Experimental hepatocarcinogenesis


The Contribution of Toll-Like Receptor Signaling to the Development of Hepatic Fibrosis and Carcinogenesis in Hepatocyte Specific TAK1 Deleted Mice

Jingyi Song, Sayaka Inokuchi, Yoonseok Roh, Ling Yang, Ekihiro Seki

Medicine, University of California, San Diego, School of Medicine, La Jolla, CA

Background: TGF-β -activated kinase 1(TAK1) is critical to activate the JNK and NF-kB signaling pathways. Gene disruption of TAK1 in hepatocytes has demonstrated that TAK1 is a key regulator of inflammation, fibrosis, cell survival, and carcinogenesis. We will examine the roles of Toll-like receptor 4, 9 (TLR4, TLR9), MyD88, IL1 receptor (IL1R), and TNF receptor (TNFR) in the development of liver fibrosis and carcinogenesis in hepatocyte-specific TAK1 deleted mice (TAK1 AH). Methods: TAK1 AH were crossed with mice deficient in TNFR, TLR4, TLR9, MyD88, and IL1R, to generate double knockout mice. Liver morphologies and functions were studied in mice at 1, 4, and 9 months of age. Results: TAK1 AH mice had increased ALT levels, inflammatory cell infiltration, collagen deposition, and development of visible liver tumors. At 1 month of age, ALT levels reduced significantly to 577U/L in TAK1ΔH/TNFR-/-, but remained at 1670U/L in TAK1ΔH/TLR4-/-, compared to 1608U/L in TAK1 AH mice. The deletion of TNFR and TLR4 in TAK1 AH mice significantly reduced macrophage infiltration assessed by immunohistochemistry (IHC) for F4/80 (35 and 39% reduction vs. TAK1 AH). Myofibroblast activation, as assessed by IHC for αSMA, was reduced in 1-month-old TAK1 AH/TNFR-/- and TAK1ΔH/TLR4-/- by 49 and 57%, respectively, compared with TAK1 AH. Liver fibrosis was reduced in 9-month-old TAK1 ΔH/TNFR-/- and TAK1ΔH/TLR4-/(54 and 77% reduction vs. TAK1 AH). Subsequent studies made in TAK1ΔH/TLR9-/- and TAK1 ΔH /MyD88-/- showed reductions in ALT levels (744 and 887 vs. 1608U/L in TAK1 AH), macrophage infiltration (46% and 24% reduction vs. TAK1 AH), myofibroblast activation (64 and 52% reduction vs. TAK1 AH) at 1 month of age, and hepatic fibrosis at 9 months of age (36 and 42% reduction vs. TAK1 ΔH). Compared with TAK1 AH mice, levels of related mRNAs such as Col1a, Timp1, Ccl2, and Tnfα were suppressed by additional deletions of TNFR, TLR4, TLR9, and MyD88 in 1-month-old mice. At 9 months of age, TAK1 AH/TNFR-/-, TAK1ΔH/TLR4-/-, TAK1ΔH/TLR9-/- and TAK1 ΔH/MyD88-/- mice displayed fewer and smaller tumors compared with TAK1 AH mice (by numbers, 4, 10, 11, 5 vs. 18 in TAK1 AH; by max sizes 1. 2, 3. 5, 4. 3, and 3. 1 vs 7. 1mm in TAK1 AH). Although 1-month-old TAK1ΔH/IL1 R mice had reduced macrophage infiltration and hepatic fibrosis (25 and 37% vs. TAK1 AH), tumor numbers and sizes were not reduced in comparison with TAK1 AH. Conclusions: TNFR, TLR4, TLR9, and MyD88 signaling may promote liver injury, inflammation, fibrosis, and compensatory proliferation in TAK1 AH mice. IL1R signaling may be critical for the formation of hepatic fibrosis but not for liver injury and carcinogenesis.


