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Background: This study evaluated health-related quality of life (HRQoL) in the RATIONALE-309 (NCT03924986) intent-to-treat (ITT) population and in a subgroup of patients with liver metastases., Methods: Patients were randomized 1:1 to tislelizumab + chemotherapy or placebo + chemotherapy. As the secondary endpoint, HRQoL was evaluated using seven selected scores from the EORTC QLQ-C30 and QLQ Head and Neck Cancer module (QLQ-H&N35)., Results: Of 263 randomized patients in the ITT population (tislelizumab + chemotherapy n = 131, placebo + chemotherapy n = 132), 43% had liver metastases (tislelizumab + chemotherapy n = 56; placebo + chemotherapy n = 57). No differences in change in selected scores on the QLQ-C30 from baseline to cycle 4 or cycle 8 were observed for the ITT or liver metastases subgroup. No differences in selected QLQ-H&N35 scores were observed between the arms from baseline to cycle 4. In the ITT population and the liver metastases subgroup, a greater reduction from baseline to cycle 8 was observed in the tislelizumab + chemotherapy arm than the placebo + chemotherapy arm in QLQ-H&N35 pain score. At cycle 8 in the liver metastases subgroup, the tislelizumab + chemotherapy arm experienced greater improvement in the QLQ-H&N35 senses problems score than the placebo + chemotherapy arm. Differences in time to deterioration between arms were not observed., Conclusions: The current findings, along with improved survival and favorable safety, suggests that tislelizumab + chemotherapy represents a potential first-line treatment for recurrent or metastatic nasopharyngeal cancer., (© 2024 The Authors. Head & Neck published by Wiley Periodicals LLC.)

Background: NDRG1, the first member of the NDRG family, is a multifunctional protein associated with carcinogenesis. Its function in human cancer is currently poorly understood. The aim of this study was to explore the importance of NDRG1 in tumor immune cell infiltration and epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma., Methods: NDRG1 expression in various cancers was analyzed using TIMER 2.0, the Human Protein Atlas (HPA), UALCAN and PrognoScan. Wound healing, Transwell, MTT and colony formation assays were performed to confirm the effects of NDRG1 on the metastasis and proliferation of HCC cells. Western blotting was used to study the effect of NDRG1 on the expression of EMT-related proteins. Signaling networks were constructed using LinkedOmics and Metascape. TIMER2.0 and TISIDB were used for comprehensive analysis of tumor-infiltrating immune cells and tumor-infiltrating lymphocytes (TILs)., Result: NDRG1 expression was higher in HCC tissue than in normal liver tissue at both the mRNA and protein levels. Overexpression of NDRG1 is associated with poor prognosis in HCC patients. Genomic analysis suggests that NDRG1 promoter hypermethylation leads to enhanced transcription, which may be one mechanism for NDRG1 upregulation in HCC. The overexpression of NDRG1 promotes the invasion, migration, and proliferation of HCC cells and induces the expression of EMT-related proteins. Immunoinfiltration analysis suggests that NDRG1 is involved in the recruitment of immune cells., Conclusions: The present study showed that NDRG1 may induce metastasis and invasion through EMT and immune cell infiltration. NDRG1 could be used as a biomarker for the diagnosis and prognosis of HCC and could be a potential therapeutic target in HCC., Competing Interests: Conflict of interest Written informed consent was obtained from all participants. Ethical approval was obtained from the Ethical Committee of Tianjin medical university. The authors declare that there are no competing interests., (Copyright © 2024 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Due to soared obesity population worldwide, hepatosteatosis is becoming a major risk factor for hepatocellular carcinoma (HCC). Undertaken molecular events during the progression of steatosis to liver cancer are thus under intensive investigation. In this study, we demonstrated that high-fat diet potentiated mouse liver AKT2. Hepatic AKT2 hyperactivation through gain-of-function mutation of Akt2 (Akt2E17K) caused spontaneous hepatosteatosis, injury, inflammation, fibrosis, and eventually HCC in mice. AKT2 activation also exacerbated lipopolysaccharide and D-galactosamine hydrochloride-induced injury/inflammation and N-Nitrosodiethylamine (DEN)-induced HCC. A positive correlation between AKT2 activity and SCD1 expression was observed in human HCC samples. Activated AKT2 enhanced the production of monounsaturated fatty acid which was dependent on SREBP1 upregulation of SCD1. Blockage of active SREBP1 and ablation of SCD1 reduced steatosis, inflammation, and tumor burden in DEN-treated Akt2 E17K mice. Therefore, AKT2 activation is crucial for the development of steatosis-associated HCC which can be treated with blockage of AKT2-SREBP1-SCD1 signaling cascade., (Copyright © 2024. Published by Elsevier Inc.)

Background: Although hepatitis B virus (HBV) infection is a major risk factor for hepatic cancer, the majority of HBV carriers do not develop this lethal disease. Additional molecular alterations are thus implicated in the process of liver tumorigenesis. Since phosphatase and tensin homolog (PTEN) is decreased in approximately half of liver cancers, we investigated the significance of PTEN deficiency in HBV-related hepatocarcinogenesis., Methods: HBV-positive human liver cancer tissues were checked for PTEN expression. Transgenic HBV, Alb-Cre and Pten fl/fl mice were inter-crossed to generate WT, HBV, Pten -/- and HBV; Pten -/- mice. Immunoblotting, histological analysis and qRT-PCR were used to study these livers. Gp73 -/- mice were then mated with HBV; Pten -/- mice to illustrate the role of hepatic tumor biomarker golgi membrane protein 73 (GP73)/ golgi membrane protein 1 (GOLM1) in hepatic oncogenesis., Results: Pten deletion and HBV transgene synergistically aggravated liver injury, inflammation, fibrosis and development of mixed hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). GP73 was augmented in HBV; Pten -/- livers. Knockout of GP73 blunted the synergistic effect of deficient Pten and transgenic HBV on liver injury, inflammation, fibrosis and cancer development., Conclusions: This mixed HCC-ICC mouse model mimics liver cancer patients harboring HBV infection and PTEN/AKT signaling pathway alteration. Targeting GP73 is a promising therapeutic strategy for cancer patients with HBV infection and PTEN alteration., (© 2024. The Author(s).)

