Rachel Guest

Hepatic progenitor cells of biliary origin with liver repopulation capacity

Hepatocytes and cholangiocytes self-renew following liver injury. Following severe injury hepatocytes are increasingly senescent, but whether hepatic progenitor cells (HPCs) then contribute to liver regeneration is unclear. Here, we describe a mouse model where the E3 ubiquitin ligase Mdm2 is inducibly deleted in more than 98% of hepatocytes, causing apoptosis, necrosis and senescence with nearly all hepatocytes expressing p21. This results in florid HPC activation, which is necessary for survival, followed by complete, functional liver reconstitution. HPCs isolated from genetically normal mice, using cell surface markers, were highly expandable and phenotypically stable in vitro. These HPCs were transplanted into adult mouse livers where hepatocyte Mdm2 was repeatedly deleted, creating a non-competitive repopulation assay. Transplanted HPCs contributed significantly to restoration of liver parenchyma, regenerating hepatocytes and biliary epithelia, highlighting their in vivo lineage potency. HPCs are therefore a potential future alternative to hepatocyte or liver transplantation for liver disease.

WNT signaling drives cholangiocarcinoma growth and can be pharmacologically inhibited

Cholangiocarcinoma (CC) is typically diagnosed at an advanced stage and is refractory to surgical intervention and chemotherapy. Despite a global increase in the incidence of CC, little progress has been made toward the development of treatments for this cancer. Here we utilized human tissue; CC cell xenografts; a p53-deficient transgenic mouse model; and a non-transgenic, chemically induced rat model of CC that accurately reflects both the inflammatory and regenerative background associated with human CC pathology. Using these systems, we determined that the WNT pathway is highly activated in CCs and that inflammatory macrophages are required to establish this WNT-high state in vivo. Moreover, depletion of macrophages or inhibition of WNT signaling with one of two small molecule WNT inhibitors in mouse and rat CC models markedly reduced CC proliferation and increased apoptosis, resulting in tumor regression. Together, these results demonstrate that enhanced WNT signaling is a characteristic of CC and suggest that targeting WNT signaling pathways has potential as a therapeutic strategy for CC.

Cell Lineage Tracing Reveals a Biliary Origin of Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma is a treatment refractory malignancy with a high mortality and an increasing incidence worldwide. Recent studies have observed that activation of Notch and AKT signaling within mature hepatocytes is able to induce the formation of tumors displaying biliary lineage markers, thereby raising the suggestion that it is hepatocytes, rather than cholangiocytes or hepatic progenitor cells that represent the cell of origin of this tumor. Here, we use a cholangiocyte-lineage tracing system to target p53 loss to biliary epithelia and observe the appearance of labeled biliary lineage tumors in response to chronic injury. Consequent to this, upregulation of native functional Notch signaling is observed to occur spontaneously within cholangiocytes and hepatocytes in this model as well as in human intrahepatic cholangiocarcinoma. These data prove that in the context of chronic inflammation and p53 loss, frequent occurrences in human disease, biliary epithelia are a target of transformation and an origin of intrahepatic cholangiocarcinoma.

Polysialic acid/neural cell adhesion molecule modulates the formation of ductular reactions in liver injury

