Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide due to its high rate of recurrence, in part because of cancer stem cell (CSC)-dependent “field cancerization”. Recently, we identified that the oncogene v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) marked CSC-like subpopulations in heterogeneous HCC and served as a therapeutic target and prognostic marker for HCC. In this study, we explored the molecular basis of upregulated MYCN gene expression in HCC cells. Liquid chromatograph time-of-flight mass spectrometry-based metabolome analysis demonstrated that the content of unsaturated fatty acids was increased in MYCN high expression (MYCN^high) CSC-like HCC cells. Inhibition of lipid desaturation using either the chemical inhibitor or siRNA/shRNA against stearoyl-CoA desaturase-1 (SCD1) suppressed cell proliferation as well as MYCN gene expression in MYCN^high HCC cells, grown as both monolayer and spheres. Further mechanistic study using RNA-seq based transcriptome analysis revealed that endoplasmic reticulum (ER) stress related signaling networks such as endocannabinoid cancer inhibition pathway were under the control of SCD1 in MYCN^high HCC cells. Furthermore, the expression of ER stress-inducible transcription suppressor cyclic AMP-dependent transcription factor (ATF3) was downregulated in MYCN^high CSC-like HCC cells and CSC-rich spheroids, which was upregulated by inhibition of lipid desaturation or treatment with acyclic retinoid (ACR). Lipid profiling using NMR spectroscopy revealed that the ACR dramatically reduced the content of unsaturated fatty acids in HCC cells. The chemical inducer of ER stress inhibited MYCN gene expression, while the chemical inhibitor of ER stress or knockdown of ATF3 gene expression partially rescued the suppression of MYCN gene expression by ACR in MYCN^high HCC cells. These data suggested that lipid desaturation-mediated ER stress signaling regulates MYCN gene expression in HCC cells and serves as a promising therapeutic target for the treatment and prevention of HCC.

Many patients in intensive care units, especially the elderly, suffer from chronic critical illness and exhibit a new pathophysiological phenotype: persistent inflammation, immunosuppression, and catabolism syndrome (PICS). Most patients with PICS have a constellation of digestive-system symptoms and gut failure. Akkermansia muciniphila (Akk) is a commensal gut bacterium that reduces inflammation, balances immune responses, modulates energy metabolism, and supports gut health. This study investigated the protective effects and underlying mechanisms of live and pasteurized Akk in treating PICS in a mouse model. PICS was induced on day 14 after performing cecal ligation and puncture (CLP) on day 1 and administrating lipopolysaccharide on day 11. Pasteurized or live Akk, or phosphate-buffered saline was administered twice daily by oral gavage for 7 days. Both live and pasteurized Akk attenuated PICS, as evidenced by reduced weight loss, and a reduction in symptoms and serum cytokine/chemokine levels. Liver and intestinal injuries were mitigated, and intestinal barrier integrity improved with Akk administration. Analysis of 16S rRNA amplicon sequences showed that Akk induced significant intestinal microbiota alterations, including increased abundance of Akk, Muribaculaceae, Parabacterbides goldsteinii, and decreased abundance of Escherichia_Shigella and Enterobacteriaceae. Collectively, Akk alleviates PICS by enhancing gut barrier function and reshaped the microbial community.

Sepsis results in lethal organ malfunction due to dysregulated host response to infection, which is a condition with increasing prevalence worldwide. Transglutaminase 2 (TG2) is a crosslinking enzyme that forms a covalent bond between lysine and glutamine. TG2 plays important roles in diverse cellular processes, including extracellular matrix stabilization, cytoskeletal function, cell motility, adhesion, signal transduction, apoptosis, and cell survival. We have shown that the co-culture of Candida albicans and hepatocytes activates and induces the translocation of TG2 into the nucleus. In addition, the expression and activation of TG2 in liver macrophages was dramatically induced in the lipopolysaccharide-injected and cecal ligation puncture-operated mouse models of sepsis. Based on these findings and recently published research, we have reviewed the current understanding of the relationship between TG2 and sepsis. Following the genetic and pharmacological inhibition of TG2, we also assessed the evidence regarding the use of TG2 as a potential marker and therapeutic target in inflammation and sepsis.

