Publications by authors named "Cédric Coulouarn"

60 Publications

The noncoding MIR100HG RNA enhances the autocrine function of transforming growth factor β signaling.

Oncogene 2021 May 4;40(21):3748-3765. Epub 2021 May 4.

Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Box 582, Biomedical Center, Uppsala University, Uppsala, Sweden.

Activation of the transforming growth factor β (TGFβ) pathway modulates the expression of genes involved in cell growth arrest, motility, and embryogenesis. An expression screen for long noncoding RNAs indicated that TGFβ induced mir-100-let-7a-2-mir-125b-1 cluster host gene (MIR100HG) expression in diverse cancer types, thus confirming an earlier demonstration of TGFβ-mediated transcriptional induction of MIR100HG in pancreatic adenocarcinoma. MIR100HG depletion attenuated TGFβ signaling, expression of TGFβ-target genes, and TGFβ-mediated cell cycle arrest. Moreover, MIR100HG silencing inhibited both normal and cancer cell motility and enhanced the cytotoxicity of cytostatic drugs. MIR100HG overexpression had an inverse impact on TGFβ signaling responses. Screening for downstream effectors of MIR100HG identified the ligand TGFβ1. MIR100HG and TGFB1 mRNA formed ribonucleoprotein complexes with the RNA-binding protein HuR, promoting TGFβ1 cytokine secretion. In addition, TGFβ regulated let-7a-2-3p, miR-125b-5p, and miR-125b-1-3p expression, all encoded by MIR100HG intron-3. Certain intron-3 miRNAs may be involved in TGFβ/SMAD-mediated responses (let-7a-2-3p) and others (miR-100, miR-125b) in resistance to cytotoxic drugs mediated by MIR100HG. In support of a model whereby TGFβ induces MIR100HG, which then enhances TGFβ1 secretion, analysis of human carcinomas showed that MIR100HG expression correlated with expression of TGFB1 and its downstream extracellular target TGFBI. Thus, MIR100HG controls the magnitude of TGFβ signaling via TGFβ1 autoinduction and secretion in carcinomas.
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http://dx.doi.org/10.1038/s41388-021-01803-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8154591PMC
May 2021

The Consumption of Cholesterol-Enriched Diets Conditions the Development of a Subtype of HCC with High Aggressiveness and Poor Prognosis.

Cancers (Basel) 2021 Apr 6;13(7). Epub 2021 Apr 6.

Área de Medicina Experimental y Traslacional, Departamento de Ciencias de la Salud, Universidad Autónoma Metropolitana-Iztapalapa, Mexico City 09340, Mexico.

Non-alcoholic fatty liver disease (NAFLD) and progression to non-alcoholic steatohepatitis (NASH) result as a consequence of diverse conditions, mainly unbalanced diets. Particularly, high-fat and cholesterol content, as well as carbohydrates, such as those commonly ingested in Western countries, frequently drive adverse metabolic alterations in the liver and promote NAFLD development. Lipid liver overload is also one of the main risk factors for initiation and progression of hepatocellular carcinoma (HCC), but detailed knowledge on the relevance of high nutritional cholesterol remains elusive. We were aimed to characterize HCC development in mice fed with a Western diet (high in lipids and cholesterol) and to identify molecular alterations that define a subtype of liver cancer induced by lipid overload. Mice under western or high cholesterol diets more frequently developed tumors with a more aggressive phenotype than animals fed with a chow diet. Associated changes involved macrophage infiltration, angiogenesis, and stemness features. RNA-seq revealed a specific gene expression signature ( and ) resembling the adverse phenotypic features and poor clinical outcomes seen in patients with HCC. In conclusion; consumption of lipid enriched diets; particularly cholesterol; could accelerate HCC development with an aggressive phenotype and poor prognosis.
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http://dx.doi.org/10.3390/cancers13071721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038696PMC
April 2021

Extracellular vesicles shed by follicular lymphoma B cells promote the polarization of bone marrow stromal cell niche.

Blood 2021 Feb 24. Epub 2021 Feb 24.

Faculté de Médecine, Rennes, France.

Follicular Lymphoma (FL) originates in the lymph nodes (LN) and infiltrates bone marrow (BM) early in the course of the disease. BM FL B cells are characterized by a lower cytological grade, a decreased proliferation, and a specific phenotypic and subclonal profile. Mesenchymal stromal cells (MSC) obtained from FL BM display a specific gene expression profile (GEP), including enrichment for a lymphoid-stromal cell signature, and an increased capacity to sustain FL B-cell growth. However, the mechanisms triggering the formation of the medullar FL permissive stromal niche have not been yet identified. In the current work, we demonstrated that FL B cells produced extracellular vesicles (EVs) that could be internalized by BM-MSC, making them more efficient to support FL B-cell survival and quiescence. Accordingly, EVs purified from FL BM plasma activated TGF-b dependent and independent pathways in BM-MSC, modified their GEP, triggering an upregulation of factors classically associated with hematopoietic stem cell niche, including CXCL12 or angiopoietin-1. Moreover, we provided the first characterization of BM FL B-cell GEP, allowing the definition of the landscape of molecular interactions they could engage with EV-primed BM-MSC. This work identified FL-derived EVs as putative mediators of BM stroma polarization and supported further investigation of their clinical interest for targeting the crosstalk between BM-MSC and malignant B cells.
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http://dx.doi.org/10.1182/blood.2020008791DOI Listing
February 2021

CRISPR screens identify tumor-promoting genes conferring melanoma cell plasticity and resistance.

EMBO Mol Med 2021 May 16;13(5):e13466. Epub 2021 Mar 16.

CNRS, IGDR (Institut de génétique et développement de Rennes)-UMR 6290, Univ Rennes, Rennes, France.

