Integrated multi-omic analysis identifies fatty acid binding protein 4 as a biomarker and therapeutic target of ischemia-reperfusion injury in steatotic liver transplantation.

BACKGROUND AND AIMS: Due to a lack of donor grafts, steatotic livers are used more often for liver transplantation (LT). However, steatotic donor livers are more sensitive to ischemia-reperfusion (IR) injury and have a worse prognosis after LT. Efforts to optimize steatotic liver grafts by identifying injury targets and interventions have become a hot issue. METHODS: Mouse LT models were established, and 4D label-free proteome sequencing was performed for four groups: normal control (NC) SHAM, high-fat (HF) SHAM, NC LT, and HF LT to screen molecular targets for aggravating liver injury in steatotic LT. Expression detection of molecular targets was performed based on liver specimens from 110 donors to verify its impact on the overall survival of recipients. Pharmacological intervention using small-molecule inhibitors on an injury-related target was used to evaluate the therapeutic effect. Transcriptomics and metabolomics were performed to explore the regulatory network and further integrated bioinformatics analysis and multiplex immunofluorescence were adopted to assess the regulation of pathways and organelles. RESULTS: HF LT group represented worse liver function compared with NC LT group, including more apoptotic hepatocytes (P < 0.01) and higher serum transaminase (P < 0.05). Proteomic results revealed that the mitochondrial membrane, endocytosis, and oxidative phosphorylation pathways were upregulated in HF LT group. Fatty acid binding protein 4 (FABP4) was identified as a hypoxia-inducible protein (fold change > 2 and P < 0.05) that sensitized mice to IR injury in steatotic LT. The overall survival of recipients using liver grafts with high expression of FABP4 was significantly worse than low expression of FABP4 (68.5 vs. 87.3%, P < 0.05). Adoption of FABP4 inhibitor could protect the steatotic liver from IR injury during transplantation, including reducing hepatocyte apoptosis, reducing serum transaminase (P < 0.05), and alleviating oxidative stress damage (P < 0.01). According to integrated transcriptomics and metabolomics analysis, cAMP signaling pathway was enriched following FABP4 inhibitor use. The activation of cAMP signaling pathway was validated. Microscopy and immunofluorescence staining results suggested that FABP4 inhibitors could regulate mitochondrial membrane homeostasis in steatotic LT. CONCLUSIONS: FABP4 was identified as a hypoxia-inducible protein that sensitized steatotic liver grafts to IR injury. The FABP4 inhibitor, BMS-309403, could activate of cAMP signaling pathway thereby modulating mitochondrial membrane homeostasis, reducing oxidative stress injury in steatotic donors.

Downregulation of N4-acetylcytidine modification in myeloid cells attenuates immunotherapy and exacerbates hepatocellular carcinoma progression.

BACKGROUND: N4-acetylcytidine (ac4C) is a conserved and abundant mRNA modification that controls protein expression by affecting translation efficiency and mRNA stability. Whether the ac4C modification of mRNA regulates hepatocellular carcinoma (HCC) development or affects the immunotherapy of HCC is unknown. METHODS: By constructing an orthotopic transplantation mouse HCC model and isolating tumour-infiltrated immunocytes, we evaluated the ac4C modification intensity using flow cytometry. Remodelin hydrobromide (REM), an ac4C modification inhibitor, was systematically used to understand the extensive role of ac4C modification in immunocyte phenotypes. Single-cell RNA-seq was performed to comprehensively evaluate the changes in the tumour-infiltrating immunocytes and identify targeted cell clusters. RNA-seq and RIP-seq analyses were performed to elucidate the underlying molecular mechanisms. Tyramide Signal Amplification (TSA) analysis on the HCC tissue microarray was performed to explore the clinical relatedness of our findings. RESULTS: Ac4C modification promoted M1 macrophage infiltration and reduced myeloid-derived suppressor cell MDSCs infiltration in HCC. The inhibition of ac4C modification induces PDL1 expression by stabilising mRNA in the myeloid cells, thereby attenuating the CTL-mediated tumour cell-killing ability. High infiltration of ac4C+CD11b+ cells is positively related to a better prognosis in patients with HCC. CONCLUSIONS: Ac4C modification of myeloid cells enhanced the HCC immunotherapy by suppressing PDL1 expression.

