[Role of endothelial cells in hepatocellular carcinoma associated with metabolic syndrome]

Le syndrome métabolique (SM) est un facteur de risque émergent de carcinome hépatocellulaire (CHC). Dans ce contexte, le CHC présente des particularités cliniques et morphologiques suggérant l’existence de mécanismes moléculaires spécifiques impliqués dans la cancérogenèse. Nous avons étudié le rôle de deux protéines dans le CHC associé au SM, une adipokine (FABP4, Fatty Acid Binding Protein 4) et une protéine de l’autophagie (ATG5). Nous avons montré que FABP4 était surexprimée dans les CHC/SM, principalement par les cellules endothéliales sinusoïdales péritumorales. La régulation endothéliale de FABP4 était médiée in vitro par le VEGF, l’insuline, le glucose, le TNFα, et l’hypoxie. FABP4 a des effets oncogéniques sur des lignées de CHC, effets annulés par son inhibiteur spécifique, le BMS309403. Nous avons identifié les microvésicules endothéliales comme vecteurs de FABP4 entre les cellules endothéliales et les hépatocytes tumoraux. In vivo, le traitement par le BMS309403 ralentissait la croissance tumorale des xénogreffes tumorales chez la souris nude BALB/c nourries avec un régime gras (RG). Dans un modèle expérimental d’invalidation des cellules endothéliales hépatiques d’ATG5 (KO ATG5), l’injection de diethylnitrosamine (carcinogène hépatique) a montré une progression tumorale accrue comparée aux souris contrôles. Dans le foie non tumoral (FNT) des souris KO ATG5, le nombre de cellules sénescentes était augmenté sans toutefois atteindre la significativité. L’analyse de prélèvements hépatiques provenant de patients avec SM montrait une surexpression de p62 (protéine de l’autophagie) dans les CHC, et une augmentation de la sénescence dans le FNT. Ce travail a mis en évidence la contribution des cellules endothéliales dans la carcinogenèse hépatique associée au SM par des mécanismes différents impliquant de nouveaux médiateurs (FABP4 et ATG5).Metabolic syndrome (MS) is a major risk factor for hepatocellular carcinoma (HCC). In this context, HCC display some peculiar features suggesting the involvement of specific molecular mechanisms of carcinogenesis. The aim of our study was to investigate the role of two proteins, an adipokine (FABP4, Fatty Acid Binding Protein 4) and a protein of autophagy (ATG5) in liver carcinogenesis associated with MS. We showed a FABP4 overexpression in HCC from patients with MS, mostly restricted to peritumoral sinusoidal endothelial cells. In vitro, FABP4 regulation in endothelial cells involved various mediators (insulin, glucose, TNFα, VEGF, hypoxia). We identified microvesicles as FABP4 carriers between endothelial and tumoral cells. FABP4 exerted oncogenic effects on hepatoma cell lines, increasing cell proliferation and migration, effects being reversed by a specific FABP4 inhibitor (BMS309403). In vivo, BMS309403 also significantly reduced tumour progression in heterotopic and orthotopic xenografted tumor cells in obese nude mice. In ATG5 liver endothelial deficient mice (KO ATG5), progression of liver tumour mice (induced by diethylnitrosamine injection) was greater compared to control mice. We observed an increase in senescent cells in the non-tumoral liver (NTL) of KO ATG5 mice, although not significant. In tissue specimen from patients with MS, p62 (protein of autophagy) expression was increased in HCC, and senescent cells were more frequent in NTL. This study highlighted the key role of sinusoidal endothelial cells in liver carcinogenesis related to SM through novel mediators, FABP4 and ATG5

MALDI Imaging, a Powerful Multiplex Approach to Decipher Intratumoral Heterogeneity: Combined Hepato-Cholangiocarcinomas as Proof of Concept

Combined hepato-cholangiocarcinomas (cHCC-CCA) belong to the spectrum of primary liver carcinomas, which include hepatocellular carcinomas (HCC) and intrahepatic cholangiocarcinomas (iCCA) at both ends of the spectrum. Mainly due to the high intratumor heterogeneity of cHCC-CCA, its diagnosis and pathological description remain challenging. Taking advantage of in situ non-targeted molecular mapping provided by MALDI (Matrix Assisted Laser Desorption Ionization) imaging, we sought to develop a multiscale and multiparametric morphological approach, integrating molecular and conventional pathological analysis. MALDI imaging was applied to five representative cases of resected cHCC-CCA. Principal component analysis and segmentations with MALDI imaging techniques identified areas related to either iCCA or HCC and also hidden tumor areas not visible microscopically. In addition, the overlap between MALDI segmentation and immunostaining provided a comprehensive description of cHCC-CCA tumor heterogeneity by identifying transitional and micro-metastatic areas. Moreover, a list of peptides derived from in silico digestion was obtained for each immunohistochemical marker and was matched within the peptide peak list acquired by MALDI. Comparison of immunostaining images with ions from in silico digestion revealed an accurate identification of iCCA and HCC areas. Our study provides further evidence on the performance of MALDI imaging in exploring intratumor heterogeneity and offering virtual multiplex immunostaining through a single acquisition

Tumoral heterogeneity of hepatic cholangiocarcinomas revealed by MALDI imaging mass spectrometry.

