Multikinase inhibitor sorafenib inhibits proliferation and angiogenesis of tumors by suppressing the Raf/MEK/ERK signaling pathway and VEGF receptor tyrosine kinase. It significantly prolongs median survival of patients with advanced hepatocellular carcinoma (HCC) but the response is disease‐stabilizing and cytostatic rather than one of tumor regression. To examine the mechanisms underlying the relative resistance in HCC, we investigated the role of autophagy, an evolutionarily conserved self‐digestion pathway, in hepatoma cells in vitro and in vivo. Sorafenib treatment led to accumulation of autophagosomes as evidenced by conversion from LC3‐I to LC3‐II observed by immunoblot in Huh7, HLF and PLC/PRF/5 cells. This induction was due to activation of autophagic flux, as there was further increase in LC3‐II expression upon treatment with lysosomal inhibitors, clear decline of the autophagy substrate p62, and an mRFP‐GFP‐LC3 fluorescence change in sorafenib‐treated hepatoma cells. Sorafenib inhibited the mammalian target of rapamycin complex 1 and its inhibition led to accumulation of LC3‐II. Pharmacological inhibition of autophagic flux by chloroquine increased apoptosis and decreased cell viability in hepatoma cells. siRNA‐mediated knockdown of the ATG7 gene also sensitized hepatoma cells to sorafenib. Finally, sorafenib induced autophagy in Huh7 xenograft tumors in nude mice and coadministration with chloroquine significantly suppressed tumor growth compared with sorafenib alone. In conclusion, sorafenib administration induced autophagosome formation and enhanced autophagic activity, which conferred a survival advantage to hepatoma cells. Concomitant inhibition of autophagy may be an attractive strategy for unlocking the antitumor potential of sorafenib in HCC.

Background and Aims: NAFLD is the most common liver disease worldwide. NASH, the progressive form of NAFLD, and advanced fibrosis are associated with poor outcomes. We searched for their noninvasive biomarkers. Approach and Results: Global RNA sequencing of liver tissue from 98 patients with biopsy‐proven NAFLD was performed. Unsupervised hierarchical clustering well distinguished NASH from nonalcoholic fatty liver (NAFL), and patients with NASH exhibited molecular abnormalities reflecting their pathological features. Transcriptomic analysis identified proteins up‐regulated in NASH and/or advanced fibrosis (stage F3‐F4), including matricellular glycoprotein thrombospondin‐2 (TSP‐2), encoded by the thrombospondin 2 (THBS2) gene. The intrahepatic THBS2 expression level showed the highest areas under the receiver operating characteristic curves (AUROCs) of 0.915 and 0.957 for diagnosing NASH and advanced fibrosis, respectively. THBS2 positively correlated with inflammation and ballooning according to NAFLD activity score, serum aspartate aminotransferase and hyaluronic acid (HA) levels, and NAFLD Fibrosis Score (NFS). THBS2 was associated with extracellular matrix and collagen biosynthesis, platelet activation, caspase‐mediated cleavage of cytoskeletal proteins, and immune cell infiltration. Serum TSP‐2 expression was measured in 213 patients with biopsy‐proven NAFLD, was significantly higher in NASH than in NAFL, and increased parallel to fibrosis stage. The AUROCs for predicting NASH and advanced fibrosis were 0.776 and 0.856, respectively, which were comparable to Fibrosis‐4 index, serum HA level, and NFS in advanced fibrosis diagnosis. Serum TSP‐2 level and platelet count were independent predictors of NASH and advanced fibrosis. Serum TSP‐2 levels could stratify patients with NAFLD according to the risk of hepatic complications, including liver cancer and decompensated cirrhotic events. Conclusions: TSP‐2 may be a useful biomarker for NASH and advanced fibrosis diagnosis in patients with NAFLD.

