The zinc finger protein Miz1 suppresses liver tumorigenesis by restricting hepatocyte-driven macrophage activation and inflammation.

Chronic inflammation plays a central role in hepatocellular carcinoma (HCC), but the contribution of hepatocytes to tumor-associated inflammation is not clear. Here, we report that the zinc finger transcription factor Miz1 restricted hepatocyte-driven inflammation to suppress HCC, independently of its transcriptional activity. Miz1 was downregulated in HCC mouse models and a substantial fraction of HCC patients. Hepatocyte-specific Miz1 deletion in mice generated a distinct sub-group of hepatocytes that produced pro-inflammatory cytokines and chemokines, which skewed the polarization of the tumor-infiltrating macrophages toward pro-inflammatory phenotypes to promote HCC. Mechanistically, Miz1 sequestrated the oncoprotein metadherin (MTDH), preventing MTDH from promoting transcription factor nuclear factor κB (NF-κB) activation. A distinct sub-group of pro-inflammatory cytokine-producing hepatocytes was also seen in a subset of HCC patients. In addition, Miz1 expression inversely correated with disease recurrence and poor prognosis in HCC patients. Our findings identify Miz1 as a tumor suppressor that prevents hepatocytes from driving inflammation in HCC.

Genetic and phenotypic difference in CD8+ T cell exhaustion between chronic hepatitis B infection and hepatocellular carcinoma.

BACKGROUND: Several recent studies published have suggested that T cell exhaustion exists both in chronic infection and cancer. However, to date, few studies have investigated their differences. Here we designed this study to explore the genetic and phenotypic difference in CD8+ T cell exhaustion between chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC). METHODS: In this study, we assayed the phenotypes and functional states of CD8+ T cells separating from human CHB tissues and HCC tissues, and re-analyse the single-cell sequencing data (GSE98638) published previously. Clustering analysis of genes was performed using the T cell exhaustion gene modules (modules 1-4) proposed by Speiseret al. RESULTS: CD8+ T cells from liver tissues of both CHB and HCC showed high levels of exhaustion markers, DOI: programmed cell death-1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and lymphocyte-activation gene 3 (LAG-3), decreased proliferation (Ki67) and cell activity (CD69), and reduced production of effector cytokines (interferon-γ, interleukin-2 and tumour necrosis factor-α). Compared with CD8+ T cells from CHB tissues, those from HCC tissue showed higher expression levels of exhaustion markers, lower levels of proliferation, cell activity and the production of effector cytokines. Cluster analysis showed that exhaustion associated genes in CHB and HCC are inclined to distribute into modules 3 while those isolated from HCC into modules 1 and 2. CONCLUSIONS: CD8+ T cell exhaustion existed both in CHB and HCC, but the phenotypes, functional states and underlying mechanisms are somewhat different between the two.

Exosomes derived from exhausted CD8+ T cells impaired the anticancer function of normal CD8+ T cells.

BACKGROUND: Previous studies suggested that diverse cells in cancer microenvironment can interact with CD8+ T cells via exosomes. We designed this study to explore the potential interaction between exhausted CD8+ T cells and normal CD8+ T cells via exosome. METHODS: Fluorescence activated cell sorting was used to get PD1+TIM3+/PD1-TIM3-CD8+ T cells. Exosomes from the cell culture medium were collected by ultracentrifugation. Microarrays were performed to analyse the lncRNA expression profile in exosomes. RESULTS: Functional exhausted CD8+ T cells could secrete vast exosomes, which can be uptake by normal CD8+ T cells, and impaired their proliferation (Ki67), cell activity (CD69) and the production of cytokines such as interferon-γ and interleukin-2. Microarray detection identified 257 candidate lncRNAs differently expressed in exosomes derived from exhausted CD8+ T cells and non-exhausted CD8+ T cells. Functional enrichment analysis indicated that these lncRNAs actively participated in the regulation of diverse process of CD8+ T cell activity, like metabolism, gene expression, biosynthetic process and so forth. CONCLUSIONS: The exosomes derived from exhausted CD8+ T cells could be uptake by non-exhausted CD8+ T cells and subsequently impaired the function of receipt cells. Exosomes secreted from exhausted CD8+ T cells have distinct lncRNA expression profiles which are significantly different from those in exosomes secreted by non-exhausted CD8+ T cells.

