter. Thiostrepton may specifically target FOXM1 to induce cytotoxic effect and could be potentially developed as a novel anticancer drug against HCC.
The following people have nothing to disclose: Huang S. Feng, Ai J. Gang, Wu Yan, Chen J. Juan, Liping Zhang
S1P dependent stellate cell production of MMP9 regulates cancer cell migration and invasion
Yan Bi, Jiachu Li, Ningling Kang, Vijay Shah;
Gastroenterology Research Unit, Mayo Clinic, Rochester, MN
Background and Aims: Liver is the most common site of metastasis for pancreatic cancer. In tumor microenvironment, stellate cells influence growth and metastasis of cancer by multiple mechanisms, including regulating extracellular matrix turnover. Sphingosine 1-phosphate (S1P) signaling has been implicated in tumorigenesis and metastasis in many cancers; however, its role in the metastasis of pancreatic cancer cells to the liver, remains unexplored. We hypothesized that S1P activates stellate cells to release paracrine factors that promote tumor cell migration and invasion that promote metastatic growth. Methods: Immortalized human or mouse stellate cells were stimulated with S1P (0.5–5 μM) or vehicle. S1 P1 or S1P2 receptor was disrupted by shRNA, siRNA, or pharmacological inhibitors. Stellate cell conditioned media was used to measure pancreatic cancer cell (PANC1) migration and invasion in Boy-den chamber and Transwell assays. mRNA levels were measured by PCR array and confirmed by real-time PCR. Gelatin zymography and luciferase assays were used for MMP9 protein and promoter activity, respectively. Results: Conditioned medium from S1 P stimulated stellate cells increased PANC1 cell migration (3.06 ± 0.36 vs 20.44 ± 1.57, p<0.05) and invasion (2.83 ± 1.06 vs 16.40 ± 2.62; p<0.05) compared to conditioned media from vehicle treated stellate cells. Targeted PCR array analysis showed that S1 P increased MMP9 mRNA levels in stellate cells, which was confirmed by real-time PCR (4.15 ± 1.1 fold; p<0.05; n=4) and MMP9 promoter activity analysis (1.51 ±0.11 fold; p<0.05, n=4). S1 P stimulated c-abl kinase phosphorylation at Y87, Y412 and Y425 in stellate cells and c-abl siRNA as well as the c-abl inhibitor Gleevac diminished S1P induced MMP9 promoter activity. Conditioned medium from S1P2 receptor knockdown stellate cells showed decreased PANC1 cell invasion and migration compared to that from S1 P1 receptor knockdown stellate cells or control cells indicating that S1P increased MMP9 production by activating S1 P2 receptor rather than the S1 P1 receptor. Finally, the MMP9 pharmacologic inhibitor (MMPI1) decreased PANC1 cell migration (18.33 ± 1.45 vs 11.08 ± 1.39; p<0.05) and invasion (7.44 ± 1.23 vs 2.00 ±0.516; p<0.05). Conclusion: S1P increases MMP9 production from stellate cells through c-abl activation, which results in enhanced migration and invasion of tumor cells. S1 P/c-Abl/MMP9 pathway in stellate cells may provide a mechanism for the growth of pancreatic cancer metastasis in liver.
The following people have nothing to disclose: Yan Bi, Jiachu Li, Ningling Kang, Vijay Shah
The function of targeted host genes determines the oncogenicity of HBV integration in hepatocellular carcinoma
Xiaojun Li, Ziwei Yang, Xiangmei Chen, Fengmin Lu;
Department of Microbiology, School of Basic Medical Sciences, Peking University Health Science Center, BeiJing, China
Objective: Hepatitis B Virus (HBV) integration into the human genome is one of the major causative factors to hepatocellular carcinoma (HCC) genesis. However, the oncogenic mechanism of HBV integration was still elusive. The aim of this study is to investigate the essential oncogenic difference(s) between HCC tumor and adjacent non-tumor tissues Methods: 1115 HBV integration sites were collected from four recent studies. Functional annotation analysis of integrated targeted host genes (ITGs) were performed using DAVID based on Gene Ontology and KEGG pathway databases. Array-based expression profiles, real-time qPCR and western blot were used to detect the expression of recurrently integration targeted genes (RTGs). The biological consequence of the overexpression of UBXN8 in HepG2 cell lines were studied in vitro. Results: HBV genomic fragments are prone to integrate in genic regions (exons, introns and promoters) and gene-dense regions. Functional annotation analysis reveals that, compared to those in adjacent non-tumor tissues, ITGs in HCC tumor tissues were significantly enriched in functional terms related to negative regulation of cell death, transcription regulation, development and differentiation, as well as pathways related to cancer. 32% of the 75 RTGs identified in this analysis expressed abnormally in HCC tissues. One of the RTGs- UBXN8 was proved to be a potential tumor suppressor through inhibiting G1/S transition of cancer cells. Conclusion: The oncogenicity of HBV integration was determined by the unction of HBV integration targeted host genes
The following people have nothing to disclose: Xiaojun Li, Ziwei Yang, Xiangmei Chen, Fengmin Lu
The Hypoxia-Induced Secretion of PDGF-BB by Hepatocellular Carcinoma Cells Increases Activated Hepatic Stellate Cell Proliferation, Migration and VEGF-A Expression
Nan Lin1, Xu Linan2;