SULT2B1-CS-DOCK2 axis regulates effector T-cell exhaustion in HCC microenvironment.

BACKGROUND AND AIMS: HCC is a malignant disease. Compared with tyrosine kinase inhibitors (the classical therapy), immune checkpoint inhibitors are more effective in the treatment of HCC, despite their limited efficacy. Among these restricted factors, exhaustion of tumor-infiltrated lymphocytes, especially CD8 + T cells, is a core event. We aimed to determine the key factors contributing to CD8 + T-cell infiltration in HCC and investigate the underlying mechanisms. APPROACH AND RESULTS: Using machine learning and multiplex immunohistochemistry analysis, we showed that dedicator of cytokinesis protein 2 (DOCK2) was a potential indicator of infiltrated CD8 + T cells in HCC. Using RNA sequencing, flow cytometry analysis, and mouse HCC models, we demonstrated that DOCK2 inactivation accounted for infiltrated CD8 + T-cell exhaustion in tumors. Using quasi-targeted metabolomics, mass spectrum, and mass cytometry by time of flight analysis, we found that cholesterol sulfate synthesized by sulfotransferase 2B1 in tumor cells suppressed DOCK2 enzymatic activity of T cells. Through virtual screening, molecular docking simulation, and experiments validation, we demonstrated that tolazamide reversed DOCK2 inactivation-mediated CD8 + T-cell exhaustion and enhanced anti-programmed death-ligand 1 antibody+apatinib immunotherapeutic effects on HCC. CONCLUSIONS: This study indicates that DOCK2 controls CD8 + T-cell infiltration in HCC, and cholesterol sulfate synthesized by sulfotransferase 2B1 in tumor cells promotes effector T-cell exhaustion. The findings suggest that the usage of conventional drugs affects immunotherapy efficacy in HCC patients.

Ten-eleven translocation-2-mediated macrophage activation promotes liver regeneration.

BACKGROUND: The remarkable regenerative capacity of the liver enables recovery after radical Hepatocellular carcinoma (HCC) resection. After resection, macrophages secrete interleukin 6 and hepatocyte growth factors to promote liver regeneration. Ten-eleven translocation-2 (Tet2) DNA dioxygenase regulates pro-inflammatory factor secretion in macrophages. In this study, we explored the role of Tet2 in macrophages and its function independent of its enzymatic activity in liver regeneration. METHODS: The model of liver regeneration after 70% partial hepatectomy (PHx) is a classic universal model for studying reparative processes in the liver. Mice were euthanized at 0, 24, and 48 h after PHx. Enzyme-linked immunosorbent assays, quantitative reverse transcription-polymerase chain reaction, western blotting, immunofluorescence analysis, and flow cytometry were performed to explore immune cell infiltration and liver regenerative capability. Molecular dynamics simulations were performed to study the interaction between Tet2 and signal transducer and activator of transcription 1 (Stat1). RESULTS: Tet2 in macrophages negatively regulated liver regeneration in the partial hepatectomy mice model. Tet2 interacted with Stat1, inhibiting the expression of proinflammatory factors and suppressing liver regeneration. The Tet2 inhibitor attenuated the interaction between Stat1 and Tet2, enhanced Stat1 phosphorylation, and promoted hepatocyte proliferation. The proliferative function of the Tet2 inhibitor relied on macrophages and did not affect hepatocytes directly. CONCLUSION: Our findings underscore that Tet2 in macrophages negatively regulates liver regeneration by interacting with Stat1. Targeting Tet2 in macrophages promotes liver regeneration and function after a hepatectomy, presenting a novel target to promote liver regeneration and function.

Arming CAR-T cells with cytokines and more: Innovations in the fourth-generation CAR-T development.