Most genetic alterations that drive melanoma development and resistance to targeted therapy have been uncovered. In contrast, and despite their increasingly recognized contribution, little is known about the non-genetic mechanisms that drive these processes. Here, we performed in vivo gain-of-function CRISPR screens and identified SMAD3, BIRC3, and SLC9A5 as key actors of BRAFi resistance. We show that their expression levels increase during acquisition of BRAFi resistance and remain high in persister cells and during relapse. The upregulation of the SMAD3 transcriptional activity (SMAD3-signature) promotes a mesenchymal-like phenotype and BRAFi resistance by acting as an upstream transcriptional regulator of potent BRAFi-resistance genes such as EGFR and AXL. This SMAD3-signature predicts resistance to both current melanoma therapies in different cohorts. Critically, chemical inhibition of SMAD3 may constitute amenable target for melanoma since it efficiently abrogates persister cells survival. Interestingly, decrease of SMAD3 activity can also be reached by inhibiting the Aryl hydrocarbon Receptor (AhR), another druggable transcription factor governing SMAD3 expression level. Our work highlights novel drug vulnerabilities that can be exploited to develop long-lasting antimelanoma therapies.
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http://dx.doi.org/10.15252/emmm.202013466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8103100PMC
May 2021

Integrative genomics highlights opportunities for innovative therapies targeting the tumor microenvironment in gallbladder cancer.

J Hepatol 2021 May 16;74(5):1018-1020. Epub 2021 Feb 16.

Inserm, Univ Rennes 1, UMR_S 1242, COSS (Chemistry, Oncogenesis Stress Signaling), Centre de Lutte contre le Cancer Eugène Marquis, Rennes, France. Electronic address:

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http://dx.doi.org/10.1016/j.jhep.2020.12.024DOI Listing
May 2021

Targeting the tumor microenvironment in cholangiocarcinoma: implications for therapy.

Expert Opin Ther Targets 2021 Feb 7;25(2):153-162. Epub 2021 Feb 7.

Inserm, Univ Rennes 1, COSS (Chemistry, Oncogenesis Stress Signaling), UMR_S 1242, Centre De Lutte Contre Le Cancer Eugène Marquis, Rennes, France.

: Cholangiocarcinoma (CCA) is a rare, deadly cancer that is characterized by an abundant desmoplastic stroma. Late diagnoses and limited available effective treatments are major problems with this malignancy. Targeting of the tumor microenvironment (TME) has emerged as a potential therapeutic strategy.: In this review, we describe the role of the various compartments of the TME in CCA and focus on the preclinical rationale for the development of innovative therapies. Relevant literature was identified by a PubMed search covering the last decade (2010-2020).: Low efficacy of surgery and cytotoxic chemotherapy emphasizes the need for new therapeutic strategies and companion biomarkers. Single-cell RNA sequencing of the stroma is yielding a critical functional characterization of TME in CCA and is paving the way for immunotherapies and cancer-associated fibroblast and extracellular matrix-oriented treatments. We believe that the development of treatments targeting the components of the TME will produce the best results if in combination with cytotoxic chemotherapy. Biomarkers should be developed to define the patient population of interest for each combination strategy.
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http://dx.doi.org/10.1080/14728222.2021.1882998DOI Listing
February 2021

Long Noncoding RNAs in Cholangiocarcinoma.

Hepatology 2021 Mar 15;73(3):1213-1226. Epub 2021 Feb 15.

Inserm, Univ Rennes, NuMeCan (Nutrition Metabolisms and Cancer), UMR_S 1241, CHU Rennes, F-35000, Rennes, France.

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http://dx.doi.org/10.1002/hep.31534DOI Listing
March 2021

Cholangiocarcinoma 2020: the next horizon in mechanisms and management.

Nat Rev Gastroenterol Hepatol 2020 09 30;17(9):557-588. Epub 2020 Jun 30.

National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, "Instituto de Salud Carlos III"), San Sebastian, Spain.

Cholangiocarcinoma (CCA) includes a cluster of highly heterogeneous biliary malignant tumours that can arise at any point of the biliary tree. Their incidence is increasing globally, currently accounting for ~15% of all primary liver cancers and ~3% of gastrointestinal malignancies. The silent presentation of these tumours combined with their highly aggressive nature and refractoriness to chemotherapy contribute to their alarming mortality, representing ~2% of all cancer-related deaths worldwide yearly. The current diagnosis of CCA by non-invasive approaches is not accurate enough, and histological confirmation is necessary. Furthermore, the high heterogeneity of CCAs at the genomic, epigenetic and molecular levels severely compromises the efficacy of the available therapies. In the past decade, increasing efforts have been made to understand the complexity of these tumours and to develop new diagnostic tools and therapies that might help to improve patient outcomes. In this expert Consensus Statement, which is endorsed by the European Network for the Study of Cholangiocarcinoma, we aim to summarize and critically discuss the latest advances in CCA, mostly focusing on classification, cells of origin, genetic and epigenetic abnormalities, molecular alterations, biomarker discovery and treatments. Furthermore, the horizon of CCA for the next decade from 2020 onwards is highlighted.
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http://dx.doi.org/10.1038/s41575-020-0310-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7447603PMC
September 2020

Molecular profiling of stroma highlights stratifin as a novel biomarker of poor prognosis in pancreatic ductal adenocarcinoma.

Br J Cancer 2020 07 7;123(1):72-80. Epub 2020 May 7.

Univ Rennes, Inserm, Inra, CHU Rennes, Service de Chirurgie Hépatobiliaire et Digestive, Institut NuMeCan (Nutrition Metabolisms and Cancer), UMR_S 1241, Rennes, France.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer worldwide, as a result of a late diagnosis and limited therapeutic options. Tumour microenvironment (or stroma) plays a key role in cancer onset and progression and constitutes an intrinsic histological hallmark of PDAC. Thus we hypothesised that relevant prognostic biomarkers and therapeutic targets can be identified in the stroma.

Methods: Laser microdissection of the stroma from freshly frozen PDAC was combined to gene expression profiling. Protein expression of candidate biomarkers was evaluated by immunohistochemistry on tissue microarrays (n = 80 tumours) and by ELISA in plasma samples (n = 51 patients).

Results: A signature made of 1256 genes that significantly discriminate the stroma from the non-tumour fibrous tissue was identified. Upregulated genes were associated with inflammation and metastasis processes and linked to NF-Kappa B and TGFβ pathways. TMA analysis validated an increased expression of SFN, ADAMTS12 and CXCL3 proteins in the stroma of PDAC. Stromal expression of SFN was further identified as an independent prognostic factor of overall (p = 0.003) and disease-free survival (DFS) (p = 0.034). SFN plasma expression was significantly associated with reduced DFS (p = 0.006).

