349 Hepatic progenitor cells express VIP receptor type 2

04D. Molecular and cellular biology

(d) Liver regeneration and experimental oncology

(d) Liver regeneration and experimental oncology

04D. M o l e c u l a r a n d c e l l u l a r b i o l o g y -

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O V E R E X P R E S S I O N OF THE Wnt/Frizzled SIGNAL'S MEMBERS: A C O M M O N AND EARLY EVENT IN HUMAN HEPATOCELLULAR C A R C I N O M A (HCC)

A. Bengochea 1, M. De Souza 1, L. Lefrancois 1, C. Trepo 1, M. Kim 3, J.R. Wands 3, R Hainaut2, J.Y. Scoazec 4, L. Vitvitski 1, R Merle 1.

I INSERM U271, Lyon, France," :IARC, Lyon, France," SLiver Researeh Cente~ Providence RI, USA," 4Laboratoire d'Anatomopathologie, Hdpital Edouard Hdrriot, Lyon, France Background and Aims: The Writ/Frizzled signaling, induced by binding of a Writ ligand (n 19) to its transmembrane receptor Frizzled (Fzd; n 10) and co-receptor LRP (n 2), and antagonized by the secreted Frizzled-related proteins (sFRP; n 4), can alternatively activate [J-catenin, JNK or PKC pathways which may control the cancerous phenotype. We previously demonstrated that FZD7 is commonly overexpressed in hepatitis B virus (HBV)-related human HCC as well as in rodent models [Merle, Gastroenterology 2004, J. Hepatol. 2005]. The aim of our work was to study the expression patterns of WNT, FZD, LRP and sFRP genes in human HCC in accordance with etiology and histological grade (Edmonson criteria). Material and Methods: Gene expression of all WNT, FZD, LRP and sFRP was assessed by quantitative real-time RT-PCR in human HCCs: tumors (T) and the corresponding nontumorous liver (NT HBV- (n 13), HCV- (n 9) or nonviral-related (NBNC) (n 11)- by comparison to normal livers (NL, n 9) (cut-off mean NL + 2SD, a 5%). FZD expression was confirmed by immunohistochemistry (IHC). Histological examination was performed from paraffin sections. Results: FZD-3, -6 and -7 were highly and commonly overexpressed in T (74%), and FZD-3 and -6 in NT (37%). IHC confirmed that these FZDs were specifically expressed by hepatocytes. In accordance with etiology, FZD-7 was mostly overexpressed in virus-related HCCs vs NBNC (54% vs 21%), as well as FZD-6 (35% vs 9%), by comparison to FZD-3. In accordance with histology, FZD-3 was mostly overexpressed in poorly differentiated tumors. LRPs were stably expressed in NL, NT and T. As secreted activators, Wnt-3 and -5A were commonly overexpressed with FZD-3, -6 and -7 in T and NT. As secreted inhibitors, sFRP1 was downregulated in more than 50% of T and NT. Conclusion: The possible activation of the Wnt/FZD signaling is the most common (89% T) and earliest (66% NT) event in human hepatocarcinogenesis independently of etiology, due to molecular interactions between 3 FZD and 2 WNT factors, added to downregulation of the sFRP1 inhibitor. However, it remains to study the specific ligand/receptor bindings and their impact on pathway activation and control of the cancerous phenotype.

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HUMAN UMBILICAL CORD (HUC) AND HUC BLOOD CD105-POSITIVE CELLS CAN DIFFERENTIATE IN VITRO INTO HEPATOCYTE-LIKE CELLS

P. Burra 1, A. Masier 1, M. Turetta 2, C. Calore 2, R. Di Liddo 2, R Bo a, S. Tomat 1, F.R Russo 1, M.T. Conconi 2, RR Parnigotto 2. 1Gastroenterology

Section, Department of Surgical and Gastroenterological Sciences, 2Department of Pharmaceutical Sciences, 3Obstetrics and Gynaecology Unit, Cittadella Hospital, Padova, Italy Background: To overcome the shortage of donors for liver transplantation, several approaches have been performed using bioartificial devices or cell