Chimeric antigen receptor T cells (CAR-T) therapy has shown great potential in tumor treatment. However, many factors impair the efficacy of CAR-T therapy, such as antigenic heterogeneity and loss, limited potency and persistence, poor infiltration capacity, and a suppressive tumor microenvironment. To overcome these obstacles, recent studies have reported a new generation of CAR-T cells expressing cytokines called armored CAR-T, TRUCK-T, or the fourth-generation CAR-T. Here we summarize the strategies of arming CAR-T cells with natural or synthetic cytokine signals to enhance their anti-tumor capacity. Moreover, we summarize the advances in CAR-T cells expressing non-cytokine proteins, such as membrane receptors, antibodies, enzymes, co-stimulatory molecules, and transcriptional factors. Furthermore, we discuss several prospective strategies for armored CAR-T therapy development. Altogether, these ideas may provide new insights for the innovations of the next-generation CAR-T therapy.

Human umbilical cord mesenchymal stem cells alleviate fatty liver ischemia-reperfusion injury by activating autophagy through upregulation of IFNγ.

Liver ischemia-reperfusion injury (IRI) is an important factor affecting the prognosis of liver transplantation, and extended criteria donors (e.g., steatosis donor livers) are considered to be more sensitive to ischemia-reperfusion injury in liver transplantation. Currently, the application of human umbilical cord mesenchymal stem cells (hMSCs) has great promise in the treatment of various injuries in the liver. This study aimed to investigate the therapeutic role and mechanism of hMSCs in fatty liver IRI. After more than 8 weeks of high-fat chow feeding, we constructed a fatty liver mouse model and established ischemic injury of about 70% of the liver. Six hours after IRI, liver injury was significantly alleviated in hMSCs-treated mice, and the expression levels of liver enzyme, inflammatory factor TNF-α, and apoptotic proteins were significantly lower than those of the control group, which were also significant in pathological sections. Transcriptomics analysis showed that IFNγ was significantly upregulated in the hMSCs group. Mechanistically, IFNγ, which activates the MAPK pathway, is a potent agonist that promotes the occurrence of autophagy in hepatocytes to exert a protective function, which was confirmed by in vitro experiments. In summary, hMSCs treatment could slow down IRI in fatty liver by activating autophagy through upregulation of IFNγ, and this effect was partly direct.

Peritoneal Gene Transfection of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand for Tumor Surveillance and Prophylaxis.

Peritoneal metastasis is very common in gastrointestinal, reproductive, and genitourinary tract cancers in late stages or postsurgery, causing poor prognosis, so effective and nontoxic prophylactic strategies against peritoneal metastasis are highly imperative. Herein, we demonstrate the first gene transfection as a nontoxic prophylaxis preventing peritoneal metastasis or operative metastatic dissemination. Lipopolyplexes of TNF-related-apoptosis-inducing-ligand (TRAIL) transfected peritonea and macrophages to express TRAIL for over 15 days. The expressed TRAIL selectively induced tumor cell apoptosis while exempting normal tissue, providing long-term tumor surveillance. Therefore, tumor cells inoculated in the pretransfected peritoneal cavity quickly underwent apoptosis and, thus, barely formed tumor nodules, significantly prolonging the mouse survival time compared with chemotherapy prophylaxis. Furthermore, lipopolyplex transfection showed no sign of toxicity. Therefore, this peritoneal TRAIL-transfection is an effective and safe prophylaxis, preventing peritoneal metastasis.

A mixed blessing for liver transplantation patients - Rapamycin.

BACKGROUND: Liver transplantation (LT) is an effective treatment option for end-stage liver disease. Mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin, are widely used post LT. DATA SOURCES: In this review, we focused on the anti-cancer activities and metabolic side effects of rapamycin after LT. The literature available on PubMed for the period of January 1999-September 2022 was reviewed. The key words were rapamycin, sirolimus, liver transplantation, hepatocellular carcinoma, diabetes, and lipid metabolism disorder. RESULTS: Rapamycin has shown excellent effects and is safer than other immunosuppressive regimens. It has exhibited excellent anti-cancer activity and has the potential in preventing hepatocellular carcinoma (HCC) recurrence post LT. Rapamycin is closely related to two long-term complications after LT, diabetes and lipid metabolism disorders. CONCLUSIONS: Rapamycin prevents HCC recurrence post LT in some patients, but it also induces metabolic disorders. Reasonable use of rapamycin benefits the liver recipients.