1 Department of Hepatobiliary Surgery, the Third Affiliated Hospital, SunYat-Sen University, Guangzhou, China; 2the sixth Affiliated Hospital, SunYat-Sen University, Guangzhou, China
Background and aims: Angiogenesis plays an important role in the proliferation and metastasis of hepatocellular carcinoma (HCC) under hypoxic tumor microenvironment. Activated hepatic stellate cells (HSCs) infiltrate the stroma of liver tumors and potently increase angiogenesis via the tumor-stromal interaction. This study aimed to investigate the paracrine effect of HCC-derived platelet-derived growth factor-BB (PDGF-BB) on HSCs under hypoxia condition. Methods: PDGF-BB expression and secretion by HepG2 cells was measured by Western-blotting or ELISA in normoxia or hypoxia. Conditioned medium (CM) from HepG2 cells was used to culture LX-2 cells. LX-2 cell proliferation, migration and VEGF-A expression were assessed by MTT assay, cell migration assay or Western-blotting and the paracrine effect of HepG2-derived PDGF-BB was determined. Results: We demonstrated that PDGF-BB expression was robustly increased in HepG2 cells exposed to hypoxia. Conditioned medium from HepG2 cells stimulated LX-2 cell proliferation, migration and VEGF-A expression. We determined that blocking PDGF-BB in HepG2-CM abolished these effects on LX-2 cells. The ectopic expression of PDGF-BB in HepG2 cells strongly affected LX-2 cell proliferation, migration and VEGF-A expression. Conclusions: Our study suggests that hypoxia-induced PDGF-BB secretion by HCC cells stimulates HSCs to accumulate and proliferate in the tumor stroma and the enhanced VEGF-A expression in HSCs may promote HCC angiogenesis.
The following people have nothing to disclose: Nan Lin, Xu Linan
Characterization of Cancer Stem Cells in Human Cholangiocarcinoma (CCA) subtypes
Alessia Torrice1, Guido Carpino2, Alice Fraveto1, Anastasia Renzi3, Maria Consiglia Bragazzi1, Felice Giuliante4, Agostino DeRose4, Vincenzo Cardinale1, Rossella Semeraro1, Paolo Onori3, Chiara Napoletano5, Antonio Franchitto3, Alfredo Cantafora1, Gian Luca Grazi6, Eugenio Gaudio3, Domenico Alvaro1;
1Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Rome, Italy; 2Health Science, University of Rome "Foro Ital-ico", Rome, Italy; 3SAIMLAL, Sapienza, University of Rome, Rome, Italy; 4Surgery, Hepatobiliary Unit, Catholic University of the Sacred Heart School of Medicine, Rome, Italy; 5Experimental Medicine, Sapienza University of Rome, Rome, Italy; 6Hepato-Biliary Surgery, Regina Elena National Cancer Institute, Rome, Italy
CCA is classified as extrahepatic (EHCCA) or intrahepatic (IHCCA). While EHCCA is only represented by a pure mucin-secreting form, the IHCCA may occur as a pure mucin (Muc-IHCCA) or as a mixed (Mixed-IHCCA) form. Cancer stem cells (CSCs) are critical for tumor formation but no information exists on CCA subtypes. Aim. To investigated CSCs in the different subtypes of human CCA. Methods. CSC markers (CD90, CD44, CD13, EpCAM, CD133, Lgr5) were investigated by immunohistochemistry (IHC), RT-PCR and Flow Cytometry (FC) in CCA samples (n= 16 resected patients) and cell lines. Different cell subpopulations were isolated from human CCA and cell lines by immunomagnetic separation and their tumorigenic potential investigated in xenografted mice. Results: CD90+ and CD90+/CD44+ (vimentin positive) were the predominant sub-populations in mucin-negative IHCCA immortalized cell lines (HuH-28, CC-LP-I) while they were negligible in mucin-positive IH- (HUCCT-1) or EH- (TFK-1, Mz-ChA-1) CCA cell lines. In contrast, EpCAM+ and EpCAM+/CD44+ (vimentin negative) were the most represented subpopulations in mucin-positive but, negligible in mucin-negative CCA cell lines. In human CCA samples, the IHC expression of Keratin-7, EpCAM and CD133 did not differ between mixed-IHCCA, Muc-IHCCA and Muc-EHCCA. In contrast, CD1 3 positivity, considered as a marker of quiescent CSCs, was prevalent in Mixed-IHCCA while LGR5 positivity predominated in Muc-IHCCA or Muc-EHCCA. Moreover, in all CCA samples, CD90 expression was mostly restricted in tumor stromal cells (αSMA+/vimentin+). In all CCA samples, vimentin expression (western blot) largely predominates with respect to E-cadherin. In cultures of human CCA samples, RT-PCR showed how vimentin, CD90, CD44 and CD13 were largely (> 10-folds; p< 0.01) more expressed than CD133, EpCAM and Lgr5. When the different CCA subtypes were compared, the CD13+ and CD44+ cell subpopulations predominated (FC and RT-PCR) in Mixed-IH with respect to Muc-IHCCA or Muc-EHCCA (p< 0.01), while the opposite was found for CD90+ cells. No difference was found between Muc-IH- or Muc-EHCCA. The tumorigenic potential (number, volume and growth curves of tumor xenografts) was: CD90+ > CD 13+ > CD133+; CD90+ and CD133+ cells from Mucin-CCA > Mixed-CCA. Conclusions: the human CCA subtypes, Mixed and Mucin, show a different profile of CSCs further confirming their patho-biological diversity. The subpopulations of CD44+ and CD1 3+ cells were more represented in the Mixed-IHCCA, while CD90+ predominated in Muc-EH- or Muc-IHCCA. The CD90+ CSC subpopulation showed the highest tumorigenic potential and should be taken in great consideration for targeted therapies.
