90 HOXA13 AND HOTTIP EXPRESSION LEVELS PREDICT PATIENTS' SURVIVAL AND METASTASIS FORMATION IN HEPATOCELLULAR CARCINOMA

ORAL PRESENTATIONS enhanced anisokaryosis and a clear increase in the occurrence of ballooned hepatocytes. In the NIPP1L-KO mice an immunostaining of the liver sections for the cholangiocyte and progenitor cell marker cytokeratine 19 revealed the beginning of an oval cell response. After one year, the liver of knockout mice showed an obvious bile duct hyperplasia which, surprisingly, was associated with the recurrence of NIPP1 expression. Interestingly, the injection of diethylnitrosamine (DENA) during 25 weeks resulted in an increased sensitivity towards the development of both hepatocellular and cholangiocarcinoma in the NIPP1L-KO mice. At this stage most hepatocytes and cholangiocytes in the NIPP1L-KO showed a normal expression of NIPP1, hinting at the replacement of damaged liver cells by NIPP1-containing cells, probably originating from progenitor cells that had escaped Cre-recombination. In conclusion, NIPP1 appears to be indispensable for cell proliferation in the mouse liver and seems required for a balanced differentiation of oval cells into cholangiocytes and hepatocytes. Nevertheless, the NIPP1L-KO mice show an increased predisposition towards the development of liver cancer, hinting at long-term effects from NIPP1-depleted cells. 88 THE FUNCTION OF Fos AND Fos~Jun DIMERS IN LIVER CANCER DEVELOPMENT R. Hamacher, L. Bakiri, E.F. Wagner. Genes, Development and Disease Group, F-BBVA – CNIO Cancer Cell Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain E-mail: [email protected] Background and Aims: Hepatocellular carcinoma (HCC) belongs to the cancer types which are associated with chronic inflammation. The crucial crosstalk between hepatocytes and non-parenchymal cells, especially immune cells, is well established and many important signaling molecules like MAPKs, NF-úB, AP-1 and STAT3 are involved. We have shown that the proto-oncogene c-Jun, a component of the dimeric AP-1 transcription factor, is required for mouse liver tumorigenesis (Eferl et al., Cell, 2003). In addition, we have demonstrated that c-Jun promotes cell survival during tumor initiation by controlling c-Fos/SIRT6-dependent expression of the anti-apoptotic protein Survivin (Min et al., Nat Cell Biol, 2012). The c-Jun partner c-Fos is frequently over-expressed in HCC, however, the in vivo function of c-Fos in liver cancer remains to be defined. Methods: The well-established chemical carcinogenesis (DEN) protocol was applied to different mouse models. Conditional deletion of c-fos in hepatocytes and non-parenchymal liver cells was achieved using Mx-cre and hepatocyte-specific Alfp-cre. Importantly, we also generated mice carrying novel tetracycline (tet)-switchable hepatocyte-specific c-fos and forced jun~fos alleles. Results: Mice with genetic inactivation of c-fos in hepatocytes displayed a significant reduction in tumor burden when subjected to chemical carcinogenesis, whereas deletion of c-fos in hepatocytes and non-parenchymal liver cells did not lead to significant alterations in HCC development. Moreover, hepatocyte-specific ectopic expression of c-Fos in adult mice led to spontaneous dysplastic lesions and promoted DEN-induced liver carcinogenesis. Interestingly, when c-Fos dimerization was restricted to a single Jun partner, the resulting c-Jun~c-Fos and JunD~c-Fos expressing mice displayed multiple cancer nodules, unlike JunB~c-Fos expressing mice. Conclusions: These results suggest a cell type-dependent role of c-Fos in liver cancer development. c-Fos appears to have an oncogenic function in hepatocytes, through dimerization with c-Jun or JunD, but not JunB. Interestingly, c-Fos shows a tumor suppressive function in non-parenchymal liver cells. Future studies will lead to a better understanding how c-Fos controls development of liver tumors and possibly influences the tumor microenvironment. This will help to identify new prognostic biomarkers and therapeutical targets.

