The TRAF2-p62 axis promotes proliferation and survival of liver cancer by activating mTORC1 pathway.

TRAF2 (Tumor necrosis factor receptor-associated factor 2) is a dual function protein, acting as an adaptor protein and a ubiquitin E3 ligase, which plays an essential role in mediating the TNFα-NFκB signal pathway. Dysregulated expression of TRAF2 has been reported in a variety of human cancers. Whether and how TRAF2 regulates the growth of liver cancer cells remains elusive. The goal of this study is to investigate potential dysregulation of TRAF2 and its biological function in liver cancer, and to elucidate the underlying mechanism, leading to validation of TRAF2 as an attractive liver cancer target. Here, we reported TRAF2 is up-regulated in human liver cancer cell lines and tissues, and high TRAF2 expression is associated with a poor prognosis of HCC patients. Proteomics profiling along with Co-immunoprecipitation analysis revealed that p62 is a new substrate of TRAF2, which is subjected to TRAF2-induced polyubiquitination via the K63 linkage at the K420 residue. A strong negative correlation was found between the protein levels of p62 and TRAF2 in human HCC samples. TRAF2 depletion inhibited growth and survival of liver cancer cells both in vitro and in vivo by causing p62 accumulation, which is partially rescued by simultaneous p62 knockdown. Mechanistically, TRAF2-mediated p62 polyubiquitylation activates the mTORC1 by forming the p62-mTORC1-Rag complex, which facilitates the lysosome localization of mTORC1. TRAF2 depletion inhibited mTORC1 activity through the disruption of interaction between p62 and the mTORC1 complex. In conclusion, our study provides the proof-of-concept evidence that TRAF2 is a valid target for liver cancer.

The Inhibition of Bruton Tyrosine Kinase Alleviates Acute Liver Failure via Downregulation of NLRP3 Inflammasome.

There is no effective treatment for acute liver failure (ALF) except for an artificial liver support system (ALSS) and liver transplant. Bruton tyrosine kinase (Btk) plays important immunoregulatory roles in the inflammatory diseases, but its possible function in ALF remains to be characterized. In this study, we detected the phosphorylation level of Btk in ALF mouse liver and analyzed the protective effects of Btk inhibitor on survival rate and liver damage in ALF mouse models. We measured the expression levels of various inflammatory cytokines in the ALF mouse liver and primary human monocytes. In addition, we examined the expression of the NLRP3 inflammasome in mouse models with or without Btk inhibition. Clinically, we observed the dynamic changes of Btk expression in PBMCs of ALSS-treated patients. Our results showed that Btk was upregulated significantly in the experimental ALF mouse models and that Btk inhibition alleviated liver injury and reduced the mortality in these models. The protective effect of Btk inhibitors on ALF mice partially depended on the suppression of NLRP3 inflammasome signaling. Clinical investigations revealed that the dynamic changes of Btk expression in PBMCs could predict the effect of ALSS treatment. Our work shows that Btk inhibition is an effective therapeutic strategy for ALF. Moreover, Btk is a useful indicator to predict the therapeutic effect of ALSS on liver failure, which might have great value in clinical practice.

Inhibition of BTK improved APAP-induced liver injury via suppressing proinflammatory macrophages activation by restoring mitochondrion function.

BACKGROUND: Acetaminophen (APAP) overdose can cause severe liver injury and APAP-induced liver injury (AILI) is one of the leading causes of acute liver failure (ALF). Bruton's tyrosine kinase (BTK) is a key tyrosine kinase in immune responses, which plays an important role in many inflammatory diseases. However, its effect on AILI is still not clear. Here, we aimed to assess the effect of BTK on AILI and explore its underlying mechanism. METHODS: In our study, western blot and immunohistochemistry were used to detect the expression of BTK in AILI. The C57BL/6 mice were used to check the protective effect of BTK inhibition on AILI and the activation of BTK was confirmed in mice macrophages treated with APAP. Immunofluorescence, immunohistochemistry, oxygen consumption rate (OCR) detection, polymerase chain reaction (PCR), flow cytometry and western blot were used to determine the role of BTK in mitochondrial dynamics and function of macrophages and the underlying mechanisms in AILI. RESULTS: Our results showed that BTK upregulated in AILI. BTK inhibition protected mice from AILI and BTK was activated in mice macrophages in response to APAP. Mechanically, BTK inhibition promoted mitochondrial fusion and restored mitochondrial function through phospholipase C gamma 2 (PLCγ2)-reactive oxygen species (ROS)-Optic Atrophy 1(OPA1) pathway in macrophages and finally suppressed the release of proinflammatory cytokines. CONCLUSIONS: In conclusion, we found that BTK inhibition protected mice from AILI by restoring the mitochondrial function of macrophages through the improvement of the mitochondrial dynamic imbalance via PLCγ2-ROS-OPA1 signaling pathway, which indicated that BTK might be a potential therapeutic target of AILI.

