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Zinc finger E-box binding homeobox 1 (ZEB1) is a transcription factor that promotes metastatic and stem cell features, which has been associated with poor prognosis in cholangiocarcinoma (CCA), a desmoplastic cancer enriched in cancer-associated fibroblasts (CAFs). We aimed to define ZEB1 regulatory functions in malignant and stromal compartments of CCA.Bioinformatic and immunohistochemical analyses were performed to determine correlations between ZEB1 and markers of progressiveness in human intrahepatic CCA (iCCA). Gain-of-function and loss-of-function models were generated in CCA cells and liver myofibroblasts as a model of CAFs. Conditioned media (CM) was used to unravel tumor-stroma interplay. In vivo experiments were performed using a xenograft CCA model. ZEB1 expression in tumor cells of human iCCA was associated with undifferentiated tumor and vascular invasion. In vitro, ZEB1 promoted epithelial-mesenchymal transition and stemness in tumor cells, leading to cell migration and spheroid formation. In vivo, ZEB1-overexpressing CCA cells formed larger tumors with more abundant stroma. Expression of cellular communication network factor 2 (CCN2, encoding connective tissue growth factor [CTGF]) was increased in tumor cells from ZEB1-overexpressing xenografts and correlated with ZEB1 expression in human tumors. In vitro, CM from ZEB1-overexpressing tumor cells or recombinant CTGF induced myofibroblast proliferation. ZEB1 was also expressed by CAFs in human CCA, and its expression correlated with CCN2 in myofibroblasts and CCA stroma. In mice, cotransplantation of CCA cells with ZEB1-depleted myofibroblasts reduced CCA progressiveness compared to CCA cells/ZEB1-expressing myofibroblasts. Furthermore, ZEB1 controls the expression of paracrine signals (i.e., HGF and IL6) in tumor cells and myofibroblasts.ZEB1 plays a key role in CCA progression by regulating tumor cell-CAF crosstalk, leading to tumor dedifferentiation and CAF activation.

International audience; More than 20 years ago, a new secreted ligand of the tumor necrosis factor (TNF) family was identified in mouse peritoneal macrophage, human tonsil and fetal liver. However, in contrast to TNF, TWEAK induced only weakly apoptosis in a panel of cell types such as hematopoietic cells, fibroblasts and cancer cells, and was thus named TWEAK for “tumor necrosis factor-like weak inducer of apoptosis”. TWEAK displays two forms, membrane-bound or soluble. As TNF, TWEAK activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) pathway and thereby inflammatory responses. Meanwhile, and independently of TWEAK, fibroblast growth factor-inducible 14 (Fn14), a type I transmembrane protein was identified as a modulator of fibroblast adhesion and migration. Soon after, it was discovered in endothelial cells (ECs) for its role in angiogenesis as the receptor of TWEAK. While Fn14 was already known for its hepatic functions including regeneration and cancer, i.e. hepatocellular carcinoma (HCC) [1], the role of TWEAK/Fn14 signaling in cholangiocarcinoma (CCA), the second more frequent liver cancer after HCC, was unknown so far. In an elegant work, Dwyer et al. [2] highlighted essential functions of TWEAK/Fn14 in intrahepatic CCA (iCCA), the anatomical subtype of CCA arising above the second-order bile ducts. In iCCA, TWEAK promotes the development of the tumor niche sustaining tumor initiation and progression, which could pave the way for a potential treatment of iCCA patients since TWEAK/Fn14 pathway is a “druggable” target.

