Hepatobiliary Cells Research Articles

RNA Sequencing of Hepatobiliary Cancer Cell Lines: Data and Applications to Mutational and Transcriptomic Profiling.

Simple SummaryResearch on gallbladder cancer (GBC) has been largely neglected and molecular GBC data is underrepresented in public databases. Cancer cell lines constitute a valuable tool to examine the mechanisms of malignant transformation and identify potential therapeutic targets. Here we use RNA sequencing to characterize 23 commercial hepatobiliary cancer cell lines, including ten GBC cell lines, and provide detailed mutation and gene expression data to the research community. We illustrate the practical utility of the released information by (1) assessing the presence of specific mutations in the investigated cancer cell lines, (2) comparing global gene expression patterns in cell lines and primary biliary tumours and (3) examining the expression levels of specific genes. The released data and showcase applications will ease the design of in vitro cell culture assays for future studies.Cancer cell lines allow the identification of clinically relevant alterations and the prediction of drug response. However, sequencing data for hepatobiliary cancer cell lines in general, and particularly gallbladder cancer (GBC), are sparse. Here, we apply RNA sequencing to characterize 10 GBC, eight hepatocellular carcinoma, and five cholangiocarcinoma (CCA) cell lines. RNA extraction, quality control, library preparation, sequencing, and pre-processing of sequencing data were implemented using state-of-the-art techniques. Public data from the MSK-IMPACT database and a large cohort of Japanese biliary tract cancer patients were used to illustrate the usage of the released data. The total number of exonic mutations varied from 7207 for the cell line NOZ to 9760 for HuCCT1. Researchers planning experiments that require TP53 mutations could use the cell lines NOZ, OCUG-1, SNU308, or YoMi. Mz-Cha-1 showed mutations in ATM, SNU308 presented SMAD4 mutations, and the only investigated cell line that showed ARID1A mutations was GB-d1. SNU478 was the cell line with the global gene expression pattern most similar to GBC, intrahepatic CCA, and extrahepatic CCA. EGFR, KMT2D, and KMT2C generally presented a higher expression in the investigated cell lines than in Japanese primary GBC tumors. We provide the scientific community with detailed mutation and gene expression data, together with three showcase applications, with the aim of facilitating the design of future in vitro cell culture assays for research on hepatobiliary cancer.

Effect of Bax Gene Expression on Cell Invasion in Hepatobiliary Carcinoma Cells

There is a growing tendency of the morbidity of hilar cholangiocarcinoma in recent years. Some studies indicate that Bax gene expression was slightly increased in adjacent tissues of cholangiocarcinoma. Therefore, it is speculated that this condition may have certain correlation with the invasive ability of cholangiocarcinoma cell. This research aimed to detect the effect of Bax on cell invasive ability. The cholangiocarcinoma cell line RBE was cultured. Bax high-expression vector and Bax small interfere RNA (Bax-siRNA) were named pLBax and pLBax-siRNA, respectively. The constructed vector was transfected into the RBE cell line by using liposome transfection followed by analysis of the relationship between Bax expression and invasive ability of cholangiocarcinoma cell. The Bax gene expression in cells transfected with both vectors showed increased and decreased expression, respectively. The RBE cell apoptosis was significantly increased after high expression of Bax, while cell invasive ability was decreased with significant differences among pLBax, pLBax-siRNA and control group (p < 0.05). Bax was downregulated and RBE cell showed reversed changes with significant differences among different groups (p < 0.05). The change of the Bax expression in cholangiocarcinoma cell can affect cell invasive ability and apoptosis. Bax expression was negatively correlated with cell invasive ability.

