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Background Brahma-related gene 1 (BRG1) is essential for embryogenesis and cellular metabolism. A deficiency of BRG1 in vivo decreases lipid droplets, but the molecular mechanism underlying its role in lipid metabolism associated with hepatocellular carcinoma (HCC) remains unknown. Aims We aimed to determine the role of BRG1 in lipid metabolism in HCC. Methods We assessed the differential expression of BRG1 in HCC and adjacent non-tumorous tissues using tissue microarrays. We stained lipid droplets in HCC cells with Bodipy fluorescence and Oil Red O, and verified BRG1 binding to the promoter region of glycosylated lysosomal membrane protein (GLMP) using chromatin immunoprecipitation. Results The expression of GLMP, a potential lipid metabolism regulator, was suppressed by BRG1 via transcriptional activity. Knockdown of BRG1 decreased lipid droplets, increased GLMP expression and altered the phosphoinositide-3-kinase adaptor protein 1 (PIK3AP1)/phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) pathway in HCC, which further GLMP knockdown partially restored. Thus, GLMP knockdown increased lipid droplets and differentially altered the PI3K/AKT pathway. Conclusions Downregulating BRG1 decreased lipid droplet deposition in HCC cells by upregulating GLMP and altering the PI3K/AKT pathway. Both BRG1 and GLMP might serve as therapeutic targets for disorders associated with dysregulated lipid metabolism, such as NAFLD and NAFLD-associated HCC.

Background T follicular helper (Tfh) cells and T follicular regulatory (Tfr) cells were newly identified as the subsets of cluster of CD4+ T cells. As major components of human immune system, they were found in tumor microenvironment and reported to play vital roles in the progression of cancer. But their clinical significance in Hepatocellular carcinoma (HCC) was not elucidated. Thus, this research aimed to investigate their prognostic value in HCC. Materials and methods A total of 210 subjects (including 110 HCC patients, 50 chronic hepatitis patients and 50 healthy individuals) were enrolled in the research. Tfh, Tfr cells and Treg cells from peripheral blood were measured by flow cytometry. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic performance of Tfr-Tfh Index (TTI) in early HCC and relapse status. Its further prognostic valve was assessed by Kaplan-Meier survival estimate and log rank tests. Results Tfh cells, Tfr cells, Treg cells and TTI were all higher in HCC patients than in chronic hepatitis patients and healthy control. TTI was found to have positive correlation with the load of HBV. The AUC of TTI for early HCC and relapse status was better than other clinical indices in HBV positive patients. An optimal cutoff point for the TTI stratified the HCC patients into high (>21.96) and low index (≤21.96) groups. High TTI was significantly correlated with recurrence. Univariate and multivariate analyses revealed TTI could be a predictor for recurrence. Moreover, it retained prognostic performance for patients with lower recurrence risk. Conclusion Our research showed that TTI could be a promising indicator for early recurrence in HCC patients with HBV infection.

Background Prognosis of hepatocellular carcinoma (HCC) remains poor despite significant recent improvement in therapy. Recent studies have reported that transglutaminase 3 (TGM3) plays an important role in several human cancer types. However, the role of TGM3 in HCC have not been previously elucidated. Methods We evaluated the role of TGM3 in regulating HCC cell proliferation, migration, and invasion. We also investigated the prognostic significance of TGM3 in an HCC cohort. Finally, we explored the signalling pathways that TGM3 regulates in HCC. Results We identified TGM3 to be overexpressed in HCC compared to normal tissues. Higher expression of TGM3 predicts poor prognosis in HCC patients. TGM3 knockdown led to decreased HCC cell proliferation, invasion, and xenograft tumour growth. TGM3 depletion inhibited AKT, extracellular signal–regulated kinase (ERK), p65, and glycogen synthase kinase 3β (GSK3β)/β-catenin activation, but promoted levels of cleaved caspase 3. Moreover, TGM3 knockdown cells had increased E-cadherin levels and decreased vimentin levels, suggesting that TGM3 contributes to epithelial–mesenchymal transition (EMT) in HCC. Conclusion Our results suggest that TGM3 controls multiple oncogenic pathways in HCC, thereby contributing to increased cell proliferation and EMT, and TGM3 potentially enhances HCC metastasis. TGM3 may serve as a novel therapeutic target in HCC.