Correct actin cytoskeleton organization is vital in the liver organ homeostasis and disease control. Rearrangements of the actin cytoskeleton may play a vital role in the bile duct cells cholangiocytes. An abnormal actin network leads to aberrant cell morphology, deregulated signaling networks and ultimately triggering the development of cholangiocarcinoma (CCA) and paving the route for cancer cell dissemination (metastasis). In this review, we will outline alterations of the actin cytoskeleton and the potential role of this dynamic network in initiating CCA, as well as regulating the course of this malignancy. Actin rearrangements not only occur because of signaling pathways, but also regulate and modify cellular signaling. This emphasizes the importance of the actin cytoskeleton itself as cause for aberrant signaling and in promoting tumorigenic phenotypes. We will highlight the impact of aberrant signaling networks on the actin cytoskeleton and its rearrangement as potential cause for CCA. Often, these exact mechanisms in CCA are limited understood and still must be elucidated. Indeed, focusing future research on how actin affects and regulates other signaling pathways may provide more insights into the mechanisms of CCA development, progression, and metastasis. Moreover, manipulation of the actin cytoskeleton organization highlights the potential for a novel therapeutic area.

International audience; Cholangiocarcinoma (CCA) results from the malignant transformation of cholangiocytes. Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are chronic diseases in which cholangiocytes are primarily damaged. Although PSC is an inflammatory condition predisposing to CCA, CCA is almost never found in the autoimmune context of PBC. Here, we hypothesized that PBC might favor CCA immunosurveillance. In preclinical murine models of cholangitis challenged with syngeneic CCA, PBC (but not PSC) reduced the frequency of CCA development and delayed tumor growth kinetics. This PBC-related effect appeared specific to CCA as it was not observed against other cancers, including hepatocellular carcinoma. The protective effect of PBC was relying on type 1 and type 2 T cell responses and, to a lesser extent, on B cells. Single-cell TCR/RNA sequencing revealed the existence of TCR clonotypes shared between the liver and CCA tumor of a PBC host. Altogether, these results evidence a mechanistic overlapping between autoimmunity and cancer immunosurveillance in the biliary tract.

The Epidermal Growth Factor Receptor (EGFR) has been targeted through the development of selective tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAb). These molecules have shown effectiveness in a subset of patients with specific genetic alterations (i.e. gain-of-function EGFR mutations or EGFR gene amplification) and have been approved for their use in non-small-cell lung cancer (NSCLC), colorectal cancer (CRC), pancreatic cancer and head and neck cancer. In addition, extensive research is being performed in many other tumour types hoping for a future approval. However, the majority of the patients show no benefit from these molecules due to primary mechanisms of resistance, already present before treatment or show disease progression upon the acquisition of drug resistance mechanisms during the treatment. At present, the majority of patients display resistance due to alterations in genes related to the EGFR signalling pathway that eventually circumvent EGFR inhibition and allow cancer progression. Thus, in this review article we focus on the molecular mechanisms underlying drug resistance via genetic alterations leading to resistance to all anti-EGFR drugs approved by the FDA and/or EMA. We also discuss novel approaches to surmount these chemoresistance modalities.

Background & aims Cholangiopathies are chronic liver diseases in which damaged cholangiocytes trigger a proinflammatory and profibrotic reaction. The nuclear vitamin D receptor (VDR) is highly expressed in cholangiocytes and exerts immune-regulatory functions in these cells. In the present study, we examined the protective function of VDR and other vitamin D signaling pathways in chronic cholangiopathy and cholangiocytes. Methods Vdr was invalidated in Abcb4 knockout mice, a widely used animal model of chronic cholangiopathy. The impact of vitamin D signaling on cholangiopathy features was examined in vivo and in cholangiocytes (primary and cell lines). Results Cholangiopathy features (i.e, cholestasis, ductular reaction and fibrosis) were aggravated in Vdr;Abcb4 double knockout mice compared to the Abcb4 simple knockout, and associated with an overexpression of proinflammatory factors. The proinflammatory phenotype of cholangiocytes was also exacerbated following VDR silencing in vitro. The expression of proinflammatory factors and the severity of cholangiopathy were reduced in the double knockout mice treated with the vitamin D analog calcipotriol or with vitamin D. In vitro, the inflammatory response to TNFα was significantly reduced by calcipotriol in biliary cells silenced for VDR, and this effect was abolished by co-silencing the plasma membrane receptor of vitamin D, protein disulfide-isomerase A3 (PDIA3). Conclusions Our results demonstrate an anti-inflammatory role of VDR signaling in cholangiocytes and cholangiopathy. They also provide evidence for PDIA3-mediated anti-inflammatory effects of vitamin D and vitamin D analog in these settings.