Conclusions: We demonstrated that gene expression changes within the stroma of PDAC correlate with tumour progression, and we identified Stratifin as a novel independent prognostic biomarker.
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http://dx.doi.org/10.1038/s41416-020-0863-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341840PMC
July 2020

A novel immunosuppression-based classification of liver tumors opens new perspectives for adapted therapeutic strategies.

EBioMedicine 2020 Apr 5;54:102737. Epub 2020 Apr 5.

Inserm, CHU Pontchaillou, 2 rue Henri Le Guilloux, F-35033 Rennes, France. Electronic address:

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http://dx.doi.org/10.1016/j.ebiom.2020.102737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136603PMC
April 2020

Molecular classification of cholangiocarcinoma.

Curr Opin Gastroenterol 2020 03;36(2):57-62

Inserm, Univ Rennes, Rennes, France.

Purpose Of Review: Cholangiocarcinoma (CCA) are heterogeneous tumors that arise from the malignant transformation of cholangiocytes along the biliary tree. CCA heterogeneity occurs at multiple levels and results in resistance to therapy and poor prognosis. Here, we review the molecular classification of CCA by focusing on the latest progresses based on genetic, epigenetic, transcriptomic and proteomic profiles. In addition, we introduce the emerging field of radiogenomics.

Recent Findings: Genome-wide integrative omics approaches have been widely reported by using large cohorts of CCA patients. Morphomolecular correlations have been established, including enrichment of FGFR2 gene fusions and IDH1/2 mutations in iCCA. A specific IDH mutant iCCA subtype displays high mitochondrial and low chromatin modifier expression linked to ARID1A promoter hypermethylation. Examples of translation of these classifications for the management of CCA have also been reported, with prediction of drug efficacy based on genetic alterations.

Summary: Although there is currently no international consensus on CCA morphomolecular classification, the recent initiatives developed under the umbrella of The European Network for the Study of Cholangiocarcinoma (ENSCCA) should favor new collaborative research. Identifying distinct molecular subgroups and developing appropriate targeted therapies will improve the clinical outcome of patients with CCA.
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http://dx.doi.org/10.1097/MOG.0000000000000611DOI Listing
March 2020

Exosomal circRNAs: new players in the field of cholangiocarcinoma.

Clin Sci (Lond) 2019 11;133(21):2239-2244

Inserm, Univ Rennes, UMR_S 1241, 35033, Rennes, France.

Cholangiocarcinoma (CCA) is a deadly cancer worldwide associated with limited therapeutic options. A recent study published in Clinical Science by Wang and colleagues [Clin. Sci. (2019) 133(18), 1935-1953] brought new perspectives to CCA management and therapy by focusing on circular RNAs (circRNAs). CircRNAs belong to an emerging class of functional non-coding RNAs (ncRNAs) regulating numerous biological processes. Notably, circRNAs have been associated with cancer onset and progression, although reports in CCA are very limited so far. In this work, the expression of circular RNA circ-0000284 (aka circHIPK3) was specifically elevated in CCA cell lines, human tumor tissues and plasma exosomes. Gain and loss of function approaches were performed to better understand the molecular mechanisms regulated by circ-0000284. Notably, the authors evaluated the role of circ-0000284 as a microRNA (miRNA) sponge. By prediction analysis and functional tests, a direct interaction was demonstrated with miR-637 that targets lymphocyte antigen-6 E (LY6E). Increased expression of circ-0000284 was associated with enhanced migration, invasion and proliferation of CCA cell lines. Interestingly, exosomal-mediated circ-0000284 was reported to exhibit pro-oncogenic effects on surrounding normal cells. Altogether, these data highlight circRNAs not only as new players in CCA pathogenesis but also as promising molecules for innovative non-invasive biomarkers, as circRNAs are enriched and stable in exosomes. Further investigations on extracellular vesicles should provide the necessary tools to improve CCA diagnosis, and move toward targeted-therapies.
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http://dx.doi.org/10.1042/CS20190940DOI Listing
November 2019

Transforming Growth Factor-Beta (TGFβ) Signaling Pathway in Cholangiocarcinoma.

Cells 2019 08 23;8(9). Epub 2019 Aug 23.

Inserm, Univ Rennes, Inra, Institut NuMeCan (Nutrition Metabolisms and Cancer), UMR_S 1241, 35033 Rennes, France.

Cholangiocarcinoma is a deadly cancer worldwide, associated with a poor prognosis and limited therapeutic options. Although cholangiocarcinoma accounts for less than 15% of liver primary cancer, its silent nature restricts early diagnosis and prevents efficient treatment. Therefore, it is of clinical relevance to better understand the molecular basis of cholangiocarcinoma, including the signaling pathways that contribute to tumor onset and progression. In this review, we discuss the genetic, molecular, and environmental factors that promote cholangiocarcinoma, emphasizing the role of the transforming growth factor β (TGFβ) signaling pathway in the progression of this cancer. We provide an overview of the physiological functions of TGFβ signaling in preserving liver homeostasis and describe how advanced cholangiocarcinoma benefits from the tumor-promoting effects of TGFβ. Moreover, we report the importance of noncoding RNAs as effector molecules downstream of TGFβ during cholangiocarcinoma progression, and conclude by highlighting the need for identifying novel and clinically relevant biomarkers for a better management of patients with cholangiocarcinoma.
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http://dx.doi.org/10.3390/cells8090960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770250PMC
August 2019

Moderate chronic ethanol consumption exerts beneficial effects on nonalcoholic fatty liver in mice fed a high-fat diet: possible role of higher formation of triglycerides enriched in monounsaturated fatty acids.

Eur J Nutr 2020 Jun 3;59(4):1619-1632. Epub 2019 Jun 3.

Univ Rennes, Inserm, Inra, Institut NUMECAN (Nutrition Metabolisms and Cancer)-UMR_S 1241, UMR_A 1341, 35000, Rennes, France.

Purpose: Several clinical studies suggested that light-to-moderate alcohol intake could alleviate nonalcoholic fatty liver disease (NAFLD), but the underlying mechanism is still poorly understood.