Hepatic stellate cells (HSCs) play vital roles in tumorigenesis and progression of hepatocellular carcinoma (HCC). However, there remains a lack of high-throughput studies on gene expression alterations in HCC cells in response to direct interactions with HSCs. In this study, we established a direct co-culture model of HSCs and HCC cells. We found that the expression of a set of miRNAs, most notably miR-1246, was triggered by HSCs. RORα was confirmed as the target gene of miR-1246. Either overexpression of miR-1246 or knockdown of RORα enhanced the proliferation, invasiveness, and metastatic capability of HCC both in vitro and in vivo, through Wnt/β-catenin pathway activation and promotion of epithelial-mesenchymal transition (EMT). Moreover, upregulation of miR-1246 and repression of RORα were prominent features of aggressive clinical HCC. The miR-1246-RORα-Wnt/β-catenin axis is a novel pathway through which HSCs accelerate HCC progression.

Background The venous thromboembolism, which may be caused by increased platelet activation, is a risk factor for tumor prognosis. We determined the platelet activation status for diagnosis and predicting postoperative prognosis of hepatocellular carcinoma. Methods We conducted a prospective study of 191 patients diagnosed with HCC at Zhongshan Hospital from April 2016 to July 2016 as well as 99 healthy people. The platelet activation status was assessed by 2 platelet markers, PAC-1 and CD62p, using flow cytometry. The patients were treated with TACE or resection and monitored for ≥6 months. The diagnostic value of marker-positive platelets was determined by the receiver operating characteristic curve and the postoperative value were analyzed using the Kaplan-Meier method and COX regression model. Results All the 3 groups with high levels of marker-positive platelets were likely to be diagnosed with HCC and the PAC-1+ percentage had the best efficacy. The univariate analysis showed that the levels of PAC-1+ and CD62p+ platelets was risker factors for poor postoperative prognosis after both TACE and resection. Moreover, the multivariate analysis revealed that the level of PAC-1+ platelets was an independent risk factor for poor prognosis. Conclusions The PAC-1+ percentage of platelets is a new indicator for diagnosis and predicting postoperative prognosis.

The complexity of liver tumorigenesis is underscored by the recently observed anti-oncogenic effects of oncoproteins, although the mechanisms are unclear. Shp2/Ptpn11 is a proto-oncogene in hematopoietic cells, and antagonizes the effect of tumor suppressor Pten in leukemogenesis. In contrast, we show here cooperative functions of Shp2 and Pten in suppressing hepatocarcinogenesis. Ablating both Shp2 and Pten in hepatocytes induced early-onset non-alcoholic steatohepatitis (NASH), and promoted genesis of liver tumor-initiating cells likely due to augmented cJun expression/activation, and elevated ROS and inflammation in the hepatic microenvironment. Inhibiting cJun partially suppressed NASH-driven liver tumorigenesis without improving NASH. SHP2 and PTEN deficiencies were detected in liver cancer patients with poor prognosis. These data depict a mechanism of hepato-oncogenesis and suggest a potential therapeutic strategy.

Summary Hepatocellular carcinoma (HCC) has high relapse and low 5-year survival rates. Single-cell profiling in relapsed HCC may aid in the design of effective anticancer therapies, including immunotherapies. We profiled the transcriptomes of ∼17,000 cells from 18 primary or early-relapse HCC cases. Early-relapse tumors have reduced levels of regulatory T cells, increased dendritic cells (DCs), and increased infiltrated CD8+ T cells, compared with primary tumors, in two independent cohorts. Remarkably, CD8+ T cells in recurrent tumors overexpressed KLRB1 (CD161) and displayed an innate-like low cytotoxic state, with low clonal expansion, unlike the classical exhausted state observed in primary HCC. The enrichment of these cells was associated with a worse prognosis. Differential gene expression and interaction analyses revealed potential immune evasion mechanisms in recurrent tumor cells that dampen DC antigen presentation and recruit innate-like CD8+ T cells. Our comprehensive picture of the HCC ecosystem provides deeper insights into immune evasion mechanisms associated with tumor relapse.