Physical oncology recognizes tissue stiffness mediated by activation of cancer-associated fibroblasts (CAF) and extracellular matrix remodeling as an active modulator of tumorigenesis, treatment resistance, and clinical outcome. Cholangiocarcinoma (CCA) is a highly aggressive and chemoresistant desmoplastic cancer enriched in CAF. CCA's stroma mechanical properties are considered responsible for its chemoresistant character. To normalize tumor mechanics, we propose a physical strategy based on remotely light-activated nanohyperthermia to modulate the tumor microenvironment. In this study, we report the use of multifunctional iron oxide nanoflowers decorated with gold nanoparticles (GIONF) as efficient nanoheaters to achieve complete tumor regression following three sessions of mild hyperthermia. The preferential uptake of GIONF by CAF allowed targeting this cell population, which resulted in a significant early reduction of tumor stiffness followed by tumor regression. In conclusion, our study highlights a spatially and temporally controlled physical strategy, GIONF-mediated photothermal therapy to deplete CAF and normalize the tumor mechanics that may apply to desmoplastic cancer and CCA treatment.

Only a fraction of cancer patients benefits from immune checkpoint inhibitors. This may be partly due to the dense extracellular matrix (ECM) that forms a barrier for T cells. Comparing 5 preclinical mouse tumor models with heterogeneous tumor microenvironments, we aimed to relate the rate of tumor stiffening with the remodeling of ECM architecture and to determine how these features affect intratumoral T cell migration. An ECM-targeted strategy, based on the inhibition of lysyl oxidase (LOX) was used. In vivo stiffness measurements were found to be strongly correlated with tumor growth and ECM crosslinking but negatively correlated with T cell migration. Interfering with collagen stabilization reduces ECM content and tumor stiffness leading to improved T cell migration and increased efficacy of anti-PD-1 blockade. This study highlights the rationale of mechanical characterizations in solid tumors to understand resistance to immunotherapy and of combining treatment strategies targeting the ECM with anti-PD-1 therapy.

International audience; Purpose of review: To give a state-of-art knowledge regarding cancer-associated fibroblasts (CAF) in cholangiocarcinoma (CCA) based both on direct evidence and studies on other desmoplastic cancers. High contingency of CAF characterizes CCA, a tumor with a biliary epithelial phenotype that can emerge anywhere in the biliary tree. Current treatments are very limited, the surgical resection being the only effective treatment but restricted to a minority of patients, whereas the remaining patients undergo palliative chemotherapy regimens. In cancer, CAF shape the tumor microenvironment, drive cancer growth and progression, and contribute to drug resistance. All these functions are accomplished through an interplay network between CAF and surrounding cells including tumor and other stromal cells, i.e. immune and endothelial cells.Recent findings: Several studies have pointed out the existence of CAF sub-populations carrying out several and opposite functions, cancer-promoting or cancer-restraining as shown in pancreatic cancer, another prototypic desmoplastic tumor in which heterogeneity of CAF is well demonstrated.Summary: New CAF functions are now emerging in pancreatic and breast cancers like the modulation of immune responses or tumor metabolism, opening new area for treatments.

Through the last decade, cold atmospheric plasma (CAP) has emerged as an innovative therapeutic option for cancer treatment. Recently, we have set up a potentially safe atmospheric pressure plasma jet device that displays antitumoral properties in a preclinical model of cholangiocarcinoma (CCA), a rare and very aggressive cancer emerging from the biliary tree with few efficient treatments. In the present study, we aimed at deciphering the molecular mechanisms underlying the antitumor effects of CAP towards CCA in both an in vivo and in vitro context. In vivo, using subcutaneous xenografts into immunocompromised mice, CAP treatment of CCA induced DNA lesions and tumor cell apoptosis, as evaluated by 8-oxoguanine and cleaved caspase-3 immunohistochemistry, respectively. The analysis of the tumor microenvironment showed changes in markers related to macrophage polarization. In vitro, the incubation of CCA cells with CAP-treated culture media (i.e., plasma-activated media, PAM) led to a dose response decrease in cell survival. At molecular level, CAP treatment induced double-strand DNA breaks, followed by an increased phosphorylation and activation of the cell cycle master regulators CHK1 and p53, leading to cell cycle arrest and cell death by apoptosis. In conclusion, CAP is a novel therapeutic option to consider for CCA in the future.