TNFR2 is a potent prognostic biomarker for post-transplant lung metastasis in patients with hepatocellular carcinoma.

Objective: Lung metastasis is a common and fatal complication of liver transplantation for hepatocellular carcinoma (HCC). The precise prediction of post-transplant lung metastasis in the early phase is of great value. Methods: The mRNA profiles of primary and paired lung metastatic lesions were analyzed to determine key signaling pathways. We enrolled 241 HCC patients who underwent liver transplantation from three centers. Tissue microarrays were used to evaluate the prognostic capacity of tumor necrosis factor (TNF), tumor necrosis factor receptor 1 (TNFR1), and TNFR2, particularly for post-transplant lung metastasis. Results: Comparison of primary and lung metastatic lesions revealed that the TNF-dependent signaling pathway was related to lung metastasis of HCC. The expression of TNF was degraded in comparison to that in para-tumor tissues (P<0.001). The expression of key receptors in the TNF-dependent signaling pathway, TNFR1 and TNFR2, was higher in HCC tissues than in para-tumor tissues (P<0.001). TNF and TNFR1 showed no relationship with patients' outcomes, whereas elevated TNFR2 in tumor tissue was significantly associated with worse overall survival (OS) and increased recurrence risk (5-year OS rate: 31.9% vs. 62.5%, P<0.001). Notably, elevated TNFR2 levels were also associated with an increased risk of post-transplant lung metastasis (hazard ratio: 1.146; P<0.001). Cox regression analysis revealed that TNFR2, Hangzhou criteria, age, and hepatitis B surface antigen were independent risk factors for post-transplant lung metastasis, and a novel nomogram was established accordingly. The nomogram achieved excellent prognostic efficiency (area under time-dependent receiver operating characteristic =0.755, concordance-index =0.779) and was superior to conventional models, such as the Milan criteria. Conclusions: TNFR2 is a potent prognostic biomarker for predicting post-transplant lung metastasis in patients with HCC. A nomogram incorporating TNFR2 deserves to be a helpful prognostic tool in liver transplantation for HCC.

E2F7 promotes mammalian target of rapamycin inhibitor resistance in hepatocellular carcinoma after liver transplantation.

The mammalian target of rapamycin (mTOR) pathway is frequently deregulated and has critical roles in cancer progression. mTOR inhibitor has been widely used in several kinds of cancers and is strongly recommended in patients with hepatocellular carcinoma (HCC) after liver transplantation (LT). However, the poor response to mTOR inhibitors due to resistance remains a challenge. Hypoxia-associated resistance limits the therapeutic efficacy of targeted drugs. The present study established models of HCC clinical samples and cell lines resistance to mTOR inhibitor sirolimus and screened out E2F7 as a candidate gene induced by hypoxia and promoting sirolimus resistance. E2F7 suppressed mTOR complex 1 via directly binding to the promoter of the TSC1 gene and stabilizes hypoxia-inducible factor-1α activating its downstream genes, which are responsible for E2F7-dependent mTOR inhibitor resistance. Clinically, low E2F7 expression could be an effective biomarker for recommending patients with HCC for anti-mTOR-based therapies after LT. Targeting E2F7 synergistically inhibited HCC growth with sirolimus in vivo. E2F7 is a promising target to reverse mTOR inhibition resistance. Collectively, our study points to a role for E2F7 in promoting mTOR inhibitor resistance in HCC and emphasizes its potential clinical significance in patients with HCC after LT.

Patient-derived xenograft models in hepatopancreatobiliary cancer.