Single cell analysis of human foetal liver captures the transcriptional profile of hepatobiliary hybrid progenitors

The liver parenchyma is composed of hepatocytes and bile duct epithelial cells (BECs). Controversy exists regarding the cellular origin of human liver parenchymal tissue generation during embryonic development, homeostasis or repair. Here we report the existence of a hepatobiliary hybrid progenitor (HHyP) population in human foetal liver using single-cell RNA sequencing. HHyPs are anatomically restricted to the ductal plate of foetal liver and maintain a transcriptional profile distinct from foetal hepatocytes, mature hepatocytes and mature BECs. In addition, molecular heterogeneity within the EpCAM+ population of freshly isolated foetal and adult human liver identifies diverse gene expression signatures of hepatic and biliary lineage potential. Finally, we FACS isolate foetal HHyPs and confirm their hybrid progenitor phenotype in vivo. Our study suggests that hepatobiliary progenitor cells previously identified in mice also exist in humans, and can be distinguished from other parenchymal populations, including mature BECs, by distinct gene expression profiles.

Single cell RNA sequencing reveals regional heterogeneity of hepatobiliary innate lymphoid cells in a tissue-enriched fashion.

IL-33 promotes type 2 immunity, epithelial repair, and tissue fibrosis by activating group 2 innate lymphoid cells (ILC2). ILC2 lack all known surface markers of mature T, B, NK, and myeloid cell lineages (Linneg), express the IL-33 receptor ST2, and release type 2 cytokines which contribute to cholangiocyte proliferation and activation of hepatic stellate cells. This pathway results in massive proliferation of the extrahepatic bile duct (EHBD) but also exacerbates liver fibrosis, suggesting that there may be tissue-specific subpopulations of IL-33-induced ILC. To determine the tissue-specific subsets of ILC in the hepatobiliary system, we analyzed CD45+Linneg mononuclear cells from IL-33 treated adult Balb/c mouse liver or EHBD by single cell RNA sequencing. Principal component analysis identified 6 major CD45+Linneg cell classes, two of which were restricted to the EHBD. One of these classes, biliary immature myeloid (BIM) cells, was predicted to interact with ILC2 by a network of shared receptor-ligand pairs. BIM highly expressed Gp49 and ST2 receptors on the cell surface while lacking surface expression of markers for mature myeloid cells. In conclusion, single cell RNA sequencing identified IL-33 responsive cell groups regionally confined to the liver or extrahepatic bile duct, including a novel population of CD45+Linneg Gp49-expressing mononuclear cells.

Human-relevant preclinical in vitro models for studying hepatobiliary development and liver diseases using induced pluripotent stem cells.

In this review, we address the potential of human-induced pluripotent stem cell-based hepatobiliary differentiation technology as a means to study human liver development and cell fate determination, and to model liver diseases in an effort to develop a new human-relevant preclinical platform for drug development.

Personalized Nutrition Therapy in Hashimoto's Thyroiditis and Herpes Zoster Oticus, Recalcitrant to Conventional Therapy: A Case Report

Personalized nutrition therapy (PNT) has been developing in terms of functional medicine. Its efficacy has been controversial. In the scope of functional medicine, the diagnostic methods to analyze blood are generally based on two kinds of perspectives; biochemistry, blood chemistry and CBC with differential counts. Out of two methods, I devised the latter of my own with imbibing the result of my Pulse Pattern Diagnosis (PPD). The PPD established by me can measure the metabolic, inflammatory, and/or hemodynamic states of major organs, using the radial artery in the wrist of each person. For example, when hepatobiliary tract cells are metabolically activated, and/or the liver is hyper-perfused, my PPD indicates correlation with the increased value of uric acid in blood analysis. The effect of vitamin B5 on liver decreases the metabolic rate and blood flow into the liver in terms of my PPD. It has been reported that vitamin B5 is important in breaking down the excess of uric acid in our body. Likewise, my blood analytic algorithm for PNT is created. I have four clinical records of two kinds of disease entities; herpes zoster oticus with acute otitis media (1 case), thyroid Hashimoto’s thyroiditis (3 cases). These cases were not recalcitrant to conventional therapy but improved after PNT. In summary, PNT may be an alternative to the invincible infectious and autoimmune diseases recalcitrant to conventional therapy.

Influence of Progenitor‐Derived Regeneration Markers on Hepatitis C Virus–Related Cirrhosis Outcome (ANRS CO12 CirVir Cohort)

Progenitor-derived regeneration depicted by K7 and EpCAM immunostaining of hepatocytes in liver biopsies of patients with compensated HCV-related cirrhosis marks a cirrhosis stage more prone to develop complications. (HEPATOLOGY 2018; 68:1534-1548).