International audience

International audience; Hepatocellular carcinoma (HCC) is one of the most common and deadly neoplasms. Insulin receptor (IR) exists in two isoforms, IR-A and IR-B, the latter being predominantly expressed in normal adult hepatocytes while IR-A is overexpressed in HCC to the detriment of IR-B. This study evaluated the biological functions associated with IR-A overexpression in HCC in relation to expression of its ligand IGF-II. The value of INSRA:INSRB ratio which was increased in ˜70% of 85 HCC was associated with stem/progenitor cell features such as cytokeratin-19 and α-fetoprotein and correlated with shorter patient survival. IGF2 mRNA upregulation was observed in 9.4% of HCC and was not associated with higher INSRA:INSRB ratios. Ectopic overexpression of IR-A in two HCC cell lines presenting a strong autocrine IGF-II secretion loop or not stimulated cell migration and invasion. In cells cultured as spheroids, IR-A overexpression promoted gene programs related to stemness, inflammation and cell movement. IR-A also increased cell line tumorigenicity in vivo after injection to immunosuppressed mice and the sphere-forming cells made a significant contribution to this effect. Altogether, these results demonstrate that IR-A is a novel player in HCC progression.

Purpose: Cholangiocarcinoma (CCA) is a desmoplastic tumor of the biliary tree in which epidermal growth factor receptor (EGFR) is overexpressed and contributes to cancer progression. Although EGFR has been envisaged as a target for therapy, treatment with tyrosine kinase inhibitors (TKI) such as erlotinib did not provide therapeutic benefit in patients with CCA, emphasizing the need to investigate resistance mechanisms against EGFR inhibition. Experimental Design: Resistant CCA cells to EGFR inhibition were obtained upon long-time exposure of cells with erlotinib. Cell signaling, viability, migration, and spheroid growth were determined in vitro, and tumor growth was evaluated in CCA xenograft models. Results: Erlotinib-resistant CCA cells displayed metastasis-associated signatures that correlated with a marked change in cell plasticity associated with an epithelial–mesenchymal transition (EMT) and a cancer stem cell (CSC)–like phenotype. Resistant cells exhibited an upregulation of insulin receptor (IR) and insulin-like growth factor (IGF) 1 receptor (IGF1R), along with an increase in IGF2 expression. IR/IGF1R inhibition reduced EMT and CSC-like traits in resistant cells. In vivo, tumors developed from resistant CCA cells were larger and exhibited a more prominent stromal compartment, enriched in cancer-associated fibroblasts (CAF). Pharmacological coinhibition of EGFR and IR/IGF1R reduced tumor growth and stromal compartment in resistant tumors. Modeling of CCA-CAF crosstalk showed that IGF2 expressed by fibroblasts boosted IR/IGF1R signaling in resistant cells. Furthermore, IR/IGF1R signaling positively regulated fibroblast proliferation and activation. Conclusions: To escape EGFR-TKI treatment, CCA tumor cells develop an adaptive mechanism by undergoing an IR/IGF1R-dependent phenotypic switch, involving a contribution of stromal cells. Clin Cancer Res; 24(17); 4282–96. ©2018 AACR.