Animal models are crucial tools for evaluating the biological progress of human cancers and for the preclinical investigation of anticancer drugs and cancer prevention. Various animals are widely used in hepatopancreatobiliary cancer research, and mouse models are the most popular. Generally, genetic tools, graft transplantation, and chemical and physical measures are adopted to generate sundry mouse models of hepatopancreatobiliary cancer. Graft transplantation is commonly used to study tumour progression. Over the past few decades, subcutaneous or orthotopic cell-derived tumour xenograft models (CDX models) have been developed to simulate distinct tumours in patients. However, two major limitations exist in CDX models. One model poorly simulates the microenvironment of tumours in humans, such as the vascular, lymphatic and immune environments. The other model loses genetic heterogeneity compared with the corresponding primary tumour. Increased efforts have focused on developing better models for hepatopancreatobiliary cancer research. Hepatopancreatobiliary cancer is considered a tumour with high molecular heterogeneity, making precision medicine challenging in cancer treatment. Developing a new animal model that can better mimic tumour tissue and more accurately predict the efficacy of anticancer treatments is urgent. For the past several years, the patient-derived xenograft model (PDX model) has emerged as a promising tool for translational research. It can retain the genetic and histological stability of their originating tumour at limited passages and shed light on precision cancer medicine. In this review, we summarize the methodology, advantages/disadvantages and applications of PDX models in hepatopancreatobiliary cancer research.

Cellular crosstalk during liver regeneration: unity in diversity.

The liver is unique in its ability to regenerate from a wide range of injuries and diseases. Liver regeneration centers around hepatocyte proliferation and requires the coordinated actions of nonparenchymal cells, including biliary epithelial cells, liver sinusoidal endothelial cells, hepatic stellate cells and kupffer cells. Interactions among various hepatocyte and nonparenchymal cells populations constitute a sophisticated regulatory network that restores liver mass and function. In addition, there are two different ways of liver regeneration, self-replication of liver epithelial cells and transdifferentiation between liver epithelial cells. The interactions among cell populations and regenerative microenvironment in the two modes are distinct. Herein, we first review recent advances in the interactions between hepatocytes and surrounding cells and among nonparenchymal cells in the context of liver epithelial cell self-replication. Next, we discuss the crosstalk of several cell types in the context of liver epithelial transdifferentiation, which is also crucial for liver regeneration. Video abstract.

Efficacy and safety of sirolimus early conversion protocol in liver transplant patients with hepatocellular carcinoma: A single-arm, multicenter, prospective study.

Mammalian target of rapamycin (mTOR) inhibitor as an attractive drug target with promising antitumor effects has been widely investigated. High quality clinical trial has been conducted in liver transplant (LT) recipients in Western countries. However, the pertinent studies in Eastern world are paucity. Therefore, we designed a clinical trial to test whether sirolimus can improve recurrence-free survival (RFS) in hepatocellular carcinoma (HCC) patients beyond the Milan criteria after LT. This is an open-labeled, single-arm, prospective, multicenter, and real-world study aiming to evaluate the clinical outcomes of early switch to sirolimus-based regimens in HCC patients after LT. Patients with a histologically proven HCC and beyond the Milan criteria will be enrolled. The initial immunosuppressant regimens are center-specific for the first 4-6 weeks. The following regimens integrated sirolimus into the regimens as a combination therapy with reduced calcineurin inhibitors based on the condition of patients and centers. The study is planned for 4 years in total with a 2-year enrollment period and a 2-year follow-up. We predict that sirolimus conversion regimen will provide survival benefits for patients particular in the key indicator RFS as well as better quality of life. If the trial is conducted successfully, we will have a continued monitoring over a longer follow-up time to estimate indicator of overall survival. We hope that the outcome will provide better evidence for clinical decision-making and revising treatment guidelines based on Chinese population data. Trial register: Trial registered at http://www.chictr.org.cn: ChiCTR2100042869.

Key factors and potential drug combinations of nonalcoholic steatohepatitis: Bioinformatic analysis and experimental validation-based study.