Biological and Clinical Aspects of an Olive Oil-Based Lipid Emulsion-A Review.

Intravenous lipid emulsions (ILEs) have been an integral component of parenteral nutrition for more than 50 years. Numerous formulations are available and are based on vegetable (soybean, olive, coconut) and animal (fish) oils. Therefore, each of these formulations has a unique fatty acid composition that offers both benefits and limitations. As clinical experience and our understanding of the effects of fatty acids on various physiological processes has grown, there is evidence to suggest that some ILEs may have benefits compared with others. Current evidence suggests that olive oil-based ILE may preserve immune, hepatobiliary, and endothelial cell function, and may reduce lipid peroxidation and plasma lipid levels. There is good evidence from a large randomized controlled study to support a benefit of olive oil-based ILE over soybean oil-based ILE on reducing infections in critically ill patients. At present there is limited evidence to demonstrate a benefit of olive oil-based ILE over other ILEs on glucose metabolism, and few data exist to demonstrate a benefit on clinical outcomes such as hospital or intensive care unit stay, duration of mechanical ventilation, or mortality. We review the current research and clinical evidence supporting the potential positive biological and clinical aspects of olive oil-based ILE and conclude that olive oil-based ILE is well tolerated and provides effective nutritional support to various PN-requiring patient populations. Olive oil-based ILE appears to support the innate immune system, is associated with fewer infections, induces less lipid peroxidation, and is not associated with increased hepatobiliary or lipid disturbances. These data would suggest that olive oil-based ILE is a valuable option in various PN-requiring patient populations.

Combination of Kras activation and PTEN deletion contributes to murine hepatopancreatic ductal malignancy

Kras mutations are among the most common genetic abnormalities in human neoplasms, including cholangiocarcinomas, pancreatic cancer and colon cancer. PTEN has previously been associated with cholangiocarcinoma development in murine models. Here, we have established novel mouse models of neoplasms by liver-specific and biliary-pancreatic Kras activation and PTEN deletion. By liver-specific disruption of PTEN and activation of Kras in mice caused rapid development of intrahepatic biliary epithelial proliferative lesions (Intrahepatic cholangiocarcinoma, ICC), which progress through dysplasia to invasive carcinoma. In contrast, Kras activation in combination with heterozygous PTEN deletion induced mixed carcinomas of liver (both ICC and hepatocellular carcinoma, HCC), whereas Kras activation alone did not induce biliary tract neoplasm. Use of Sox9-Cre-LoxP-based approach to coordinately delete PTEN and activate Kras in the adult mouse resulted in not only development of low-grade biliary lesions (ICC and extrahepatic bile duct carcinoma, ECC) but also pancreatic carcinomas. Our data provide a functional link between PTEN gene status, hepatobiliary cell fate, and HCC, biliary carcinoma, pancreatic cancer pathogenesis, and present novel genetically engineered mouse models of PTEN loss-driven malignancy.

FRI-413 - Human hepatobiliary progenitor cells as new candidates for liver cell therapy

FRI-413 - Human hepatobiliary progenitor cells as new candidates for liver cell therapy

Acute opisthorchiasis: What about the clinical manifestation?

Opisthorchiasis or liver fluke infestation is a common parasitic infestation in tropical countries. In chronic infection, the malignant transformation of hepatobiliary cell can be seen. Its high prevalence in Indochina is relating to the high incidence of cholangiocarcinoma in that area. Nevertheless, the acute clinical problem due to opisthorchiasis is also existed but little mentioned. In this short article, the authors review and discuss on the clinical manifestation of acute opisthorchiasis.