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transplantation. Nowadays, the search of alternative sources of hepatocytes represents an important goal in order to obtain large number of fully functional cells without immunogenic potential. Aim: to verify that human umbilical cord blood (HUCB) and umbilical cord (HUC) itself could contain MSCs able to differentiate into hepatocytelike cells. Material and Methods: Primary cultures were obtained from enzymatic digestion of subendothelium of umbilical vein and from HUCB by density gradient separation. Both the cultures were enriched in CD105-positive subset by immunoseparation and characterized by immunofluorescence. Then, osteogenic and adipogenic differentiations were performed using inductive media. To obtain hepatocyte-like cells, CD105-positive cultures were seeded either on Matrigel and PuraMatrix gel and grown in presence of hepatic growth factors. Results: The CD105-positive cultures obtained from both HUC and HUCB showed high proliferative potential and mesenchymal morphology. They were positive for mesenchymal markers (CD105, 90, 54), but negative for hemopoietic (CD45, 34, 38) and endothelial (CD31 and KDR) ones. At day 7 for HUC and day 14 for HUCB, calcium salt deposits revealed by Von Kossa staining were well visible inside the cultures grown with osteogenic medium. Moreover, cells from HUCB and HUC contained several lipidic droplets, stained with Oil Red O, at day 9 and day 18, respectively, thus confirming that cells were adipocyte-like cells. HUC 105-positive cells cultured on Matrigel expressed hepatic markers, such as AFP, HNF4~ (day 14), albumin and c-met (day 30). After 12 days in hepatogenic medium, cultures obtained from HUCB and encapsulated into PuraMatrix presented immunoreactivity for HNF4a. Conclusions: Our preliminary results implicate HUC and HUCB as a promising source of MSCs able to differentiate into osteoblast-, adipocyte-, and hepatocyte-like cells. Nevertheless, to ascertain the clinical relevance of this finding, further in vitro and in vivo investigations must be performed.

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HEPATIC P R O G E N I T O R CELLS EXPRESS VIP R E C E P T O R TYPE 2

D. Cassimanl, R. De Vos2, N. Sinelli 2, S. Vander Borght2, B. Petersen 3, I. Bockx 1, E Aertsen2, L. Libbrecht2, F. Nevens 1, T. Roskams2.

1Department of Hepatology, 2Department of Mo@ology, KU Leuven, Belgium," 3Department of Pathology, Immunology and Laboratory Medicine, University of Florida, USA Background: We recently showed that hepatic progenitor cells (HPC) express muscarinic acetylcholine receptor subtype 3 and that diseased, surgically vagotomised livers show a significantly lower number of HPC. (D. Cassiman et al. Am J Pathol 2002) To further elaborate on this, we examined whether HPC also express receptors for vasoactive intestinal peptide (VIP), which, besides acetylcholine, is also an important parasympathetic neurotransmitter. VIP expressing nerves have been shown to be present in the liver. Methods: We performed immunohistochemistry for VIP receptor subtype 1 and 2 (VIPR1 and 2), on paraffin and cryostat sections of normal and diseased human (n 18) and rat liver (models of acute and chronic toxic and biliary liver disease). We also performed RT-PCR for VIPR1 and 2 on total RNA from purified rat HPC, since isolated, purified human HPC are not available to perform these tests. Results: Immunohistochemical staining with an antibody raised against the VIPR2 receptor revealed the presence of intensely staining HPC and reactive bile ductules on paraffin-embedded and frozen tissue sections of human liver. VIPR2 staining on rat liver did not show any immunoreactivity on paraffin-embedded or frozen tissue sections. Human and rat paraffin-embedded or frozen tissue sections did not show any staining with any of the VIPR1 receptor antibodies tested. The presence of VIPR2 mRNA in HPC was confirmed by RT-PCR on total RNA of purified rat HPC. RT-PCR for VIPR1 and muscarinic acetylcholine receptor 3 were also found to be positive in this cell population.