China accounted for 45.3% of new cases of primary liver cancer (PLC) worldwide in 2020. While variations in PLC incidence between different regions of China and decreasing incidence in overall China have been reported, incidence patterns have not been thoroughly explored by region. We examined the nearly status and temporal trends of PLC incidence in different geographical regions in China and project future trends. The age-standardized incidence rate (ASR) was estimated for 1978 to 2012 by different geographical regions and gender in China. Age-period-cohort model was adopted to evaluate age and birth cohort effects on the temporal trend of five registries of China (Hong Kong, Shanghai, Jiashan, Harbin and Zhongshan), Bayesian age-period-cohort model was adopted to project future trends for 2013 to 2032. PLC incidence in China exhibits marked geographical disparity, with the highest incidence in Southwest China, and gender differences being particularly pronounced in South China. While other registries exhibited decreasing trend, Zhongshan exhibited an increasing trend, with the cohort effect showing a marked upward trend for females born in 1916 to 1949 and males born in 1916 to 1962. During 2013 to 2032, the ASR appears to increase by 86.9% for men and 40.0% for women in Zhongshan, while the remaining registries will decline by around 50%. Since the high incidence of hepatitis B virus infection in early birth cohort, recent rise of nonviral risk factors and the severe aging of the Chinese population, it may be critical to tailor future prevention and control strategies for PLC to the distribution of risk factors in different geographical regions., (© 2023 UICC.)

Objectives: Most signatures are constructed on the basis of RNA or protein expression levels. The value of vascular invasion-related signatures based on lncRNA pairs, regardless of their specific expression level in hepatocellular carcinoma (HCC), is not yet clear., Methods: Vascular invasion-related differentially expressed lncRNA (DElncRNA) pairs were identified with a two-lncRNA combination strategy by using a novel modeling algorithm. Based on the optimal cutoff value of the ROC curve, patients with HCC were classified into high- and low-risk subgroups. We used KM survival analysis to evaluate the overall survival rate of patients in the high- and low-risk subgroups. The independent indicators of survival were identified using univariate and multivariate Cox analyses., Results: Five pairs of vascular invasion-related DElncRNAs were selected to develop a predictive model for HCC. High-risk subgroups were closely associated with aggressive clinicopathological characteristics and genes, chemotherapeutic sensitivity, and highly expressed immune checkpoint inhibitors., Conclusions: We identified a signature composed of 5 pairs of vascular invasion-related lncRNAs that does not require absolute expression levels of lncRNAs and shows promising clinical predictive value for HCC prognosis. This predictive model provides deep insight into the value of vascular invasion-related lncRNAs in prognosis., (© 2024. The Author(s).)

Background: The use of regorafenib in the treatment of hepatocellular carcinoma (HCC) is widespread. Albumin-Bilirubin (ALBI) has been shown to be a potential prognostic marker for regorafenib treatment, but its prognostic value remains controversial. Therefore, we conducted a meta-analysis to investigate the value of the baseline ALBI grade in predicting the efficacy and survival outcomes of HCC patients after regorafenib treatment., Methods: PubMed, Embase, Cochrane library, Web of Science, CNKI, Wan Fang Data, and Vip Database were searched from January 2010 to October 2022. Studies treating HCC patients with regorafenib and with ALBI as a categorical variable, overall survival (OS) and progression-free survival (PFS) as outcome indicators were included. After applying Newcastle-Ottawa Scale (NOS) to evaluate the quality of the included studies, Review Manager 5.4 was used to statistically analyze. Chi-square Q test and I 2 statistics were used to detect heterogeneity. Funnel plot asymmetry, Egger's and Begg's test were used to evaluate publication bias., Results: A total of 12 studies, comprising 1,918 patients, were included in the meta-analysis. The included studies were all evaluated as high quality. Compared to the high-grade baseline ALBI group, patients in the low-grade group had a longer survival time after receiving regorafenib and also more suitable for regorafenib treatment [odds ratio (OR) = 6.50, 95% confidence interval (CI): 2.22-18.96, P < 0.01]. The low-grade baseline ALBI group before sorafenib treatment was significantly correlated with better OS [hazard ratio (HR) = 2.36, 95% CI: 1.68-3.31, P < 0.00001] and PFS (HR = 1.56, 95% CI: 1.16-2.08, P = 0.003). Likewise, the low-grade baseline ALBI group before regorafenib was also significantly correlated with better OS (HR = 1.56, 95% CI: 1.15-2.13, P = 0.005) and PFS (HR = 2.06, 95% CI: 1.37-3.11, P = 0.0005). In addition, the ALBI grade was significantly correlated with disease control rate (DCR) (OR = 2.90, 95% CI: 1.45-5.79, P = 0.003), but not the objective response rate (OR = 1.98, 95% CI: 0.71-5.46, P = 0.19)., Conclusions: The baseline ALBI grade could be a valuable prognostic indicator for predicting response and outcomes in HCC patients treated with regorafenib., (© 2023. BioMed Central Ltd., part of Springer Nature.)

