A Lymphotoxin-Driven Pathway to Hepatocellular Carcinoma

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A lymphotoxin-driven pathway to hepatocellular carcinoma Johannes Haybaeck1,*, Nicolas Zeller1,*,#, Monika Julia Wolf1, Achim Weber2, Ulrich Wagner3, Michael Odo Kurrer4, Juliane Bremer1, Giandomenica Iezzi5, Rolf Graf6, Pierre-Alain Clavien6, Robert Thimme7, Hubert Blum7, Sergei A. Nedospasov8, Kurt Zatloukal9, Ramzan Mohammad10, Sandra Ciesek11, Thomas Pietschmann11, Patrice N. Marche10, Michael Karin12, Manfred Kopf5, Jeffrey L. Browning13, Adriano Aguzzi1,* and Mathias Heikenwalder1,*. 1

2

Department of Pathology, Institutes of Neuropathology and Clinical Pathology , University Hospital Zurich, CH 8091 Zurich, Switzerland.

3

Functional Genomics Center Zurich, University Zurich, CH 8057 Zurich, Switzerland.

4

Department of Pathology, Cantonal Hospital Aarau, CH 5001 Aarau, Switzerland.

5

Institute of Integrative Biology, Molecular Biomedicine, Swiss Federal Institute of Technology (ETH),

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Zurich, Schlieren, CH 8952 Schlieren, Switzerland. 6

Swiss HPB (Hepato-Pancreatico-Biliary) Center, Department of Surgery, University Hospital Zurich, CH 8091 Zurich, Switzerland.

7

Department of Internal Medicine, University of Freiburg, D 79095 Freiburg, Germany.

8

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.

9

Institute of Pathology, Medical University of Graz, A 8036 Graz, Austria.

10

INSERM & Université Joseph Fourier-Grenoble, Unité 823, Institut Albert Bonniot UJF Site Santé BP 170 La Tronche, F 38042 Grenoble, France.

11

Division of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), D-30625 Hannover, Germany.

12

University of California, San Diego and University of California, Los Angeles, CA 92093-0723, USA.

13

Department of Immunobiology, Biogen Idec, Cambridge, MA 02142, USA.

#

Current address: Department of Pathology, University of Freiburg, Institute of Neuropathology, D79106 Freiburg, Germany.

Correspondence:

Mathias Heikenwalder Institute of Neuropathology University Hospital Zurich Schmelzbergstrasse 12 CH-8091 Zurich, Switzerland Phone: +41 (44) 255-2106 FAX:

+41 (44) 255-4402

E-mail: [email protected]

Additional Footnotes: * These authors contributed equally. Running title: Sustained hepatic LT signaling causes HCC.

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Summary Hepatitis B and C viruses (HBV, HCV) cause chronic hepatitis and hepatocellular carcinoma (HCC) by poorly understood mechanisms. We show that the cytokines lymphotoxin (LT) ,  and their receptor (LTR) are significantly increased in HBV- or HCV-induced hepatitis and HCC. Liver-specific LT,  expression in mice causes liver inflammation and HCC suggesting a causal link between sustained hepatic LT expression, hepatitis and HCC. Development of HCC, composed in part of A6+ oval cells, depends on lymphocytes and IKappa B kinase expressed by hepatocytes but not on TNFR1. In vivo LTR stimulation

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implicates hepatocytes as the major LT-responsive liver cells and LTR inhibition in LTtransgenic mice with hepatitis suppresses HCC formation. Thus, sustained LT signaling may represent a hitherto unknown pathway involved in hepatitis induced HCC.

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Significance Pharmacological inhibition of LTR signaling reduces pathogen- and concavalin A-induced liver injury while LT signaling on hepatocytes is beneficial during liver regeneration. We demonstrate that sustained hepatic LT expression in mice can be injurious causing chronic hepatitis and HCC. Enhanced hepatic LTR signaling might be of potential clinical relevance since LTR and its ligands are drastically increased in human HBV- and HCV-induced hepatitis and HCC when compared to normal livers or non-viral, benign liver diseases. Thus, hepatic LT signaling might be advantageous if transiently active during liver regeneration, but

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detrimental if chronically triggered. Based on the data presented in this study, suppression of hepatic LTR signaling might be beneficial in liver diseases with chronic LT, LT or LIGHT overexpression.

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Introduction A causal relationship between chronic hepatitis, hepatocellular damage and regeneration with fibrosis and carcinogenesis is well established from epidemiological studies (Berasain et al., 2009; El-Serag and Rudolph, 2007). Various etiologies, including chronic alcohol consumption, chronic drug abuse, autoimmune disorders, exposure to toxins (e.g. aflatoxin B) or infections with hepatotropic viruses (e.g. HBV, HCV) can lead to chronic hepatitis, liver fibrosis and cirrhosis. HBV- and HCV-infections are by far the most common cause of chronic hepatitis in humans (Malhi et al., 2006). Chronic HBV- and HCV-infections are