The following people have nothing to disclose: Jingyi Song, Sayaka Inokuchi, Yoonseok Roh, Ling Yang, Ekihiro Seki


High Mobility Group Box 1(HMGB1) Triggers Progenitor Expansion and Hepatocarcinogenesis in the Chronically Injured Liver

Peter Huebener, Pradere Jean-Philippe, Geum-Youn Gwak, Robert Schwabe

Medicine, Columbia University, New York, NY

BACKGROUND: Acute liver injury or acute loss of liver mass typically trigger regeneration by multiple rounds of hepatocyte division. In contrast, chronic liver injury not only induces regeneration from mature hepatocytes, but also promotes an expansion of progenitor cells that are capable to form hepatocytes. The mechanisms by which these progenitors sense injury and expand are not well understood. AIM: Here we seek to test the hypothesis that high mobility group box 1(HMGB1), a damage-associated molecular pattern (DAMP), signals the presence of injury to progenitor cells thereby triggering their expansion. METHODS: We floxed exons 2-4 of the HMGB1 gene and generated mice with a hepatocyte-specific HMGB1 deletion using Albumin-Cre (HMGB1 AHep) or AAV8-TBG-Cre. Chronic liver injury and liver progenitor cell expansion were induced by concomitant hepatocyte-specific deletion of the MAP3-kinase TAK1 (HMGB1 ΔHep/TAK1 ΔHep), or by feeding mice a diet containing 0.1% DDC for 3. 5 weeks. Hepatocarcinogenesis was induced by DEN-injection at day 15 p. p. followed by weekly CCl4-injections. RESULTS: Tak1 ΔHep mice exhibit spontaneous liver injury leading to fibrosis and a strong expansion of the cytokeratin (CK)-positive progenitor cell compartment at 8 weeks of age. Despite a similar extent of liver injury and fibrosis in both groups, HMGB1 ΔHep/Tak1 AHep exhibit a profound attenuation of the liver progenitor cell response as reflected by a 90% reduction in CK-positive cells (p<0.001) as well as a 57-94% reduction of progenitor cell markers AFP, H19 and CD133 (all p<0.05). Similar observations were made in the DDC diet model, with a 68% reduction of CK staining after 3. 5 weeks and >70% reduction of AFP and H19 (p<0.05) in HMGB1 ΔHep mice. Hepatocyte-specific deletion of HMGB1 by AAV8-TBG-Cre also strongly suppressed progenitor expansion after the DDC diet (p<0.001), confirming that the observed phenotype is mediated by lack of HMGB1 in hepatocytes and not in liver progenitor cells. Deletion of HMGB1 candidate receptors TLR4 and RAGE revealed a key role for RAGE, but not TLR4, in the expansion of progenitors as seen by a 70% reduction of CK-positive cells in RAGE-/- mice in the DDC model (p<0.05). Finally, HMGB1 AHep are largely protected from inflammatory hepatocarcinogenesis following DEN+CCl4 as demonstrated by a 65% reduction in tumor number and a profoundly reduced liver/bodyweight ratio in HMGB1 Δhep mice (both p<0.001). CONCLUSION: Progenitor expansion in chronic liver injury is mediated by HMGB1 and its receptor RAGE. Moreover, HMGB1 contributes to hepatocarcinogenesis in the chronically injured liver.


The following people have nothing to disclose: Peter Huebener, Pradere JeanPhilippe, Geum-Youn Gwak, Robert Schwabe


Activated β-catenin and Yap Synergize to Promote Hepatoblastoma Formation in Mice

Satdarshan (paul) S. Monga1, Lili Zhou1, Kari Nejok-Bowen1, Sarangarajan Ranganahan1, Junyon Tao2, Diego Calvisi4, Xin Chen2,3

1Pathology and Medicine, University of Pittsburgh, SOM' pittsburgh, FA; 2Bioengineering ond Therapeutic Sciences,University of California, San Francisco, CA; 3Liver Center, University of California, Son Francisco, CA; 4Pathology, University of Greifswald, Greitswald, Germany