The following people have nothing to disclose: Alessia Torrice, Guido Carpino, Alice Fraveto, Anastasia Renzi, Maria Consiglia Bragazzi, Felice Giuliante, Agostino DeRose, Vincenzo Cardinale, Rossella Semeraro, Paolo Onori, Chiara Napoletano, Antonio Franchitto, Alfredo Cantafora, Gian Luca Grazi, Eugenio Gaudio, Domenico Alvaro
Bmi-1 Up-regulated Rat Oval Cells Generated Poorly Differentiated Hepatocellular Carcinoma and Tumor Endothelial Cells in Vivo
Mansheng Zhu, Rui Zhang, Wen-rui Wu, Hong Zeng, Xiujing Yue, Lei-bo Xu, Chao Liu;
Department of Hepato-Pancreato-Biliary Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China
Background: Oval cells are adult liver progenitor cells whose role in hepatocarcinogenesis remains obscure. Wnt/β-catenin, reported to constitute a positive feedback loop with the differentiation monitor Bmi1, contributes to hepatocarcinogenesis, while Notch 1 serves as its antagonizer. In this study, we tried to elucidate the multipotency of oval cells. Methods: Bmi1 was stably transfected into SD rat oval cells. The Bmi1 high oval cells were injected subcutaneously into Balb/c nude mice. The Bmi1 normal oval cells were used as control. Transplanted tumors were taken for pathological analysis and immunohisto-chemical assessment using monoclonal antibodies to CD34, AFP, CK18 and CK19. RT-PCR was applied to determine Wnt/β-catenin and Notch1 gene expression. Notch1 was silenced by siRNA. Tube formation assay was performed with Matrigel. Results: Bmi1high oval cells generated tumors in nude mice (9/14) and formed tube-like structures. The tumors turned out to be poorly differentiated hepatocellular carcinoma, positive for AFP and CK1 8, negative for CK1 9. Unexpectedly, some of the tumor endothelium was positive for anti-rat CD34, whereas the counterpart could form neither tumors nor tube-like structures. Wnt/β-catenin expression in Bmi1high oval cells was 2 folds that of the control. Their Notch1 expression, however, was 3 times higher. After silencing notch1 expression, the tube forming capacity was significantly impaired. Conclusion: Bmi1 up-regulated oval cells may be capable of hepatocarcinogenesis by enhancing Wnt/β-catenin expression, and tube formation in vitro and endothelial transdifferentiation in vivo by Notch1 expression, respectively.
Figure1. rCD34 (A) and mCD34 (B) positive endothelium (transverse arrow); Bmi1, Wnt and Notch1 expression (RT-PCR) in oval cells (C); tube formation assay, D: Bmi1 high; E: control ; F: Notch1-siRNA;G: HUVECs.
The following people have nothing to disclose: Mansheng Zhu, Rui Zhang, Wen-rui Wu, Hong Zeng, Xiujing Yue, Lei-bo Xu, Chao Liu
A Quantitative Proteomic Study on Glucose-6-Phosphate Dehydrogenase in the Development of Hepatitis B Virus-Associated Human Hepatocellular Carcinoma
Huaidong Hu1,2, Yixuan Yang1,2, Peng Hu1,2, Hongmin Zhang1,2, Hong Li1,2, Dazhi Zhang1,2, Hong Ren1,2;
1Department of Infectious Diseases, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China; 2Key Laboratory of Molecular Biology for Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Approximately 564,000 individuals are expected to be diagnosed with HCC per year. HCC is especially frequent in Asia due to a high prevalence of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. In China, HCC has been ranked as the second most frequent fatal cancer since the 1990s, and the majority of HCCs in China are caused by HBV infection. The aim of this study was to identify disease-related host proteins, which may be useful targets for future therapeutic protocols. Methods: To identify the proteins involved in HCC carcinogenesis and novel therapeutic targets, we used iTRAQ and mass spectrometry analysis to study the differentially expressed proteins in tumor and adjacent non-tumor tissue samples. Samples from 9 hepatitis B virus-associated HCC patients were analyzed. Results: In total, 1 607 unique proteins were identified and 222 proteins were found to be differentially expressed in tumor samples compared with that in non-tumor samples. Several differentially expressed proteins were further validated by real-time quantitative RT-PCR, Western Blot and immunohistochemical analysis. Functional analysis of glucose-6-phosphate dehydrogenase(G6PD), one of the highly expressed proteins in tumor samples, was carried out with small interfering (si)RNA-based silencing transfection methods. siRNA-mediated G6PD silencing reduced HBV replication by nearly 5-fold through the interferon (IFN) signaling pathway. Furthermore, silencing G6PD expression suppressed the migration and invasion of HCC cells in vitro. Conclusion: In summary, our results provide information on the mechanism of HCC development, and suggest that suppression of the G6PD expression may be a promising strategy in the development of novel cancer therapeutic drugs. Acknowledgements: This research was supported by the National Natural Science Foundation of China (81171560, 30930082, 81171561, 30972584), the National Science and Technology Major Project of China (2008ZX10002-006, 2012ZX1002007001, 2011ZX09302005, 2012ZX09303001-001, 2012ZX10002003), The National High Technology Research and Development Program of China(201 1AA0201 1 1), the Key Project of Chongqing Science and Technology Commission (cstc2012gg-yyjsB1 0007), the Chongqing Natural Science Foundation(cstc2011 jjA1 0025), the Medical Research Fund by Chongqing Municipal Health Bureau (2009-1-71).