89 A TRANSPOSON-BASED PRIMARY TUMOR RESECTION MODEL OF INTRAHEPATIC CHOLANGIOCARCINOMA (ICC) WITH EXTRAHEPATIC DISTANT METASTASES 1 E. Gurlevik ¨ , B. Fleischmann-Mundt1 , N. Armbrecht1 , T. Longerich2 , 1 1 N. Woller , A. Kloos1 , L. Zender3 , M. Manns1 , F. Kuhnel ¨ , S. Kubicka1 . 1 Gastroenterology, Hepatology, and Endocrinology, Medical School Hannover, Hannover, 2 Institute for Pathology, University of Heidelberg, Heidelberg, 3 Dept. of Internal Medicine I, University Hospital Tuebingen, Tuebingen, Germany E-mail: [email protected] Background and Aims: Intrahepatic cholangiocarcinoma (ICC) is a tumor with worldwide increasing incidence and dismal prognosis. Although surgical resection (R0) of the primary tumor is the only potential curative treatment, most of the patients die because of frequent tumor recurrence and outgrowth of metastases after surgery. Mouse tumor resection models are urgently needed to reflect the clinical relevant situation and investigate novel therapies in the context of ICC. In this study, we established a corresponding endogenously induced murine tumor model of resectable, single locus ICC formation. Methods: We investigated tumor development at a defined intrahepatic locus by injection of Sleeping Beauty-based, oncogenic transposon plasmids into the liver lobe followed by subsequent electroporation. The plasmids included the constitutively activate KRasG12V oncogene together with a plasmid for Cre-recombinase and were applied in p53-fl/fl mice to induce the p53-knockout. This setup was chosen since molecular pathogenesis of ICC frequently involves both KRas-activation and p53-aberrations. Results: Mice developed a single intrahepatic tumor lesion within 3–7 weeks following electroporation. Develoment of ICC was verified by histological analysis. Molecular analysis after electroporation of defined lineage-specific fluorescent reporter plasmids provided evidence for hepatocytes as origin of ICC formation. At the time of primary tumor growth no formation of metastases in the lung could be detected. But formation of satellites close to the primary tumor and vascular invasion could be observed as an indicator of early metastasis. After R0-resection of the primary tumor we were able to prolong median survival with the observation of local disease recurrence, peritoneal carcinomatosis and lung metastases. This resection model of the endogenous, intrahepatic tumor induction allows preclinical testings of adjuvant therapies. In conclusion, we have successfully developed a murine model of endogenously induced ICC with a single, resectable, primary tumor. This model has favorable characteristic for the study of recurrence patterns after resection and the mechanisms of metastasis. It also holds promise for preclinical evaluation of novel multimodal or adjuvant therapies to prevent recurrence and outgrowth of metastases after R0-resection. 90 HOXA13 AND HOTTIP EXPRESSION LEVELS PREDICT PATIENTS’ SURVIVAL AND METASTASIS FORMATION IN HEPATOCELLULAR CARCINOMA L. Quagliata1 , M. Matter1 , S. Piscuoglio1 , Z. Makowska2 , M. Heim2 , L. Tornillo1 , C. Cillo3 , L. Terracciano1 . 1 Molecular Pathology Unit, Institute of Pathology, University Hospital Basel, 2 DMB, University of Basel, Basel, Switzerland; 3 Biology Lab, University of Naples ‘Federico II’, Naples, Italy E-mail: [email protected] Background: The epigenetic mechanisms controlled by the polycombs and trithoraxs genes as well as the HOX genes transcriptional factors family have been linked to the genesis and evolution of liver cancer. Previously, we observed that among the HOX genes, HOXA13 is highly deregulated in hepatocellular