Hepatoblastoma (HB) is the major type of pediatric malignant liver tumor. Activating mutations of p-catenin have been implicated in the majority of the HB cases. However, expression of oncogenic, mutant forms of p-catenin fails to induce HB development in mice, suggesting that additional molecular events are required to promote HB development in association with pcatenin mutation. Yap is the major oncogene downstream of Hippo kinases and has been implicated in liver tumor development. We hypothesize that activating mutations in both pcatenin and Yap pathways are necessary for development of HB. Analysis of a large collection of human HB and HCC sampies revealed that while activation of p-catenin and Yap was found to be independent in human HCC specimens, around 6180% of HB samples show concomitant activation of p-catenin and Yap in two independent datasets. Hydrodynamic transfection of either an activated form of p-catenin (AN90-p-catenin) or Yap (YapS127A) plasmids alone failed to induce liver tumor formation in mice. In contrast, co-transfection of AN90-p-catenin and YapS127A (p-catenin/Yap) led to rapid liver tumor formation as early as 3 weeks post injection and mortality around 11 weeks. Histological analysis showed that p-catenin/Yap tumors resembled embryonal HB. Tumor cells exhibited nuclear colocalization of p-catenin and Yap proteins as well as upregulation of p-catenin target genes, such as c-Myc and cyclin D1, as well as Yap target genes, including CTGF and Jag1. Similar to p-catenin knockdown, silencing of Yap also led to a decrease in TOPflash reporter activity in HepG2 HB cells. Knockdown of pcatenin also led to decrease in YAP targets such as CYR61. Thus, coordinated activation of Yap and p-catenin is sufficient for rapid HB development in the mouse and could therefore be a major mechanism of human HB pathogenesis.


Satdarshan (Paul) S. Monga - Consulting: Bristol Myers Squibb, Phase Rx, Merck

The following people have nothing to disclose: Lili Zhou, Kari Nejak-Bowen, Sarangarajan Ranganathan, Junyan īao, Diego Calvisi, Xin Chen


Novel Genetic Model of Cholangiocarcinoma in Mice

Nataliya Rozumilovo1, Daisaku Yomodo1, Steven Bronk1, Jun Li2, Jorge A. Bezerro2, Xin Chen3, Gregory J. Gores1

1Division of Gastroenterology ond Hepatology, Mayo Clinic, Rochester, MN; 2Cincinnoti Children's Hospital Medical Center Cincinnati, OH; 3Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA

Background and Aims: Cholangiocarcinoma (CCA) is a lethal neoplasm originating from the biliary epithelium. Mouse models of CCA are suboptimal limiting advances from preclinical studies. The AKT cell survival and Hippo cell growth controlling pathways are important for CCA progression. Yes-associated protein (YAP) is a main transcriptional factor of the Hippo signaling network. In humans, CCA risk factors include liver inflammation and fibrosis, and the recently identified IL-1 family member, IL-33 had been shown to promote liver inflammation and fibrosis. Our aim was to generate a novel animal model of CCA incorporating these molecular pathways. Methods: Ectopic oncogene expression in the biliary tract was accomplished by the Sleeping Beauty transposon transfection system with transduction of constitutively active AKT (myr-AKT) and YAP (YapS127A). Intrabiliary injection of the transposontransposase complex was coupled with lobar bile duct ligation in CL57/BL6 mice. After injection, animals were treated i. p. with either vehicle or IL-33 for three consecutive days. Animals were sacrificed on day 60 and examined for the presence of tumors and tumor burden. The tissue from normal liver and tumors was studied with immunohistochemistry (IHC) for markers of biliary lineage (S〇X9), cholangiocarcinoma (pancytokeratin), hepatocellular carcinoma (HepPar1), neovascularization (CD34); and phospho-AKT and YAP. Results: All animals in the group where constitutive activation of AKT and YAP in the biliary epithelium was coupled with IL-33 injection developed advanced tumors with intrahepatic metastases; in contrast, none of the animals injected with transposons without i. p. IL-33 developed cancers. Tumors exhibited neoplastic glands with loss of nuclear polarity and desmoplasia on pathologic examination. Tumors strongly expressed S〇X9 and pancytokeratin but were negative for HepPar1, a marker of hepatocellular cells. Mimicking CCA in humans, tumors demonstrated strong expression of CD34 localized to the tumor periphery. Conclusion: In mice, the transposase-mediated transduction of constitutively active AKT and YAP in the biliary epithelium leads to development of aggressive tumors expressing markers and morphological features of cholangiocarcioma. Bile duct proliferation with fibrosis induced by IL-33 is a prerequisite for oncogene-induced development of this animal model of CCA.