The following people have nothing to disclose: Huaidong Hu, Yixuan Yang, Peng Hu, Hongmin Zhang, Hong Li, Dazhi Zhang, Hong Ren
T-cell Factor-4 isoforms directly regulate Bcl-xL expression in human hepatocellular carcinoma (HCC) cells
Hironori Koga1, Miran Kim2, Anna Nakamura3, Hirohisa Yano4, Toru Nakamura1, Takato Ueno3, Takuji Torimura3, Jack R. Wands2, Michio Sata1;
1Division of Gastroenterology, Department of Medicine, Kurume University, Kurume, Japan; 2Liver Research Center, Brown University, Providence, RI; 3Liver Cancer Research Division, Kurume University, Kurume, Japan; 4Department of Pathology, Kurume University, Kurume, Japan
Background: The T-cell factor (TCF)-4 is a key transcriptional protein activated by Wnt/β-catenin signaling. Previously we identified 14 TCF-4 isoforms derived from human HCC cell lines. The TCF-4J and K pair have been characterized based on the presence (K) or absence (J) of a SxxSS motif. Furthermore, we demonstrated thatTCF-4J conferred high tumorigenic potential to HCC cells in contrast to TCF-4K (PLoS ONE 2012). However, the anti-apoptotic protein Bcl-xL was much expressed in TCF-4K-overexpressing HCC cells than the level in TCF-4J-over-expressing cells, suggesting that the SxxSS was involved in Bcl-xL expression (AASLD 2012, #885). Indeed, Wnt/β-catenin signaling needs to control cell apoptosis during embryogenesis and carcinogenesis, possible direct interaction between the TCF-4 isoforms and the bcl-xL promoter region was suggested. Thus, the AIM of this study was to assess the protein-DNA interaction and its functional consequences by using ChIP assay and luciferase-reporter assay, respectively. Methods: The human HCC cell lines HAK-1A and HAK-1B were used. HAK-1B was an aggressive sister cell line derived from HAK-1A. TCF-4K mutants (269A, 272A, and 273A) were prepared with conversion of serine (S) in the SxxSS motif to alanine (A) by site-directed mutagenesis. HAK-1A-derived stable clones overex-pressing TCF-4J (J cells), K (K cells), and K-mutants (269A, 272A, and 273A cells, respectively) were established. Western blot analysis and real-time RT-PCR were employed to evaluate protein and mRNA expression levels, respectively. ChIP assay was performed by using SimpleChIP assay kit (Cell Signaling Technology). Two primer pairs for bcl-xL promoter region (BCL2L1 (-)01 Kb and BCL2L1 (-)02Kb) were obtained from QIA-GEN. The promoter assay was done by using LightSwitch Luciferase Assay System (SWITCHGEAR GENOMICS). Results: Robust expression of Bcl-xL protein was found in HAK-1 B cells, in contrast to its low expression in HAK-1 A cells. Consistently, the mRNA level in HAK-1 B was 2-fold of that in HAK-1 A. In ChIP assay, clear binding of TCF-4 with bcl-xL promoter regions was confirmed, encouraging us to compare the binding affinity in J cells, K cells, 269 cells, and control cells. As a result, significant TCF-4-DNA interaction was found in K cells, and, of note, the interaction was abolished in 269A cells. In the promoter assay, K cells showed the highest luciferase activity. Conclusions: The findings suggest that TCF-4 directly regulates the Bcl-xL expression. We suggest that phosphorylation at serine 269 in the TCF-4K isoform is critical to fine-tune anti-apoptotic potential through increasing the Bcl-xL expression in HCC.
Michio Sata - Speaking and Teaching: MSD K.K., Chugai Pharmaceutical Co., Ltd.