The incidence and mortality of liver cancer, mostly hepatocellular carcinoma (HCC), have increased during the last two decades, partly due to persistent inflammation in the lipid-rich microenvironment associated with lifestyle diseases, such as obesity. Gangliosides are sialic acid-containing glycosphingolipids known to be important in the organization of the membrane and membrane protein-mediated signal transduction. Ganglioside synthesis is increased in several types of cancers and has been proposed as a promising target for cancer therapy. Here, we provide evidence that ganglioside synthesis was increased in the livers of an animal model recapitulating the features of activation and expansion of liver progenitor-like cells and liver cancer (stem) cells. Chemical inhibition of ganglioside synthesis functionally suppressed proliferation and sphere growth of liver cancer cells, but had no impact on apoptotic and necrotic cell death. Proteome-based mechanistic analysis revealed that inhibition of ganglioside synthesis downregulated the expression of AURKA, AURKB, TTK, and NDC80 involved in the regulation of kinetochore metaphase signaling, which is essential for chromosome segregation and mitotic progression and probably under the control of activation of TP53-dependent cell cycle arrest. These data suggest that targeting ganglioside synthesis holds promise for the development of novel preventive/therapeutic strategies for HCC treatment.

Transglutaminase 2 (TG2) is a multifunctional protein that promotes or suppresses tumorigenesis, depending on intracellular location and conformational structure. Acyclic retinoid (ACR) is an orally administered vitamin A derivative that prevents hepatocellular carcinoma (HCC) recurrence by targeting liver cancer stem cells (CSCs). In this study, we examined the subcellular location-dependent effects of ACR on TG2 activity at a structural level and characterized the functional role of TG2 and its downstream molecular mechanism in the selective depletion of liver CSCs. A binding assay with high-performance magnetic nanobeads and structural dynamic analysis with native gel electrophoresis and size-exclusion chromatography-coupled multi-angle light scattering or small-angle X-ray scattering showed that ACR binds directly to TG2, induces oligomer formation of TG2, and inhibits the transamidase activity of cytoplasmic TG2 in HCC cells. The loss-of-function of TG2 suppressed the expression of stemness-related genes, spheroid proliferation and selectively induced cell death in an EpCAM+ liver CSC subpopulation in HCC cells. Proteome analysis revealed that TG2 inhibition suppressed the gene and protein expression of exostosin glycosyltransferase 1 (EXT1) and heparan sulfate biosynthesis in HCC cells. In contrast, high levels of ACR increased intracellular Ca²⁺ concentrations along with an increase in apoptotic cells, which probably contributed to the enhanced transamidase activity of nuclear TG2. This study demonstrates that ACR could act as a novel TG2 inhibitor; TG2-mediated EXT1 signaling is a promising therapeutic target in the prevention of HCC by disrupting liver CSCs.

Sepsis is the leading cause of death in hospitalized patients and is characterized by a dysregulated inflammatory response to infection and multiple organ failure, including the liver. Transglutaminase 2 (TG2) is a multifunctional enzyme that exhibits transamidase, GTPase, and integrin-binding activities and has opposing roles in the regulation of cell growth, differentiation, and apoptosis. TG2 plays both pathogenic and protective roles in liver diseases, revealing the need to examine the activities of TG2. Here, we introduced an ex vivo imaging approach to examine the in vivo transamidase activity of TG2 based on the combination of intraperitoneal injection of 5-biotinamidopentylamine (5BAPA), a biotinylated substrate for TG2, and fluorescent streptavidin staining in frozen liver sections. Increased 5BAPA signals was observed in the livers of lipopolysaccharide (LPS) and cecal ligation and puncture (CLP)-induced sepsis mice. Pharmacological inhibition of TG2 activity ameliorated LPS-induced liver injury. 5BAPA signals were observed in TG2-expressing and F4/80-positive midzonal macrophages, providing direct evidence that activated macrophages are the major cellular source of active TG2 in the livers of sepsis mice. Further studies focusing on the activation of 5BAPA-stained midzonal macrophages may improve understanding of the molecular pathophysiology and the development of therapeutic strategies for sepsis.