Methods: Mice fed a high-fat diet (HFD) were submitted or not to moderate ethanol intake for 3 months (ca. 10 g/kg/day) via drinking water. Biochemical, analytical and transcriptomic analyses were performed in serum and liver.

Results: Serum ethanol concentrations in ethanol-treated HFD mice comprised between 0.5 and 0.7 g/l throughout the experiment. NAFLD improvement was observed in ethanol-treated HFD mice as assessed by reduced serum transaminase activity. This was associated with less microvesicular and more macrovacuolar steatosis, the absence of apoptotic hepatocytes and a trend towards less fibrosis. Liver lipid analysis showed increased amounts of fatty acids incorporated in triglycerides and phospholipids, reduced proportion of palmitic acid in total lipids and higher desaturation index, thus suggesting enhanced stearoyl-coenzyme A desaturase activity. mRNA expression of several glycolytic and lipogenic enzymes was upregulated. Genome-wide expression profiling and gene set enrichment analysis revealed an overall downregulation of the expression of genes involved in collagen fibril organization and leukocyte chemotaxis and an overall upregulation of the expression of genes involved in oxidative phosphorylation and mitochondrial respiratory chain complex assembly. In addition, mRNA expression of several proteasome subunits was upregulated in ethanol-treated HFD mice.

Conclusions: Moderate chronic ethanol consumption may alleviate NAFLD by several mechanisms including the generation of non-toxic lipid species, reduced expression of profibrotic and proinflammatory genes, restoration of mitochondrial function and possible stimulation of proteasome activity.
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http://dx.doi.org/10.1007/s00394-019-02017-1DOI Listing
June 2020

Expression of long non-coding RNA ANRIL predicts a poor prognosis in intrahepatic cholangiocarcinoma.

Dig Liver Dis 2019 09 27;51(9):1337-1343. Epub 2019 Apr 27.

Inserm, Univ Rennes, Inra, Institut NuMeCan (Nutrition Metabolisms and Cancer), CHU Rennes, Centre de Lutte contre le Cancer Eugène Marquis, Rennes, France. Electronic address:

Background: Intrahepatic cholangiocarcinoma (iCCA) is a deadly cancer worldwide associated with an increased incidence, limited therapeutic options and absence of reliable prognostic biomarkers. Long non-coding RNAs (lncRNA) emerge as relevant biomarkers in cancer being associated with tumor progression. However, lncRNA have been poorly investigated in iCCA.

Aim: To identify lncRNA significantly associated with the survival of patients with iCCA after tumor resection for curative intent.

Methods: Gene expression profiling and Q-RT-PCR were performed from a cohort of 39 clinically well-annotated iCCA. Univariate Cox proportional hazards model with Wald Statistic was used to identify lncRNA significantly associated with overall (OS) and/or disease-free (DFS) survival.

Results: A signature made of 9 lncRNA was identified to be significantly (P < 0.05) associated with OS and DFS, including 4 lncRNA (lnc-CDK9-1, XLOC_l2_009441, CDKN2B-AS1, HOXC13-AS) highly expressed in poor prognosis iCCA and 5 lncRNA (lnc-CCHCR1-1, lnc-AF131215.3.1, lnc-CBLB-5, COL18A1-AS2, lnc-RELL2-1) highly expressed in better prognosis iCCA. We further validated CDKN2B-AS1 (ANRIL) as a poor prognosis biomarker, not only in iCCA, but also in hepatocellular carcinoma, kidney renal clear cell carcinoma and uterine corpus endometrial carcinoma.

Conclusions: We report a prognosis lncRNA signature in iCCA and the clinical relevance of CDKN2B-AS1 (ANRIL) overexpression in several cancers.
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http://dx.doi.org/10.1016/j.dld.2019.03.019DOI Listing
September 2019

Signalling networks in cholangiocarcinoma: Molecular pathogenesis, targeted therapies and drug resistance.

Liver Int 2019 05;39 Suppl 1:43-62

Inserm, Univ Rennes, Inra, Institut NuMeCan (Nutrition Metabolisms and Cancer), Rennes, France.

Cholangiocarcinoma (CCA) is a deadly disease. While surgery may attain cure in a minor fraction of cases, therapeutic options in either the adjuvant or advanced setting are limited. The possibility of advancing the efficacy of therapeutic approaches to CCA relies on understanding its molecular pathogenesis and developing rational therapies aimed at interfering with oncogenic signalling networks that drive and sustain cholangiocarcinogenesis. These efforts are complicated by the intricate biology of CCA, which integrates not only the driving force of tumour cell-intrinsic alterations at the genetic and epigenetic level but also pro-tumorigenic cues conveyed to CCA cells by different cell types present in the rich tumour stroma. Herein, we review our current understanding of the mechanistic bases underpinning the activation of major oncogenic pathways causative of CCA pathogenesis. We subsequently discuss how this knowledge is being exploited to implement rationale-based and genotype-matched therapeutic approaches that predictably will radically transform CCA clinical management in the next decade. We conclude by highlighting the mechanisms of therapeutic resistance in CCA and reviewing innovative approaches to combat resistance at the preclinical and clinical level.
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http://dx.doi.org/10.1111/liv.14102DOI Listing
May 2019

GDF11 exhibits tumor suppressive properties in hepatocellular carcinoma cells by restricting clonal expansion and invasion.

Biochim Biophys Acta Mol Basis Dis 2019 06 16;1865(6):1540-1554. Epub 2019 Mar 16.