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Conclusion: HPC express VIPR2 protein (human) and mRNA (rat), which allows them to respond to the VIP secreted by the parasympathetic nerve endings in the liver. This suggests a direct interaction between the autonomic liver innervation and HPC, which could be of importance for HPC function, survival and/or proliferation and hence for liver regeneration. This is especially relevant for liver transplant patients, who carry a denervated liver.

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A M P H I R E G U L I N A NOVEL D E T E R M I N A N T IN THE RESISTANCE OF HCC CELLS TO A P O P T O S I S I N D U C E D B Y TGF-I~ A N D CYTOSTATIC D R U G S J. Castillo 1, E. Erroba 1, M.J. Perugorria 1, M. Santamaria 1, D.C. Lee 2, J. Prieto 1, C. Berasain 1, M.A. Avila 1. 1Division of Gene Therapy and

Hepatology. CIMA, University of Navarra, Pamplona, Spain," 2Department of Bioehemistry and Biophysics'. University of North Carolina, School of Medicine, Chapel Hill, NC, USA Background and Aims: Hepatocellular carcinoma is a major cause of cancer-related deaths. Current treatments are not effective, and the identification of relevant pathways and novel therapeutic targets are much needed. Increasing evidences point to the activation of the epidermal growth factor receptor as an important mechanism in the development of hepatocarcinoma. We previously described that amphiregulin, a ligand of the epidermal growth factor receptor, is not expressed in healthy liver but is upregulated during chronic liver injury, the background on which most liver tumors develop. Now we have studied the expression and role of amphiregulin in human hepatocarcinoma. Methods: Amphiregulin expression and function was studied in human liver tumors and cell lines. Results: Amphiregulin is expressed in human hepatocellular carcinoma tissues and cell lines, and behaves as a mitogenic and antiapoptotic growth factor for hepatocarcinoma cells. We provide several lines of evidence, including amphiregulin silencing by siRNAs or inhibition of amphiregulin by neutralizing antibodies, demonstrating the existence of an amphiregulin-mediated autocrine loop that contributes to the transformed phenotype. Indeed, interference with endogenous amphiregulin production resulted in reduced constitutive epidermal growth factor receptor signaling, inhibition of cell proliferation, anchorage-independeut growth, and enhanced apoptosis. Moreover, knockdown of amphiregulin potentiated transforming growth factor-~ and doxorubicin-induced apoptosis. Conversely, overexpression of amphiregulin in SK-Hepl cells enhanced their proliferation rate, anchorage-independent growth, drug resistance and in vivo tumorigenic potential. Conclusions: These observations suggest that amphiregulin is involved in the acquisition of neoplastic traits in the liver, and thus constitutes a novel therapeutic target in human hepatocarcinoma.

IS C O M M O N L Y E X P R E S S E D B Y C A N C E R O U S FS• Frizzled-7 HEPATOCYTES IN VIRUS A N D N O N - V I R U S RELATED HUMAN HEPATOCELLULAR CARCINOMAS: C O R R E L A T I O N S WITH P A T H O L O G I C A L FEATURES A N D I~-CATENIN/P53 MUTATION STATUS M.M. de Souzal, A. Bengochea 1, E. Leroux2, L. Lefrancois 1, O. Galy 1, I. Chemin 1, C. Trepo 1, L. Vitvitski 1, J.Y. Scoa~zec3, P. Hainaut2 , P. Merle 1.

I INSERM U271, 2IARC, 3INSERM U45, Lyon, France Background and Aims: We previously demonstrated that the Frizzled-7 receptor (FZD7) is commonly overexpressed in hepatitis B vires (HBV)related human hepatocellular carcinoma (HCC) and can activate the [J-catenin pathway and control motility of cancerous hepatocytes [Merle et al., Gastroenterology 2004] as well as in rodent models [Merle et al., J. Hepatol. 2005]. However, we did not know its expression pattern in