DNA sensor proteins play an important role in transducing DNA signals to induce immune responses that initiate inflammation or clear pathogens. It has been previously shown that several DNA sensors are involved in regulating tumor biology and/or cancer immunology. However, a systemic analysis of DNA sensor expression and its correlation with prognosis has not been conducted. Here, we analyzed messenger RNA expression and protein abundance in liver cancer databases and found that the genes of 5 DNA sensors (POLR3A, PRKDC, DHX9, cGAS, and MRE11) were consistently upregulated in tumor tissue. Moreover, the expression of these DNA sensor genes correlated with patient survival. Using a gene alterations analysis, we discovered that patients with genetically altered DNA sensors had significantly lower survival compared with an unaltered group. Furthermore, receiver-operating characteristic curves confirmed that the signatures of the 5 DNA sensors were independent prognostic factors in hepatocellular carcinoma. Tumor-infiltrating immune cell analysis revealed that expression of all 5 DNA sensors correlated with the amount of B cells, CD8 T cells, CD4 T cells, Tregs, DCs, Mϕs, and neutrophils. Surprisingly, 4 of the DNA sensors (POLR3A, PRKDC, DHX9, and MRE11) were inversely correlated with the amount of γδ T cells. Gene set enrichment analysis showed that all 5 DNA sensor genes were enriched for oxidative phosphorylation and xenobiotic metabolism. These results suggest that expression of these DNA sensors is associated with a unique immune profile and metabolic regulation in hepatocellular carcinoma., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The development of human hepatocellular carcinoma (HCC) is a multistep process that is accompanied by progressive changes in the liver microenvironment, including immune evasion and angiogenesis. Lysyl oxidase-like 2 (LOXL2) has been suggested to contribute to tumour progression and metastasis; however, the underlying mechanism remains unclear. The purpose of the present study was to explore the relationship between LOXL2 and immune infiltration and vasculogenic mimicry (VM) and to identify the role of LOXL2 in HCC diagnosis prognosis evaluation., Methods: The Cancer Genome Atlas (TCGA), UALCAN, GEPIA and Kaplan-Meier plotter databases were used to analyse LOXL2 expression and perform survival analysis. The Tumour Immune Estimation Resource (TIMER) was used to analyse immune cell infiltration, immune cell biomarkers and immune checkpoints. Immunohistochemistry (IHC) of 201 HCC samples was used to confirm the expression of LOXL2 and its relationship with VM. Coimmunoprecipitation (co-IP) and gain- and loss-of-function studies were performed to confirm the molecular mechanism of LOXL2 in VM., Results: The expression of LOXL2 in HCC was higher than that in normal tissues at both the mRNA and protein levels. High expression of LOXL2 was associated with a poorer prognosis of HCC. The genetic alteration rate of LOXL2 was 5%. LOXL2 was positively related to immune cell infiltration and immune checkpoints (PD-1 and CTLA-4) in HCC. Co-IP showed that LOXL2 can interact directly with IQGAP1. Both gain- and loss-of-function studies showed that LOXL2 significantly induced cell migration, invasion and VM formation when IQGAP1 was upregulated., Conclusions: LOXL2 is involved in immune cell infiltration and promotes VM by upregulating IQGAP1. LOXL2 can be used as a novel biomarker for HCC diagnosis and prognosis prediction., Competing Interests: Declaration of Competing Interests The authors declare that there are no competing interests., (Copyright © 2022. Published by Elsevier Ltd.)

Interleukin (IL)-35, a new member of the IL-12 family, exerts immunosuppressive effects in the hepatic microenvironment. Innate immune cells, such as γδ T cells, have vital roles in hepatic diseases, including acute and chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). In the current study, we focused on the effects and mechanisms of IL-35 on the local immune status of γδ T cells, especially in liver tumors. Based on CCK8 assay and immunofluorescence results, we showed that exogenous IL-35 stimulation of γδ T cells attenuated proliferative ability and killing functions against Hepa1-6 or H22 cells. Flow cytometry results showed that exogenous IL-35 stimulated the expression of programmed cell death 1 (PDCD1) and lymphocyte activation gene 3 (LAG3) in γδ T cells. The exogenous IL-35 stimulated group also showed impairment of cytotoxic cytokine secretion. In addition, stat5a revealed a significant increase after IL-35 stimulation of γδ T cells screened via transcription factor based on PCR array analysis. Furthermore, bioinformatics analysis revealed that stat5a-related tumor-specific genes were mainly involved in immune regulatory pathways. Correlation analysis indicated that stat5a expression was significantly and positively correlated with tumor immune cell infiltration, and pdcd1 and lag3 expression. Finally, bioinformatics analysis using the TCGA and GSE36376 HCC datasets corroborated the significant positive correlation between IL-35 and stat5a. Taken together, overexpressed IL-35 promoted exhaustion and impaired the anti-tumor function of γδ T cells in HCC. Targeting IL-35 might be a promising means to enhance the antitumor therapy of γδ T cells, which would significantly improve prognosis., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Ring finger protein 31 (RNF31) has been found to play an important role in tumor immunity. However, the role of RNF31 in liver hepatocellular carcinoma (LIHC) has not been reported. Therefore, we investigated the expression and prognostic value of RNF31 in patients with LIHC and explored its relationship with immune cell infiltration. The Cancer Genome Atlas liver hepatocellular carcinoma (TCGA-LIHC) dataset was downloaded to analyse the impact of RNF31 on the prognosis and immune cell infiltration of LIHC. The Tumor Immune Estimation Resource (TIMER) database was used to analyse the correlation between RNF31 and tumor immune cell infiltration in LIHC. Additionally, we analysed the relationship between RNF31 and tumor necrosis factor (TNF) as well as the interferon-gamma (IFN-γ) signaling pathway. The expression of RNF31 in LIHC was significantly higher than that in normal tissues. Increased RNF31 expression was associated with decreased overall survival (OS) and relapse-free survival (RFS). An increase in RNF31 expression was closely related to the infiltration levels of immune cells (e.g., natural killer (NK) cells, CD8 + T cells, and B cells). RNF31 was also positively correlated with the expression of immune checkpoint genes in LIHC. Moreover, RNF31 may participate in TNF and IFN-γ signaling pathways. In conclusion, RNF31 is a potentially valuable prognostic biomarker in LIHC. RNF31 is also associated with immune cell infiltration in LIHC. RNF31 may be a potential target for immunotherapy of LIHC., (© 2023. The Author(s).)