Departamento de Ciencias de la Salud, Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, Mexico; Laboratorio de Medicina Experimental, Unidad de Medicina Translacional, Instituto de Investigaciones Biomédicas, UNAM/Instituto Nacional de Cardiología Ignacio Chavez, Mexico City, Mexico. Electronic address:

Growth differentiation factor 11 (GDF11) has been characterized as a key regulator of differentiation in cells that retain stemness features, despite some controversies in age-related studies. GDF11 has been poorly investigated in cancer, particularly in those with stemness capacity, such as hepatocellular carcinoma (HCC), one of the most aggressive cancers worldwide. Here, we focused on investigating the effects of GDF11 in liver cancer cells. GDF11 treatment significantly reduced proliferation, colony and spheroid formation in HCC cell lines. Consistently, down-regulation of CDK6, cyclin D1, cyclin A, and concomitant upregulation of p27 was observed after 24 h of treatment. Interestingly, cell viability was unchanged, but cell functionality was compromised. These effects were potentially induced by the expression of E-cadherin and occludin, as well as Snail and N-cadherin repression, in a time-dependent manner. Furthermore, GDF11 treatment for 72 h induced that cells were incapable of sustaining colony and sphere capacity in the absent of GDF11, up to 5 days, indicating that the effect of GDF11 on self-renewal capacity is not transient. Finally, in vivo invasion studies revealed a significant decrease in cell migration of hepatocellular carcinoma cells treated with GDF11 associated to a decreased proliferation judged by Ki67 staining. Data show that exogenous GDF11 displays tumor suppressor properties in HCC cells.
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http://dx.doi.org/10.1016/j.bbadis.2019.03.003DOI Listing
June 2019

The bidirectional crosstalk between metastatic uveal melanoma cells and hepatic stellate cells engenders an inflammatory microenvironment.

Exp Eye Res 2019 04 13;181:213-222. Epub 2019 Feb 13.

Inserm, Université de Rennes 1, UMR 1241, Nutrition, Métabolismes et Cancer (NuMeCan), Rennes, France; Département d'ophtalmologie, Faculté de Médecine, Université Laval, Québec, Canada; Centre Universitaire d'ophtalmologie-Recherche and Axe Médecine Régénératrice, Centre de Recherche du CHU de Québec-Université Laval, Québec, Canada; Centre de Recherche en Organogénèse Expérimentale de l'Université Laval/LOEX, Québec, Canada; Service d'ophtalmologie, CHU de Rennes, Rennes, France. Electronic address:

Uveal melanoma is the most common primary ocular neoplasm in adults. It is peculiar for its hematogenous dissemination and its high propensity to spread to the liver. Current treatments rarely prolong patient survival. We hypothesized that metastatic uveal melanoma cells modulate the function of surrounding hepatic stellate cells to facilitate their own growth and survival. This study was conducted to investigate the role of the hepatic microenvironment on uveal melanoma aggressiveness. We demonstrated that the paracrine signaling of surrounding hepatic stellate cells have more transcriptional impact on metastatic uveal melanoma cells. Upregulated transcripts were linked to inflammation and included several interleukins. The uveal melanoma-stellate cell crosstalk induced as well the expression of transmembrane integrins. In addition, the interleukin-6 receptor inhibitor Tocilizumab did not reduce the growth of uveal melanoma cells. Our results provide evidence that inflammatory mediators are key players in the homing of uveal melanoma cells to the liver. The bidirectional crosstalk between uveal melanoma cells and hepatic stellate cells involved pro-fibrogenic interleukins. The inflammatory characteristics of the metastatic microenvironment might offer relevant therapeutic opportunities in uveal melanoma.
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http://dx.doi.org/10.1016/j.exer.2019.02.012DOI Listing
April 2019

SOX9 expression decreases survival of patients with intrahepatic cholangiocarcinoma by conferring chemoresistance.

Br J Cancer 2018 11 13;119(11):1358-1366. Epub 2018 Nov 13.

Department of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.

Background: Sex-determining region Y-box (SRY-box) containing gene 9 (SOX9) expression confers cancer stem cell features. However, SOX9 function in intrahepatic cholangiocarcinoma (iCCA) is unknown. This study investigated the effects and underlying mechanisms of SOX9 in iCCA.

Methods: SOX9 expression in 59 iCCA patients was examined by immunohistochemistry. The association between SOX9 expression and clinical outcome was evaluated. Gene signature and biological functions of SOX9 in iCCA were examined in vitro.

Results: iCCA patients with high SOX9 expression had shorter survival time than those with low SOX9. In patients receiving chemotherapy, median survival time in patients with low and high levels of SOX9 were 62 and 22 months, respectively. In vitro, gemcitabine increased SOX9 expression in iCCA cells. When SOX9 was knocked down, gemcitabine-induced apoptosis was markedly increased. Silencing SOX9 significantly inhibited gemcitabine-induced phosphorylation of checkpoint kinase 1, a key cell cycle checkpoint protein that coordinates the DNA damage response and inhibited the expression of multidrug resistance genes. Microarray analyses showed that SOX9 knockdown in CCA cells altered gene signatures associated with multidrug resistance and p53 signalling.

Conclusions: SOX9 governs the response of CCA cells to chemotherapy. SOX9 is a biomarker to select iCCA patients eligible for efficient chemotherapy.
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http://dx.doi.org/10.1038/s41416-018-0338-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265288PMC
November 2018

Transforming Growth Factor-β and Axl Induce CXCL5 and Neutrophil Recruitment in Hepatocellular Carcinoma.

Hepatology 2019 01 20;69(1):222-236. Epub 2018 Dec 20.

Department of Medicine I, Institute of Cancer Research, Comprehensive Cancer Center Vienna, Medical University of Vienna, Vienna, Austria.

Transforming growth factor (TGF)-β suppresses early hepatocellular carcinoma (HCC) development but triggers pro-oncogenic abilities at later stages. Recent data suggest that the receptor tyrosine kinase Axl causes a TGF-β switch toward dedifferentiation and invasion of HCC cells. Here, we analyzed two human cellular HCC models with opposing phenotypes in response to TGF-β. Both HCC models showed reduced proliferation and clonogenic growth behavior following TGF-β stimulation, although they exhibited differences in chemosensitivity and migratory abilities, suggesting that HCC cells evade traits of anti-oncogenic TGF-β. Transcriptome profiling revealed differential regulation of the chemokine CXCL5, which positively correlated with TGF-β expression in HCC patients. The expression and secretion of CXCL5 was dependent on Axl expression, suggesting that CXCL5 is a TGF-β target gene collaborating with Axl signaling. Loss of either TGF-β or Axl signaling abrogated CXCL5-dependent attraction of neutrophils. In mice, tumor formation of transplanted HCC cells relied on CXCL5 expression. In HCC patients, high levels of Axl and CXCL5 correlated with advanced tumor stages, recruitment of neutrophils into HCC tissue, and reduced survival. Conclusion: The synergy of TGF-β and Axl induces CXCL5 secretion, causing the infiltration of neutrophils into HCC tissue. Intervention with TGF-β/Axl/CXCL5 signaling may be an effective therapeutic strategy to combat HCC progression in TGF-β-positive patients.
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http://dx.doi.org/10.1002/hep.30166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590451PMC
January 2019

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

Clin Cancer Res 2018 09 1;24(17):4282-4296. Epub 2018 May 1.