In severe liver injury, ductular reactions (DRs) containing bipotential hepatic progenitor cells (HPCs) branch from the portal tract. Neural cell adhesion molecule (NCAM) marks bile ducts and DRs, but not mature hepatocytes. NCAM mediates interactions between cells and surrounding matrix; however, its role in liver development and regeneration is undefined. Polysialic acid (polySia), a unique posttranslational modifier of NCAM, is produced by the enzymes, ST8SiaII and ST8SiaIV, and weakens NCAM interactions. The role of polySia with NCAM synthesizing enzymes ST8SiaII and ST8SiaIV were examined in HPCs in vivo using the choline‐deficient ethionine‐supplemented and 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine diet models of liver injury and regeneration, in vitro using models of proliferation, differentiation, and migration, and by use of mouse models with gene defects in the polysialyltransferases (St8sia 2+/−4+/−, and St8sia2−/−4−/−). We show that, during liver development, polySia is required for the correct formation of bile ducts because gene defects in both the polysialyltransferases (St8sia2+/−4+/− and St8sia2−/−4−/− mice) caused abnormal bile duct development. In normal liver, there is minimal polySia production and few ductular NCAM+ cells. Subsequent to injury, NCAM+ cells expand and polySia is produced by DRs/HPCs through ST8SiaIV. PolySia weakens cell‐cell and cell‐matrix interactions, facilitating HGF‐induced migration. Differentiation of HPCs to hepatocytes in vitro results in both transcriptional down‐regulation of polySia and cleavage of polySia‐NCAM. Cleavage of polySia by endosialidase (endoN) during liver regeneration reduces migration of DRs into parenchyma. Conclusion: PolySia modification of NCAM+ ductules weakens cell‐cell and cell‐matrix interactions, allowing DRs/HPCs to migrate for normal development and regeneration. Modulation of polySia levels may provide a therapeutic option in liver regeneration. (Hepatology 2014;60:1727–1740)

Wnt signalling modulates transcribed-ultraconserved regions in hepatobiliary cancers

Objective Transcribed-ultraconserved regions (T-UCR) are long non-coding RNAs which are conserved across species and are involved in carcinogenesis. We studied T-UCRs downstream of the Wnt/β-catenin pathway in liver cancer. Design Hypomorphic Apc mice (Apcfl/fl) and thiocetamide (TAA)-treated rats developed Wnt/β-catenin dependent hepatocarcinoma (HCC) and cholangiocarcinoma (CCA), respectively. T-UCR expression was assessed by microarray, real-time PCR and in situ hybridisation. Results Overexpression of the T-UCR uc.158− could differentiate Wnt/β-catenin dependent HCC from normal liver and from β-catenin negative diethylnitrosamine (DEN)-induced HCC. uc.158− was overexpressed in human HepG2 versus Huh7 cells in line with activation of the Wnt pathway. In vitro modulation of β-catenin altered uc.158− expression in human malignant hepatocytes. uc.158− expression was increased in CTNNB1-mutated human HCCs compared with non-mutated human HCCs, and in human HCC with nuclear localisation of β-catenin. uc.158− was increased in TAA rat CCA and reduced after treatment with Wnt/β-catenin inhibitors. uc.158− expression was negative in human normal liver and biliary epithelia, while it was increased in human CCA in two different cohorts. Locked nucleic acid-mediated inhibition of uc.158− reduced anchorage cell growth, 3D-spheroid formation and spheroid-based cell migration, and increased apoptosis in HepG2 and SW1 cells. miR-193b was predicted to have binding sites within the uc.158− sequence. Modulation of uc.158− changed miR-193b expression in human malignant hepatocytes. Co-transfection of uc.158− inhibitor and anti-miR-193b rescued the effect of uc.158− inhibition on cell viability. Conclusions We showed that uc.158− is activated by the Wnt pathway in liver cancers and drives their growth. Thus, it may represent a promising target for the development of novel therapeutics.