Clinical Value of 18F-FDG PET/CT Scan and Cytokine Profiles in Secondary Hemophagocytic Lymphohistiocytosis in Idiopathic Inflammatory Myopathy Patients: A Pilot Study.

Background: Secondary hemophagocytic lymphohistiocytosis (sHLH) is a rare but fatal complication in idiopathic inflammatory myopathy (IIM) patients. The clinical value of radiological manifestations and serum cytokines remain unknown in this systemic crisis. This study aims to investigate the clinical value of PET/CT scan and cytokine profiles in predicting and understanding sHLH in IIM patients. Methods: Adult IIM patients who were admitted to the four divisions of the First Affiliated Hospital, Zhejiang University School of Medicine (FAHZJU) from January 1, 2017 to December 31, 2020 were reviewed. PET/CT scan, cytokine profiles, and other factors of patients who met the inclusion and exclusion criteria were collected and analyzed. Results: Sixty-nine out of 352 IIM patients were finally enrolled into the study. Ten patients developed sHLH and 70.0% of them died within 6 months. After false discovery rate (FDR) correction and multivariate logistic regression analysis, increased serum interferon (IFN)-γ level (p = 0.017), higher spleen mean standard uptake value (SUVmean, p = 0.035), and positivity of anti-MDA5 antibody (p = 0.049) were found to be significantly correlated with development of sHLH in IIM patients. The combination of serum IFN-γ, spleen SUVmean, and anti-MDA5 antibody found a balanced and satisfying predictor with a cutoff value of 0.047 and AUC of 0.946. A moderate correlation was identified between ferritin and spleen SUVmean (p = 0.001, r = 0.380) as well as serum IFN-γ(p = 0.001, r = 0.398). Before FDR correction, higher bilateral lung SUVmean (p = 0.034) and higher colon/rectum SUVmean (p = 0.013) were also observed in IIM patients who developed sHLH. By narrowing down to IIM patients with sHLH, anti-MDA5-antibody-positive DM patients tended to suffer from unfavorable outcome (p = 0.004) in Kaplan-Meier survival analysis. Conclusion: Increased serum level of IFN-γ, elevated splenic FDG uptake, and positivity of anti-MDA5 antibody were significantly correlated with development of sHLH in IIM patients. Lung and lower digestive tract might also be affected due to systemic immune activation in IIM patients with sHLH. In addition, splenic FDG uptake, in combination with serum IFN-γand anti-MDA5 antibody, was found valuable in predicting development of sHLH in IIM patients. Among IIM patients with sHLH, anti-MDA5-antibody-positive DM patients showed higher tendency for unfavorable outcome.

The lungs were on fire: a pilot study of 18F-FDG PET/CT in idiopathic-inflammatory-myopathy-related interstitial lung disease.

BACKGROUND: Interstitial lung disease (ILD) and its rapid progression (RP) are the main contributors to unfavourable outcomes of patients with idiopathic inflammatory myopathy (IIM). This study aimed to identify the clinical value of PET/CT scans in IIM-ILD patients and to construct a predictive model for RP-ILD. METHODS: Adult IIM-ILD patients who were hospitalized at four divisions of the First Affiliated Hospital, Zhejiang University School of Medicine (FAHZJU), from 1 January 2017 to 31 December 2020 were reviewed. PET/CT scans and other characteristics of patients who met the inclusion and exclusion criteria were collected and analysed. RESULTS: A total of 61 IIM-ILD patients were enrolled in this study. Twenty-one patients (34.4%) developed RP-ILD, and 24 patients (39.3%) died during follow-up. After false discovery rate (FDR) correction, the percent-predicted diffusing capacity of the lung for carbon monoxide (DLCO%, P = 0.014), bilateral lung mean standard uptake value (SUVmean, P = 0.014) and abnormal mediastinal lymph node (P = 0.045) were significantly different between the RP-ILD and non-RP-ILD groups. The subsequent univariate and multivariate logistic regression analyses verified our findings. A "DLM" model was established by including the above three values to predict RP-ILD with a cut-off value of ≥ 2 and an area under the curve (AUC) of 0.905. Higher bilateral lung SUVmean (P = 0.019) and spleen SUVmean (P = 0.011) were observed in IIM-ILD patients who died within 3 months, and a moderate correlation was recognized between the two values. CONCLUSIONS: Elevated bilateral lung SUVmean, abnormal mediastinal lymph nodes and decreased DLCO% were significantly associated with RP-ILD in IIM-ILD patients. The "DLM" model was valuable in predicting RP-ILD and requires further validation.