The following people have nothing to disclose: Hironori Koga, Miran Kim, Anna Nakamura, Hirohisa Yano, Toru Nakamura, Takato Ueno, Takuji Torimura, Jack R. Wands
BTLA up-regulation associated with impaired cytotoxic CD8+ T-cell function and disease progression of patients with hepatocellular carcinoma
Jun-Liang Fu, Yan Chen, Zheng Zhang, Fu-Sheng Wang;
Research Center of Biological Therapy, Beijing 302 Hospital, Beijing, China
B- and T-lymphocyte attenuator (BTLA) negatively regulates immune responses; however, it remains unknown the expression profile and functional role of BTLA in patients with hepatocellular carcinoma (HCC). Here, we enrolled 150 HCC patients, 33 liver cirrhosis (LC) patients, and 45 healthy individuals in this study. We found that BTLA expression was progressively up-regulated on cytotoxic CD8+ T cells in peripheral blood and correlated with disease progression in HCC patients. And tumor-infiltrating cytotoxic CD8+ T cells had higher BTLA expression compared with non-tumor regions. Further analysis revealed that BTLA expression was negatively correlated with granzyme and perforin expression in CD8+ T cells both in peripheral blood and liver. And BTLA+CD8+ T-cell proliferation and degranulation were significantly decreased compared with BTLA-CD8+ T-cell. Importantly, increased BTLA expression on cytotoxic CD8+ T cells were associated with high recurrence rates in patients with hepatocellular carcinoma. These novel findings suggest that BTLA-mediated inhibitory function may play an important role in disease progression of HCC, and represent both a potential prognostic marker and a therapeutic target for the treatment of HCC.
The following people have nothing to disclose: Jun-Liang Fu, Yan Chen, Zheng Zhang, Fu-Sheng Wang
Hypermethylation induced downregulation of miR-9 lead to activation of Jak-STAT3 pathway through directly targeting IL-6 in hepatocellular carcinoma
Jiangbo Zhang, Yongfeng Wang, Xiangmei Chen, Fengmin Lu;
Department of Microbiology, School of Basic Medical Sciences, Peking University Health Science Center, BeiJing, China
Backgroud: The aberration status of miR-9 has been reported to be involved in a variety of human cancers, but its roles in hepa-tocarcinogenesis has rarely been discussed. In this study, we investigated the status of miR-9 and elaborated its tumor suppressor role in the development of HCC. Methods: Methylation specific DNA enzymes digestion method and methylated DNA quantification assay were used to detect the promoter methylation status of the three precursor genes of miR-9. Taqman microRNA Assay was used to determine the expression of miR-9 quantitatively; the expressions of its potential target genes were detected in 40 paired of HCC tumorous and corresponding adjacent non-tumorous tissues, as well as in normal liver tissues by real-time qPCR. U6 promoter driven vectors containing miR-9 precursor were used to explore its impact on cell migration, proliferation and colony formation in vitro. Array-based mRNA expression profiles of the transcriptome of HepG2 cells overexpressing miR-9 were used to analysis the function of miR-9. The in silico predicted target genes of miR-9 were testified by dual-luciferase reporting assay, real-time qPCR, and further confirmed by ELISA or Western blot. Results: Mir-9-1, a precursor of miR-9, was hypermethylated in 43% (37/87) of the HCC tissues; and miR-9 was down regulated in 43% (17/40) of the HCC tissues. Ectopic expression of miR-9 could restrain the migration, proliferation and colony formation efficiency of HCC cells in vitro. Four novel direct miR-9 targets (CKAP2, IL-6, TC10, and HSPC159) were identified. The ectopic expression of IL-6 was able to reverse the tumor-suppressor property of miR-9 through the activation of Jak-STAT3 pathway and the subsequent up-regulation of SOCS1 and VEGFA. Conclusions: Our study identified the frequent pro-moter-hypermethylation and down expression of miR-9 in HCC. IL-6 is confirmed as a novel target of miR-9 and miR-9 may exert its tumor suppressive capacity through the miR-9/IL-6/Jak-STAT3 pathway.