hepatitis C virus (HCV)- and nonviral (NBNC)-related human HCCs, and its relationship with tumor grading and mutation profiles for [J-catenin and p53 genes that are two key players in human hepatocarcinogenesis. Methods: 34 paired (tumor: T, and surrounding nontumorous liver: pT) samples of human HCCs (8 HBV, 13 HCV, 13 NBNC) and 9 controls (NL: normal liver parenchyma surrounding benign lesions) giving a cutoff value (mean• obtained from surgical resections were tested. Pathological examination was performed. FZD7 expression was assessed by quantitative real-time RT-PCR and immunohistochemistry (IHC). We identified TP53 gene mutations by PCR/RFLP/sequencing in exons 5 9, and [J-catenin mutations by DHPLC/sequencing in exon 3. Results: IHC clearly showed that FZD7 was specifically expressed by hepatocyte cells in HCC tissues. By QRT-PCR, FZD7 was upregulated in 38% HBV-, 54% HCV- and 23% NBNC-related T by comparison to NL (~ 5%); in 75% HBV-, 92% HCV- and 69% NBNC-HCC when comparing T versus pT; and rarely in pT. FZD7 upregulation did not correlate with either tumor grade (Edmonson criteria), or p53 mutation status (24% of HCCs). By contrast, FZD7 upregulation tended to be more commonly associated with wild-type versus mutated [J-catenin status. Conclusion: FZD7 upregulation involves specifically hepatocyte cells and is a common event in human HCC-T, independently of tumor grade and p53 mutation status. However it tends to be more frequent in virusassociated HCCs with wild-type [J-catenin status. These data underline that FZD7 might be a good candidate as molecular target for therapeutic intervention in a substantial proportion of human HCCs.

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ANALYSIS OF THE H O M E O B O X T R A N S C R I P T I O N FACTOR Prox-1 E X P R E S S I O N IN RAT LIVER D E V E L O P M E N T A N D IN DIFFERENT M O D E L S OF LIVER DAMAGE AND REGENERATION

J. Dudasl., A. Elmaouhoub 1, T. Mansuroglu1, D. Batusic 1, M. Papoutsi 2, K. Tron 1, B. Saile 1, J. Wilting 2, G. Ramadori 1. 1Department of

Gastroenterology and Endocrinology, 2Children 1' Hospital, Pediatrics L Georg-August-University G6ttingen, Germany Background and Aims: Prox-1, a homeobox transcription factor, is expressed in hepatoblasts and adult hepatocytes. Its function in hepatocyte development is still unknown. Methods: Prox-1 protein localisation and mRNA expression was examined in livers of rat embryos, fetuses at embryonic days (ED) 10 20, newborn and normal adult rats and in models of rat liver damage and regeneration: 1. Acute and chronic liver damage induced by carbon tetrachloride (CC14), 2. Conventional 2/3 partial hepatectomy (PH), 3. PH in combination with 2-acetyl-aminofluorene (AAF) treatment (AAF/PH). Livers from muscular turpentine oil injected and Sham operated rats were used as acute phase controls. Results: Prox-1 was found in the nuclei of endodermal cells, and in clustered cells of the liver anlage at embryonic day (ED) 10, at ED 14 18 the number of the Prox-l-positive cells increased. Prox-l-positive cells initially have shown cytokeratin (CK)-19 too. However, beginning at ED16 not all the CK-19 positive cells were Prox-1 positive. Later the hepatoblasts and hepatocytes become CK-19 negative, and CK-19positive cells were Prox-1 negative. Prox-1 remained a stable marker of hepatocytes, and absent from bile ducts, while the latter were CK-19 and -7 positive in the adult liver. In models of liver damage Prox-1 remained detectable in hepatocytes. In models of liver regeneration rare double positive (CK-7 + Prox-1 or CK-19 + Prox-1) cells were observed, but they did not express ~-fetoprotein. In models of liver damage and regeneration a Prox-1 mRNA expression decreased, followed by a restoration. CK-19 expression significantly decreased after CC14-induced liver damage, and was not affected significantly after PH or turpentine oil injection. In AAF/PH a remarkable increase of CK-19 and AFP gene expressions were observed at days 7 11 after PH followed by a decrease, this regulation pattern was not found for Prox-1.