MMP-21 is a newly identified member of the matrix metalloproteinase family and has been reported to regulate both embryonic development and tumor progression. However, the roles of MMP-21 in hemofiltrate C-C chemokine (HCC) remain largely unclear. In this study, we used western blot, qPCR and immunohistochemistry (IHC) to determine the upregulation of MMP-21 in HCC tissues, and showed that the increase in MMP-21 was associated with vascular invasion and poor prognosis. Although changing levels of MMP-21 in HCC cell lines had no significant effect on cell migration or invasion abilities in in vitro transwell tests, both IHC analysis and in vivo mouse models proved that upregulated MMP-21 promoted metastasis. Functional enrichments of MMP-21 using The Cancer Genome Atlas (TCGA) data suggested that MMP-21 might regulate metastasis via macrophages. Further experiments proved that MMP-21 enhanced macrophage recruitment by increasing CCL-14 levels and promoted M2-type polarization of macrophage by elevating the expression of CSF-1 and FGF-1. Taken together, this study revealed that MMP-21 controlled the tumor microenvironment remodeling and functional regulation of macrophages to regulate HCC metastasis., (© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Semaphorin7a (SEMA7A), a membrane-anchored member of the semaphorin protein family, could be involved in a diverse range of immune responses via its receptor integrin β1. Recently, we reported that the SEMA7A R148W mutation (a gain-of-function mutation, Sema7a R145W in mice) is a risk factor for progressive familial intrahepatic cholestasis and nonalcoholic fatty liver disease via upregulated membrane localization. In this study, we demonstrated that integrin β1 is a membrane receptor for nuclear factor NF-kappa-B p105 (NF-κB p105) and a critical mediator of inflammation. Integrin β1 could interact with the C-terminal domain of NF-κB p105 to promote p50 generation and stimulate the NF-κB p50/p65 signalling pathway, upregulate TNF-α and IL-1β levels, and subsequently render hepatocytes more susceptible to inflammation. The induction of integrin β1 depends on elevated Sema7a membrane localization. Moreover, we revealed elevated levels of Sema7a WT (SEMA7A WT ) in hepatocellular carcinoma (HCC) patients and an HCC mouse model. In line with our findings, the NF-κB p50/p65 pathway could also be activated by high Sema7a expression and repressed by integrin β1 silencing. In conclusion, our findings suggest that the Sema7a R145W (SEMA7A R148W ) mutation and high Sema7a WT (SEMA7A WT ) expression both activate the NF-κB p50/p65 pathway via integrin β1 and play a crucial role in inflammatory responses. Video Abstract., (© 2023. The Author(s).)

Objective: It is not well determined whether liver metastasis is a prognostic factor for survival of metastatic non-small cell lung cancer (NSCLC) patients who received immune checkpoint inhibitors (ICIs). We compared the efficacy of ICIs in patients with NSCLC with or without liver metastases, aiming to evaluate the impact of liver metastasis on survival of NSCLC., Methods: We systematically searched Pubmed, Embase, and the Cochrane library databases for randomized controlled trials (RCTs) on the efficacy of ICIs in the treatment of NSCLC patients with or without liver metastases. The duration of this search was from January 1, 2000 to June 1, 2022. The reviewers screened the literature, extracted data and conducted quality assessment, and used RevMan 5.4 software and Stata 14 to perform analyses., Results: A total of 17 RCTs were included, published from 2019 to 2022. For NSCLC patients with liver metastases, the risk of disease progression decreased by 36% (HR = 0.64; 95% CI: 0.55-0.75; P  < .01) when treated with ICIs, and the death risk (HR = 0.82; 95% CI: 0.72-0.94; P  < .01) was also reduced after ICIs treatment. For those without liver metastases, they had significantly improved PFS (HR = 0.56; 95% CI: 0.52-0.60; P  < .01) and OS (HR = 0.73; 95% CI: 0.67-0.80; P  < .01), compared to those of the control group. Subgroup analysis of OS in liver metastases patients suggested that OS benefit was associated with treatment strategy (for anti-PD-L1 plus chemotherapy versus chemotherapy, HR=1.04; 95% CI: 0.81-1.34; P = .75)., Conclusion: For NSCLC patients with or without liver metastases, ICIs administration could improve both PFS and OS, especially for those without liver metastases. More RCTs are essential to verify these findings.

Sorafenib (SFN) is a multi-kinase inhibitor drug for the treatment of advanced hepatocellular carcinoma (HCC), but its limited efficacy is a major obstacle to the clinical outcomes of patients with HCC. We aimed to explore a novel molecular mechanism underlying the chemosensitivity of HCC to SFN, and to identify a promising therapeutic target for HCC treatment. In this study, bioinformatic analysis revealed that DDX24 was associated with poor survival in HCC cases, and significantly related to the pathways modulating tumor development. DDX24 regulated HCC cell proliferation and migration potentials. Moreover, reduction of DDX24 promoted the sorafenib-mediated inhibition of HCC cell growth and migration, the elevation of sorafenib-induced HCC cell apoptosis. DDX24 overexpression suppressed the inhibitory effect of SFN on cell proliferation and migration and reduced the apoptosis induced by SFN. Further, DDX24, combined with SFN treatment, presented a synergistic enhancement of the sensitivity of SFN to the growth and migration of HCC cells via AKT/ERK and the epithelial-mesenchymal transition (EMT) pathways, and that it modulated apoptosis via the caspase/PARP pathway. Mechanistically, SNORA18 served as a target gene for DDX24, regulating the chemosensitivity of sorafenib-treated HCC cells. Furthermore, SNORA18 knockdown or overexpression could partially reverse the inhibition or elevation of cell viability, colony formation and migration induced by DDX24 in sorafenib-treated HCC cells, respectively. Collectively, our results suggest that DDX24 regulates the chemosensitivity of HCC to SFN by mediating the expression of SNORA18, which may act as an effective therapeutic target for improving SFN efficiency in HCC treatment.