Objective Epstein–Barr virus (EBV) is a well-recognized oncogenic virus that can induce host cell metabolic reprogramming and tumorigenesis by targeting vital metabolic enzymes or regulators. This study aims to explore the role of wild-type isocitrate dehydrogenase 2 (IDH2) in metabolic reprogramming and tumorigenesis induced by EBV-encoded latent membrane protein 1 (LMP1). Methods Mechanistic dissection of wild-type IDH2 in EBV-LMP1-induced tumorigenesis was investigated using western blotting, real-time polymerase chain reaction (PCR), immunochemistry, chromatin immunoprecipitation (ChIP), and luciferase assay. The role of wild-type IDH2 was examined by cell viability assays/Sytox Green staining in vitro and xenograft assays in vivo. Results IDH2 over-expression is a prognostic indicator of poorer disease-free survival for patients with head and neck squamous cell carcinoma (HNSCC). IDH2 expression is also upregulated in nasopharyngeal carcinoma (NPC, a subtype of HNSCC) tissues, which is positively correlated with EBV-LMP1 expression. EBV-LMP1 contributes to NPC cell viability and xenograft tumor growth mediated through wild-type IDH2. IDH2-dependent changes in intracellular α-ketoglutarate (α-KG) and 2-hydroxyglutarate (2-HG) contribute to EBV-LMP1-induced tumorigenesis in vitro and in vivo. Elevated serum 2-HG level is associated with high EBV DNA and viral capsid antigen-immunoglobulin A (VCA-IgA) levels in patients with NPC. A significantly positive correlation exists between serum 2-HG level and regional lymph node metastases of NPC. EBV-LMP1 enhances the binding of c-Myc with the IDH2 promoter and transcriptionally activates wild-type IDH2 through c-Myc. Targeting IDH2 decreased intracellular 2-HG levels and survival of EBV-LMP1-positive tumor cells in vitro and in vivo. Conclusions Our results demonstrate that the EBV-LMP1/c-Myc/IDH2WT signaling axis is critical for EBV-dependent metabolic changes and tumorigenesis, which may provide new insights into EBV-related cancer diagnosis and therapy., Graphical abstract Image 1

Hepatocarcinogenesis is a complicated process that is affected by a variety of microenvironmental factors, such as secretory chemokines and cell-extracellular matrix (ECM). Retinoic acid receptor-related orphan receptor (ROR)-α has been shown to attenuate tumor invasiveness by inducing suppressive cell microenvironment, and its low expression was associated with a worse prognosis in HCC patients. In the present study, we attempted to investigate the role and mechanism of the dominant transcript of ROR-α, ROR-α-1, in HCC development and progression. Among the four transcripts (ROR-α-1/-2/-3/-4), overexpression of ROR-α-1 dramatically suppressed the capacity of MHCC97H cells to proliferate, migrate and invade. We analyzed the differentially expressed genes in ROR-α-1-overexpressed and non-overexpressed MHCC97H cells, performed Gene Ontology (GO) enrichment analysis on these differentially-expressed genes, and found out that factors involved in the tumor microenvironment and ECM are related to the anti-tumor effects of ROR-α-1. Among these factors, chemokine CXCL5 was significantly downregulated by ROR-α-1 overexpression. Overexpression of ROR-α-1 remarkably inhibited the capacity of HCC cells to proliferate, migrate, invade, and downregulated the protein levels of β-catenin, c-Myc, Cyclin D1, and N-cadherin, suggesting the tumor-suppressive role of ROR-α-1 in MHCC97H cells. Moreover, overexpression of CXCL5 dramatically attenuated the suppressive effects of cell proliferation, migration and invasion induced by ROR-α-1 overexpression in MHCC97H, suggesting that ROR-α-1 exerts its anti-tumor effects via downregulating CXCL5. In conclusion, we demonstrate the tumor-suppressive role of ROR-α-1 in MHCC97H cells and that ROR-α-1 might play a tumor-suppressive role via regulation of chemokine CXCL5.