Sorbonne University, INSERM, Saint-Antoine Research Center (CRSA), Paris, France.

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. 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 , and tumor growth was evaluated in CCA xenograft models. 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. , 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. 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. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-3725DOI Listing
September 2018

Co-exposure to benzo[a]pyrene and ethanol induces a pathological progression of liver steatosis in vitro and in vivo.

Sci Rep 2018 04 13;8(1):5963. Epub 2018 Apr 13.

Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) - UMR_S 1085, F-35000, Rennes, France.

Hepatic steatosis (i.e. lipid accumulation) and steatohepatitis have been related to diverse etiologic factors, including alcohol, obesity, environmental pollutants. However, no study has so far analyzed how these different factors might interplay regarding the progression of liver diseases. The impact of the co-exposure to the environmental carcinogen benzo[a]pyrene (B[a]P) and the lifestyle-related hepatotoxicant ethanol, was thus tested on in vitro models of steatosis (human HepaRG cell line; hybrid human/rat WIF-B9 cell line), and on an in vivo model (obese zebrafish larvae). Steatosis was induced prior to chronic treatments (14, 5 or 7 days for HepaRG, WIF-B9 or zebrafish, respectively). Toxicity and inflammation were analyzed in all models; the impact of steatosis and ethanol towards B[a]P metabolism was studied in HepaRG cells. Cytotoxicity and expression of inflammation markers upon co-exposure were increased in all steatotic models, compared to non steatotic counterparts. A change of B[a]P metabolism with a decrease in detoxification was detected in HepaRG cells under these conditions. A prior steatosis therefore enhanced the toxicity of B[a]P/ethanol co-exposure in vitro and in vivo; such a co-exposure might favor the appearance of a steatohepatitis-like state, with the development of inflammation. These deleterious effects could be partly explained by B[a]P metabolism alterations.
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http://dx.doi.org/10.1038/s41598-018-24403-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899096PMC
April 2018

AXIN deficiency in human and mouse hepatocytes induces hepatocellular carcinoma in the absence of β-catenin activation.

J Hepatol 2018 06 7;68(6):1203-1213. Epub 2018 Mar 7.

INSERM, U1016, Institut Cochin, F-75014 Paris, France; CNRS, UMR8104, F-75014 Paris, France; Université Paris Descartes, F-75014 Paris, France; Equipe labellisée LNCC, France; Centre de Recherche sur l'Inflammation-Inserm UMR 1149-Université Paris Diderot, Paris, France. Electronic address:

Background & Aims: The Wnt/β-catenin pathway is the most frequently deregulated pathway in hepatocellular carcinoma (HCC). Inactivating mutations of the gene encoding AXIN1, a known negative regulator of the Wnt/β-catenin signaling pathway, are observed in about 10% of HCCs. Whole-genome studies usually place HCC with AXIN1 mutations and CTNNB1 mutations in the group of tumors with Wnt/β-catenin activated program. However, it has been shown that HCCs with activating CTNNB1 mutations form a group of HCCs, with a different histology, prognosis and genomic signature to those with inactivating biallelic AXIN1 mutations. We aimed to elucidate the relationship between CTNNB1 mutations, AXIN1 mutations and the activation level of the Wnt/β-catenin program.

Methods: We evaluated two independent human HCC datasets for the expression of a 23-β-catenin target genes program. We modeled Axin1 loss of function tumorigenesis in two engineered mouse models and performed gene expression profiling.

Results: Based on gene expression, we defined three levels of β-catenin program activation: strong, weak or no activation. While more than 80% CTNNB1-mutated tumors were found in the strong or in the weak activation program, most of the AXIN1-mutated tumors (>70%) were found in the subgroup with no activation. We validated this result by demonstrating that mice with a hepatocyte specific AXIN1 deletion developed HCC in the absence of β-catenin induction. We defined a 329-gene signature common in human and mouse AXIN1 mutated HCC that is highly enriched in Notch and YAP oncogenic signatures.

Conclusions: AXIN1-mutated HCCs occur independently of the Wnt/β-catenin pathway and involve Notch and YAP pathways. These pathways constitute potentially interesting targets for the treatment of HCC caused by AXIN1 mutations.

Lay Summary: Liver cancer has a poor prognosis. Defining the molecular pathways involved is important for developing new therapeutic approaches. The Wnt/β-catenin pathway is the most frequently deregulated pathway in hepatocellular carcinoma (HCC). Mutations of AXIN1, a member of this pathway, represent about 10% of HCC mutations. Using both human HCC collections and engineered mouse models of liver cancers with AXIN1 mutation or deletion, we defined a common signature of liver tumors mutated for AXIN1 and demonstrate that these tumors occur independently of the activation of the Wnt/β-catenin pathway.
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http://dx.doi.org/10.1016/j.jhep.2017.12.018DOI Listing
June 2018

A novel transforming growth factor beta-induced long noncoding RNA promotes an inflammatory microenvironment in human intrahepatic cholangiocarcinoma.

Hepatol Commun 2018 03 30;2(3):254-269. Epub 2018 Jan 30.

Institut National de la Santé et de la Recherche Médicale, INRA, Université de Rennes, CHU Rennes, UMR 1241, Nutrition Metabolisms and Cancer, Service de Chirurgie Hépatobiliaire et Digestive, Biosit, Biogenouest, Core Facility H2P2 and CRB Santé Rennes France.

