Laura Fouassier

Expert consensus document: Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA)

Cholangiocarcinoma (CCA) is a heterogeneous group of malignancies with features of biliary tract differentiation. CCA is the second most common primary liver tumour and the incidence is increasing worldwide. CCA has high mortality owing to its aggressiveness, late diagnosis and refractory nature. In May 2015, the “European Network for the Study of Cholangiocarcinoma” (ENS-CCA: www.enscca.org or www.cholangiocarcinoma.eu) was created to promote and boost international research collaboration on the study of CCA at basic, translational and clinical level. In this Consensus Statement, we aim to provide valuable information on classifications, pathological features, risk factors, cells of origin, genetic and epigenetic modifications and current therapies available for this cancer. Moreover, future directions on basic and clinical investigations and plans for the ENS-CCA are highlighted.

Epithelial-mesenchymal transition in cholangiocarcinoma: From clinical evidence to regulatory networks

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 centre 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 analysed 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.

Role of the PDZ-scaffold protein NHERF1|[sol]|EBP50 in cancer biology: from signaling regulation to clinical relevance

The transmission of cellular information requires fine and subtle regulation of proteins that need to interact in a coordinated and specific way to form efficient signaling networks. The spatial and temporal coordination relies on scaffold proteins. Thanks to protein interaction domains such as PDZ domains, scaffold proteins organize multiprotein complexes enabling the proper transmission of cellular information through intracellular networks. NHERF1/EBP50 is a PDZ-scaffold protein that was initially identified as an organizer and regulator of transporters and channels at the apical side of epithelia through actin-binding ezrin-moesin-radixin proteins. Since, NHERF1/EBP50 has emerged as a major regulator of cancer signaling network by assembling cancer-related proteins. The PDZ-scaffold EBP50 carries either anti-tumor or pro-tumor functions, two antinomic functions dictated by EBP50 expression or subcellular localization. The dual function of NHERF1/EBP50 encompasses the regulation of several major signaling pathways engaged in cancer, including the receptor tyrosine kinases PDGFR and EGFR, PI3K/PTEN/AKT and Wnt-β-catenin pathways.

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.

Immunohistochemical profile of ezrin and radixin in human liver epithelia during fetal development and pediatric cholestatic diseases.

AIM Ezrin and radixin are actin-binding proteins that contribute to the integrity of epithelia. Abnormalities of bile secretion occur primarily in cholestatic liver diseases and are associated with changes in cell cytoskeleton. Expression of these proteins during liver development and in cholestatic liver diseases remains poorly investigated. METHODS Ezrin and radixin expression was analyzed in fetal, adult and pediatric cholestatic human liver (i.e. biliary atresia, sclerosing cholangitis) by immunohistochemistry. RESULTS In adult and fetal livers, ezrin was expressed exclusively in the cells of the biliary lineage (i.e. biliary epithelial cells and ductal cells) whereas radixin was located not only in hepatocytes but also in cells of the biliary lineage. In the lobule of mature livers, radixin displayed a zonal distribution with predominant expression in the periportal region. In cholestatic diseases, both proteins were expressed in cells of the ductular reaction. An aberrant expression of ezrin was detected in hepatocytes of cirrhotic nodules with a CK7-positive pattern and in malignant hepatocytes in a course of cholestatic disease toward cancer. CONCLUSIONS Among the components of the liver epithelial cells, ezrin was exclusively expressed in biliary phenotype cells, while radixin was found in biliary and hepatocytic lineages, with a periportal zonal expression. In cholestatic diseases, ezrin was expressed in hepatocytes supporting the appearance of a biliary phenotype.

Role of ErbB/HER family of receptor tyrosine kinases in cholangiocyte biology

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).

Ezrin finds its groove in cholangiocytes

Ezrin was first detected in epidermal growth factor-treated skin epithelial cells as a protein-tyrosine kinase substrate prior to its purification and characterization as a structural component of microvilli of chicken intestinal epithelial cell brush borders (1, 2). Subsequently, other closely related proteins including radixin and moesin were identified, that together with ezrin constitute the ERM proteins family. ERM family members have the ability to cross-link proteins of the plasma membrane with the sub-cortical cytoskeleton and were shown to be important for the stabilization of the cell cortex structure and regulation of several signal transduction pathways (3).