Notch3 drives development and progression of cholangiocarcinoma

Significance Clinical outcomes in cholangiocarcinoma (CC) are poor; few patients are candidates for curative resection, and palliative chemotherapy produces only modest effects on survival. With an increasing incidence, new targets are urgently needed. Notch has been identified as having potential to induce CC when transgenically overexpressed, and this study aimed to characterize how endogenous Notch might drive tumorigenesis. We identify the atypical receptor Notch3 as differentially overactivated in CCs in humans, rats, and mice, with genetic deletion significantly reducing CC growth. Notch3 sustains tumor cell survival through PI3k/Akt activation via a noncanonical mechanism independent of Recombinant Signal Binding Protein for Immunoglobulin Kappa J Region (RBPJ), presenting an opportunity to target the pathway without disrupting classical Notch and bypassing toxicities associated with γ-secretase inhibitors. The prognosis of cholangiocarcinoma (CC) is dismal. Notch has been identified as a potential driver; forced exogenous overexpression of Notch1 in hepatocytes results in the formation of biliary tumors. In human disease, however, it is unknown which components of the endogenously signaling pathway are required for tumorigenesis, how these orchestrate cancer, and how they can be targeted for therapy. Here we characterize Notch in human-resected CC, a toxin-driven model in rats, and a transgenic mouse model in which p53 deletion is targeted to biliary epithelia and CC induced using the hepatocarcinogen thioacetamide. We find that across species, the atypical receptor NOTCH3 is differentially overexpressed; it is progressively up-regulated with disease development and promotes tumor cell survival via activation of PI3k-Akt. We use genetic KO studies to show that tumor growth significantly attenuates after Notch3 deletion and demonstrate signaling occurs via a noncanonical pathway independent of the mediator of classical Notch, Recombinant Signal Binding Protein for Immunoglobulin Kappa J Region (RBPJ). These data present an opportunity in this aggressive cancer to selectively target Notch, bypassing toxicities known to be RBPJ dependent.

Understanding liver regeneration to bring new insights to the mechanisms driving cholangiocarcinoma

Cancer frequently arises in epithelial tissues subjected to repeated cycles of injury and repair. Improving our understanding of tissue regeneration is, therefore, likely to reveal novel processes with inherent potential for aberration that can lead to carcinoma. These highly conserved regenerative mechanisms are increasingly understood and in the liver are associated with special characteristics that underlie the organ’s legendary capacity for restoration of size and function following even severe or chronic injury. The nature of the injury can determine the cellular source of epithelial regeneration and the signalling mechanisms brought to play. These observations are shaping how we understand and experimentally investigate primary liver cancer, in particular cholangiocarcinoma; a highly invasive malignancy of the bile ducts, resistant to chemotherapy and whose pathogenesis has hitherto been poorly understood. Interestingly, signals that drive liver development become activated in the formation of cholangiocarcinoma, such as Notch and Wnt and may be potential future therapeutic targets. In this review, we summarise the work which has led to the current understanding of the cellular source of cholangiocarcinoma, how the tumour recruits, sustains and is educated by its supporting stromal environment, and the tumour-derived signals that drive the progression and invasion of the cancer. With few current treatments of any true efficacy, advances that will improve our understanding of the mechanisms driving this aggressive malignancy are welcome and may help drive therapeutic developments.

P0282 : The long non coding RNA UC.158 modulates growth of Wnt/β;-catenin driven hepatocellular carcinoma (HCC)

model would use human liver, or a biologically relevant 3D in vitro model of human liver cells grown on an extracellular matrix (ECM) derived from healthy liver tissue. The aim of this study was to develop a rapid protocol for the decellularisation of small samples of human liver and demonstrate repopulation with cultured human liver cell lines, namely hepatocarcinoma (HepG2), metastatic adenocarcinoma (SKHep-1) and hepatic stellate (LX2) cells. Methods: Liver tissue cubes (5×5×5mm, i.e. 0.125 cm) were dissected from human livers unsuitable for transplantation. The decellularisation of the human liver cubes was completed within 3 hours of incubation and agitation in our decellularization medium (1. deionized water, 2. detergents and 3. enzymes [trypsin]), and following optimization of pre-existing protocols for animal tissues. The decellularization efficiency was determined by immunohistochemistry for ECM components and residual DNA, scanning electron microscopy, as well as DNA and ECM protein quantification. The liver cube scaffolds were seeded and repopulated by HepG2, SK-Hep-1 and LX2 for up to 21 days.