Intrahepatic cholangiocarcinoma (iCCA) is a deadly liver primary cancer associated with poor prognosis and limited therapeutic opportunities. Active transforming growth factor beta (TGFβ) signaling is a hallmark of the iCCA microenvironment. However, the impact of TGFβ on the transcriptome of iCCA tumor cells has been poorly investigated. Here, we have identified a specific TGFβ signature of genes commonly deregulated in iCCA cell lines, namely HuCCT1 and Huh28. Novel coding and noncoding TGFβ targets were identified, including a TGFβ-induced long noncoding RNA (TLINC), formerly known as cancer susceptibility candidate 15 (CASC15). TLINC is a general target induced by TGFβ in hepatic and nonhepatic cell types. In iCCA cell lines, the expression of a long and short TLINC isoform was associated with an epithelial or mesenchymal phenotype, respectively. Both isoforms were detected in the nucleus and cytoplasm. The long isoform of TLINC was associated with a migratory phenotype in iCCA cell lines and with the induction of proinflammatory cytokines, including interleukin 8, both and in resected human iCCA. TLINC was also identified as a tumor marker expressed in both epithelial and stroma cells. In nontumor livers, TLINC was only expressed in specific portal areas with signs of ductular reaction and inflammation. Finally, we provide experimental evidence of circular isoforms of TLINC, both in iCCA cells treated with TGFβ and in resected human iCCA. : We identify a novel TGFβ-induced long noncoding RNA up-regulated in human iCCA and associated with an inflammatory microenvironment. ( 2018;2:254-269).
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http://dx.doi.org/10.1002/hep4.1142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831019PMC
March 2018

Landscape of genomic alterations in hepatocellular carcinoma: current knowledge and perspectives for targeted therapies.

Hepatobiliary Surg Nutr 2017 Dec;6(6):404-407

Inserm, Inra, Univ Rennes, UMR 1241, Nutrition Metabolisms and Cancer, Rennes, France.

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http://dx.doi.org/10.21037/hbsn.2017.10.02DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5756761PMC
December 2017

Local Anesthetics Inhibit the Growth of Human Hepatocellular Carcinoma Cells.

Anesth Analg 2017 11;125(5):1600-1609

From the *INSERM, UMR 991, and Université de Rennes 1, Rennes, France; †CHU Rennes, Pôle Anesthésie et Réanimation, Inserm CIC 1414, Rennes, France; and ‡CHU Rennes, Clinical Pharmacology Department and Inserm CIC 1414, Université de Rennes 1, Rennes, France.

Background: Hepatocellular carcinoma (HCC) is an aggressive cancer with limited therapeutic options. Retrospective studies have shown that the administration of local anesthetics (LAs) during cancer surgery could reduce cancer recurrence. Besides, experimental studies reported that LAs could inhibit the growth of cancer cells. Thus, the purpose of this study was to investigate the effects of LAs on human HCC cells.

Methods: The effects of 2 LAs (lidocaine and ropivacaine) (10 to 10 M) were studied after an incubation of 48 hours on 2 HCC cell lines, namely HuH7 and HepaRG. Cell viability, cell cycle analysis, and apoptosis and senescence tests were performed together with unsupervised genome-wide expression profiling and quantitative real-time polymerase chain reaction for relevant genes.

Results: We showed that LAs decreased viability and proliferation of HuH7 cells (from 92% [P < .001] at 5 × 10 M to 40% [P = .02] at 10 M with ropivacaine and from 87% [P < .001] to 37% [P = .02] with lidocaine) and HepaRG progenitor cells (from 58% at 5 × 10 M [P < .001] to 29% at 10 M [P = .04] with lidocaine and 59% [P < .001] with ropivacaine 5 × 10 M) in concentration-dependent manner. LAs have no effect on well-differentiated HepaRG. Ropivacaine decreased the mRNA level of key cell cycle regulators, namely cyclin A2, cyclin B1, cyclin B2, and cyclin-dependent kinase 1, and the expression of the nuclear marker of cell proliferation MKI67. Lidocaine had no specific effect on cell cycle but increased by 10× the mRNA level of adenomatous polyposis coli (P < .01), which acts as an antagonist of the Wnt/β-catenin pathway. Both LAs increased apoptosis in Huh7 and HepaRG progenitor cells (P < .01).

Conclusions: The data demonstrate that LAs induced profound modifications in gene expression profiles of tumor cells, including modulations in the expression of cell cycle-related genes that result in a cytostatic effect and induction of apoptosis.
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http://dx.doi.org/10.1213/ANE.0000000000002429DOI Listing
November 2017

The release of pro-inflammatory cytokines is mediated via mitogen-activated protein kinases rather than by the inflammasome signalling pathway in keratinocytes.

Clin Exp Pharmacol Physiol 2017 Jul;44(7):827-838

MATRISCIENCE SAS, Paris Santé Cochin, Paris, France.

Toll-like receptors (TLRs) are expressed in the skin and airway epithelial tissues, which are the most important sites of host-pathogen interactions. TLRs recognize the 3-D structures of pathogen-associated molecules and are therefore useful markers of the innate immune response. Here, we investigated the role of lipopolysaccharides and monosodium urate (MSU) crystals in the activation of the TLR and NOD-like receptor (NLR) pathways in human keratinocytes. Analysis of the inflammasome compounds revealed that NOD-like receptor P3 and TLR4, both of which are components of inflammasome complexes involved in the activation of interleukin (IL)-1β, were not expressed in keratinocytes. Transcriptomic analysis showed that the combination of MSU and lipopolysaccharide priming did not elicit significant results compared to MSU treatment, which induced the expression of TLR2, IL-6 and IL-8/chemokine (C-X-C motif) ligand 8 CXCL8 in the keratinocyte cell line HaCaT. Furthermore, MSU promoted the phosphorylation of extracellular signal-regulated kinase 1/2 and MAPK14/p38α mitogen-activated protein kinases. We concluded that MSU stimulates a pro-inflammatory response in keratinocytes via mitogen-activated protein kinase pathway to induce production of IL-8/CXCL8 chemokine (C-X-C motif) ligand 8 and TLR2.
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http://dx.doi.org/10.1111/1440-1681.12765DOI Listing
July 2017

Integrative Genomic Analysis Identifies the Core Transcriptional Hallmarks of Human Hepatocellular Carcinoma.

Cancer Res 2016 11 12;76(21):6374-6381. Epub 2016 Sep 12.

INSERM, UMR 991, Liver Metabolisms and Cancer, University of Rennes, Rennes, France.