The tumor suppressor p53 has been implicated in the pathogenesis of non-cancer-related conditions such as insulin resistance, cardiac failure, and early aging. In addition, accumulation of p53 has been observed in the hepatocytes of individuals with fibrotic liver diseases, but the significance of this is not known. Herein, we have mechanistically linked p53 activation in hepatocytes to liver fibrosis. Hepatocyte-specific deletion in mice of the gene encoding Mdm2, a protein that promotes p53 degradation, led to hepatocyte synthesis of connective tissue growth factor (CTGF; the hepatic fibrogenic master switch), increased hepatocyte apoptosis, and spontaneous liver fibrosis; concurrent removal of p53 completely abolished this phenotype. Compared with wild-type controls, mice with hepatocyte-specific p53 deletion exhibited similar levels of hepatocyte apoptosis but decreased liver fibrosis and hepatic CTGF expression in two models of liver fibrosis. The clinical significance of these data was highlighted by two observations. First, p53 upregulated CTGF in a human hepatocellular carcinoma cell line by repressing miR-17-92. Second, human liver samples showed a correlation between CTGF and p53-regulated gene expression, which were both increased in fibrotic livers. This study reveals that p53 induces CTGF expression and promotes liver fibrosis, suggesting that the p53/CTGF pathway may be a therapeutic target in the treatment of liver fibrosis.

Background and Aims: Immunotherapy has become the standard‐of‐care treatment for hepatocellular carcinoma (HCC), but its efficacy remains limited. To identify immunotherapy‐susceptible HCC, we profiled the molecular abnormalities and tumor immune microenvironment (TIME) of rapidly increasing nonviral HCC. Approaches and Results: We performed RNA‐seq of tumor tissues in 113 patients with nonviral HCC and cancer genome sequencing of 69 genes with recurrent genetic alterations reported in HCC. Unsupervised hierarchical clustering classified nonviral HCCs into three molecular classes (Class I, II, III), which stratified patient prognosis. Class I, with the poorest prognosis, was associated with TP53 mutations, whereas class III, with the best prognosis, was associated with cadherin‐associated protein beta 1 (CTNNB1) mutations. Thirty‐eight percent of nonviral HCC was defined as an immune class characterized by a high frequency of intratumoral steatosis and a low frequency of CTNNB1 mutations. Steatotic HCC, which accounts for 23% of nonviral HCC cases, presented an immune‐enriched but immune‐exhausted TIME characterized by T cell exhaustion, M2 macrophage and cancer‐associated fibroblast (CAF) infiltration, high PD‐L1 expression, and TGF‐β signaling activation. Spatial transcriptome analysis suggested that M2 macrophages and CAFs may be in close proximity to exhausted CD8+ T cells in steatotic HCC. An in vitro study showed that palmitic acid‐induced lipid accumulation in HCC cells upregulated PD‐L1 expression and promoted immunosuppressive phenotypes of cocultured macrophages and fibroblasts. Patients with steatotic HCC, confirmed by chemical‐shift MR imaging, had significantly longer PFS with combined immunotherapy using anti–PD‐L1 and anti‐VEGF antibodies. Conclusions: Multiomics stratified nonviral HCCs according to prognosis or TIME. We identified the link between intratumoral steatosis and immune‐exhausted immunotherapy‐susceptible TIME.

Although the tumor microenvironment plays an important role in tumor growth, it is not fully understood what role hepatic stellate cells (HSCs) play in the hepatocellular carcinoma (HCC) microenvironment. A high-fat-diet following streptozotocin was administered to HSC-specific Atg7-deficient (GFAP-Atg7KO) or GDF15-dificient (GFAP-GDF15KO) mice. LX-2 cells, a human HSC cell line, were cultured with human hepatoma cells. In the steatohepatitis-based tumorigenesis (STAM) model, GFAP-Atg7KO mice formed fewer and smaller liver tumors than their wild-type littermates. Mixed culture of LX-2 cells and hepatoma cells promoted LX-2 cell autophagy and hepatoma cell proliferation, which were attenuated by Atg7 knockout in LX-2 cells. Hepatoma cell xenograft tumors grew rapidly in the presence of LX-2 cells, but Atg7 knockout in LX-2 cells abolished this growth. RNA-sequencing revealed that LX-2 cells cultured with HepG2 cells highly expressed growth differentiation factor 15 (GDF15), which was abolished by Atg7 knockout in LX-2 cells. GDF15 knockout LX-2 cells did not show a growth-promoting effect on hepatoma cells either in vitro or in the xenograft model. GDF15 deficiency in HSCs reduced liver tumor size caused by the STAM model. GDF15 was highly expressed and GDF15-positive non-parenchymal cells were more abundant in human HCC compared to non-cancerous parts. Single-cell RNAseq showed that GDF15-positive rates in HSCs were higher in HCC than in background liver. Serum GDF15 levels were high in HCC patients and increased with tumor progression. In the HCC microenvironment, an increase of HSCs that produce GDF15 in an autophagy-dependent manner may be involved in tumor progression.