BACKGROUND: Nonalcoholic fatty liver disease and its advanced stage, nonalcoholic steatohepatitis (NASH), are the major cause of hepatocellular carcinoma (HCC) and other end-stage liver disease. However, the potential mechanism and therapeutic strategies have not been clarified. This study aimed to identify potential roles of miRNA/mRNA axis in the pathogenesis and drug combinations in the treatment of NASH. METHODS: Microarray GSE59045 and GSE48452 were downloaded from the Gene Expression Omnibus and analyzed using R. Then we obtained differentially expressed genes (DE-genes). DAVID database was used for Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis. Protein-protein interaction (PPI) networks were used for the identification of hub genes. We found upstream regulators of hub genes using miRTarBase. The expression and correlation of key miRNA and its targets were detected by qPCR. Drug Pair Seeker was employed to predict drug combinations against NASH. The expression of miRNA and hub genes in HCC was identified in the Cancer Genome Atlas database and Human Protein Atlas database. RESULTS: Ninety-four DE-genes were accessed. GO and KEGG analysis showed that these predicted genes were linked to lipid metabolism. Eleven genes were identified as hub genes in PPI networks, and they were highly expressed in cells with vigorous lipid metabolism. hsa-miR-335-5p was the upstream regulator of 9 genes in the 11 hub genes, and it was identified as a key miRNA. The hub genes were highly expressed in NASH models, while hsa-miR-335-5p was lowly expressed. The correlation of miRNA-mRNA was established by qPCR. Functional verification indicated that hsa-miR-335-5p had inhibitory effect on the development of NASH. Finally, drug combinations were predicted and the expression of miRNA and hub genes in HCC was identified. CONCLUSIONS: In the study, potential miRNA-mRNA pathways related to NASH were identified. Targeting these pathways may be novel strategies against NASH.

Molecular phenotypes reveal heterogeneous engraftments of patient-derived hepatocellular carcinoma xenografts.

OBJECTIVE: Patient-derived xenograft (PDX) models provide a promising preclinical platform for hepatocellular carcinoma (HCC). However, the molecular features associated with successful engraftment of PDX models have not been revealed. METHODS: HCC tumor samples from 76 patients were implanted in immunodeficient mice. The molecular expression was evaluated by immunohistochemistry. Patient and tumor characteristics as well as tumor molecular expressions were compared for PDX engraftment using the Chi-square test. The independent prediction parameters were identified by logistic regression analyses. RESULTS: The engraftment rate for PDX models from patients with HCC was 39.47% (30/76). Tumors from younger patients and patients with elevated preoperative alpha-fetoprotein level had higher engraftment rates. Tumors with poor differentiation and vascular invasion were related to engraftment success. The positive expression of CK19, CD133, glypican-3 (GPC3), and Ki67 in tumor samples was associated with engraftment success. Logistic regression analyses indicated that GPC3 and Ki67 were two of the strongest predictors of PDX engraftment. Tumors with GPC3/Ki67 phenotypes showed heterogeneous engraftment rates, with 71.9% in GPC3+/Ki67+ tumors, 30.8% in GPC3-/Ki67+ tumors, 15.0% in GPC3+/Ki67- tumors, and 0 in GPC3-/Ki67- tumors. CONCLUSIONS: Successful engraftment of HCC PDXs was significantly related to molecular features. Tumors with the GPC3+/Ki67+ phenotype were the most likely to successfully establish HCC PDXs.

Systematic review: risk prediction models for recurrence of hepatocellular carcinoma after liver transplantation.