Inactivation of p120 catenin in mice disturbs intrahepatic bile duct development and aggravates liver carcinogenesis

p120 catenin (p120ctn) is required for the stability of classic cadherins at the cell surface and is thought to play a central role in modulating cell–cell adhesion. Cytoplasmic p120ctn promotes cell motility, and probably other activities, by modulating the activities of RhoA, Rac and Cdc42. E-cadherin is expressed in periportal but not in perivenous hepatocytes. In contrast, all hepatocytes of normal mouse liver express N-cadherin. Cholangiocytes express exclusively E-cadherin. Mice with p120ctn ablation in hepatocytes and cholangiocytes (p120LiKO mice) were generated by Cre-loxP technology. Livers were examined by histological, immunohistochemical, ultrastructural and serum analysis to determine the effect of the p120ctn ablation on liver structure and function. Mouse hepatocyte differentiation and homeostasis were not impaired. However, hepatoblasts differentiated abnormally into hybrid hepato-biliary cells, ductal plate structures were irregular in p120LiKO newborns, and further development of intrahepatic bile ducts was severely impaired. In adults, enrichment of ductular structures was accompanied by portal inflammation and fibrosis. p120LiKO mice did not spontaneously develop hepatocellular carcinoma but initiation of hepatocarcinogenesis by diethylnitrosamine was accelerated. In summary: p120ctn has a critical role in biliary differentiation and is a potent suppressor of liver tumor growth.

Select Effects of IL-13 in Driving Tissue Repair and Fibrosis

Fibrosis is a common complication of solid organ transplantation, but whether it is due to maladaptive cell proliferation for tissue repair, direct fibroblast activation or a combination of both is not clear. Interleukin-13 (IL-13) has been associated with bronchiolitis obliterans syndrome (BOS) following lung transplantation in the clinic, and its inactivation in a mouse model of BOS was found to reduce fibro-obliteration (Keane et al.). Similarly, IL-13 has been linked with fibrosis of various organs, including the asthmatic lung, the skin in atopic dermatitis and the colon in ulcerative colitis, with clinical trials ongoing to test the therapeutic efficacy of targeting IL-13 in these disorders. In a recent issue of Immunity, Gieseck et al. use mouse models of progressive liver disease to dissect the precise effect of IL-13 on various hepatic cell subsets. Their results have implications for chronic transplant rejection. Fibroproliferative disease of the liver, which occurs following chronic viral or parasitic infections, or chronic rejection of hepatic allografts can include aberrant proliferation of hepatobiliary progenitor cells and bile duct epithelial cells (cholangiocytes) as well as a recruitment of inflammatory cells to the biliary ducts (ductular reaction [DR]), progressive development of ductal strictures, hepatic steatosis and fibrosis. The authors track these individual features in mouse models of schistosoma infection and chronic liver overexpression of IL-13 (following hydrodynamic injection of an IL-13 plasmid to transfected liver cells). Quite strikingly, abrogating expression of the IL-4Rα (a chain necessary for IL-4 and IL-13 receptor signaling) selectively in stellate cells (the liver-resident fibroblasts) using Il-4raflox/floxPdgfrbWT/cre mice, completely prevented development of fibrosis in both the chronic infection and sterile models. In contrast, steatosis and DR were unaffected. Conversely, eliminating IL-13 signaling only in hepatocytes and/or biliary cells using Il4raflox/floxAlbWT/cre mice reduced steatosis and DR without affecting fibrosis. The authors further ascribed these effects to IL-13–dependent rather than IL-4–dependent signals. The authors also found that biliary strictures observed in the IL-13 overexpression model were not due to fibrosis but likely caused by excessive ductal proliferation. The initial source of IL-13 in this disease is not certain, but both type 2 innate lymphoid cells (ILC2) and Th2 cells can make IL-13, and the authors demonstrate that IL-13 signaling in fibroblasts and hepatocytes promotes recruitment of eosinophils that in turn may further IL-13 production and pathological progression after initial injury. This paper clearly demonstrates the direct and distinct effect of IL-13 in different cell types and separates the processes of liver regeneration and liver fibrosis. The former, driven by hepatocytes and biliary cell response to IL-13, is desirable to repair liver injuries, but if triggered excessively, can lead to biliary stenosis and occlusions which themselves can lead to cholestatic steatosis. Gieseck and colleagues postulate that a reduction in bile output to the intestine results in malabsorption of fat from the diet, which may trigger a lipogenic program in hepatocytes to compensate for this reduction in dietary fat. In contrast, fibrosis is driven by the direct response from stellate cells to IL-13 and appears completely independent from DR and its accompanying inflammatory cells. Combined elimination of IL-13 signaling in stellate cells, hepatocytes and cholangiocytes may improve all features of liver fibroproliferative disease, although some level of tissue proliferation is likely important in response to acute injury. Another publication by the same group suggests that combined inhibition of IL-13 and interferon-γ (IFN-γ) can reduce not only the fibrotic but also the inflammatory features of shistosoma-induced liver disease (Ramalingam et al.). It is tempting to speculate that a similar approach may be beneficial to prevent chronic transplant rejection. In fact, extensive bile duct epithelial damage can occur during the ischemic period of organ harvest, which may trigger excessive tissue repair and fibrosis. Thus, IL-13 expression levels have the potential to be both a predictive biomarker of chronic rejection as well as a therapeutic target (perhaps in concert with IFN-γ) to limit liver remodeling and progressive dysfunction following transplantation. Dr. Alegre is a professor in the Department of Medicine at the University of Chicago.