Long non-coding RNA LUCAT1 promotes tumourigenesis by inhibiting ANXA2 phosphorylation in hepatocellular carcinoma.

Long non-coding RNAs (lncRNAs) play essential roles in diverse biological processes; however, current understanding of the mechanism underlying the regulation of tumour proliferation and metastasis is limited. Lung cancer-associated transcript 1 (LUCAT1) has been reported in a variety of human cancers, while its role in hepatocellular carcinoma (HCC) remains unclear. This study aimed to determine the biological role and underlying mechanism of LUCAT1 on progression and metastasis in HCC cells and clinical specimens. Our results demonstrated that LUCAT1 was up-regulated in HCC tissues and cells. Loss- and gain-of-function studies revealed that LUCAT1 promotes the proliferation and metastasis of HCC cells in vitro and in vivo. Furthermore, RNA pulldown and Western blot assays indicated that LUCAT1 inhibited the phosphorylation of Annexin A2 (ANXA2) to reduce the degradation of ANXA2-S100A10 heterotetramer (AIIt), which in turn accelerated the secretion of plasminogen into plasmin, thereby resulting in the activation of metalloprotease proteins. In conclusion, we propose that LUCAT1 serves as a novel diagnostic and therapeutic target for HCC.

TOX promotes the exhaustion of antitumor CD8+ T cells by preventing PD1 degradation in hepatocellular carcinoma.

BACKGROUND & AIMS: The thymocyte selection-associated high mobility group box protein (TOX) plays a vital role in T cell development and differentiation, however, its role in T cell exhaustion was unexplored. Here, we aim to investigate the role of TOX in regulating the antitumor effect of CD8+ T cells in hepatocellular carcinoma. METHODS: Fully functional, partially and severely exhausted tumor-infiltrating CD8+ T cells were sorted by flow cytometry and subjected to transcriptome sequencing analysis. Upregulated TOX expression was validated by flow cytometry. The antitumor function of CD8+ T cells with TOX downregulation or overexpression was studied in a mouse HCC model and HCC patient-derived xenograft mouse model. Transcriptome sequencing analysis was performed in TOX-overexpressing and control CD8+ T cells. The mechanism underlying the TOX-mediated regulation of PD1 expression was studied by laser confocal detection, immune co-precipitation and flow cytometer. RESULTS: TOX was upregulated in exhausted CD8+ T cells in hepatocellular carcinoma. TOX downregulation in CD8+ T cells inhibited tumor growth, increased CD8+ T cell infiltration, alleviated CD8+ T cell exhaustion and improved the anti-PD1 response of CD8+ T cells. The mechanism behind this involved the binding of TOX to PD1 in the cytoplasm, which facilitated the endocytic recycling of PD1, thus maintaining abundant PD1 expression at the cell surface. High expression of TOX in peripheral CD8+ T cells correlated with poorer anti-PD1 responses and prognosis. CONCLUSIONS: TOX promotes CD8+ T cell exhaustion in hepatocellular carcinoma by regulating endocytic recycling of PD1. Downregulating TOX expression in CD8+ T cells exerts synergistic effects with anti-PD1 therapy, highlighting a promising strategy for cancer immunotherapy. LAY SUMMARY: Abundant TOX expression in CD8+ T cells impairs their antitumor function in hepatocellular carcinoma. Mechanically, TOX reduces PD1 degradation and promotes PD1 translocation to the cell surface in CD8+ T cells, thus maintaining high PD1 expression at the cell surface. Downregulating TOX expression improves the antitumor function of CD8+ T cells, which shows the synergetic role of anti-PD1 therapy, highlighting a promising strategy for enhancement of cancer immunotherapy.

Prognostic Value of Phosphotyrosine Phosphatases in Hepatocellular Carcinoma.

BACKGROUND/AIMS: During the occurrence and progression of hepatocellular carcinoma (HCC), phosphotyrosine phosphatases (PTPs) are usually described as tumor suppressors or proto-oncogenes, and to some degree are correlated with the prognosis of HCC. METHODS: A total of 321 patients from the Cancer Genome Atlas (TCGA) database and 180 patients from our validated cohort with hepatocellular carcinoma were recruited in this study. Kaplan-Meier, univariate and multivariate Cox proportional hazards model were used to evaluate the risk factors for survival. Quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC) were applied to detect the expression levels of PTP genes. RESULTS: After screening the data of TCGA, we identified five PTPs as HCC overall survival related PTP genes, among which only three (PTPN12, PTPRN, PTPN18) exhibited differential expression levels in our 180 paired HCC and adjacent tissues (P< 0.001). Further analysis revealed that expression of PTPN18 was positively, but PTPRN was negatively associated with prognosis of HCC both in TCGA cohort and our own cohort. As to PTPN12, results turned out to be opposite according to HBV status. In detail, higher expression of PTPN12 was associated with better outcome in HBV group but worse prognosis in Non-HBV group. CONCLUSION: Our results suggested that PTPN12, PTPRN and PTPN18 were independent prognostic factors in HCC.

