Although metabolism is profoundly altered in human liver cancer, the extent to which experimental models, e.g. cell lines, mimic those alterations is unresolved. Here, we aimed to determine the resemblance of hepatocellular carcinoma (HCC) cell lines to human liver tumours, specifically in the expression of deregulated metabolic targets in clinical tissue samples. We compared the overall gene expression profile of poorly-differentiated (HLE, HLF, SNU-449) to well-differentiated (HUH7, HEPG2, HEP3B) HCC cell lines in three publicly available microarray datasets. Three thousand and eighty-five differentially expressed genes in ≥2 datasets (P < 0.05) were used for pathway enrichment and gene ontology (GO) analyses. Further, we compared the topmost gene expression, pathways, and GO from poorly differentiated cell lines to the pattern from four human HCC datasets (623 tumour tissues). In well- versus poorly differentiated cell lines, and in representative models HLE and HUH7 cells, we specifically assessed the expression pattern of 634 consistently deregulated metabolic genes in human HCC. These data were complemented by quantitative PCR, proteomics, metabolomics and assessment of response to thirteen metabolism-targeting compounds in HLE versus HUH7 cells. We found that poorly-differentiated HCC cells display upregulated MAPK/RAS/NFkB signaling, focal adhesion, and downregulated complement/coagulation cascade, PPAR-signaling, among pathway alterations seen in clinical tumour datasets. In HLE cells, 148 downregulated metabolic genes in liver tumours also showed low gene/protein expression – notably in fatty acid β-oxidation (e.g. ACAA1/2, ACADSB, HADH), urea cycle (e.g. CPS1, ARG1, ASL), molecule transport (e.g. SLC2A2, SLC7A1, SLC25A15/20), and amino acid metabolism (e.g. PHGDH, PSAT1, GOT1, GLUD1). In contrast, HUH7 cells showed a higher expression of 98 metabolic targets upregulated in tumours (e.g. HK2, PKM, PSPH, GLUL, ASNS, and fatty acid synthesis enzymes ACLY, FASN). Metabolomics revealed that the genomic portrait of HLE cells co-exist with profound reliance on glutamine to fuel tricarboxylic acid cycle, whereas HUH7 cells use both glucose and glutamine. Targeting glutamine pathway selectively suppressed the proliferation of HLE cells. We report a yet unappreciated distinct expression pattern of clinically-relevant metabolic genes in HCC cell lines, which could enable the identification and therapeutic targeting of metabolic vulnerabilities at various liver cancer stages.

Proteomics-based clinical studies have been shown to be promising strategies for the discovery of novel biomarkers of a particular disease. Here, we present a study of hepatocellular carcinoma (HCC) that combines complementary two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography (LC-MS)-based approaches of quantitative proteomics. In our proteomic experiments, we analyzed a set of 14 samples (7 × HCC versus 7 × nontumorous liver tissue) with both techniques. Thereby we identified 573 proteins that were differentially expressed between the experimental groups. Among these, only 51 differentially expressed proteins were identified irrespective of the applied approach. Using Western blotting and immunohistochemical analysis the regulation patterns of six selected proteins from the study overlap (inorganic pyrophosphatase 1 (PPA1), tumor necrosis factor type 1 receptor-associated protein 1 (TRAP1), betaine-homocysteine S-methyltransferase 1 (BHMT)) were successfully verified within the same sample set. In addition, the up-regulations of selected proteins from the complements of both approaches (major vault protein (MVP), gelsolin (GSN), chloride intracellular channel protein 1 (CLIC1)) were also reproducible. Within a second independent verification set (n = 33) the altered protein expression levels of major vault protein and betaine-homocysteine S-methyltransferase were further confirmed by Western blots quantitatively analyzed via densitometry. For the other candidates slight but nonsignificant trends were detectable in this independent cohort. Based on these results we assume that major vault protein and betaine-homocysteine S-methyltransferase have the potential to act as diagnostic HCC biomarker candidates that are worth to be followed in further validation studies.

Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that some viruses can manipulate the infection process by packing specific viral and cellular components into exosomes, small nanometer-sized (30–150 nm) vesicles secreted from various cells. However, the impact of HBV replication on the content of exosomes produced by hepatocytes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in the protein content of exosomes secreted from HepAD38 cells with or without HBV replication. Exosomes were isolated from supernantants of HepAD38 cells cultured with or without doxycycline (dox) and their purity was confirmed by transmission electron microscopy (TEM) and Western immunoblotting assays. Ion-intensity based label-free LC-MS/MS quantitation technologies were applied to analyze protein content of exosomes from HBV replicating cells [referred as HepAD38 (dox⁻)-exo] and from HBV nonreplicating cells [referred as HepAD38 (dox⁺)-exo]. A total of 1412 exosomal protein groups were identified, among which the abundance of 35 proteins was significantly changed following HBV replication. Strikingly, 5 subunit proteins from the 26S proteasome complex, including PSMC1, PSMC2, PSMD1, PSMD7 and PSMD14 were consistently enhanced in HepAD38 (dox⁻)-exo. Bioinformatic analysis of differential exosomal proteins confirmed the significant enrichment of components involved in the proteasomal catabolic process. Proteasome activity assays further suggested that HepAD38 (dox⁻)-exo had enhanced proteolytic activity compared with HepAD38 (dox⁺)-exo. Furthermore, human peripheral monocytes incubated with HepAD38 (dox⁻)-exo induced a significantly lower level of IL-6 secretion compared with IL-6 levels from HepAD38 (dox⁺)-exo. Irreversible inhibition of proteasomal activity within exosomes restored higher production of IL-6 by monocytes, suggesting that transmission of proteasome subunit proteins by HepAD38 (dox⁻)-exo might modulate the production of pro-inflammatory molecules in the recipient monocytes. These results revealed the composition and potential function of exosomes produced during HBV replication, thus providing a new perspective on the role of exosomes in HBV-host interaction.