Isoquercitrin induces apoptosis and autophagy in hepatocellular carcinoma cells via AMPK/mTOR/p70S6K signaling pathway.

Hepatocellular carcinoma (HCC) is an aggressive malignancy with high rates of metastasis and relapse. Isoquercitrin (ISO), a natural flavonoid present in the Chinese bayberry and other plant species, reportedly exerts notable inhibitory effects on tumor cell proliferation, though the mechanism is unknown. In the present study, we exposed HepG2 and Huh7 human liver cancer cells to ISO and examined the roles of autophagy and apoptosis in ISO-mediated cell death. We found that ISO exposure inhibited cell viability and colony growth, activated apoptotic pathway, and triggered dysregulated autophagy by activating the AMPK/mTOR/p70S6K pathway. Autophagy inhibition using 3-methyladenine (3-MA) or Atg5-targeted siRNA decreased the Bax/Bcl-2 ratio, caspase-3 activation, and PARP cleavage and protected cells against ISO-induced apoptosis. Moreover, autophagy inhibition reversed the upregulation of AMPK phosphorylation and downregulation of mTOR and p70S6K phosphorylation elicited by ISO. By contrast, application of a broad-spectrum caspase inhibitor failed to inhibit autophagy in ISO-treated cells. These data indicate that ISO simultaneously induced apoptosis and autophagy, and abnormal induction of autophagic flux contributed to ISO-triggered caspase-3-dependent apoptosis.

Genetic polymorphisms of interleukin-6 influence the development of hepatitis B virus-related liver cirrhosis in the Han Chinese population.

BACKGROUND: Interleukin-6 (IL-6) plays an important role in chronic inflammation. Thus, we aimed to investigate the effects of IL-6 polymorphisms on predicting the progression of hepatitis B virus (HBV)-r elated liver cirrhosis. METHODS: A cross-sectional study was conducted to analyse IL-6 polymorphisms and serum levels of IL-6 in HBV-infected patients at different clinical phases and in healthy controls. IL-6 polymorphisms were detected by the TaqMan PCR method, and plasma IL-6 levels were assessed by ELISA. RESULTS: Our analysis included 182 chronic hepatitis B (CHB) patients, 190 HBV-infected liver cirrhosis cases, 125 inactive HBsAg carriers, and 246 healthy controls. Seven SNPs in IL-6 including rs10499563, rs17147230, rs1800796, rs2069837, rs1524107, rs2066992, and rs2069852 were analysed. In a haplotype analysis between HBV-infected liver cirrhosis cases and CHB patients, inactive HBV carriers or healthy controls, haplotype CT in block 1 and haplotype GGCGG in block 2 were associated with liver cirrhosis (P <0.05). Moreover, the genotype or allele frequencies were significantly different in IL-6 rs10499563 and rs2069837 when HBV-infected liver cirrhosis patients were compared with CHB patients, inactive HBV carriers or healthy controls. A further study found that compared with that in the healthy controls, inactive HBV carriers or CHB patients, plasma IL-6 was elevated in HBV-infected liver cirrhosis patients. CONCLUSION: In conclusion, the IL-6 rs10499563 and rs2069837 polymorphisms are associated with incidence of liver cirrhosis may through their effects on IL-6 expression and these two single nucleotide polymorphisms can be used as potential prognostic markers of HBV-related liver cirrhosis.

Effects of BTK signalling in pathogenic microorganism infections.