The γδ T cell subset was discovered over 30 years ago, yet continues to be an exciting and challenging component of the adaptive immune response. While γδ T cells represent a very small fraction of all T cells in humans, γδ T cells have a vital effect on human immunity, serving as a bridge between the innate and adaptive immune systems. The characteristics of γδ T cells include recognition of non-MHC restrictive antigens, as well as the ability to secrete an abundance of cytokines, suggesting that γδ T cells have high antitumor activity. As such, they have gained ample attention with respect to tumor immunotherapy in the last decade. The γδ T cell subset comprises up to ∼15-20% of the T-lymphocyte population in the liver, although the liver is recognized as an immune organ with primary immune functions, the role of γδ T cells in liver disease has not been established. Herein, we present a comprehensive overview of molecular mechanisms underlying immune γδ T cell activity in liver disease, including immune liver injury, viral hepatitis, cirrhosis, and hepatocellular carcinoma, and review γδ T cell-based clinical immunotherapeutic approaches., (©2022 Society for Leukocyte Biology.)

Background: Currently, the molecular mechanism of the interaction between lncRNAs and microRNAs (miRNAs) and the target of miRNAs in tumor vasculogenic mimicry (VM) formation have not been clarified. Our aim is to study the interaction between lncRNA n339260 and miRNA30e-5p in the formation of VM., Methods: Animal xenografts were established, 104 hepatocellular carcinoma (HCC) patients' frozen tissues were obtained and HCC cells in vitro were used to observe the role of n339260 in HCC progression., Results: In vivo experiment showed lncRNA n339260 promoted tumor growth and VM formation. LncRNA n339260 and miRNA30e-5p were found to be associated with VM formation, metastasis and survival time in HCC patients. In vitro experiment showed that LncRNA n339260 could inhibit miRNA30e-5p expression and TP53INP1 was found to be the downstream targets of miRNA30e-5p. Snail, MMP2, MMP9, VE-cadherin, vimentin and N-cadherin overexpression and the downregulation of TP53INP1 and E-cadherin were observed in HCCLM3 and HepG2 cells overexpressing lncRNA n339260 or in cells with decreased expression of miRNA30e-5p., Conclusion: LncRNA n339260 promotes the development of VM, and lncRNA n339260 may enhance Snail expression by decreasing the expression of miRNA30e-5p, thereby reducing TP53INP1 expression. Therefore, a potential lncRNA n339260- miRNA30e-5p- TP53INP1 regulatory axis was associated with HCC progression., (© 2022. Japanese Society of Gastroenterology.)

CTNNB1 , encoding β-catenin protein, is the most frequently altered proto-oncogene in hepatic neoplasms. In this study, we studied the significance and pathological mechanism of CTNNB1 gain-of-function mutations in hepatocarcinogenesis. Activated β-catenin not only triggered hepatic tumorigenesis but also exacerbated Tp53 deletion or hepatitis B virus infection-mediated liver cancer development in mouse models. Using untargeted metabolomic profiling, we identified boosted de novo pyrimidine synthesis as the major metabolic aberration in β-catenin mutant cell lines and livers. Oncogenic β-catenin transcriptionally stimulated AKT2, which then phosphorylated the rate-limiting de novo pyrimidine synthesis enzyme CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase) on S1406 and S1859 to potentiate nucleotide synthesis. Moreover, inhibition of β-catenin/AKT2-stimulated pyrimidine synthesis axis preferentially repressed β-catenin mutant cell proliferation and tumor formation. Therefore, β-catenin active mutations are oncogenic in various preclinical liver cancer models. Stimulation of β-catenin/AKT2/CAD signaling cascade on pyrimidine synthesis is an essential and druggable vulnerability for β-catenin mutant liver cancer.

Background: Hepatocellular carcinoma (HCC) is highly aggressive with a poor prognosis and survival rate. Certain ANGPTL members have been implicated in tumor progression. However, the relevance of the ANGPTL gene family to HCC remains poorly understood. In this study, we explored the role of ANGPTLs in the prognosis of HCC., Methods: From the CCLE database, we studied the expression of ANGPTLs in a range of cancer cell lines. The UCSC, HCCDB, and Human Protein Atlas databases were used to analyze the differences in mRNA and protein expression of ANGPTLs in HCC tissues. Additionally, the correlation between ANGPTL mRNA and methylation levels and clinicopathological features were assessed in the TCGA database. The correlation between ANGPTL mRNA and overall survival was determined by the Kaplan-Meier plotter. cBioPortal database was used to analyze ANGPTL genomic alterations. Genes associated with ANGPTLs were determined by enrichment with KEGG. Moreover, the differentially expressed genes of ANGPTLs were analyzed by the LinkedOmics database, and the KEGG pathway and miRNA targets of ANGPTLs were also enriched., Results: There was a significant correlation between the ANGPTL members (excluding ANGPTL2) and the prognosis of HCC patients according to the Kaplan-Meier plotter analysis ( p < 0.05). ANGPTL1 was the gene with the highest mutation frequency. ANGPTLs are involved in certain pathways that may influence the development of HCC., Conclusion: In summary, the expression of some members of ANGPTLs was significantly correlated with HCC prognosis, suggesting that the ANGPTL gene family members may be promising molecular markers for HCC treatment and prognosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yun Bai et al.)