The following people have nothing to disclose: Jiangbo Zhang, Yongfeng Wang, Xiangmei Chen, Fengmin Lu
Modulation of systemic immune response after radiofre-quency ablation of early HCC developed on compensated cirrhosis. Preliminary results of a pilot prospective monocentric study in 43 patients
Jean-Charles Nault3, 1, Nathalie Barget2, Lucie Del Pozo2, Valerie Bourcier3, Francoise Gondois-Rey4, Bernadette Barbarat4, Gisele Nkontchou3, Veronique Grando3, Pierre Nahon3, Marianne Ziol5, Jean Claude Trinchet3, Olivier Seror6, Daniel R. Olive4, Nathalie Ganne-Carrie3;
1Inserm U674, Paris, France; 2Liver CRB, Jean Verdier hospital APHP, Bondy, France; 3Liver unit, Jean Verdier hospital APHP, Bondy, France; 4Inserm UMR891, Institut Paoli Cal-mette, Marseille, France; 5Pathology department, Jean Verdier Hospital, Bondy, France; 6Radiological department, Jean Verdier Hospital, Bondy, France
Introduction: Immunity is involved in antitumor defense. Tumor necrosis induced by hyperthermia could elicit an immunogenic cell death and stimulate the immune system by releasing Damage-associated Molecular Pattern molecules. Our hypothesis is that immune system against dying cells could mediate a decrease of tumor recurrence. In order to analyze the systemic immune response before and after radiofrequency ablation (RFA) of hepatocellular carcinoma (HCC) and the correlation with tumor relapse, we have performed a pilot exploratory prospective study. Material and methods: since January 2011, we have consecutively included all voluntary patients treated by a first RFA for solitary HCC of less than 5 cm (BCLC 0/A) developed on compensated cirrhosis in our institution. We collected additional blood samples (21 ml) the day before RFA (D0), at day 1 (D1) and day 30 (D30) in order to study immune cells and perform phenotypic and functional analysis of NK cells, dendritic cells and T lymphocytes. Statistical analysis was performed using paired non-parametric Wilcoxon test. Results: 123 blood samples of 43 patients were analyzed. The success rates of immune cells collection were 26% at D0, 20% at D1 and 1 8% at D30, the phenotypic analysis was performed on 95 samples of 31 patients (77%) and the functional analysis on 53 samples of 22 patients (50 %). At D1, we observed an increase of T regulatory lymphocytes (P=0.02), a decrease of plasmocytic dendritic cell (P=0.0013), an increase of NK CD56 dim cells (P=0.04) and a decrease of NK CD56 bright cells (P=0.02). All these early changes were transient, since a return at the baseline phenotype was observed at D30. We also characterized surface marker of cell activation: NKG2D decreased at D1 then returned to baseline levels at D30 in T (P=0.0001) and NK cells (P=0.001); NKP46 decreased from D1 to D30 (P=0.0020) on NK cells. CD69 decreased at D1 on T cells (P=0.0066) and increased at D30 (P=0.04) on NK cells. Functional analysis showed an increased production of inter-feron gamma by NK cells after stimulation by IL8 at D1 and D30 (CD56 bright>CD56 Dim) and a non-significant reduced ability of NK cells to degranulate at D1 and D30. Conclusion: This pilot prospective study supports the hypothesis that radiofrequency ablation could induce an early systemic immune response. Analysis of additional patients and correlation with tumor relapse are on-going.
Marianne Ziol - Grant/Research Support: Echosens
Nathalie Ganne-Carrie - Advisory Committees or Review Panels: Roche, MSD; Speaking and Teaching: BMS, Gilead
The following people have nothing to disclose: Jean-Charles Nault, Nathalie Bar-get, Lucie Del Pozo, Valerie Bourcier, Francoise Gondois-Rey, Bernadette Barbarat, Gisele Nkontchou, Veronique Grando, Pierre Nahon, Jean Claude Trinchet, Olivier Seror, Daniel R. Olive
Differential effects of sorafenib on liver and hepatocellular carcinoma fibrosis mediated by SDF1α/CXCR4 axis and Gr-1 + myeloid cell infiltration
Yunching Chen1, Yuhui Huang1, Thomas Reiberger1, Peigen Huang1, Gregory Y. Lauwers2, Andrew X. Zhu3, Rakesh K. Jain1, Dan G. Duda1;
1Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA; 2Department of Pathology, Massachusetts General Hospital, Boston, MA; 3Department of Medicine, Massachusetts General Hospital, Boston, MA
Sorafenib - a broad kinase inhibitor - is a standard therapy for hepatocellular carcinoma (HCC), and has been proposed as an anti-fibrosis approach to prevent liver cirrhosis, an underlying pathology in HCC patients. However, the effects of sorafenib on tumor fibrosis - and its consequences on treatment resistance - remain unknown. Here, we show that sorafenib has differential effects on tumor fibrosis versus liver fibrosis in murine models of liver disease. Sorafenib treatment intensifies tumor hypoxia, which increases stromal-derived factor 1α (SDF1α) expression - in cancer and stromal cells - and Gr-1+ myeloid cell infiltration in ortotopically implanted and in spontaneous HCC. SDF1α/CXCR4 pathway directly promotes hepatic stellate cell (HSC) differentiation and activation via MAP kinase pathway. SDF1α increases the survival of HSCs after treatment with sorafenib as well as their α-SMA and expression of Collagen I, resulting in increased tumor fibrosis. Moreover, Gr-1 + myeloid cells mediate HSC differentiation/activation in a paracrine manner. CXCR4 inhibition in combination with sorafenib treatment prevents the increase in tumor fibrosis -despite elevated hypoxia - in part by reducing Gr-1+ myeloid cell infiltration, and inhibits HCC growth. Similarly, antibody blockade of Gr-1 also reduces tumor fibrosis and inhibits HCC growth when combined with sorafenib treatment. Thus, blocking SDF1α/CXCR4 or Gr-1 + myeloid cell infiltration may be a novel approach to inhibit HCC resistance to sorafenib by targeting pro-fibrosis signals activated by sorafenib treatment.
Model of tumor-associated fibrosis regulation by SDF1α/CXCR4 pathway in HCC. Sorafenib has differential effects of fibrosis in the tumor versus the surrounding liver. These effects are the result of increased intratumoral hypoxia, SDF1α expression and Gr-1 + myeloid cell infiltration. Blocking CXCR4 prevents Gr-1+ myeloid cell infiltration and hepatic stellate cells differentiation and activation, and synergizes with the anti-tumor effects of sorafenib.
