Orlando Musso

Human hepatocellular carcinomas (HCCs) with cancer stem cell (CSC) features are a subclass of therapeutically challenging cancers. We recently showed that retrodifferentiation of hepatic cancer cells into CSC-like cells leads to metabolic reprogramming and chemoresistance. The molecular mechanisms whereby differentiated cancer cells switch towards a CSC phenotype are poorly understood. By studying metabolic reprogramming associated with HCC cell plasticity, we identified an unsuspected role of peroxisome proliferator-activated receptor (PPAR)γ in hepatic CSC phenotype acquisition. Gene expression and metabolic analyses performed throughout the cell differentiation/retrodifferentiation process of human HepaRG and HBG-BC2 HCC cells show that metabolic reprogramming in hepatic CSCs is associated with a fragmented mitochondrial network, decreased respiration, de novo lipogenesis, and fatty acid oxidation, but increased glycolysis and lipid storage. Mitochondrial genes downregulated in HepaRG-CSCs are also downregulated in the STEM HCC subclass. While PPARα is the main isoform in differentiated hepatic cells, we find high PPARγ expression in hepatic CSCs. Accordingly, nuclear localization of PPARγ is detected in human HCC tumors, and PPARγ ^high /PPARα ^low expression is associated with the STEM HCC subclass and a poor outcome in human HCC cohorts. PPARγ silencing or/and inhibition of its target gene pyruvate dehydrogenase kinase 4 reactivates cell respiration, increases reactive oxygen species production and sensitizes hepatic CSCs to chemotherapy. Conversely, PPARα activation synergizes with chemotherapy to induce cell death. Targeting PPARγ, a key regulator of metabolic reprogramming and stemness in hepatic CSCs, or modulating the PPARγ/PPARα balance that finely tunes the differentiation/retrodifferentiation process in HCC deserves further investigation for anti-tumor therapy.

Liver cancers show high interindividual and intratumor heterogeneity. Among them, hepatocellular carcinoma (HCCs) represents approximately 90% of liver cancers, followed by intrahepatic cholangiocarcinoma (iCCA; ~10 to 15%), childhood hepatoblastoma, angiosarcoma and hemangioendothelioma (< 1%). More than 80% of HCCs arise in a backdrop of chronic inflammatory liver diseases of diverse etiologies. These underlying liver diseases are major determinants of geographic diversity of HCCs. Across the world, substantial differences in the prevalence of chronic viral hepatitides, alcohol misuse, Metabolic Disfunction-Associated Steatotic Liver Disease (MASLD) and exposure to toxic substances are frequently related to social and economic inequalities. Vulnerable populations are more frequently exposed to infections such as hepatitis B and C viruses that, combined with other risk factors, lead to both vertical and horizontal transmission and, in turn, impact on age and sex-related diversity. In this review, we describe the global landscape of risk factors leading to HCC: MASLD, chronic hepatitis B and C infections, alcohol misuse, exposure to other toxic substances and genetic predispositions. We describe their combined effects on the clinical and epidemiological features of HCCs around the globe. Clinical presentation, incidence and mortality rates of HCCs show therefore great geographic heterogeneity, which is also related to the inequalities in the gross domestic product per capita, the socio-demographic index, the access to health care resources and to the implementation of policies for surveillance and screening of patients at risk. Awareness of the biological and geopolitical sources of HCC diversity will hopefully lead to more efficient international cooperation in the prevention and early management of chronic liver diseases and HCC.

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Weighted Gene Correlation Network Analysis (WGCNA) is used to build weighted gene networks representing direct interconnections among genes. This method is useful to identify gene modules associated with biological functions, revealing core functional differences between samples. Here, we describe a step-by-step methodology to build a WGCNA network associated with a phenotype of interest. The results can be visualized using Cytoscape and other available software or used as a basis for further functional enrichment analyses. Key words Transcriptomics Liver cancer Tumor heterogeneity Fatty liver disease

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.

Angiotensin-converting enzyme 2 (ACE2) is the receptor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causing Coronavirus disease 2019 (COVID-19). Transmembrane serine protease 2 (TMPRSS2) is a coreceptor. Abnormal hepatic function in COVID-19 suggests specific or bystander liver disease. Because liver cancer cells express the ACE2 viral receptor, they are widely used as models of SARS-CoV-2 infection in vitro. Therefore, the purpose of this study was to analyze ACE2 and TMPRSS2 expression and localization in human liver cancers and in non-tumor livers. We studied ACE2 and TMPRSS2 in transcriptomic datasets totaling 1503 liver cancers, followed by high-resolution confocal multiplex immunohistochemistry and quantitative image analysis of a 41-HCC tissue microarray. In cancers, we detected ACE2 and TMPRSS2 at the biliary pole of tumor hepatocytes. In whole mount sections of five normal liver samples, we identified ACE2 in hepatocyte’s bile canaliculi, biliary epithelium, sinusoidal and capillary endothelial cells. Tumors carrying mutated β-catenin showed ACE2 DNA hypomethylation and higher mRNA and protein expression, consistently with predicted β-catenin response sites in the ACE2 promoter. Finally, ACE2 and TMPRSS2 co-expression networks highlighted hepatocyte-specific functions, oxidative stress and inflammation, suggesting a link between inflammation, ACE2 dysfunction and metabolic breakdown.

Osteopontin (OPN) expression correlates with tumor progression in many cancers, including hepatocellular carcinoma (HCC); however, its role in the onset of HCC remains unclear. We hypothesized that increased hepatocyte‐derived OPN is a driver of hepatocarcinogenesis. Analysis of a tissue microarray of 366 human samples revealed a continuous increase in OPN expression during hepatocarcinogenesis. In patients with cirrhosis, a transcriptome‐based OPN correlation network was associated with HCC incidence along 10 years of follow‐up, together with messenger RNA (mRNA) signatures of carcinogenesis. After diethylnitrosamine (DEN) injection, mice with conditional overexpression of Opn in hepatocytes (Opn ^Hep transgenic [Tg]) showed increased tumor burden. Surprisingly, mice with conditional ablation of Opn in hepatocytes (Opn ^ΔHep) expressed a similar phenotype. The acute response to DEN was reduced in Opn ^ΔHep, which also showed more cancer stem/progenitor cells (CSCs, CD44⁺AFP⁺) at 5 months. CSCs from Opn ^Hep Tg mice expressed several mRNA signatures known to promote carcinogenesis, and mRNA signatures from Opn ^Hep Tg mice were associated with poor outcome in human HCC patients. Treatment with rOPN had little effect on CSCs, and their progression to HCC was similar in Opn ^−/− compared with wild‐type mice. Finally, ablation of Cd44, an OPN receptor, did not reduce tumor burden in Cd44 ^−/− Opn ^Hep Tg mice. Conclusions: Hepatocyte‐derived OPN acts as a tumor suppressor at physiological levels by controlling the acute response to DEN and the presence of CSCs, while induction of OPN is pro‐tumorigenic. This is primarily due to intracellular events rather that by the secretion of the protein and receptor activation.