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frequently associated with hepatocellular carcinoma (HCC) development, the most prevalent primary human liver cancer (El-Serag, 2007), and except for HBV-infections, liver cirrhosis precedes HCC in most cases. The exact mechanisms driving chronic hepatitis-induced liver cancer remain elusive. Among others, aberrant expression of cytotoxic cytokines is thought to be critically involved (Greten and Karin, 2004; Karin, 2006; Lee et al., 2005; Lowes et al., 2003; Maeda and Omata, 2008; Vainer et al., 2008). The pro-inflammatory and homeostatic cytokines LT and LT are members of the tumor necrosis factor (TNF) superfamily. Under physiological conditions LTs are expressed by activated T-, B-, NK- and lymphoid tissue inducer cells (Fu et al., 1998; Ware, 2005) and are crucial for organogenesis and maintenance of lymphoid tissue (Alimzhanov et al., 1997; Koni et al., 1997; Mackay et al., 1997; Rennert et al., 1996). Whereas LT contains a transmembrane domain, LT is soluble. Consequently, LT can exist as membrane bound heterotrimers (LT1 2 or LT21) interacting with LTR or as soluble secreted homotrimers (LT3) triggering TNF receptor 1, 2 (TNFR1, TNFR2) and the herpes virus entry mediator receptor (HVEM) (Browning et al., 1997; Ware, 2005). LTR and TNFR1 signaling can be activated by the HCV core protein (Chen et al., 1997; Zhu et al., 1998) involving the canonical or non-canonical NF-B signaling pathways (Ware, 2005; You et al., 1999). Furthermore, HBV- or HCV-infections lead to increased hepatic LT expression in vivo and in vitro (Lee et al., 2005; Lowes et al., 2003) and HCV replication has been demonstrated to 4|Page

depend on components of the LTR signaling pathway in vitro (Ng et al., 2007). Besides, IL6 and IL-1 induce LTexpression in hepatoma cell lines and oval cells (Subrata et al., 2005). LTs can directly act on hepatocytes which physiologically express high levels of LTR but little LT (Browning and French, 2002). T-cell-derived LT and LIGHT (LT-like, exhibits inducible expression, competes with HSV glycoprotein D for HVEM, expressed by T lymphocytes) signaling to hepatocytes controls lipoprotein homeostasis (Lo et al., 2007). In addition, LT signaling is important for liver regeneration through T-cell-derived LT expression

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(Tumanov et al., 2008) and regulates hepatic stellate cell function and wound healing (Ruddell et al., 2008). Thus, hepatic LTR signaling is crucial for controlling liver homeostasis in both health and disease. The observation that chronic inflammatory stimuli promote HCC formation has been recapitulated in various mouse models. Ablation of the multi-drug resistance gene 2 (mdr2) induces cholestatic hepatitis and liver cancer (Pikarsky et al., 2004) and application of the chemical carcinogen diethylnitrosamine (DEN) causes acute liver injury and HCC (Maeda et al., 2005). Experiments with transgenic mice expressing the hepatitis B surface antigen (HBsAg) specifically in the liver demonstrate that chronic immune-mediated liver cell injury is critical for HCC formation (Nakamoto et al., 1998). Triggering TNFR1 or LTR induces the classical and alternative NF-B signaling pathways, which are linked to inflammation-induced carcinogenesis (Greten and Karin, 2004; Karin, 2006). However, the precise role of these pathways in HCC pathogenesis is controversial (Vainer et al., 2008). Mice lacking IKappa B kinase specifically in hepatocytes (Ikkhep) exhibit a marked increase in DEN-induced HCC formation (Maeda et al., 2005) suggesting a protective function of IKK in HCC development. In contrast, NF-B signaling promotes HCC development in mdr2-/- mice and anti-TNF treatment is protective (Pikarsky et al., 2004). Interestingly, mice with a hepatocyte-specific deletion of IKK (also called NEMO) develop steatohepatitis and HCC (Luedde et al., 2007). Consequently, the role of NF-B signaling in 5|Page

hepatocarcinogenesis might depend on the mouse model and the type or degree of liver inflammation and injury (Vainer et al., 2008). Therefore, more research is needed to understand the role of NF-B signaling in HCC formation. Here we report that LTR and its ligands are drastically upregulated in chronic hepatitis B, C and in HCC. Immunohistochemistry and mRNA expression analysis revealed that LTs are expressed on both, lymphocytes and hepatocytes. Consistent with this result, challenge with infectious, cell culture derived HCV caused upregulation of LT, LT, LIGHT, LTR and various chemokines by a human hepatocyte cell line in vitro.

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Sustained hepatic LT,  expression in transgenic mice caused chronic hepatitis and HCC formation independent of viral infection. Hepatitis and HCC development were prevented by the ablation of lymphocytes or hepatocyte-specific removal of IKK, yet HCC induction was TNFR1- independent. In vivo LTR stimulation with an agonistic LTR antibody identified hepatocytes as the major LT responsive liver cells and pharmacological inhibition of LTR signaling in LT-transgenic mice with hepatitis reduced liver inflammation and prevented HCC. Our results demonstrate that sustained hepatic LT signaling is critically involved in hepatitis and HCC development.

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Results Upregulation of LT, LT and LTR in HBV or HCV infected human livers and in HCC The specific role of LT signaling in the pathogenesis of virally-induced hepatitis and HCC formation is currently ill-defined. We first analyzed transcriptional levels of LT, LT, LIGHT, TNF, LTR and TNFR1 in human HBV- or HCV-induced chronic hepatitis and HCC or in non-viral HCC compared to healthy liver specimens (Fig. 1; Supplemental Fig. S1). LT, LT and LTR mRNA expression was increased on average ~27 to 210 fold in HBV- or HCVinduced hepatitis and HCC (P