As a cytoplasmic protein tyrosine kinase, Bruton's tyrosine kinase (Btk) is widely considered as a vital kinase in many aspects of different physiologic processes. It is engaged in many important signalling pathways related to the immune response, such as the B cell receptor pathway, pattern-recognition receptor pathway, and triggering receptor expressed on myeloid cell pathway. Recent studies have increasingly focused on the important role of Btk in various inflammatory diseases, which are related to Btk expression in myeloid innate immune cells, such as macrophages, dendritic cells and neutrophils. Although some investigations have explored the role of Btk in microbial infections, many aspects remain elusive, and some of the results are opposite and controversial. Considering the complicated and multiple roles of Btk in the immune system, we summarized the engagement of Btk signalling in various pathogenic microorganism infections, the possible mechanisms involved and its therapeutic potential in the control of infectious diseases.

Serum Procalcitonin Correlates with Renal Function in Hepatitis B Virus-Related Acute-on-Chronic Liver Failure.

BACKGROUND/AIMS: To investigate the relationship between elevated serum procalcitonin (PCT) and renal function in hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF). METHODS: HBV-ACLF patients (n = 201) presenting to the State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University, from January 2013 to November 2016 were categorized into three groups according to serum PCT levels: (i) normal group (n = 74) had PCT of ≤ 0.5 ng/mL; (ii) elevated group (n = 85) had PCT in the range 0.5-1.0 ng/mL; and (iii) highly elevated group (n = 42) had PCT of > 1.0 ng/mL. Thirty-five cases received standard care after admission. Serum PCT levels and renal function were determined during a two-week follow-up. RESULTS: Significant increases in serum creatinine (Cr) were recorded in male and female patients in the elevated group and highly elevated group compared with the normal group (P < 0.05). In addition, serum Cr levels in male and female patients were significantly higher in the highly elevated group than in the elevated group (P < 0.05). The glomerular filtration rate (GFR) was significantly lower in the highly elevated group (P < 0.05) and this group had the highest risk of altered Cr (45.9% in males; 80% in females) and abnormal GFR (37.5%). Serum PCT levels correlated significantly with all renal function parameters including homocysteine (Hcy), GFR, Cr, blood urea nitrogen, uric acid, and cystatin C at baseline and during treatment. Univariate and multivariate analyses indicated that serum PCT was a strong predictor of renal dysfunction. CONCLUSION: Serum PCT is closely related to renal dysfunction in HBV-ACLF.

Hepatic Macrophages are the Cell Source of Hepatic Procalcitonin in Acute Liver Failure.

BACKGROUND/AIMS: Serum procalcitonin (PCT) is elevated in acute liver failure (ALF), but the expression of PCT in the liver has not been elucidated. We aimed to clarify the regulation of hepatic PCT expression and the cell sources in ALF. METHODS: Human monocytic leukemia line U937 cells were treated with 12-O-tetradecanoylphorbol-l3-acetate (PMA) (100 ng/ mL) for 24 h to induce activated macrophages. In the presence of lipopolysaccharide (LPS, 1 µg/mL), activated macrophages and human hepatocyte line L02 cells were incubated with LPS or co-cultured for 0, 2, 6, and 24 h. In an in vivo experiment, male C57BL/6 mice were challenged with intraperitoneal LPS/D-galactosamine (LPS/D-GalN). Serum liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using an automatic chemical analyzer. Inflammatory mediators were measured by real-time PCR and liver histology was examined by hematoxylin-eosin (HE) staining and immunohistochemistry (IHC). RESULTS: LPS induced the upregulation of PCT mRNA in U937-activated macrophages but not in L02 cells. When co-cultured with L02 cells, the expression of PCT mRNA of activated macrophages was upregulated compared to controls; however, the activated macrophages did not induce the expression of PCT mRNA in L02 cells in the presence of LPS. Moreover, serum liver enzymes (ALT, AST), inflammation, necrosis, and hepatic expression of PCT were significantly elevated in the LPS/D-GalN-challenged ALF mouse model. IHC revealed that PCT expression was co-localized with hepatic macrophages. CONCLUSIONS: Hepatic PCT expression is upregulated in ALF. Hepatic macrophages but not hepatocytes are the cell source of hepatic PCT expression.

0404 inhibits hepatocellular carcinoma through a p53/miR-34a/SIRT1 positive feedback loop.