We and others have shown that MPM (micropeptide in mitochondria) regulates myogenic differentiation and muscle development. However, the roles of MPM in cancer development remain unknown. Here we revealed that MPM was downregulated significantly in human hepatocellular carcinoma (HCC) tissues and its decrease was associated with increased metastasis potential and HCC recurrence. Gain- and loss-of-function investigations disclosed that in vitro migration/invasion and in vivo liver/lung metastasis of hepatoma cells were repressed by restoring MPM expression and increased by silencing MPM. Mechanism investigations revealed that MPM interacted with NDUFA7. Mitochondrial complex I activity was inhibited by overexpressing MPM and enhanced by siMPM, and this effect of siMPM was attenuated by knocking down NDUFA7. The NAD + /NADH ratio, which was regulated by complex I, was reduced by MPM but increased by siMPM. Treatment with the NAD + precursor nicotinamide abrogated the inhibitory effect of MPM on hepatoma cell migration. Further investigations showed that miR-17-5p bound to MPM and inhibited MPM expression. miR-17-5p upregulation was associated with MPM downregulation in HCC tissues. These findings indicate that a decrease in MPM expression may promote hepatoma metastasis by increasing mitochondrial complex I activity and the NAD + /NADH ratio., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Hepatocellular carcinoma is the world's leading cause of cancer-related death. Chronic hepatitis B virus and hepatitis C virus infection cause liver cancer. The aim of this study was to investigate the relationship between statins and the risk of hepatocellular carcinoma in patients with hepatitis B or C., Methods: We systematically searched Web of Science, Embase, PubMed, Cochrane Library, China National Knowledge Infrastructure, and Wanfang Database from their inception to January 2019. We included studies that reported the hepatocellular carcinoma incidence among hepatitis B virus- or hepatitis C virus-infected patients or hepatitis B virus- or hepatitis C virus-related cirrhotic patients, evaluated and clearly defined exposure to statins, provided effective comparison groups, and reported risk estimates. Inclusion was not otherwise restricted. Summary relative risk estimates with 95% CIs were calculated using a random-effects model., Results: Meta-analysis of 10 studies showed that statin users had a significantly lower risk of hepatocellular carcinoma (relative risk = 0.47, 95% CI = 0.38-0.56) with significant heterogeneity. In 7 hepatitis studies, using statin was associated with a 53% reduction in the incidence of hepatocellular carcinoma (relative risk = 0.47, 95% CI = 0.43-0.50) with substantial heterogeneity. In 3 cirrhosis studies, the incidence of hepatocellular carcinoma in statin users was significantly reduced by 55% (relative risk = 0.45, 95% CI = 0.30-0.61) with no heterogeneity., Conclusion: Statins reduce the hepatocellular carcinoma risk among patients infected with hepatitis B virus or hepatitis C virus. This chemoprotective association is more pronounced in hepatitis B virus or hepatitis C virus-associated cirrhotic patients.

Background and Objectives: Abnormal activation of the PI3K/AKT pathway is closely related to tumor occurrence, development and angiogenesis. PI3K, as a key protein in the PI3K/Akt pathway, has different subtypes that play diverse roles in various tumors. The aim of this study was to examine the roles of different PI3K protein subtypes (PI3Kp110α, PI3Kp110β, and PI3Kp110δ) in the metastasis, angiogenesis and prognosis of hepatocellular carcinoma (HCC)., Methods: The roles of different PI3K protein subtypes in the metastasis, angiogenesis and prognosis of HCC were assessed by immunohistochemical staining of 97 HCC tissues and the STRING database., Results: Our results showed that PI3Kp110α and PI3Kp110δ were associated with HCC metastasis and angiogenesis. Patients with high expression of PI3Kp110α and PI3Kp110δ had a worse prognosis and shorter survival time, respectively, than those with low expression, whereas these effects were not observed for PI3Kp110β. Cox regression analysis showed that PI3Kp110α and clinical stage were independent risk factors for the overall survival of HCC patients., Conclusions: PI3Kp110α and PI3Kp110δ promoted HCC metastasis and angiogenesis via the PI3K/AKT pathway, and PI3Kp110α was an independent risk factor for HCC patients. These findings provide valuable insights for the prognosis evaluation and the selection of subtype inhibitors of HCC patients., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The solute carrier (SLC) superfamily genes encode more than 300 members that are responsible for the transmembrane transportation of many essential endogenous and exogenous compounds ranging from nutrients to drugs. SLCs are highly expressed in metabolic organs such as the liver, regulating the homeostasis of metabolites and the disposition of drugs. In contrast to their well-studied roles in physiological and pharmacological processes, little is known about the relationship between SLCs and cancer progression. Here, we aimed to explore the potential role of SLCs in progression and prognosis of hepatocellular carcinoma (HCC), one of the most commonly diagnosed cancers and leading causes of death worldwide. By performing bioinformatics analyses of HCC dataset from The Cancer Genome Atlas database, we identified three novel signature SLCs (SLC51B, SLC22A15, and SLC2A1) that are indicative of poor prognosis. Further functional analyses suggested the potential regulation of the three prognostic SLCs on cell proliferation and metastasis. Subsequent knockdown experiments performed in HCC cell lines showed that all three prognostic SLCs positively regulated the proliferation of HCC cells, among which SLC22A15 and SLC2A1 were required for migration and invasion of the cells, demonstrating remarkable consistency with the roles identified by bioinformatics methods in HCC. Therefore, our study provides a novel prognostic biomarker for HCC and reveals the significant roles of SLCs in HCC progression, which might have been undervalued in the past., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background and Aims: Hepatocellular carcinoma (HCC) is an aggressive malignancy which is often associated with a complex tumor microenvironment attributable to etiology-induced cellular inflammation. γδ T cells are known to detect and react to chronic inflammation, which is linked to cancer development, progression, and metastasis. Our recent genomic study revealed an increased infiltration of several immune cell types, including γδ T cells, in tumor microenvironments of a Thai HCC subtype associated with a good prognosis., Approach and Results: Here, we quantified the amount of γδ T cells using a γδ T-cell-specific gene signature in 247 Chinese HCC patients. We also validated the γδ T-cell signature in American HCC patients. Additionally, such an association was only found in tumor transcriptomic data, but not in adjacent nontumor transcriptomic data, suggesting a selective enrichment of γδ T cells in the tumor microenvironment. Moreover, the γδ T-cell signature was positively correlated with the expression of natural killer cell receptor genes, such as NKG2D and cytolytic T-cell genes granzymes and perforin, suggesting a stronger T-cell-mediated cytotoxic activity. Furthermore, we found that the γδ T-cell-specific gene expression is positively correlated with the expression of chemokine (C-C motif) ligand 4 (CCL4)/chemokine (C-C motif) ligand 5 (CCL5) and C-C chemokine receptor type 1 (CCR1)/C-C chemokine receptor type 5 (CCR5), the receptors for γδ T cells. We validated these results using immunohistochemical analysis of formalin-fixed, paraffin-embedded tumor biopsies from 182 HCC patients. Moreover, we found evidence of CCL4/CCL5-mediated recruitment of γδ T cells both in vitro and in a murine orthotopic Hepa1-6 HCC model., Conclusions: We propose that CCL4/CCL5 may interact with their receptor, CCR1/CCR5, which may facilitate the recruitment of γδ T cells from peripheral blood or peritumor regions to the tumor regions. Consequently, an increasing infiltration of γδ T cells in tumors may enhance antitumor immunity and improve patients' prognosis., (© 2020 by the American Association for the Study of Liver Diseases.)