Thomas Reiberger - Grant/Research Support: Gilead, Gilead, Phenex; Speaking and Teaching: Roche, MSD, Roche, MSD
Rakesh K. Jain - Board Membership: XTuit, H&Q Healthcare Investors, H&Q Life Sciences Investors; Consulting: Enlight Biosciences, Noxxon, SynDevRx, WebMD, Zyngenia; Grant/Research Support: Dyax, MedImmune, Roche; Stock Shareholder: Enlight Biosciences, SynDevRx, XTuit
Dan G. Duda - Advisory Committees or Review Panels: Hexal
The following people have nothing to disclose: Yunching Chen, Yuhui Huang, Peigen Huang, Gregory Y. Lauwers, Andrew X. Zhu
EpCAM Positive Hepatocellular Carcinoma Cell Subpopulation is Highly Tumorigenic
Benoit Lacoste, Grégory Merlen, Valérie-Ann Raymond, Marc Bilodeau;
Centre de Recherche du CHUM, Montreal, QC, Canada
Hepatocellular carcinoma (HCC) occurs mainly on livers with a chronic liver injury process such as viral hepatitis or long-standing steatohepatitis. HCC tumors are known to be heterogeneous and thus composed of cell subpopulations with different behaviours. Our laboratory has isolated by in vivo selection a highly tumorigenic murine cell line (dt-Hepa1-6) issued from the Hepa1-6 parental cell line. While Hepa1-6 cells only have few EpCAM positive cells (0.9±0.1%) and limited ability to form liver tumors after intrasplenic (IS) injection in C57bl6 mice, dt-Hepa1-6 are enriched in EpCAM positive cells (35±1%) and lead to systematic liver tumor development. We also observed higher survivin and β-catenin mRNA expression in dt-Hepa1-6 cells compared to Hepa1-6 cells (survivin:1 .0±0.1 vs 0.6±0.0fold changes (fc); P<0.05, β-catenin: 1.0±0.2 vs 0.2±0.1fc; P<0.05). In order to determine the potential role of EpCAM expression in HCC tumorigenicity, we separated 2 cell populations from the dt-Hepa1-6 cell line by flow cytometry on the basis of their EpCAM membrane expression. After cell sorting and 4 passages, C57bl6 mice were injected IS with 1M cells and sacrificed 21 days later. EpCAMmRNA and membrane protein expression were determined on cell aliquots before injection. At sacrifice, macroscopic tumor load (>0.5mm) was counted, RNA was extracted from whole liver and analyzed by qRT-PCR for alpha-foetoprotein (AFP). Cell sorting led to 2 cell lines according to their EpCAM expression: EpCAM+ (87±3%) and EpCAM- (15±1%); these 2 were compared to the parental dt-Hepa1-6 cell line (35±1%). EpCAMmRNA expression paralleled the membranous protein expression of EpCAM (EpCAM+:16.2±1.3, EpCAM-:4.5±0.2, dt-Hepa1-6:8.7±1.0fc). No significant difference was observed in AFPm-RNA expression between cell lines. Tumor load was higher in mice injected with EpCAM+ than EpCAM- cells (1093±74 vs 472±100 tumors; P<0.01) and dt-Hepa1-6 cells led to results that lied just in between (832±89; P<0.05 vs both groups). Total liver AFPmRNA, as an alternative measure of tumor load, paralleled those described above (EpCAM+:877±1 40 vs EpCAM-:279±36 vs dt-Hepa1-6:435±20fc; all comparisons P<0.05). β-catenin and survivin mRNA expressions were similar between dt-Hepa1-6, EpCAM- and EpCAM+ cells (β-catenin:1.0±0.2 vs 1.2±0.2 vs 1 .1±0.2fc; survivin:1 .0±0.1 vs 1.1±0.1 vs 1. 1 ±0.1 fc). Cell doubling time did not differ between EpCAM- and EpCAM+ cell line (33.8±0.7 vs 31.7±1.0h); this was confirmed by thymidine-3H incorporation (277±3 vs 275±10CPM/mg protein). Conclusion: EpCAM+ HCC cells have an increased ability to grow in vivo and thus have a higher tumorigenic profile in comparison to EpCAM-cells.