Hepatocellular carcinoma (HCC) has a high relapse rate and low 5-year survival rate. We generated > 20,000 full-length single-cell transcriptomes from 19 primary or relapsed HCC patients, determining unique features of the tumor microenvironment in relapsed as compared to primary HCC. Relapsed tumors have reduced regulatory T cells, and increased dendritic cells (DCs) and infiltrated CD8+ T cells. CD8+ T cells from relapsed tumors show group 2 innate lymphoid cell like state, with high naive, low cytotoxic and exhausted signals. The abundance of distinct DC subtypes is associated with good prognosis in primary or relapsed HCC, while FCN1+ monocytes are associated with poor prognosis only in relapsed HCC. Differentially expressed genes and interactions analyses reveal potential mechanisms driving immune evasion and tumor survival in relapsed and primary HCC. Together these results provide a compressive understanding of the ecosystem in primary and relapsed tumors, guiding future immune therapy strategies in HCC.

Background There have been progressive increases in both the incidence and death rates of female patients with hepatocellular carcinoma (HCC). Our objective was to investigate the clinicopathologic characteristics and prognostic factors influencing the recurrence and survival of female patients with HCC. Methods We performed a retrospective analysis of 459 consecutive female and 2,936 male patients with HCC who underwent curative resection. Multivariate competing risks analyses with Bonferroni correction were used to evaluate independent prognostic factors. Results Female patients had a better overall survival rate (P = .001) than male patients, but a survival benefit was only observed in female patients with tumor-node-metastasis stage I diseases compared with male patients of the same stage (P = .023). Female patients less often had multiple tumors, vascular invasion, and larger tumors. Although female patients had a greater prevalence of increased serum alpha-fetoprotein (AFP), AFP and tumor number had prognostic significance only for male but not for female patients. The incidence of recurrence in female patients was not different than male patients (P = .130). Vascular invasion and serum γ-glutamyl transpeptidase level were independent risk factors for early recurrence of female patients, whereas AFP and γ-glutamyl transpeptidase level were independent risk factors for late recurrence. After curative treatment for recurrence, female patients still had a better overall survival than male patients (P = .025). Conclusion Female patients had a less invasive tumor phenotype and different prognostic factors from male patients. AFP had no prognostic value in female patients. Estrogen may have a protective effect against early- but not late-stage HCC. Female patients have a better outcome after curative resection of recurrent HCC.

Background & Aims The pathogenesis of intrahepatic cholangiocarcinoma (ICC), the second most common hepatic cancer, is poorly understood, and the incidence of ICC is increasing worldwide. We searched for mutations in human ICC tumor samples and investigated how they affect ICC cell function. Methods We performed whole exome sequencing of 7 pairs of ICC tumors and their surrounding nontumor tissues to detect somatic alterations. We then screened 124 pairs of ICC and nontumor samples for these mutations, including 7 exomes. We compared mutations in PTPN3 with tumor recurrence in 124 patients and PTPN3 expression levels with recurrence in 322 patients (the combination of both in 86 patients). The functional effects of PTPN3 variations were determined by RNA interference and transgenic expression in cholangiocarcinoma cell lines (RBE, HCCC-9810, and Huh28). Results Based on exome sequencing, pathways that regulate protein phosphorylation were among the most frequently altered in ICC samples and genes encoding protein tyrosine phosphatases (PTPs) were among the most frequently mutated. We identified mutations in 9 genes encoding PTPs in 4 of 7 ICC exomes. In the prevalence screen of 124 paired samples, 51.6% of ICCs contained somatic mutations in at least 1 of 9 PTP genes; 41.1% had mutations in PTPN3. Transgenic expression of PTPN3 in cell lines increased cell proliferation, colony formation, and migration. PTPN3L232R and PTPN3L384H, which were frequently detected in ICC samples, were found to be gain-of-function mutations; their expression in cell lines further increased cell proliferation, colony formation, and migration. ICC-associated variants of PTPN3 altered phosphatase activity. Patients whose tumors contained activating mutations or higher levels of PTPN3 protein than nontumor tissues had higher rates of disease recurrence than patients whose tumors did not have these characteristics. Conclusions Using whole exome sequencing of ICC samples from patients, we found that more than 40% contain somatic mutations in PTPN3. Activating mutations in and high expression levels of PTPN3 were associated with tumor recurrence.