Hepatic inflammatory response is a risk factor for liver cancer initiation and progression. Interleukin (IL)-35 is the newest member of the IL-12 cytokine family, and has been reported to play an essential role in the immunosuppressive liver microenvironment. Herein we focus on the expression profiles of IL-35 in hepatocellular carcinoma (HCC) and effects on local immune status. HCC transcriptome array data were downloaded from Gene Expression Omnibus (GEO). Analysis was performed by BRB-Array Tools and Ingenuity Pathway Analysis (IPA) software. Serum IL-35 level was detected by AimPlet bead-based immunoassay. In-situ IL-35 detection was performed by immunohistochemical staining and Western blot. The n-vitro effect of IL-35 on CD4 + or CD8 + T cell function was detected by flow cytometry. Our results showed that there were large amounts of IL-35 expressed in HCC serum and tumor tissues. IL-35 expression affects the transcript of thousands of genes, most differentially expressed genes (DEGs), in tumor tissues correlated with T cell immunity. The IL-35 high-expression group exhibited enhancement of regulatory T cells (T regs ) and impairment of cytolytic T cells. In-vitro experiments proved that exogenous IL-35 stimulated the expression of programmed cell death 1 (PD-1) and lymphocyte activation gene-3 (LAG3) in CD4 + and CD8 + T cells. In addition, the stimulating effect was time-dependent. Furthermore, IL-35 inhibited interferon (IFN)-γ secretion by CD4 + and CD8 + T cells. Elevated IL-35 had an immune suppressive role in HCC tumor microenvironments through affecting inhibitor receptor expression and cytokine secretion of CD4 + and CD8 + T cells. Dissection of the precise targets and underlying molecular mechanisms would mean alternative treatments for HCC patients., (© 2020 British Society for Immunology.)

Hepatocellular carcinoma (HCC) is a common malignant tumor with insidious onset and rapid progression. Its treatment is often difficult owing to tumor resistance. In this study, we aimed to understand the different biological characteristics, gene expression profiles, and drug resistance mechanisms of HCC cells cultured under different conditions. A conventional adherence method and a liquid overlay technique were used to prepare two- and three-dimensional cultures of Bel-7402 and 5-fluorouracil (5-Fu)-resistant Bel-7402 (Bel-7402/5-Fu) cells. Morphological characteristics were assessed via microscopy, and cell cycle distribution and apoptotic rate were obtained using flow cytometry. Cell sensitivity to different concentrations of drugs was detected with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Gene expression profiles and signal transduction pathways of Bel-7402 and Bel-7402/5-Fu cells under different culture conditions were determined using gene chips. Cells in three-dimensional culture were suspended and they grew into dense multicellular spheroid (MCS) structures, aggregating with each other. In contrast to cells in the two-dimensional culture, cell cycle arrest was observed in MCSs. The sensitivity of Bel-7402 cells in the two-dimensional culture to drugs at high concentrations was significantly higher than that of cells in the three-dimensional culture (p < .05). The apoptotic rate of Bel-7402 and Bel-7402/5-Fu cells was also higher in the two-dimensional culture (p < .05). Signal transduction pathway analysis showed that after Bel-7402 cells acquired resistance to 5-Fu, CCND1, MCM2, and MCM3 gene expression was upregulated in the G1 to S cell cycle control signal transduction pathway, CDKN1C and CCNG2 gene expression was downregulated, and MCM2 and MCM3 gene expression was upregulated in the DNA replication signal transduction pathway. Therefore, the liquid overlay technique is a simple, low-cost procedure to successfully construct three-dimensional culture models of HCC. This study provides new information and methods for exploring the molecular mechanisms of liver cancer resistance, clinical treatment, development of molecular information, and interventional prevention., (© 2021 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