The functional role of Notch3 in intrahepatic cholangiocarcinoma

Abstract Background Clinical outcomes from intrahepatic cholangiocarcinoma (ICC) continue to be dismal in the face of an increasing worldwide incidence. Notch signalling is crucial for faithful embryological development of the biliary tree and is upregulated during biliary regeneration after injury in the adult liver. We hypothesised that Notch could directly drive tumorigenesis in ICC, a malignancy frequently arising on a background of chronic inflammation and cycles of epithelial regeneration. As such, Notch inhibition might represent a novel therapeutic target in this treatment-refractory disease. Methods In this preclinical study, we used human cholangiocarcinoma specimens, human cell lines, and rodent models to profile Notch pathway expression in ICC across three species with PCR array, real-time quantitative PCR, western blotting, and immunohistochemistry. We used γ-secretase inhibitors and RNA interference to investigate the phenotypic and molecular effects of pan-Notch inhibition in vitro and in a chemically induced in-situ model in the rat. Short hairpin RNA species stably inhibited specific Notch receptors in vitro and in xenografted human cell lines with proto-oncogene PCR arrays used to identify signalling pathways through which Notch functions. We developed a novel transgenic murine model of ICC with Cre-loxP technology to target loss of the tumour suppressor p53 to the biliary epithelia (cytokeratin 19-positive cells) on a background of chronic inflammation using the chemical carcinogen thioacetamide. Findings The Notch pathway was globally upregulated in ICC in human, rat, and mouse specimens. In particular, there was activation of the Notch3 receptor paralog, which was expressed in clusters of malignant ductal epithelia as well as in the desmoplastic stroma. A thioacetamide model of rat ICC was used to observe a progressive upregulation of the pathway during disease development—in particular the ligand Jagged1 and the Notch3 receptor. Pan-Notch inhibition with the γ-secretase inhibitor DAPT resulted in an attenuation of tumour growth in vitro and in vivo. These effects were recapitulated using specific inhibition of the Notch3 receptor using the small molecule inhibitor Trichostatin A as well as RNA interference. Epithelial inhibition of Notch3 was associated with a reduction in PI3K/AKT activity and associated cell survival. Interpretation The Notch3 receptor represents a specific driver of tumorigenesis in ICC, and acts independently of the effector of the canonical Notch pathway RPBJk to stimulate PI3k/AKT-mediated cell survival. Specific Notch3 ablation results in attenuation of malignancy both in vitro and in vivo. Further studies are needed to assess whether Notch3 inhibition could be an adjuvant treatment to existing and currently ineffective chemotherapeutic agents. In view of the known effects of global Notch inhibition to impeding biliary regeneration, targeting Notch3 might be an exciting therapeutic opportunity for patients with disease potentially amenable for surgical resection. Funding Wellcome Trust, Cancer Research UK.

P64 THE ULTRACONSERVED NON CODING RNA UC.158 IS DOWNSTREAM OF THE WNT/b-CATENIN PATHWAY IN LIVER CANCERS

P64 THE ULTRACONSERVED NON CODING RNA UC.158 IS DOWNSTREAM OF THE WNT/b-CATENIN PATHWAY IN LIVER CANCERS V. Paulus-Hock, A. Lampis, G. Ferrari, L. Boulton, R. Guest, D. Athineos, T. Jamieson, A. Veronese, R. Visone, R. Evans, G.J. Feng, T. Dale, M. Negrini, S. Forbes, T. Patel, O. Sansom, N. Valeri, C. Braconi. Institute of Cancer Sciences, University of Glasgow, Glasgow, Division of Molecular Pathology, Institute of Cancer Research, London, Scottish Centre for Regenerative Medicine, MRC Centre for Regenerative Medicine, Edinburgh, Beatson Institute for Cancer Research, Glasgow, United Kingdom; Aging Research Center, University of Chieti, Chieti, Italy; School of Biosciences, University of Cardiff, Cardiff, United Kingdom; University of Ferrara, Ferrara, Italy; Mayo Clinc, Jacksonville, FL, United States; Division of Cancer Therapeutics, Institute of Cancer Research, London, United Kingdom E-mail: chiarabraconi@gmail.com