The ErbB/HER family comprises four distinct tyrosine kinase receptors, EGFR/ErbB1/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4, which trigger intracellular signals at the origin of essential cellular functions, including differentiation, proliferation, survival, and migration. Epithelial cells, named cholangiocytes, that line intrahepatic and extrahepatic bile ducts, contribute substantially to biliary secretory functions and bile transport. Although ErbB receptors have been widely studied in cholangiocarcinoma (CCA), a malignancy of the biliary tract, knowledge of these receptors in biliary epithelium physiology and in non-malignant cholangiopathies is far from complete. Current knowledge suggests a role for epidermal growth factor receptor (EGFR) in cholangiocyte specification and proliferation, and in hepatocyte transdifferentiation into cholangiocytes during liver regeneration to restore biliary epithelium integrity. High expression and activation of EGFR and/or ErbB2 were recently demonstrated in biliary lithiasis and primary sclerosing cholangitis, two cholangiopathies regarded as risk factors for CCA. In CCA, ErbB receptors are frequently overexpressed, leading to tumor progression and low prognosis. Anti-ErbB therapies were efficient only in preclinical trials and have suggested the existence of resistance mechanisms with the need to identify predictive factors of therapy response. This review aims to compile the current knowledge on the functions of ErbB receptors in physiology and physiopathology of the biliary epithelium. (Hepatology 2018;67:762-773).

Intrahepatic cholangiocarcinoma (CCA) is characterized by an abundant desmoplastic environment. Poor prognosis of CCA has been associated with the presence of alpha-smooth muscle actin (α-SMA)-positive myofibroblasts (MFs) in the stroma and with the sustained activation of the epidermal growth factor receptor (EGFR) in tumor cells. Among EGFR ligands, heparin-binding epidermal growth factor (HB-EGF) has emerged as a paracrine factor that contributes to intercellular communications between MFs and tumor cells in several cancers. This study was designed to test whether hepatic MFs contributed to CCA progression through EGFR signaling. The interplay between CCA cells and hepatic MFs was examined first in vivo, using subcutaneous xenografts into immunocompromised mice. In these experiments, cotransplantation of CCA cells with human liver myofibroblasts (HLMFs) increased tumor incidence, size, and metastastic dissemination of tumors. These effects were abolished by gefitinib, an EGFR tyrosine kinase inhibitor. Immunohistochemical analyses of human CCA tissues showed that stromal MFs expressed HB-EGF, whereas EGFR was detected in cancer cells. In vitro, HLMFs produced HB-EGF and their conditioned media induced EGFR activation and promoted disruption of adherens junctions, migratory and invasive properties in CCA cells. These effects were abolished in the presence of gefitinib or HB-EGF-neutralizing antibody. We also showed that CCA cells produced transforming growth factor beta 1, which, in turn, induced HB-EGF expression in HLMFs. Conclusion: A reciprocal cross-talk between CCA cells and myofibroblasts through the HB-EGF/EGFR axis contributes to CCA progression. (Hepatology 2013; 58:2001–2011)

International audience; Cholangiocarcinoma (CCA) is an aggressive tumor with a poor prognosis due to its late clinical presentation and the lack of effective non-surgical therapies. Unfortunately, most of the patients are not eligible for curative surgery owing to the presence of metastases at the time of diagnosis. Therefore, it is important to understand the steps leading to cell dissemination in patients with CCA. To metastasize from the primary site, cancer cells must acquire migratory and invasive properties by a cell plasticity-promoting phenomenon known as epithelial-mesenchymal transition (EMT). EMT is a reversible dynamic process by which epithelial cells gradually adopt structural and functional characteristics of mesenchymal cells, and has lately become a center of attention in the field of metastatic dissemination. In the present review, we aim to provide an extensive overview of the current clinical data and the prognostic value of different EMT markers that have been analyzed in CCA. We summarize all the regulatory networks implicated in EMT from the membrane receptors to the main EMT-inducing transcription factors (SNAIL, TWIST and ZEB). Furthermore, since a tumor is a complex structure not exclusively formed by tumor cells, we also address the prominent role of the main cell types of the desmoplastic stroma that characterizes CCA in the regulation of EMT. Finally, we discuss the therapeutic considerations and difficulties faced to develop an effective anti-EMT treatment due to the redundancies and bypasses among the pathways regulating EMT.