Marc Bilodeau - Grant/Research Support: Merck; Speaking and Teaching: Merck, Vertex
The following people have nothing to disclose: Benoit Lacoste, Grégory Merlen, Valérie-Ann Raymond
Sorafenib suppresses CD90+ cancer stem cells to inhibit hepatocellular carcinoma metastasis
Mariko Yoshida, Taro Yamashita, Naoki Oishi, Kouki Nio, Sha Sha Zeng, Takehiro Hayashi, Yoshimoto Nomura, Tomoyuki Hayashi, Hikari Okada, Hajime Sunagozaka, Hajime Takatori, Masao Honda, Shuichi Kaneko;
kanazawa univercity, kanazawa, Japan
Background: Cancer stem cells (CSCs) are considered a pivotal target for the eradication of hepatocellular carcinoma (HCC). We recently reported that CSC markers EpCAM and CD90 are independently expressed in primary HCCs and HCC cell lines. EpCAM+ cells share features with tumorigenic epithelial stem cells, whereas CD90+ cells share those of metastatic vascular endothelial cells (Yamashita T, et al., Hepatology 2013). Here we explored the effect of sorafenib on these distinct liver CSCs. Methods: Primary HCC cells obtained from surgically resected specimens and HCC cell lines Huh1, Huh7, Hep3B, HLE, HLF, and SK-Hep-1 were treated with sorafenib in vitro and characterized. Cell proliferation was analyzed by MTS assay, gene and protein expression was evaluated by qRT-PCR and Western blotting, and the frequency of EpCAM/CD90 expressing CSCs was determined byfluorescence-activated cell sorting (FACS). CSC characteristics were evaluated by spheroid formation, invasion assays, and tumorigenicity in immune deficient mice. Time-lapse image analysis was performed to monitor the effect of sorafenib on cell motility. Results: Sorafenib inhibited cell proliferation in cell lines containing CD90+ CSCs (HLE, HLF, and SK-Hep-1) more than in those containing EpCAM+ CSCs (Huh1, Huh7, and Hep3B). Furthermore, sorafenib attenuated CSC characteristics more in CD90+ cells than in EpCAM+ cells. FACS analysis of primary HCCs and HCC cell lines indicated that sorafenib treatment resulted in a reduction in CD90+ and increase in EpCAM+ CSC populations. This effect was possibly mediated through inhibition of c-Kit signaling. Time-lapse image analysis indicated that co-culture of EpCAM+ Huh7 cells with CD90+ HLF cells enhanced their mobility in vitro, and this effect was completely abolished by sorafenib treatment. In vivo, non-metastatic EpCAM+ Huh7 cells could metastasize to the lung when subcutaneously co-injected with
CD90+ HLF cells in NOD/SCID mice. Conclusions: Sorafenib may target CD90+ CSCs responsible for distant organ metastasis through inhibition of c-Kit signaling in HCC. Suppression of CD90+ CSCs and vascular endothelial cells may explain the survival benefit of sorafenib treatment without apparent tumor shrinkage in HCC patients.
Mariko Yoshida - Grant/Research Support: Bayer
The following people have nothing to disclose: Taro Yamashita, Naoki Oishi, Kouki Nio, Sha Sha Zeng, Takehiro Hayashi, Yoshimoto Nomura, Tomoyuki Hayashi, Hikari Okada, Hajime Sunagozaka, Hajime Takatori, Masao Honda
Expression Of Specific Glucose Transporters Is Upregu-lated In Hepatitis B Virus-Associated Hepatocellular Carcinoma
Jung Wha Chung, Sung Wook Yang, Sang Soo Lee, Sukho Hong, Seong Min Chung, Eun Sun Jang, Jin-Wook Kim, Sook-Hyang Jeong;
Internal medicine, Seoul National University Bundang Hospital, Gyeonggi-do, Republic of Korea
Background and aims: Increased glycolysis in the presence of oxygen (Warburg effect) is commonly observed in rapid-growing human cancer cells. Glucose transport across the plasma membrane, the first rate-limiting step for glucose metabolism, is mediated by glucose transporter (GLUT) proteins. However, the expression of class 1 GLUT family in hepatocellular carcinoma (HCC), typical glycolytic tumor, is not fully elucidated yet. The aims of this study were to elucidate the pattern of GLUT expression in human HCC tissues, and to determine the effect of GLUT knockdown in vitro. Methods: Twenty-nine HCC tissues and matched non-tumorous liver tissues were obtained from surgical specimens of chronic hepatitis B patients from February 2010 to March 2013. Expressions of GLUT-1, GLUT-2, GLUT-3 and GLUT-4 were quantified by qPCR and normalized to GAPDH. HBV pregenomic RNA of matched tissue samples were measured by real-time PCR. Clinical, radiologic and pathologic correlation was made with GLUT expression profiles. HepAD38 cells were treated with siRNA against GLUT-1, GLUT-2, GLUT-3 and GLUT-4 in order to assess the effect of GLUT knockdown on the cell proliferation and apoptosis. Results: At least one glucose transporter was over-expressed in HCC compared to non-neoplastic tissue in most patients (28/29). There were significant correlations between expression of GLUT-2, GLUT-3 and GLUT-4 (p <0.005). Overexpression of GLUT-2, GLUT-3 and GLUT-4 was correlated with low tumor necrosis and increased fatty change. Expressions of GLUT-4 and that of GLUT-2 were negatively correlated with level of serum PIVKA-II. Knockdown of GLUT-2, GLUT-3 and GLUT-4 with siRNA suppressed cell proliferation by 33.1%, 55.0% and 66.4%, respectively, and increased cell death / apoptosis. Conclusions: HBV-related HCC usually over-express one or more GLUTs. Knockdown of GLUT-2, GLUT-3, or GLUT-4 induces tumor cell death, suggesting their potential role as therapeutic targets.
The following people have nothing to disclose: Jung Wha Chung, Sung Wook Yang, Sang Soo Lee, Sukho Hong, Seong Min Chung, Eun Sun Jang, Jin-Wook Kim, Sook-Hyang Jeong