DNA-damaging agents have been used in cancer chemotherapy for a long history. Unfortunately, chemotherapeutic treatment strategies against hepatocellular carcinoma (HCC) are still ineffective. We screened a novel DNA-damaging compound, designated as 0404, by using time-dependent cellular response profiling (TCRP) based on unique DNA-damage characteristics. We used human HCC cell lines and HCC xenograft mouse model to analyze the anti-cancer effects of 0404. Transcriptome and miRNA arrays were used to verify the anti-cancer mechanism of 0404. It was confirmed that p53 signaling pathway was crucial in 0404 anti-cancer activity and the expression of miR-34a, a key tumor-suppressive miRNA, was up-regulated in 0404-treated HepG2 cells. MiR-34a expression was also down-regulated in HCCs compared with corresponding non-cancerous hepatic tissues. We further identified the mechanisms of 0404 in HepG2 cells. 0404 increased miR-34a expression and acylation p53 protein levels and decreased SIRT1 protein levels in a concentration-dependent manner. The sensitivity of HepG2 cells to 0404 was significantly decreased by transfection with miR-34a inhibitors and SIRT1 protein levels were up-regulated by miR-34a inhibition. Our findings show that 0404 is probably an attractive agent for treating HCC, especially in HCC with wide type (WT) p53, through forming a p53/miR-34a/SIRT1 signal feedback loop to promote cell apoptosis.

MiR-122 modification enhances the therapeutic efficacy of adipose tissue-derived mesenchymal stem cells against liver fibrosis.

Mesenchymal stem cell (MSC) transplantation alone may be insufficient for treatment of liver fibrosis because of complicated histopathological changes in the liver. Given that miR-122 plays an essential role in liver fibrosis by negatively regulating the proliferation and transactivation of hepatic stellate cells (HSCs), this study investigated whether miR-122 modification can improve the therapeutic efficacy of adipose tissue-derived MSCs in treating liver fibrosis. MiR-122-modified AMSCs (AMSC-122) were constructed through lentivirus-mediated transfer of pre-miR-122. MiR-122-modified AMSCs expressed high level of miR-122, while they retained their phenotype and differentiation potential as naïve AMSCs. AMSC-122 more effectively suppressed the proliferation of and collagen maturation in HSCs than scramble miRNA-modified AMSCs. In addition, AMSC-derived exosomes mediated the miR-122 communication between AMSCs and HSCs, further affecting the expression levels of miR-122 target genes, such as insulin-like growth factor receptor 1 (IGF1R), Cyclin G(1) (CCNG1) and prolyl-4-hydroxylase α1 (P4HA1), which are involved in proliferation of and collagen maturation in HSCs. Moreover, miR-122 modification enhanced the therapeutic efficacy of AMSCs in the treatment of carbon tetrachloride (CCl4 )-induced liver fibrosis by suppressing the activation of HSCs and alleviating collagen deposition. Results demonstrate that miR-122 modification improves the therapeutic efficacy of AMSCs through exosome-mediated miR-122 communication; thus, miR-122 modification is a new potential strategy for treatment of liver fibrosis.

Direct targeting sperm-associated antigen 9 by miR-141 influences hepatocellular carcinoma cell growth and metastasis via JNK pathway.

BACKGROUND: The aberrant expression of sperm-associated antigen 9 (SPAG9) is associated with numerous cancers, including hepatocellular carcinoma (HCC). The exploration of molecules and mechanisms regulating SPAG9 expression may provide new options for HCC therapy. METHODS: MiRNA target prediction programs were used to explore SPAG9-targeted miRNAs. SPAG9 and miR-141 expression were detected in HCC tissues and cell lines by Western blot and real-time PCR. Dual-luciferase reporter assay was utilized to validate SPAG9 as a direct target gene of miR-141. Cell proliferation, invasion, and migration assays were used to determine whether miR-141-mediated regulation of SPAG9 could affect HCC progression. RESULTS: An inverse correlation was observed between SPAG9 and miR-141 expression in HCC tissues and cell lines. Dual-luciferase reporter assay further showed that SPAG9 was a direct target gene of miR-141. The ectopic expression of miR-141 could markedly suppress SPAG9 expression in HCC cells. MiR-141 overexpression also resulted in significantly reduced cell proliferation, invasion, and migration, and imitation of the SPAG9 knockdown effects on HCC cells. Furthermore, SPAG9 restoration in miR-141-expressing cells sufficiently attenuated the tumor-suppressive effects of miR-141. Finally, JNK activity was found to be reduced by miR-141 overexpression the same way as by SPAG9 silencing. The overexpression of SPAG9 lacking its 3'-UTR significantly restored JNK activity and its downstream genes in miR-141-transfected HCC cells. CONCLUSION: MiR-141 suppression may cause aberrant expression of SPAG9 and promote HCC tumorigenesis via JNK pathway.