SummaryIn hepatocellular carcinoma (HCC), biomarkers for prediction of prognosis and response to immunotherapy such as interferon-α (IFN-α) would be very useful in the clinic. We found that expression of retinoic acid-inducible gene-I (RIG-I), an IFN-stimulated gene, was significantly downregulated in human HCC tissues. Patients with low RIG-I expression had shorter survival and poorer response to IFN-α therapy, suggesting that RIG-I is a useful prognosis and IFN-α response predictor for HCC patients. Mechanistically, RIG-I enhances IFN-α response by amplifying IFN-α effector signaling via strengthening STAT1 activation. Furthermore, we found that RIG-I deficiency promotes HCC carcinogenesis and that hepatic RIG-I expression is lower in men than in women. RIG-I may therefore be a tumor suppressor in HCC and contribute to HCC gender disparity.

Background & aims Phosphatidylethanolamine-binding protein 1 (PEBP1, also RKIP) plays a pivotal role in cancer by regulating multiple cellular signaling processes and suppressing metastasis in animal models. We examined whether PEBP1 expression in hepatocellular carcinoma (HCC) correlated with the risk of recurrence and survival after resection. Methods A randomly selected cohort of 240 Chinese HCC patients, predominantly hepatitis B related, formed the basis of the study. PEBP1 expression levels were evaluated by immunohistochemistry and real-time reverse-transcriptase PCR. Survival analysis was performed by univariate and multivariate analyses. The results were further validated in an independent series of 403 patients. The relevance of PEBP1 to phospho-ERK was determined by Western blot analysis on clinical samples and hepatoma cell lines. Results PEBP1, prevalently down-regulated in HCC, was significantly associated with tumor invasive characteristics (such as vascular invasion, lack of encapsulation, poor differentiation and large size). Both PEBP1 protein and mRNA levels were independent predictors for tumor recurrence (hazard ratio (HR)=1.877, p =0.001; HR=2.633, p =0.001; respectively), and patient survival (HR=1.796, p =0.004; HR=1.730, p =0.044; respectively). The prognostic value of PEBP1 was then confirmed in the validation cohort. In addition, Western blot suggested that loss of PEBP1 led to hyperactivity of MAPK signaling. Conclusions Down-regulation of PEBP1 in HCC indicated aggressive tumor behaviors and predicted a worse clinical outcome, which may be a useful biomarker to identify the patients at high risk of post-operative recurrence.

Epidermal growth factor receptor variant III (EGFRvIII), the most common EGFR mutation, is associated with cell migration of glioblastoma multiforme (GBM) cases; however, the mechanism has not been elucidated. In this study, we found that the EGFRvIII-promoted glioma cell migration was closely linked to high levels of tyrosine phosphorylation in focal adhesion kinase (FAK) Y397. We also demonstrated that EGFRvIII formed a complex with FAK, resulting in enhanced tyrosine phosphorylation levels of FAK Y397 and EGFR Y1068. After knockdown of FAK expression via anti-FAK shRNA, the U87ΔEGFR cell migration was significantly inhibited, accompanying with the reduced phosphorylation levels of extracellular signal-regulated kinase (ERK1/2). Furthermore, the role of ERK1/2 in FAK-regulated cell migration was confirmed. Taken together, our results suggest that FAK and its downstream molecule ERK were involved in EGFRvIII-promoted glioma cell migration in U87ΔEGFR cells.