Dietary fiber alleviates alcoholic liver injury via Bacteroides acidifaciens and subsequent ammonia detoxification.

The gut microbiota and diet-induced changes in microbiome composition have been linked to various liver diseases, although the specific microbes and mechanisms remain understudied. Alcohol-related liver disease (ALD) is one such disease with limited therapeutic options due to its complex pathogenesis. We demonstrate that a diet rich in soluble dietary fiber increases the abundance of Bacteroides acidifaciens (B. acidifaciens) and alleviates alcohol-induced liver injury in mice. B. acidifaciens treatment alone ameliorates liver injury through a bile salt hydrolase that generates unconjugated bile acids to activate intestinal farnesoid X receptor (FXR) and its downstream target, fibroblast growth factor-15 (FGF15). FGF15 promotes hepatocyte expression of ornithine aminotransferase (OAT), which facilitates the metabolism of accumulated ornithine in the liver into glutamate, thereby providing sufficient glutamate for ammonia detoxification via the glutamine synthesis pathway. Collectively, these findings uncover a potential therapeutic strategy for ALD involving dietary fiber supplementation and B. acidifaciens.

Experimental models of fatty liver diseases: Status and appraisal.

Fatty liver diseases, including alcohol-associated liver disease (ALD) and nonalcoholic fatty liver disease nonalcoholic fatty liver disease (NAFLD), affect a large number of people worldwide and become one of the major causes of end-stage liver disease, such as liver cirrhosis and hepatocellular carcinoma (HCC). Unfortunately, there are currently no approved pharmacological treatments for ALD or NAFLD. This situation highlights the urgent need to explore new intervention targets and discover effective therapeutics for ALD and NAFLD. The lack of properly validated preclinical disease models is a major obstacle to the development of clinical therapies. ALD and NAFLD models have been in the development for decades, but there are still no models that recapitulate the full spectrum of ALD and NAFLD. Throughout this review, we summarize the current in vitro and in vivo models used for research on fatty liver diseases and discuss the advantages and limitations of these models.

Mucosal-Associated Invariant T Cells in the Digestive System: Defender or Destroyer?

Mucosal-associated invariant T (MAIT) cells are a subset of innate T lymphocytes that express the semi-invariant T cell receptor and recognize riboflavin metabolites via the major histocompatibility complex class I-related protein. Given the abundance of MAIT cells in the human body, their role in human diseases has been increasingly studied in recent years. MAIT cells may serve as targets for clinical therapy. Specifically, this review discusses how MAIT cells are altered in gastric, esophageal, intestinal, and hepatobiliary diseases and describes their protective or pathogenic roles. A greater understanding of MAIT cells will provide a more favorable therapeutic approach for digestive diseases in the clinical field.

Interplay Between Liver Type 1 Innate Lymphoid Cells and NK Cells Drives the Development of Alcoholic Steatohepatitis.

BACKGROUND & AIMS: Liver contains high frequency of group 1 innate lymphoid cells (ILC), which are composed of comparable number of type 1 ILC (ILC1) and natural killer (NK) cells in steady state. Little is known about whether and how the interaction between ILC1 and NK cells affects the development of alcoholic liver disease. METHODS: A mouse model of chronic alcohol abuse plus single-binge (Gao-Binge model) was established. The levels of alanine aminotransferase/aspartate aminotransferase, hepatic lipid, and inflammatory cytokines or neutrophils were measured to evaluate the degree of liver injury, steatosis, and inflammation. Flow cytometric analysis, cell depletion, or adoptive transfer were used to interrogate the interaction between ILC1 and NK cells. RESULTS: Upon chronic alcohol consumption, NK cells, but not ILC1, underwent apoptosis, resulting in ILC1 dominance among group 1 ILC. Interleukin (IL) 17A expression was up-regulated, and increased IL17A was mainly derived from liver ILC1 after chronic alcohol feeding. Either depletion of ILC1 or neutralization of IL17A could significantly attenuate liver steatosis, inflammation, and injury in alcohol-fed mice. In contrast, normalization of the ILC1/NK cells ratio through NK cells transfer or expanding NK cells had a significant hepatoprotection against alcohol-induced steatohepatitis. Furthermore, NK cell-derived interferon gamma exerted a protective function via inhibiting IL17A production by liver ILC1 during alcoholic steatohepatitis. CONCLUSIONS: This is the first study showing that the interplay between liver ILC1 and NK cells occurs and drives the development of alcoholic steatohepatitis. Our findings support further exploration of liver ILC1 or NK cells as a therapeutic target for the treatment of alcohol-associated liver disease.

