Background and Aims: HCC, the third leading cause of cancer-related death, arises in the context of liver fibrosis. Although HCC is generally poorly fibrogenic, some tumors harbor focal intratumor extracellular matrix (ECM) deposits called “fibrous nests.” To date, the molecular composition and clinical relevance of these ECM deposits have not been fully defined. Approach and Results: We performed quantitative matrisome analysis by tandem mass tags mass spectrometry in 20 human cancer specific matrisome (HCCs) with high or low-grade intratumor fibrosis and matched nontumor tissues, as well as in 12 livers from mice treated with vehicle, carbon tetrachloride, or diethylnitrosamine. We found 94 ECM proteins differentially abundant between high and low-grade fibrous nests, including interstitial and basement membrane components, such as several collagens, glycoproteins, proteoglycans, enzymes involved in ECM stabilization and degradation, and growth factors. Pathway analysis revealed a metabolic switch in high-grade fibrosis, with enhanced glycolysis and decreased oxidative phosphorylation. Integrating the quantitative proteomics with transcriptomics from HCCs and nontumor livers (n = 2,285 samples), we identified a subgroup of fibrous nest HCCs, characterized by cancer-specific ECM remodeling, expression of the WNT/TGFB (S1) subclass signature, and poor patient outcome. Fibrous nest HCCs abundantly expressed an 11-fibrous-nest–protein signature, associated with poor patient outcome, by multivariate Cox analysis, and validated by multiplex immunohistochemistry. Conclusions: Matrisome analysis highlighted cancer-specific ECM deposits, typical of the WNT/TGFB HCC subclass, associated with poor patient outcomes. Hence, histologic reporting of intratumor fibrosis in HCC is of clinical relevance.

Hepatocellular carcinoma (HCC) is the 3rd cause of cancer-related death worldwide. Most cases arise in a background of chronic inflammation, extracellular matrix (ECM) remodeling, severe fibrosis and stem/progenitor cell amplification. Although HCCs are soft cellular tumors, they may contain fibrous nests within the tumor mass. Thus, the aim of this study was to explore cancer cell phenotypes in fibrous nests. Combined anatomic pathology, tissue microarray and real-time PCR analyses revealed that HCCs (n=82) containing fibrous nests were poorly differentiated, expressed Wnt pathway components and target genes, as well as markers of stem/progenitor cells, such as CD44, LGR5 and SOX9. Consistently, in severe liver fibroses (n=66) and in HCCs containing fibrous nests, weighted correlation analysis revealed a gene network including the myofibroblast marker ACTA2, the basement membrane components COL4A1 and LAMC1, the Wnt pathway members FZD1; FZD7; WNT2; LEF1; DKK1 and the Secreted Frizzled Related Proteins (SFRPs) 1; 2 and 5. Moreover, unbiased random survival forest analysis of a transcriptomic dataset of 247 HCC patients revealed high DKK1, COL4A1, SFRP1 and LAMC1 to be associated with advanced tumor staging as well as with bad overall and disease-free survival. In vitro, these genes were upregulated in liver cancer stem/progenitor cells upon Wnt-induced mesenchymal commitment and myofibroblast differentiation. In conclusion, fibrous nests express Wnt target genes, as well as markers of cancer stem cells and mesenchymal commitment. Fibrous nests embody the specific microenvironment of the cancer stem cell niche and can be detected by routine anatomic pathology analyses.

// <![CDATA[ $('.header-date').hide();$('#titleAuthors').hide();$('#abstractHeader').hide(); // ]]> Sihem Mebarki1,2, Romain Désert1,2, Laurent Sulpice1,2,8, Marie Sicard1,2, Mireille Desille1,2,6, Frédéric Canal3,4,5, Hélène Dubois-Pot Schneider1,2, Damien Bergeat1,2,8, Bruno Turlin1,2,6, Pascale Bellaud7, Elise Lavergne1,2, Rémy Le Guével9, Anne Corlu1,2,9, Christine Perret3,4,5, Cédric Coulouarn1,2, Bruno Clément1,2 and Orlando Musso1,2 1 Inserm, UMR991, Liver Metabolisms and Cancer, Rennes, France 2 Université de Rennes 1, Rennes, France 3 Inserm, Institut Cochin, Paris, France 4 Cnrs, UMR8104, Paris, France 5 Université Paris Descartes, Sorbonne Paris Cité, Paris, France 6 CHU de Rennes, Centre de Ressources Biologiques Santé, Rennes, France 7 Université de Rennes 1, UMS 18 Biosit, Biogenouest, Rennes, France 8 CHU de Rennes, Department of Gastrointestinal and Hepatobiliary Surgery, Rennes, France 9 Université de Rennes 1, UMS 18 Biosit, Biogenouest, ImPACcellCore Facility, Rennes, France Correspondence to: Orlando Musso, email: // <![CDATA[ mail("orlando.musso","inserm",18,"") // ]]> Keywords: Wnt; β-catenin; epithelial-mesenchymal transition; LGR5; CD44; Gerotarget Received: December 01, 2015 Accepted: April 22, 2016 Published: May 13, 2016

Hepatocellular carcinoma (HCC) is the 3rd leading cause of cancer-related death worldwide. More than 80% of HCCs arise within chronic liver disease resulting from viral hepatitis, alcohol, hemochromatosis, obesity and metabolic syndrome or genotoxins. Projections based on Western lifestyle and its metabolic consequences anticipate a further increase in incidence, despite recent breakthroughs in the management of viral hepatitis. HCCs display high heterogeneity of molecular phenotypes, which challenges clinical management. However, emerging molecular classifications of HCCs have not yet formed a unified corpus translatable to the clinical practice. Thus, patient management is currently based upon tumor number, size, vascular invasion, performance status and functional liver reserve. Nonetheless, an impressive body of molecular evidence emerged within the last 20 years and is becoming increasingly available to medical practitioners and researchers in the form of repositories. Therefore, the aim this work is to review molecular data underlying HCC classifications and to organize this corpus into the major dimensions explaining HCC phenotypic diversity. Major efforts have been recently made worldwide toward a unifying "clinically-friendly" molecular landscape. As a result, a consensus emerges on three major dimensions explaining the HCC heterogeneity. In the first dimension, tumor cell proliferation and differentiation enabled allocation of HCCs to two major classes presenting profoundly different clinical aggressiveness. In the second dimension, HCC micro-environment and tumor immunity underlie recent the-rapeutic breakthroughs prolonging patients' survival. In the third dimension, metabolic reprogramming, with the recent emergence of subclass-specific metabolic profiles, may lead to adaptive and combined therapeutic approaches. Therefore, here we review recent molecular evidence, their impact on tumor histopathological features and clinical behavior and highlight the remaining challenges to translate our cognitive corpus into patient diagnosis and allocation to therapeutic options.