Recurrence of hepatocellular carcinoma (HCC) after liver transplantation (LT) is a significant clinical problem associated with poor surgical outcomes. This study aims to summarize the current evidence on risk prediction models of HCC recurrence after LT. PubMed and EMBASE were searched to May 25, 2019, for relevant articles. Studies originally designed to develop or validate a risk prediction model for HCC recurrence after LT were included. Two independent authors summarized the study characteristics and evaluated the risk of bias and applicability concerns in the included studies. From 26 included studies, 18 original risk prediction models were determined, but only five models were externally validated. The average number of predictors involved in the construction of risk models was three. The most frequently employed predictors were alpha-fetoprotein, tumor size, vascular invasion, tumor number, tumor differentiation, and neutrophil-lymphocyte ratio. Most studies showed good discriminatory performance (AUC >0.75). The overall quality of the included studies was generally low. Most of the original models lacked the highly recommended external and prospective validation in diverse populations. The AFP model was the well-validated preoperative risk model that can stratify patients into high- and low-risk groups.

Glutamine synthetase promotes tumor invasion in hepatocellular carcinoma through mediating epithelial-mesenchymal transition.

AIM: Glutamine synthetase (GS) levels increase gradually with the development of hepatocellular carcinogenesis. In this study, we aimed to investigate the clinical significance of GS and the underlying mechanism of GS promoting hepatocellular carcinoma (HCC) invasion. METHODS: Serum concentration of GS and α-fetoprotein (AFP) in HCC patients, liver cirrhosis patients, and healthy individuals were detected. The GS-mRNA level and its prognostic value were explored in an independent HCC cohort from The Cancer Genome Atlas database. GS expression in HCC tissue and matched para-tumor tissue was determined. The effect of GS on HCC invasion was assessed in vitro and in vivo. RESULTS: The serum GS and AFP level in HCC patients was higher than that in healthy controls and liver cirrhosis patients. The area under the receiver operating characteristic curve for HCC diagnosis was 0.848 and 0.861 for GS and AFP, respectively. The area under the receiver operating characteristic curve of GS for diagnosis of AFP-negative HCC was 0.913. Combining GS with AFP achieved a diagnostic sensitivity and specificity of 82.5% and 93%, respectively. The GS level was higher in tumor tissues than that in para-tumor tissues. High GS expression was associated with poor prognosis of moderately differentiated HCC patients. In vitro, GS exerted an influence on HCC cell migration by mediating epithelial-mesenchymal transition. The lung and liver metastatic model of HCC further confirmed that GS expression affected the invasion of HCC cells in vivo. CONCLUSIONS: GS is a useful biomarker for HCC diagnosis, especially for AFP-negative patients. In addition, GS affects HCC metastasis through mediating epithelial-mesenchymal transition.

Ubiquitin-Specific Protease 22/Silent Information Regulator 1 Axis Plays a Pivotal Role in the Prognosis and 5-Fluorouracil Resistance in Hepatocellular Carcinoma.

BACKGROUND: Ubiquitin-specific protease 22 (USP22) is described as a key subunit of the Spt-Ada-Gcn5 acetyl transferase complex, which plays an important role in the prognosis and resistance to chemotherapy drugs in hepatocellular carcinoma (HCC). Silent information regulator 1 (SIRT1) is a member of the sirtuin family that is deubiquitinated by USP22. However, it is still unknown whether USP22 and SIRT1 co-expression is associated with disease progression and 5-Fluorouracil (5-FU) resistance in HCC. METHODS: 141 patients who received hepatectomy at our hospital from January 2010 to December 2014 were enrolled in this study. The expression of USP22 and SIRT1 was detected by immunohistochemical staining. Clinicopathological features, including age, gender, tumor number, tumor size, tumor differentiation, tumor stage, alpha-fetoprotein and microscopic vascular invasion, were assessed. Further experiments confirmed the role of SIRT1 in 5-FU drug resistance in vivo. RESULTS: Immunohistochemical staining showed that the high expression of USP22 and SIRT1 was frequently observed in HCC tissues relative to normal liver tissues. Overexpression of USP22 is associated with microscopic vascular invasion (MVI). Further analysis showed that the co-expression of USP22 and SIRT1 was more effective in predicting the prognosis of HCC. The SIRT1 inhibitor EX-527 dramatically inhibited the expression of Cyclin B1 and resistance-associated protein 3 (MRP3) to reduce 5-FU drug resistance in vivo. CONCLUSION: These findings suggest that the co-expression of USP22 and SIRT1 is significantly associated with unfavorable HCC progression. The inhibition of SIRT1 in vivo could be valuable in improving 5-FU drug sensitivity and inhibiting tumor cell proliferation and inducing apoptosis.