Endoplasmic reticulum stress in innate immune cells - a significant contribution to non-alcoholic fatty liver disease.

Liver disease and its complications affect millions of people worldwide. NAFLD (non-alcoholic fatty liver disease) is the liver disease associated with metabolic dysfunction and consists of four stages: steatosis with or without mild inflammation (NAFLD), non-alcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. With increased necroinflammation and progression of liver fibrosis, NAFLD may progress to cirrhosis or even hepatocellular carcinoma. Although the underlying mechanisms have not been clearly elucidated in detail, what is clear is that complex immune responses are involved in the pathogenesis of NASH, activation of the innate immune system is critically involved in triggering and amplifying hepatic inflammation and fibrosis in NAFLD/NASH. Additionally, disruption of endoplasmic reticulum (ER) homeostasis in cells, also known as ER stress, triggers the unfolded protein response (UPR) which has been shown to be involved to inflammation and apoptosis. To further develop the prevention and treatment of NAFLD/NASH, it is imperative to clarify the relationship between NAFLD/NASH and innate immune cells and ER stress. As such, this review focuses on innate immune cells and their ER stress in the occurrence of NAFLD and the progression of cirrhosis.

Downregulation of TPX2 impairs the antitumor activity of CD8+ T cells in hepatocellular carcinoma.

Targeting key genes that play dominant roles in T cell dysfunction is an efficient strategy for cancer immunotherapy. Here, we aimed to investigate the role of TPX2 in the antitumor effect of CD8 + T cells in hepatocellular carcinoma (HCC). Flow cytometry was used to assay the level of cell surface markers and cytokines in T cells, through which we found that TPX2 was downregulated in HCC-infiltrating CD8 + T cells. TPX2 depletion restricted the antitumor activity of CD8 + T cells, and TPX2 overexpression increased the antitumor effect of CD8 + T cells in tumor-bearing Cd8-/- mice. TPX2 overexpression improved the antitumor function of human CD8 + T cells and response to anti-PD-1 therapy in an HCC patient-derived xenograft (PDX) mouse model with or without anti-PD-1 therapy. In mechanism, TPX2 promotes the phosphorylation of P65, thus increases the level of p-P65 in nuclear, and p-P65 binds to the promoter of CXCR5, activates its transcription, and increases the level of CXCR5 on CD8 + T cells in a TPX2-dependent way. In conclusion, TPX2 maintains the antitumor effect of CD8 + T cells in HCC by regulating CXCR5 via the NF-κB signaling pathway. Increased TPX2 expression in CD8 + T cells exerts synergistic effects with anti-PD-1 therapy, suggesting a promising immunotherapeutic method in HCC.

Microwave-Assisted Ablation Improves the Prognosis of Patients With Hepatocellular Carcinoma Undergoing Liver Resection.

OBJECTIVE: We evaluated microwave-assisted liver resection for hepatocellular carcinoma. PATIENTS AND METHODS: We enrolled 79 patients in this study, and microwave ablation was used for liver resection. Patients were randomized to group A (50.6%; n = 40), liver resection without microwave ablation, or group B (49.4%; n = 39), liver resection performed using microwave ablation. Data were analyzed for statistical significance. RESULTS: Of the participants enrolled, 60 were male, and the participant's average age was 59.32 ± 10.34 years. The mean overall tumor diameter was 4.39 (2.00) cm, and this did not differ between groups. Intraoperative blood loss in group B was significantly less than that in group A ( P < .001). No differences were reported between the 2 groups regarding surgical time ( P = .914), postoperative morbidity ( P = .718), and late postoperative complications ( P = .409). Postoperative drainage volume for group B was less than that of group A on the first ( P = .005) and third ( P = .019) day after surgery. The time of postoperative hospitalization in group B was significantly shorter than that in group A ( P < .001). Local recurrence was noted in 18.99% of cases (n = 15) in group B, which is less than that of group A ( P = 0.047), while in group B distant metastasis is less but not statistically significant ( P = 0.061). The 1-year and 3-year cumulative survival rates were 57% and 93.7%, respectively. CONCLUSIONS: The curative effects of liver resection combined with microwave ablation during operation are superior to only liver resection in the treatment of primary liver cancer.