International audienceCholangiocarcinoma (CC) is the second most common primary malignancy of the liver. Although all CC derive from biliary epithelial cells, two main subtypes, hilar (H), and peripheral (P) CC are described. The objective of the study was to compare, using MALDI imaging mass spectrometry (MALDI IMS), in situ proteomic profiles of H- and P-CC in order to assess whether these subtypes may express different markers and to describe their respective localizations. Twenty-seven CC (16 P-CC and 11 H-CC) were subjected to MALDI IMS. Proteomic data were submitted to a dedicated cross-classification comparative design, enabling comparison of the entire generated spectra. Immunohistochemistry was performed for validation. Comparative analysis yielded a list of 19 differential protein peaks for the two subtypes, 14 of which were overexpressed in H-CC and five in P-CC. Among H-CC protein markers, most discriminant were human neutrophil peptides 1-3 that were expressed mainly by tumor cells and S100 proteins (A6 and A11) that were restricted to the stromal area. In P-CC, thymosin β4 was diffusely overexpressed. These results highlight the potential of MALDI IMS to discover new relevant biomarkers of CC and to characterize the heterogeneity of the two different subtypes

Imaging mass spectrometry provides fingerprints for distinguishing hepatocellular carcinoma from cirrhosis.

International audienceMALDI imaging mass spectrometry (MALDI IMS) is a powerful tool for comprehending the spectrum of peptides/proteins expressed in tissue sections. The aim of the present study was to investigate, using MALDI IMS, the proteome of hepatocellular carcinomas (HCC) and to compare it with peritumoral cirrhosis so as to characterize new biomarkers of HCC. Frozen liver tissues corresponding to HCC and background cirrhosis (n = 30) were selected and subjected to MALDI IMS. We found a set of proteins/peptides with a differential intensity level that most accurately delineated cancer from adjacent cirrhotic tissue. Using a support vector machine algorithm, we generated a classification model in the train set that enabled segmenting images from the independent validation set and that in most cases matched histologic analysis. The most discriminating peak (m/z 8565) more intense in HCC was characterized as the monomeric ubiquitin. An immunohistochemical study in a large series of HCC/cirrhosis sampled on tissue microarray supported that ubiquitin was overexpressed in HCC. We demonstrated also that this increase was not related to an upregulation of ubiquitin gene transcription in HCC, thus suggesting a post-transcriptional mechanism. This approach might provide a new tool for diagnosis of difficult HCC cases and an opportunity for identifying candidate biomarkers

Imaging mass spectrometry reveals modified forms of histone H4 as new biomarkers of microvascular invasion in hepatocellular carcinomas

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Progression from cirrhosis to cancer is associated with early ubiquitin post-translational modifications: identification of new biomarkers of cirrhosis at risk of malignancy

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The histone acetyltransferase hMOF promotes vascular invasion in hepatocellular carcinoma

International audienceBACKGROUND & AIMS:Vascular invasion is a major prognostic factor in hepatocellular carcinoma (HCC). We previously identified histone H4 acetylated at lysine 16 (H4K16ac), a histone modification involved in transcription activation, as a biomarker of microvascular invasion (mVI) in HCC. This study aimed to investigate the role of hMOF, the histone acetyltransferase responsible for H4K16 acetylation, in the process of vascular invasion in HCC.METHODS:hMOF expression was assessed by RT-qPCR and immunohistochemistry in a retrospective series of HCC surgical samples, and correlated with the presence of mVI. The functional role of hMOF in HCC vascular invasion was investigated in vitro in HCC cell lines using siRNA, transcriptomic analysis and transwell invasion assay, and in vivo using a Zebrafish embryo xenograft model.RESULTS:We found that hMOF was significantly upregulated at the protein level in HCC with mVI, compared with HCC without mVI (P < .01). Transcriptomic analysis showed that hMOF downregulation in HCC cell line lead to significant downregulation of key genes and pathways involved in vascular invasion. These results were confirmed by transwell invasion assay, where hMOF downregulation significantly reduced HCC cells invasion. Finally, hMOF downregulation significantly reduced tumour cell intravasation and metastasis in vivo.CONCLUSIONS:Altogether, these results underpin a critical role for hMOF in vascular invasion in HCC, via transcription activation of key genes involved in this process. These data confirm the major role of epigenetic alterations in HCC progression, and pave the way for future therapies targeting hMOF in HCC