Jorge A. Bezerra - Grant/Research Support: Molecular Genetics Laboratory,

Gregory J. Gores - Advisory Committees or Review Panels: Bayer, Chugia, Daiichi, Generon, Conatus, IntegraGen

The following people have nothing to disclose: Nataliya Razumilava, Daisaku Yamada, Steven Bronk, Jun Li, Xin Chen


Genomics-based directions for targeted therapy in cholangiocarcinoma

Jesper B. Andersen, Valentino M. Factor, Snorri S. Thorgeirsson

NCI, Nat. Inst of Health, Bethesda, MD

Cholangiocarcinoma (CCA) is a treatment-refractory disease with heterogeneous underlying pathobiology and no current approved therapy. The incidence of CCA is rising with an estimated 400% in the next decade prompting the urgency for new clinical options. We use translational genomics to identify novel targets by integrating data obtained from the transcriptomes of 104 human CCAs with whole exome sequencing (WES) and molecular characterization. For WES, the matched tumor/normal pairs (n=15) were selected from unique subgroups with either KRAS(G12D), IDH1(R132G) or undetermined genetic status. The identified targets were validated in 13 CCA lines with known transcriptomes to test the optimal therapy for the homogeneous patient groups. The effect of inhibiting C〇X2 (by celecoxib), HER2 (by trastuzumab and lapatinib), AKT (by MK2206) and NOTCH (by y-secretase) were examined in vitro and in CCA xenografts. We showed that patients with a poor survival displayed a strong up-regulation of EGFR, HER2 and MET expression as well as a frequent and prominent deregulation of NOTCH, COX2, MAPK and AKT pathways underscroring the significance of genomicsbased medicine for selecting optimal therapy for the defined patient subgroups. しsing CCA lines in concordance with molecularly diverse CCAs, we found that lapatinib was significantly more effective than trastuzumab in suppressing tumor cell growth. A combination of celecoxib and trastuzumab suppressed C〇X2 and AKT activities and inhibited growth of TKIresistant lines in vitro (P<0.0001). In the xenograft model of TKI-resistant CCA, co-administration of celecoxib and trastuzumab for 4 weeks (P<0.004) reduced tumor growth, increased the inhibition rate (IR)=0.75 (R2=0.95, P<0.02) along with duration of treatment, and improved the overall survival (HR=6. 26, P<0.02) compared to mono-therapies or control groups. The effect of celecoxib was related to the level of AKT inhibition. Inhibiting AKT with MK2206 for three weeks decreased the tumor volume as compared to control mice (P<0.0001), with 3/8 mice demonstrating a complete block of tumor growth in response in to anti-AKT therapy. Among the patients with the worst clinical prognosis (P<0.0007, X2=1 7. 2), we identified a unique subgroup with increased NOTCH1 expression and a broad deregulation of downstream NOTCH signaling. Targeting NOTCH signaling using ۷-secretase inhibitors was effective in 13 CCA lines, indicating a potential use for ۷-secretase inhibitors to treat CCA. Our study demonstrates that targeting C〇X as a component of the underlying inflammation may sensitize CCAs to TKIs and increase efficacy of targeting AKT, MAPK or NOTCH.