Integrative genomics helped characterize molecular heterogeneity in hepatocellular carcinoma (HCC), leading to targeted drug candidates for specific HCC subtypes. However, no consensus was achieved for genes and pathways commonly altered in HCC. Here, we performed a meta-analysis of 15 independent datasets (n = 784 human HCC) and identified a comprehensive signature consisting of 935 genes commonly deregulated in HCC as compared with the surrounding nontumor tissue. In the HCC signature, upregulated genes were linked to early genomic alterations in hepatocarcinogenesis, particularly gains of 1q and 8q. The HCC signature covered well-established cancer hallmarks, such as proliferation, metabolic reprogramming, and microenvironment remodeling, together with specific hallmarks associated with protein turnover and epigenetics. Subsequently, the HCC signature enabled us to assess the efficacy of signature-relevant drug candidates, including histone deacetylase inhibitors that specifically reduced the viability of six human HCC cell lines. Overall, this integrative genomics approach identified cancer hallmarks recurrently altered in human HCC that may be targeted by specific drugs. Combined therapies targeting common and subtype-specific cancer networks may represent a relevant therapeutic strategy in liver cancer. Cancer Res; 76(21); 6374-81. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-16-1559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660733PMC
November 2016

De novo HAPLN1 expression hallmarks Wnt-induced stem cell and fibrogenic networks leading to aggressive human hepatocellular carcinomas.

Oncotarget 2016 Jun;7(26):39026-39043

Inserm, UMR991, Liver Metabolisms and Cancer, Rennes, France.

About 20% hepatocellular carcinomas (HCCs) display wild-type β-catenin, enhanced Wnt signaling, hepatocyte dedifferentiation and bad outcome, suggesting a specific impact of Wnt signals on HCC stem/progenitor cells. To study Wnt-specific molecular pathways, cell fates and clinical outcome, we fine-tuned Wnt/β-catenin signaling in liver progenitor cells, using the prototypical Wnt ligand Wnt3a. Cell biology assays and transcriptomic profiling were performed in HepaRG hepatic progenitors exposed to Wnt3a after β-catenin knockdown or Wnt inhibition with FZD8_CRD. Gene expression network, molecular pathology and survival analyses were performed on HCCs and matching non-tumor livers from 70 patients by real-time PCR and tissue micro-array-based immunohistochemistry. Wnt3a reprogrammed liver progenitors to replicating fibrogenic myofibroblast-like cells displaying stem and invasive features. Invasion was inhibited by 30 nM FZD7 and FZD8 CRDs. Translation of these data to human HCCs revealed two tight gene networks associating cell surface Wnt signaling, stem/progenitor markers and mesenchymal commitment. Both networks were linked by Hyaluronan And Proteoglycan Link Protein 1 (HAPLN1), that appeared de novo in aggressive HCCs expressing cytoplasmic β-catenin and stem cell markers. HAPLN1 was independently associated with bad overall and disease-free outcome. In vitro, HAPLN1 was expressed de novo in EPCAM¯/NCAM+ mesoderm-committed progenitors, upon spontaneous epithelial-mesenchymal transition and de-differentiation of hepatocyte-like cells to liver progenitors. In these cells, HAPLN1 knockdown downregulated key markers of mesenchymal cells, such as Snail, LGR5, collagen IV and α-SMA. In conclusion, HAPLN1 reflects a signaling network leading to stemness, mesenchymal commitment and HCC progression.
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http://dx.doi.org/10.18632/oncotarget.9346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129911PMC
June 2016

Cholangiocarcinoma stem-like subset shapes tumor-initiating niche by educating associated macrophages.

J Hepatol 2017 01 1;66(1):102-115. Epub 2016 Sep 1.

Center for Autoimmune Liver Diseases, Humanitas Clinical and Research Center, Rozzano, Italy; Program for Autoimmune Liver Diseases, International Center for Digestive Health, Department of Medicine and Surgery, University of Milan-Bicocca, Italy. Electronic address:

Background & Aims: A therapeutically challenging subset of cells, termed cancer stem cells (CSCs) are responsible for cholangiocarcinoma (CCA) clinical severity. Presence of tumor-associated macrophages (TAMs) has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche.

Methods: CCA cells were cultured in 3D conditions to generate spheres. CCA sphere analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed. The in vitro and in vivo effect of CCA spheres on macrophage precursors was tested after culturing healthy donor cluster of differentiation (CD)14 with CCA-sphere conditioned medium.

Results: CCA spheres engrafted in 100% of transplanted mice and revealed a significant 20.3-fold increase in tumor-initiating fraction (p=0.0011) and a sustained tumorigenic potential through diverse xenograft-generations. Moreover, CCA spheres were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. Next, fluorescence-activated cell sorting analysis showed that in the presence of CCA sphere conditioned medium, CD14 macrophages expressed key markers (CD68, CD115, human leukocyte antigen-D related, CD206) indicating that CCA sphere conditioned medium was a strong macrophage-activator. Gene expression profile of CCA sphere activated macrophages revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated macrophages from CCA resections recapitulated a similar molecular phenotype of in vitro-educated macrophages. Consistent with invasive features, the largest CD163 set was found in the tumor front of human CCA specimens (n=23) and correlated with a high level of serum cancer antigen 19.9 (n=17). Among mediators released by CCA spheres, only interleukin (IL)13, IL34 and osteoactivin were detected and further confirmed in CCA patient sera (n=12). Surprisingly, a significant association of IL13, IL34 and osteoactivin with sphere stem-like genes was provided by a CCA database (n=104). In vitro combination of IL13, IL34, osteoactivin was responsible for macrophage-differentiation and invasion, as well as for in vivo tumor-promoting effect.

Conclusion: CCA-CSCs molded a specific subset of stem-like associated macrophages thus providing a rationale for a synergistic therapeutic strategy for CCA-disease.

Lay Summary: Immune plasticity represents an important hallmark of tumor outcome. Since cancer stem cells are able to manipulate stromal cells to their needs, a better definition of the key dysregulated immune subtypes responsible for cooperating in supporting tumor initiation may facilitate the development of new therapeutic approaches. Considering that human cholangiocarcinoma represents a clinical emergency, it is essential to move to predictive models in order to understand the adaptive process of macrophage component (imprinting, polarization and maintenance) engaged by tumor stem-like compartment.
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http://dx.doi.org/10.1016/j.jhep.2016.08.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522599PMC
January 2017