CC motif chemokine ligand 16 inhibits the progression of liver cirrhosis via inactivating hepatic stellate cells.

BACKGROUND: Liver cirrhosis results from many forms of chronic damage, characterized by accumulation of extracellular matrix. The present study aimed to explore a potential non-invasive biomarker and its mechanism in the progression of liver cirrhosis. METHODS: Gene Expression Omnibus (GEO) dataset (GSE15654, n = 216) was analyzed to screen genes associated with progression of liver cirrhosis. A total of 181 plasma samples, including healthy control (HC, n = 20), chronic hepatitis B (CHB, n = 77) and HBV-related liver cirrhosis (LC, n = 84), were enrolled for validation. In vitro and in vivo experiments were employed for the mechanistic investigation. RESULTS: GEO dataset analysis showed that relatively low mRNA-expression of CC motif chemokine ligand 16 (CCL16) was associated with elevated Child-Pugh score (P = 0.034) and worse prognosis (P = 0.025). Plasma CCL16 level decreased in a stepwise pattern, with a median concentration of 10.29, 6.57 and 4.47 ng/mL in the HC, CHB and LC groups, respectively (P<0.001). Low plasma CCL16 was significantly related to hepatic dysfunction both in the CHB and LC groups (P<0.05). Combination of CCL16 and ALT showed improved distinguishing capability for LC compared to either alone. In vitro, CCL16 expression was downregulated by lipopolysaccharide and hypoxia. Overexpression of CCL16 from human normal liver cell line (LO2) reduced the extracellular matrix associated proteins (Col1 and Col4) in human hepatic stellate cell line (LX-2). In vivo, the pathological feature of cirrhosis was alleviated by the hepatocyte-specific expression of CCL16. CONCLUSIONS: CCL16 could be a feasible plasma marker to predict the occurrence and progression of liver cirrhosis. CCL16 might impact liver cirrhosis through inactivating hepatic stellate cells.

Hangzhou criteria as downstaging criteria in hepatocellular carcinoma before liver transplantation: A multicenter study from China.

BACKGROUND: The downstaging of hepatocellular carcinoma (HCC) has been confirmed to benefit liver transplantation (LT) patients whose tumors are beyond the transplantation criteria. Milan criteria (MC), a tumor size and number-based assessment, is currently used as the endpoint in these patients. However, many studies believe that tumor biological behavior should be added to the evaluation criteria for downstaging efficacy. Hence, this study aimed to explore the feasibility of Hangzhou criteria (HC), which introduced tumor grading and alpha-fetoprotein in addition to tumor size and number, as an endpoint of downstaging. METHODS: We performed a multicenter and retrospective study of 206 patients accepted locoregional therapy (LRT) as downstaging/bridge treatment prior to LT in three centers of China. RESULTS: Recipients were divided into four groups: failed downstaging to the HC (group A, n = 46), successful downstaging to the HC (group B, n = 30), remained within the HC all the time (group C, n = 113), and tumor progressed (group D, n = 17). The 3-year HCC recurrence probabilities of groups B and C were not significantly different (10.3% vs. 11.6%, P = 0.87). The HCC recurrent rate was significantly higher in group A (52.3%) compared with that in group B/C (P < 0.05). Seven patients (7/76, 9.2%) whose tumor exceeded the the HC were successfully downstaged to the MC, and 39.5% (30/76) to the the HC. In group B, 23 patients remained beyond the MC and their survivals were as well as those of patients within the MC. CONCLUSIONS: Compared to the MC, HC downstaging criteria can give more HCC patients access to LT and furthermore, the outcome of these patients is the same as those matching MC downstaging criteria. Hangzhou downstaging criteria therefore is applicable in clinical practice.