Keratin 23 is a stress-inducible marker of mouse and human ductular reaction in liver disease

Keratins (K) constitute the epithelial intermediate filaments. Among them, K7/K19 are widely used markers of the regenerative liver response termed ductular reaction (DR) that consists of activated biliary epithelial cells (BECs) and hepatic progenitor cells (HPCs) and correlates with liver disease severity. In the present study we aimed to characterize K23 in the liver. We analyzed the expression and localization of K23 in the digestive system under basal conditions as well as in various human and mouse liver diseases/stress models. Cell culture studies were used to study factors regulating K23 expression. In untreated mice, K23 was restricted to biliary epithelia. It was (together with K7/K19) markedly upregulated in three different DR/cholestatic injury models, i.e., multidrug resistance protein 2 (Mdr2) knockouts, animals treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine or subjected to bile duct ligation. K23 levels correlated with the DR marker Fn14 and immunofluorescence staining showed a distinct co-localization with K7/K19. In chronic human liver disease, K23 expression increased in patients with a more prominent inflammation/fibrosis. A dramatic upregulation (>200times) was observed in patients with acute liver failure (ALF) and end-stage primary biliary cholangitis (PBC). Patients with alcoholic liver cirrhosis displayed increased K23 serum levels. In primary hepatocytes as well as hepatobiliary cell lines, treatment with TNF-related weak inducer of apoptosis (TWEAK), and the type I acute phase inducer interleukin (IL)-1β but not the type II inducer IL-6 elevated K23 expression. K23 represents a specific, stress-inducible DR marker, whose levels correlate with liver disease severity. K23 may represent a useful non-invasive DR marker. Ductular reaction represents a basic response to liver injury and correlates with liver disease severity. Our study identifies K23 as a novel ductular reaction marker in mice and humans.

Mitogen-activated protein kinase-activated protein kinase 2 mediates resistance to hydrogen peroxide-induced oxidative stress in human hepatobiliary cancer cells

The development and progression of liver cancer are characterized by increased levels of reactive oxygen species (ROS). ROS-induced oxidative stress impairs cell proliferation and ultimately leads to cell death. Although liver cancer cells are especially resistant to oxidative stress, mechanisms of such resistance remain understudied. We identified the MAPK-activated protein kinase 2 (MK2)/heat shock protein 27 (Hsp27) signaling pathway mediating defenses against oxidative stress. In addition to MK2 and Hsp27 overexpression in primary liver tumors compared to adjacent nontumorous tissues, the MK2/Hsp27 pathway is activated by hydrogen peroxide-induced oxidative stress in hepatobiliary cancer cells. MK2 inactivation or inhibition of MK2 or Hsp27 expression increases caspase-3 and PARP cleavage and DNA breaks and therefore cell death. Interestingly, MK2/Hsp27 inhibition decreases antioxidant defenses such as heme oxygenase 1 through downregulation of the transcription factor nuclear factor erythroid-derived 2-like 2. Moreover, MK2/Hsp27 inhibition decreases both phosphorylation of epidermal growth factor receptor (EGFR) and expression of its ligand, heparin-binding EGF-like growth factor. A new identified partner of MK2, the scaffold PDZ protein EBP50, could facilitate these effects through MK2/Hsp27 pathway regulation. These findings demonstrate that the MK2/Hsp27 pathway actively participates in resistance to oxidative stress and may contribute to liver cancer progression.