14-3-3ζ delivered by hepatocellular carcinoma-derived exosomes impaired anti-tumor function of tumor-infiltrating T lymphocytes.

Increasing evidence shows that the anti-tumor functions of tumor-infiltrating T lymphocytes (TILs) were inhibited significantly, but the underlying mechanisms remain not fully understood. In this study, we found that 14-3-3ζ expression was up-regulated in hepatocellular carcinoma (HCC) cells and in TILs. TILs with 14-3-3ζ high-expression (14-3-3ζhigh) exhibited impaired activation (CD69), proliferation (Ki67) and anti-tumor functions compared to 14-3-3ζ low expression (14-3-3ζlow) TILs. Flow cytometry assay showed that compared with 14-3-3ζlow CD8+T cells, 14-3-3ζhigh ones exhibited higher frequency of exhausted phenotypes as measured by inhibitory receptors such as PD-1, TIM-3, LAG3, and CTLA-4. 14-3-3ζ overexpression inhibited the activity and proliferation of peripheral blood CD3+ T cells, deviated the differentiation of naive T cells from effector T cells to regulatory T cells. Moreover, we found that 14-3-3ζ expression levels in TILs correlated positively with those in HCC cells. Naive T cells co-cultured with HCC cells or the visible components of culture medium of HCC cells exhibited increased 14-3-3ζ expression. Stochastic optical reconstruction microscopy (STORM) and confocal assay showed that 14-3-3ζ-containing exosomes derived from HCC cells could be swallowed by T cells, suggesting that 14-3-3ζ might be transmitted from HCC cells to TILs at least partially through exosomes. In conclusion, our study for the first time demonstrated that 14-3-3ζ is up-regulated in and inhibited the anti-tumor functions of tumor-infiltrating T cells in HCC microenvironment and that 14-3-3ζ might be transmitted from HCC cells to T cells at least partially through exosomes.

Kinesin superfamily protein expression and its association with progression and prognosis in hepatocellular carcinoma.

OBJECTIVES: In this study, we characterized the expression of 32 other kinesin superfamily proteins (KIFs) and analyzed their association with the progression and prognosis of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: The data from 295 HCC patients from The Cancer Genome Atlas were included in the study. An independent t-test was used to compare the KIF levels in HCC and adjacent tissues. Pearson's Chi-square test was used to assess the relationships of KIF expression with tumor biomarkers and clinicopathological parameters. Kaplan-Meier plots and log-rank tests were used to analyze survival, and univariate and multivariate analyses were used to identify independent prognostic factors. RESULTS: The expressions of 32 KIFs were compared between HCC and adjacent nontumor tissues. Among them, 12 KIFs showed no statistical significance, 17 KIFs were upregulated, and three KIFs were downregulated in tumor tissues. The levels of some KIFs were markedly correlated with that of biomarkers for the S phase and proliferation. KIF2A and KIFC3 expression was positively associated with biomarkers for cell invasion and migration. Some KIF overexpression was significantly associated with neoplastic pathological grade and tumor-node-metastasis staging. Furthermore, KIF2C, KIF4A, and KIF11 overexpression were significantly associated with shorter relapse-free survival times. KIF2A, KIF2C, KIF3A, KIF4B, KIF11, KIF15, KIFC1, and KIFC3 overexpression was associated with shorter overall survival (OS) times, whereas higher expression of KIF19 was associated with a longer OS time. Further multivariate analyses suggested that only KIF4B was an independent prognostic factor for HCC. CONCLUSIONS: Most overexpressions of abnormal KIFs were significantly associated with HCC progression and prognosis, indicating that KIFs could be prognostic and therapeutic biomarkers for HCC. However, it is necessary to further study the function of KIFs and their mechanisms involved in HCC.