Cholestatic disorders may arise from liver ischemia (e.g., in liver transplantation) through various mechanisms. We have examined the potential of hypoxia to induce changes in the expression of hepatobiliary transporter genes. In a model of arterial liver ischemia subsequent to complete arterial deprivation of the rat liver, the mRNA levels of VEGF, a hypoxia-inducible gene, were increased fivefold after 24 h. The pattern of VEGF-induced expression and ultrastructural changes, including swelling of the endoplasmic reticulum, indicated that hypoxia affected primarily cholangiocytes, but also hepatocytes, predominantly in the periportal area. Serum and bile analyses demonstrated liver dysfunction of cholestatic type with reduced bile acid biliary excretion. Fluorescence-labeled ursodeoxycholic acid used as a tracer displayed no regurgitation, eliminating bile leakage as a significant mechanism of cholestasis in this model. In liver tissue, a marked reduction in the mRNA levels of Na+-taurocholate-cotransporting polypeptide (Ntcp), bile salt export protein (Bsep), and multidrug resistance-associated protein 2 (Mrp2) and an increase in those of Cftr were detected before bile duct proliferation occurred. In cultured hepatocytes, a nontoxic hypoxic treatment caused a decrease in the mRNA and protein expression of Ntcp, Bsep, and Mrp2 and in the mRNA levels of nuclear factors involved in the transactivation of these genes, i.e., HNF4α, RXRα, and FXR. In bile duct preparations, hypoxic treatment elicited an increase in Cftr transcripts, along with a rise in cAMP, a major regulator of Cftr expression and function. In conclusion, hypoxia triggers a downregulation of hepatocellular transporters, which may contribute to cholestasis, whereas Cftr, which drives secretion in cholangiocytes, is upregulated.

International audience; 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. Besides to MK2 and Hsp27 overexpression in primary liver tumors compared to adjacent non-tumorous tissues, 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 (HO-1) through down-regulation of the transcription factor nuclear factor-erythroid-derived 2-like 2 (Nrf2). Moreover, MK2/Hsp27 inhibition decreases both phosphorylation of epidermal growth factor receptor (EGFR) and expression of its ligand, heparin-binding EGF-like growth factor (HB-EGF). A new identified partner of MK2, the scaffold PDZ-protein EBP50, could facilitate these effects through MK2/Hsp27 pathway regulation. These findings demonstrate that MK2/Hsp27 pathway actively participates in resistance to oxidative stress and may contribute to liver cancer progression.

Aims Ezrin connects proteins from the plasma membrane to the subcortical cytoskeleton, and contributes to epithelial integrity by interacting with the cell–cell adhesion molecule E-cadherin. In the liver, ezrin is restricted to cholangiocytes, where it regulates biliary secretory functions. During carcinogenesis, ezrin expression is impaired and associated with enhancement of cell migratory activity in cancer cells; therefore, we aimed to analyse ezrin in cholangiocarcinogenesis. Methods and results Ezrin expression was evaluated by immunohistochemistry on tissue microarrays from 94 surgical specimens of intrahepatic cholangiocarcinoma (CCA), and correlated with clinicopathological factors and E-cadherin expression. Ezrin function was also analysed in human CCA cell lines. In CCA, ezrin was negative/weakly expressed in 49 cases (52%) and moderately/strongly expressed in 45 cases (48%), mostly in cell cytoplasm. The negative/weak expression of ezrin was more frequent in peripheral than in perihilar CCA (P = 0.002), and was associated with high tumour size (P = 0.001), low mucus secretion (P = 0.042), the presence of satellite nodules (P = 0.024), and ectopic cytoplasmic expression of E-cadherin (P = 0.005). In vitro, silencing of ezrin in CCA cells caused internalization of E-cadherin and favoured cell migration. Conclusions Ezrin is down-regulated during cholangiocarcinogenesis, and its loss results in a more aggressive phenotype.