Overcoming the tumor microenvironment: the role of nanohyperthermia

Abnormal tumor mechanics promotes cancer progression & treatment resistance Cancer cells sense and respond to mechanical cues in tumors. Mechanobiology has recently emerged as a key determinant in malignant tumor development and progression involving complex cellular crosstalk mechanisms. The majority of mechanical stimuli stem from the tumor microenvironment (TME). Indeed, the TME represents a dynamic and evolutive niche comprising supportive stromal cells, soluble factors, vascular networks and the structural extracellular matrix (ECM) architecture to provide a tumor permissive soil [1,2]. Excessive accumulation and aberrant architecture of the ECM are the hallmarks of most solid tumors. ECM remodeling is derived from cancer-associated fibroblasts and their activated phenotype, which results in an increased proliferative activity and enhanced secretion of ECM proteins such as type I collagen, hyaluronic acid, tenascin C and fibronectin [3,4]. The excessive and aberrant deposition of collagen around the tumor mass as well as its linear, long and highly reticulated networks significantly contribute to the abnormal tumor stiffening in comparison to healthy tissue [5]. Importantly, a rigid stroma has been correlated to an adverse prognosis in several human carcinomas [6]. Increasing evidence suggests a tight relationship between ECM remodeling and stiffening, cellular mechanosignaling, tissue inflammation and the induction of the epithelio-mesenchymal transition [7]. Altered mechanics also regulate the tumor behavior and progression via the capacity of cell invasiveness, migration and their resistance to apoptosis. A number of mechanosignaling pathways have been elucidated recently to link the cancer biology to mechanical cues. For instance, stiffness was reported to be sensed through β4 integrin, Rac1 and the PI3K pathway modulating the induction of malignant phenotypes [8,9]. Increased ECM stiffness also regulates the expression of tumor suppressor genes through a miRNAmediated transcriptional circuit. Mechanotransduction pathways are shown to mediate the physical effects of the microenvironment on tumor cell progression. In addition to biological outcomes, the TME and its physical anomalies also dramatically affect the efficiency of anticancer treatment. The aberrantly abundant and dense ECM, high interstitial pressure, chaotic vessel organization and enhanced solid stress are physical features of the TME that dramatically restrict the transport of cytotoxic therapeutic agents [10–12]. Drugs as well as nanovectors are blocked by those physical barriers in their diffusion to tumor cells, thereby creating drug-free sanctuaries that contribute to the emergence of drug resistance. In the same way, immune cells, such as T lymphocytes, show higher difficulty to migrate toward Overcoming the tumor microenvironment: the role of nanohyperthermia

The IGF2/IR/IGF1R Pathway in Tumor Cells and Myofibroblasts Mediates Resistance to EGFR Inhibition in Cholangiocarcinoma

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.

Rac1 and EMT: a dangerous liaison?

Rac1 was originally identified in 1989 in human platelets as a substrate of the botulinum toxin from which its name derives: Ras-related C3 botulinum toxin substrate (1). Rac1 belongs to the Rho family of small guanosine triphosphatases (GTPases), and is ubiquitously expressed. It exists in two conformational states, an inactive GDP-bound form and an active GTP-bound form. The transition from one form to another occurs upon stimuli and depends on GTPase-activating proteins (GAPs), which inactivate Rac1, and guanine nucleotide exchange factors (GEFs), which convert Rac1 to its active form. Among GEFs, the trimeric complex SOS1/EPS8/ABI1 plays a role in Rac regulation. Dissection of the complex shows that SOS1 is central because it supports the catalytic component of the complex, and both ABI1, also named EPS8 SH3 domain binding protein, and EPS8 with its SOS1 binding domain, allow the cohesion of this complex. Functionally, Rac1 was the subject of numerous studies highlighting diverse and broad cellular functions since its discovery. Rac1 serves as a conformational switch in several signal transduction pathways at the origin of its biological functions. One of its primary function identified, far away the most characterized, is the regulation of actin cytoskeleton organization and dynamics, in the migratory structures such as filopodia and lamellipodia. Subsequently, other Rac1 functions were evidenced including roles in cell polarity, gene expression, cell-cycle progression, and cell survival.