Aim Sirolimus (SRL) acts as a primary immunosuppressant or antitumor agent. The aim of the present study was to evaluate the influence of SRL on the recurrence rate and survival of patients after orthotopic liver transplantation (OLT) for hepatocellular carcinoma (HCC) exceeding the Milan criteria. Materials and Methods We retrospectively examined 73 consecutive patients who underwent OLT for HCC exceeding the Milan criteria from March 2004 through December 2005. Among them, 27 patients were treated with SRL-based immunosuppressive protocols after OLT, and 46 patients by an FK506-based protocol. Statistical analysis was based on the intent-to-treat method. Results The 2 groups were comparable in all clinicopathologic parameters. The mean overall survival was 594 ± 35 days in the SRL group and 480 ± 42 days in the FK506 group ( P = .011); the mean disease-free survival period was 519 ± 43 days in the SRL group and 477 ± 48 days in the FK506 group ( P = .234). Multivariate analysis revealed Child's status ( P = .004) and immunosuppressive protocol ( P = .015) were the significant factors affecting overall survival. Only microvascular invasion ( P = .004) was significantly associated with disease-free survival. Among 24 surviving patient in the SRL group, 2 patients had SRL discontinued for toxicity; 10 had SRL monotherapy immunosuppression. Conclusion The SRL-based immunosuppressive protocol improved the overall survival of patients after OLT for HCC exceeding the Milan criteria, probably by postponing recurrence and with better tolerability.

CC chemokine receptor 1 (CCR1) has an important role in the recruitment of leukocytes to the site of inflammation. The migration and metastasis of tumor cells shares many similarities with leukocyte trafficking, which is mainly regulated by chemokine receptor-ligand interactions. CCR1 is highly expressed in hepatocellular carcinoma (HCC) cells and tissues with unknown functions. In this study, we silenced CCR1 expression in the human HCC cell line HCCLM3 using artificial microRNA (miRNA)-mediated RNA interference (RNAi) and examined the invasiveness and proliferation of CCR1-silenced HCCLM3 cells and the matrix metalloproteinase (MMP) activity. The miRNA-mediated knockdown expression of CCR1 significantly inhibited the invasive ability of HCCLM3 cells, but had only a minor effect on the cellular proliferation rate. Moreover, CCR1 knockdown significantly reduced the secretion of MMP-2. Together, these findings indicate that CCR1 has an important role in HCCLM3 invasion and that CCR1 might be a new target of HCC treatment.

Early hepatic artery thrombosis (HAT) after orthotopic liver transplantation remains a significant cause of graft loss and patient death. The most effective treatment approach is still controversial. The purpose of this study was to assess the effect of continuous transcatheter arterial thrombolysis in the treatment of early HAT. Routine posttransplant color Doppler imaging (CDI) was performed to monitor hepatic artery blood flow. HAT was confirmed by arterial angiography in suspected cases. HAT was identified in 8 patients (8/287, 2.8%) which occurred on days 2 to 19 (mean, 5.2 days) after liver transplantation. Patients with HAT were treated with continuous transcatheter arterial thrombolysis using urokinase. Successful revascularization through thrombolysis was obtained in all eight cases. One patient died of a pulmonary infection at 2 months after liver transplantation. Another patient underwent retransplantation because of resistant allograft rejection and recurrence of HAT 6 months after the first operation, but died from multiple system organ failure 2 months later. The other six patients remained in good health during the follow-up period of 3 to 27 months. Our results demonstrate that CDI is an effective method to monitor the occurrence of early HAT after liver transplantation. Furthermore, continuous transcatheter arterial thrombolysis with urokinase could be a rational therapeutic approach to rescue the allograft following early HAT diagnosis confirmed by arterial angiography.