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ORAL PRESENTATIONS carcinoma (HCC). More recently, a lincRNA located at the 5 end of the HOXA locus (in physical contiguity with HOXA13), named HOTTIP, has been identified. HOTTIP binds the WDR5/MLL complexes driving gene transcription along the entire HOXA locus. In this study we aimed to evaluate the impact of HOTTIP and HOXA13 deregulation on HCC pathogenesis. Methods: Total RNA extracted from 60-paired biopsies obtained from HCC patients was used to quantify HOTTIP/HOXA13 expression levels via qRT-PCR and subjected to global transcriptome analysis. HOTTIP/HOXA13 expression levels have been correlated with patients’ clinicopathological data. Non HCC-conditions (normal liver donor, liver inflammation, steatosis, cirrhosis) samples have been used as controls. Results: qRT-PCR data confirmed that HOXA13 is highly deregulated in HCC with no major alteration found in non HCC-conditions. Furthermore, we outlined that HOTTIP is also deregulated in HCC but not in non HCC-conditions and that its expression directly correlates with HOXA13 levels. We also found that virus-related (HBC and/or HCV) HCC samples present the highest expression levels of both HOTTIP and HOXA13 among HCC patients. In addition, we found that both HOTTIP and HOXA13 expression levels predict patients’ overall survival as well as metastasis formation. Finally, the global transcriptome analysis revealed that HOTTIP/HOXA13 overexpression in HCC identifies a specific subset of genes mostly involved in mRNA processing. In vitro siRNA experiments against HOXA13/HOTTIP in HCC cell lines further validated the strong interplay between HOTTIP and HOXA13. Conclusions: We demonstrated for the first time that HOTTIP expression directly correlates with HOXA13 levels in HCC. Moreover, we reported that HOTTIP/HOXA13 deregulation as a key feature in HCC. Finally, we outlined HOTTIP/HOXA13 as predictive markers of HCC patients’ outcome and incidence metastasis formation. 91 IMMUNOTHERAPY WITH VECTOR ENCODING ALPHA-FETOPROTEIN FUSED WITH PROTEASOME TARGETING SIGNAL PROVIDES NOTABLE PROTECTIVE IMMUNITY AGAINST HEPATOCELLULAR CARCINOMA IN MICE A.V. Morozov1 , V.A. Morozov2 , T.M. Astakhova3 , V.L. Karpov1 . 1 W.A. Engelhardt Institute of Molecular Biology RAS, Moscow, Russia; 2 Robert Koch Institute, Berlin, Germany; 3 N.K Koltsov Institute of Developmental Biology RAS, Moscow, Russia E-mail: [email protected] Background and Aims: Alpha-fetoprotein (AFP) is a marker of hepatocellular carcinoma (HCC). DNA vaccines against AFP were shown to generate strong immune response. Previously we demonstrated that DNA vaccine bearing HIV-1 reverse transcriptase (RT) gene and mouse ornithine decarboxylase (ODC) degradation signal induced a strong Th1 immune response against RT HIV-1 in mice. It was suggested that the DNA vaccine bearing AFP+ODCsignal directed for degradation in proteasome, would induce strong CD8+CTL response against tumor cells expressing AFP and might retard or even prevent HCC appearance. Materials and Methods: Vectors expressing murine AFP (pmAFP), mAFP+ODC degradation signal without AFP exportation signal (pDAFPODCsignal) and a number of other constructs were designed. Protein expression was examined in transfected HEK 293T cells. Proteasomal degradation was evaluated by the cycloheximide chase, proteasome inhibition assay and immunofluorescence. Th1 immune response was assessed by ELISA. Tumors in C57BL mice were induced by subcutaneous admittance of 2x105 hepatoma cells (Hepa 1–6 cell line), or by injection of diethylnitrosamine (DENA). Three trials were performed. “Therapeutic” trial – 14 days after tumor cell challenge mice were vaccinated intramuscularly with 100 mg of plasmid. “Prevention” trial – mice were vaccinated four times (50 mg, 2 week intervals). Two weeks after the last vaccination S40