The following people have nothing to disclose: Jesper B. Andersen, Valentina M. Factor, Snorri S. Thorgeirsson


SYK(L) and its splicing variant SYK(S) exert opposing roles in hepatocellular carcinoma

Jian Hong1,2, Yunfei Yuan1, Jianping Wang3, Yadi Liao1, Ruhai Zou1, Chuonlong Zhu2, Binkui Li1'Yi Liang1, pinzhu Huong1, Wenyu Lin2, Jia Le Dai4, Raymond T. Chung2

1State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Chino; 2Gastroirnestinal Unit, Massachusetts General Hospital, Harvard Medicol School, Boston, MA; 3Deporfment of Surgery, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; 4Division of Pharmacy, The University of Texas M. D. Anderson Cancer Center, Houston, TX

Background / Aims: The spleen tyrosine kinase (SYK) has been reported as a novel biomarker for human hepatocellular carcinoma (HCC). The biological contribution of the two SYK isoforms SYK(L) and SYK(S) to HCC are not well characterized. In previous investigation (J Clin Invest 2012; 122: 2165), we demonstrated that SYK(L) suppressed HCC proliferation and development. In this study, we sought to evaluate the association of expression patterns of SYK(L) and SYK(S) with prognosis in HCC, and to explore the biological effect of SYK isoforms on HCC. Methods: We used qPCR, immunoblotting and immunohistochemistry to study the expression levels of SYK(L) and SYK(S) in human HCC tissue and hepatoma cell lines. The expression levels of SYK(L) and SYK(S) in HCC specimens was compared with clinicopathologic variables and patient survival information. Matrige丨 assay and orthotopic animal models of athymic mice were used to evaluate the effect of SYK on metastasis in vitro and in vivo, respectively. Results: SYK(L) was downregulated in 38% (58/152), and SYK(S) was detectable in 40% (61/152) of the HCC samples but not in normal liver tissue samples without cirrhosis (0/30). SYK(S) was significantly correlated with pathological parameters characteristic of tumor metastasis, including multiple tumors (P=0.003), absent or incomplete tumor capsule (P=0.041) and vascular invasion (P=0.001). Expression of SYK(S) promoted tumor growth, and suppressed apoptosis of HCC cells, phenotypes that opposed those induced by SYK(L). Expression of SYK(L) significantly decreased the number of metastatic foci in lung (P=0.004) in athymic mice. In contrast, SYK(S) increased the number of lung metastases (P=0.017). In human clinical specimens (n=152), SYK(S) expression significantly correlated (P=0.002) with the reduction of E-cadherin, a hallmark of epithelial-mesenchymal transition (EMT) associated with HCC metastasis. In contrast, there was no significant association between SYK(L) and E-cadherin (P=0.869). The patients with SYK(L+/S-) tumors had a significantly longer overall survival (OS) and time to recurrence (TTR) than those with SYK(L-/S-) or SYK(L+/S+) tumors (P <0.001). Multivariate analyses showed that among all factors associated with TTR and OS, decreased SYK(L) and presence of SYK(S) expression were the two most powerful independent prognostic markers. Conclusions: Our results suggest that SYK(S) enhances HCC tumor invasion through induction of EMT. However, SYK(L) inhibits HCC metastasis. Down-regulated SYK(L) or added SYK(S) expression are strong predictors of poor survival in HCC patients, indicative of the possible need for aggressive therapeutic intervention.


Raymond ī. Chung - Advisory Committees or Review Panels: Idenix; Consulting: Enanta; Grant/Research Support: Gilead, Merck, Mass Biologic, Gilead

The following people have nothing to disclose: Jian Hong, Yunfei Yuan, Jianping Wang, Yadi Liao, Ruhai Zou, Chuanlong Zhu, Binkui Li, Yi Liang, Pinzhu Huang, Wenyu Lin, Jia Le Dai