Background: Cholangiocarcinoma is the second most frequent primary malignant tumor of the liver, accounting for 10-15% of all primary liver cancers. Cholangiocarcinoma grows at the expense of epithelial cells that line intra- and extra-hepatic bile ducts. The adaptor protein EBP50 (Ezrin-radixin-moesin Binding Phosphoprotein 50) binds and regulates transmembrane transporters and receptors including Epidermal Growth Factor Receptor (EGFR). EBP50 and EGFR are both expressed in the normal biliary epithelium. While EGFR participates in biliary carcinogenesis, implication of EBP50 in this cancer is unknown. The aim of the study was to determine the potential links between EBP50 and EGFR in human biliary carcinoma. Methods: In vitro studies were performed in the human biliary epithelial cell line, Mz-ChA-1, that retains epithelial phenotype and endogenous expression of EBP50 and EGFR. The role of EBP50 was examined using siRNA. Cell scattering and migration were evaluated using E-cadherin immunostaining and Boyden chamber, respectively. For in vivo study, 112 patients (mean age: 59.5±11 years, 55 females and 57 males) who underwent surgical resection for hilar (n=53) and peripheral (n=59) cholangiocarcinoma in one single institution were analyzed retrospectively. Immunohistochemistry on tissue microarrays was performed to assess EBP50 and EGFR expression. Results: EGF activates EGFR and the three main signaling pathways, ERK1/2, STAT3 and Akt, in Mz-ChA-1 biliary cancer cells. Inhibition of EBP50 in EGF-treated cells increases and maintains activation of EGFR, and of ERK1/2 and STAT3 but not Akt. Enhanced EGFR activation in response to EBP50 depletion confers sensitivity to EGFR tyrosine kinase inhibitor, gefitinib. Functionally, upon EGF, EGFR drives cell scattering by inducing disruption of adherens junctions. In addition, EGF activates formation of lamellipodia structures and therefore cell migration. Interestingly, inhibition of EBP50 in non EGF-stimulated cell has the same effects on cell scattering and migration as those observed with EGF alone. Cell dispersion due to EBP50 inhibition is abolished in the presence of gefitinib. Moreover, impact on cell scattering and migration is even greater when both EGF addition and EBP50 siRNA are combined. In vivo, delocalization of EBP50 normally expressed beneath the apical membrane is associated with EGFR expression in human biliary carcinoma tissues (p=0.002). Conclusion: These data provide evidence for an essential role of EBP50 in repressing EGFR-induced signaling pathway and migration. EBP50 at the membrane may be critical to confine cancer progression by preventing migration of biliary cancer cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5205.

Background & Aims In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis: a regulated form of necrotic cell death mediated by the receptor-interacting protein kinase (RIPK) 1. Herein, we assessed the potential for RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL) to act as therapeutic targets and markers of activity in NAFLD. Methods C57/BL6J-mice were fed a normal chow diet or a high-fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in HFD-fed mice and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD. Results When used as either a prophylactic or curative treatment for HFD-fed mice, RIPA-56 caused a downregulation of MLKL and a reduction of liver injury, inflammation and fibrosis, characteristic of non-alcoholic steatohepatitis (NASH), as well as of steatosis. This latter effect was reproduced by treating primary human steatotic hepatocytes with RIPA-56 or necrosulfonamide, a specific inhibitor of human MLKL, and by knockout (KO) of Mlkl in fat-loaded AML-12 mouse hepatocytes. Mlkl-KO led to activation of mitochondrial respiration and an increase in β-oxidation in steatotic hepatocytes. Along with decreased MLKL activation, Ripk3-KO mice exhibited increased activities of the liver mitochondrial respiratory chain complexes in experimental NASH. In patients with NAFLD, serum concentrations of RIPK1 and MLKL increased in correlation with activity. Conclusion The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis via an MLKL-dependent mechanism that, at least partly, involves an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD. Lay summary There are currently no pharmacological treatment options for non-alcoholic fatty liver disease (NAFLD), which is now the most frequent liver disease. Necroptosis is a regulated process of cell death that can occur in hepatocytes during NAFLD. Herein, we show that RIPK1, a gatekeeper of the necroptosis pathway that is activated in NAFLD, can be inhibited by RIPA-56 to reduce not only liver injury, inflammation and fibrosis, but also steatosis in experimental models. These results highlight the potential of RIPK1 as a therapeutic target in NAFLD.