animals were challenged with tumor cells. “DENA therapeutic trial” – infant mice received 25 mg/g DENA and were immunized with the constructs 4 times with 2 week intervals starting at 3 months of age and at the age of 10 months the animals were sacrificed. At the end of each trial tumors, livers and serum were examined. Results: Protein DAFPODCsignal degraded fast and specific in the proteasomes of transfected cells (half-life 1.5–2h). Preventive vaccination with pDAFPODCsignal yielded 5-fold reduction in mean tumor volume compared to the non-immunized group on day 77 after tumor cell challenge. pDAFPODCsignal impaired growth of HCC by 35% in animals with tumors induced by DENA. Conclusions: Immunotherapy with the DNA encoding AFP and proteasome targeting tag induce not only prominent immune response, but most important a significant retardation of tumor growth in vaccinated animals, becoming a promising candidate vaccine for the HCC prevention. 92 FORCED IL-6 SIGNALING IS SUFFICIENT TO PROMOTE MALIGNANT TRANSFORMATION IN TELOMERASE-IMMORTALIZED HUMAN FETAL HEPATOCYTES FOLLOWING CHALLENGE WITH OXIDATIVE STRESS D. Heim1 , A.C. Parplys2 , S. Rose-John3 , A.W. Lohse1 , E. Dikomey2 , H. Wege1 . 1 Gastroenterology and Hepatology, 2 Laboratory of Radiobiology and Experimental Radiooncology, University Medical Center Hamburg-Eppendorf, Hamburg, 3 Institute of Biochemistry, Christian-Albrechts-Universit¨ at zu Kiel, Kiel, Germany E-mail: [email protected] Background and Aims: HCC is the most prevalent cancer associated with chronic inflammation. Telomere stabilization is considered a prerequisite and early event in multi-step hepatocarcinogenesis; however, hTERT-mediated telomerase reactivation alone does not induce transformation. In chronic inflammation, further downstream oncogenic events are driven by genotoxic reactive oxygen species (ROS) in the context of pro-proliferative signaling pathways. In this project, we investigated the antioxidant response and cellular transformation events in untransformed hTERTimmortalized human fetal hepatocytes (FH-hTERT) with forced activation of IL-6 signaling. Methods: To activate the IL-6 signaling pathway, we generated clones with stable expression of a constitutively active gp130 (Lgp130), responsible for downstream signal transduction. Derived single cell-clones were characterized and challenged with H2 O2 following glutathione depletion by buthioninesulfoximine (BSO). Quantification of ROS was performed by FACS (Carboxy-H2 DCFDA) and DNA-double strand breaks were determined by fluorescent staining for gamma-H2AX. DNA damage and antioxidant response were assessed by qRT-PCR. Finally, to monitor malignant transformation, we determined anchorage-independent growth in soft agar as an established in vitro marker. Results: Stable transfection with L-gp130 resulted in enhanced phosphorylation of STAT3 and MAPK1/2 with upregulation of CCND1. These molecular changes did not induce a transformed phenotype in FH-hTERT/L-gp130 cells and, interestingly, even somewhat suppressed proliferation. Treatment with H2 O2 /BSO resulted in 2- to 3-fold higher ROS levels in FH-hTERT/L-gp130 clones compared to control FH-hTERT cells. In contrast, p21 was not induced and suppressed in some clones in comparison to FH-hTERT control cells. Surprisingly, FH-hTERT/L-gp130 clones developed colony growth capabilities in soft agar with a frequency of up to 20 colonies per 5,000 seeded cells only after challenge with H2 O2 /BSO (in comparison, HuH-7 without treatment 91±12, FH-hTERT with and without treatment no colony formation, FH-hTERT/L-gp130 without treatment no colony formation). As possible mechanism,

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