Portal inflammation during NAFLD is frequent and associated with the early phases of putative hepatic progenitor cell activation

AimsWe investigated whether portal tract inflammation observed in non-alcoholic fatty liver disease (NAFLD) is associated with hepatic progenitor cell compartment activation, as thoroughly evaluated with different markers of the staminal...

Piperine prevents cholesterol gallstones formation in mice

Biliary cholesterol may contribute to the formation of cholesterol gallstones, and regulation of these levels could be a useful therapeutic strategy for gallstones disease. Piperine (PA) is a potential cholesterol lowering agent. In this study, we assessed the effect and mechanism of PA in preventing cholesterol gallstones formation induced by feeding lithogenic diet containing high cholesterol levels to mice. C57BL/6 inbred mice were fed lithogenic or chow diets for 10 weeks, with or without PA (15, 30 and 60mg/kg) or ursodeoxycholic acid (UDCA, 60mg/kg) administration. Cholesterol, phospholipids and crystals in bile, the lipid in serum, pathological changes and proteins expression in liver were analyzed. The results showed that PA could decrease the cholesterol potency and crystals in bile, reduce total cholesterol (TC), triglycerides (TG) and increase high-density lipoprotein/low-density lipoprotein (HDL/LDL) levels in serum. Furthermore, PA treatment reduced liver lipid peroxidation and protected hepatobiliary cells from liver injury by decreasing malondialdehyde (MDA) and increasing superoxide dismutase (SOD). In addition, PA inhibited the expression of ATP-binding cassette transporters G5/8 (ABCG5/8) and liver X receptor (LXR) in liver, and reduced cholesterol transport from the hepatocytes to the gallbladder. It may be the mechanism of PA in preventing cholesterol gallstones formation. PA as a potential drug for prevention cholesterol gallstones merits further investigation.

Hepato-biliary stem cells: facts and fancies

The canals of Hering and stem cells At the close of the 20 century, a decades long questions was finally settled: both rodent and human livers had facultative hepato-biliary stem cells. The stem cell niche in which these cells’ activities were identified was the canal of Hering (CoH) [1] – the link between hepatocyte canaliculi and the biliary tree. This structure had been functionally demonstrated by Ewald Hering in 1857, but its anatomic structure was merely inferred, but not visualized. Hering’s suggested that the link was located at the limiting plate, where hepatic parenchyma encountered portal tract stroma. His initial drawings depicted that hypothesis. The first direct imagings of the CoH were via electron microscopy, which confirmed that the CoH, as Hering intuited, was comprised of hepatocytes on one side and the smallest cholangiocytes on the other, but these high powered, ultrastructural views could not really explore the canal’s tissue level anatomic structure. Immunohistochemistry (for biliary keratins) and serial sectioning to create a three dimensional analysis of CoH in human livers solved the question [2]. Human CoH were not as small as thought and not restricted to the limiting plate; rather, they were delicate structures that extended across the limiting plate, into the periportal parenchymal regions, as much as a third of the way toward the central vein (but no further). It was from these structures that new hepatocytes were seen to be arising in the setting of severe acute injury (e.g. acetaminophen toxicity) [2] and in chronic liver diseases of all kinds (reviewed in [3]). These findings led to significant paradigm shifts. For hepato-biliary regeneration, settling of the argument as to whether there actually were liver stem cells potentiated explosive creativity in exploring their physiologic behavior and theraperutic potential. For pathology, the ubiquitous, but poorly understood “ductular reactions” (DR) – seen in all severe acute injuries and in all advanced stage chronic liver disease – were now understood to be tissue activations of the hepatobiliary stem cell niche [3].

Cellular Inhibitor of Apoptosis (cIAP)-Mediated Ubiquitination of Phosphofurin Acidic Cluster Sorting Protein 2 (PACS-2) Negatively Regulates Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Cytotoxicity

Lysosomal membrane permeabilization is an essential step in TRAIL-induced apoptosis of liver cancer cell lines. TRAIL-induced lysosomal membrane permeabilization is mediated by the multifunctional sorting protein PACS-2 and repressed by the E3 ligases cIAP-1 and cIAP-2. Despite the opposing roles for PACS-2 and cIAPs in TRAIL-induced apoptosis, an interaction between these proteins has yet to be examined. Herein, we report that cIAP-1 and cIAP-2 confer TRAIL resistance to hepatobiliary cancer cell lines by reducing PACS-2 levels. Under basal conditions, PACS-2 underwent K48-linked poly-ubiquitination, resulting in PACS-2 proteasomal degradation. Biochemical assays showed cIAP-1 and cIAP-2 interacted with PACS-2 in vitro and co-immunoprecipitation studies demonstrated that the two cIAPs bound PACS-2 in vivo. More importantly, both cIAP-1 and cIAP-2 directly mediated PACS-2 ubiquitination in a cell-free assay. Single c-Iap-1 or c-Iap-2 gene knock-outs in mouse hepatocytes did not lead to PACS-2 accumulation. However, deletion of both cIAP-1 and cIAP-2 reduced PACS-2 ubiquitination, which increased PACS-2 levels and sensitized HuH-7 cells to TRAIL-induced lysosomal membrane permeabilization and apoptosis. Correspondingly, deletion of cIAPs sensitized wild-type, but not PACS-2-deficient hepatocarcinoma cells or Pacs-2−/− mouse hepatocytes to TRAIL-induced apoptosis. Together, these data suggest cIAPs constitutively downregulate PACS-2 by polyubiquitination and proteasomal degradation, thereby restraining TRAIL-induced killing of liver cancer cells.

Perinodular ductular reaction/epithelial cell adhesion molecule loss in small hepatic nodules.

To investigate if loss of epithelial cell adhesion molecule (EpCAM) is associated with microinvasion in hepatocellular carcinomas (HCCs) in the presence of chronic hepatitis B. The expression of EpCAM, cytokeratin 7 (CK7) and CK19 in 112 hepatic nodules was studied, including 20 HCCs with nodules ≤ 3 cm, 26 HCCs with nodules > 3 cm, 20 high-grade dysplastic nodules, 26 cirrhotic, large regenerative nodules and 20 cases of cirrhosis. Membranes of ductular reaction (DR) hepatobiliary cells, interlobular bile duct and some hepatic cells were positive for EpCAM expression. Active expression of DR/EpCAM was observed in the majority of noninvasive nodules (50/66, 75.76%); however, expression was absent in the major area of invasion in HCCs (42/46, 91.30%). DR/EpCAM loss in HCCs ≤ 3 cm was higher than in high-grade dysplastic nodules (HGDNs) (P < 0.05), cirrhotic, large regenerative nodules and cirrhosis (P < 0.01). Furthermore, patients (20 HCCs ≤ 3 cm, 26 HCCs > 3 cm, 20 HGDNs) with DR/EpCAM expression had a higher overall survival rate (P < 0.01) and lower early recurrence rate (P < 0.01). DR/EpCAM expression showed a close relationship with DR/CK7 and DR/CK19 expression (P < 0.01). The area under the receiver operating characteristic (ROC) curve of DR/EpCAM was similar to that of DR/CK7 and DR/CK19 (P > 0.05). The diagnostic specificity and diagnostic accuracy were both increased when DR/EpCAM, DR/CK7 and DR/CK19 were combined (P < 0.01). DR/EpCAM loss may be a useful marker for determining microinvasion in HCCs ≤ 3 cm, but also for predicting prognosis.