The Chinese formula Pien Tze Huang (PZH) has been used to treat hepatocellular carcinoma (HCC) and showed positive clinical effects. However, the antitumor mechanism of PZH in HCC remains unclear. In this study, HCC xenograft Balb/c mice were treated with PZH; then, proteomics detection and Ingenuity Pathway Analysis (IPA) were used to analyze the differentiated phosphorylated proteins in tumor tissues. The results indicated that PZH could inhibit tumor weight by 50.76%. Eighty-four upregulated and 11 downregulated phosphorylated proteins were identified in PZH-treated mice. Twenty signaling pathways were associated with inflammation (including the IL-6 and TNFR1/2 pathways), cancer growth (including the p53 and FAK pathways), and the cell cycle (including the G2/M and G1/S checkpoint regulation pathways). Moreover, TNF- α , IL-6, and several typical differentially expressed phosphorylated proteins (such as p-CCNB1, p-FOXO3, and p-STAT3) in tumor tissues, tumor cell viability, and cell cycle arrest assay in vitro further verify the results of IPA. These results revealed that PZH achieved antitumor activity in HCC; the underlying mechanisms of which were mainly through regulating the inflammation-associated cytokine secretion, cancer growth pathways, and induction of G2/M arrest. These data provided the potential molecular basis for PZH to act as a therapeutic drug or a supplement to chemotherapy drugs for human HCC in the future., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020 Dancai Fan et al.)

Hepatocellular carcinoma (HCC) is a prevalent malignant tumour with high global morbidity and mortality associated with its multiple aetiologies, poor prognosis, resistance to chemotherapeutic drugs and high rate of recurrence. Here, we evaluated a gene therapy strategy that targets HCC using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system in HCC cell lines and in an in vivo human HCC xenograft mouse model. Apolipoprotein E (ApoE)-modified liposomes were used for targeted gene delivery to the tumour tissue, and the survivin promoter was used to drive HSVtk expression in HCC cells. Our results showed that the survivin promoter was specifically activated in tumour cells, and HSVtk was expressed selectively in tumour cells. In combination with GCV treatment, HSVtk expression resulted in the inhibition of HCC cell proliferation via enhanced apoptosis. In addition, tail vein injection of ApoE-HSVtk significantly suppressed the growth of xenograft tumours through an apoptosis-dependent pathway and extended the survival time of tumour-bearing mice. In summary, this study illustrates an effective cancer-specific gene therapy strategy for HCC that can be further developed for future clinical trials.

Hepatocellular carcinoma (HCC) is one of the most common types of cancer worldwide, and there is currently no effective therapeutic strategy in clinical practice. Gene therapy has great potential for decreasing tumor-induced mortality but has been clinically limited because of the lack of tumor-specific targets and insufficient gene transfer. The study of targeted transport of therapeutic genes in HCC treatment seems to be very important. In this study, we evaluated a gene therapy approach targeting HCC using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system in HCC cell lines and in an in vivo human HCC xenograft mouse model. GP73-modified liposomes targeted gene delivery to the tumor tissue, and the survivin promoter drove HSVtk expression in the HCC cells. Our results showed that the survivin promoter was specifically activated in tumor cells and HSVtk was expressed selectively in tumor cells. Combined with GCV treatment, HSVtk expression resulted in suppression of HCC cell proliferation via enhancing apoptosis. Moreover, tail vein injection of GP73-HSVtk significantly suppressed the growth of xenograft tumors through an apoptosis-dependent pathway and extended the survival of tumor-bearing mice without damaging the mice liver functions. Taken together, this study demonstrates an effective cancer-specific gene therapy strategy using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system for HCC that can be further developed for future clinical trials., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Lysyl oxidase-like 2 (LOXL2) has shown to promote metastasis and poor prognosis in hepatocellular carcinoma (HCC). Also, we have previously reported that vasculogenic mimicry (VM) is associated with invasion, metastasis and poor survival in HCC patients. In the present study, we investigated molecular function of LOXL2 in HCC and VM. We used the immunohistochemical and CD31/periodic acid-Schiff double staining to detect the relationship between LOXL2 and VM formation. We performed the gain and loss of function studies and analysed the migratory, invasion and tube formation in HCC cell lines. We analysed the function of LOXL2 in VM formation and HCC metastasis both in vitro and in vivo. We have showed that LOXL2 was overexpression in HCC and was positively correlated with tumour grade, metastasis, VM formation and poor survival in 201 HCC patients. Secondly, our studies have showed that LOXL2 overexpression in HCC cells significantly promoted migration, invasion and tube formation. Finally, we found that LOXL2 may increase SNAIL expression, thereby enabling VM. Our study indicated that LOXL2 may promote VM formation and tumour metastasis by collaborating with SNAIL in HCC. What's more, the overexpression of LOXL2 indicated a poor prognosis in HCC patients., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

CHI3L1 (YKL40) is a secreted glycoprotein and elevated serum CHI3L1 level has been proved to be associated with poor prognosis in many human cancers. However, the mechanism of how CHI3L1 causes poor prognosis in cancers is still unknown. Here, considering that CHI3L1 is a liver specific/enriched protein, we use hepatocellular carcinoma as a model to study the function of CHI3L1. We showed that, both in vivo and in vitro, overexpression of CHI3L1 could promote liver cancer cells growth, migration and invasion. We then used RNA-seq to analyze the expression profiles of CHI3L1 overexpressed in two HCC cell lines and found that CHI3L1 overexpression affected genes that were involved in cell-cell adhesion, extracellular exosome and adherens junction. Western blot analysis further revealed that CHI3L1 could activate TGF-β signal pathways. Our data added new understanding of the mechanism of CHI3L1's action. 1) CHI3L1 promoted cancer cell proliferation by regulating cell cycles; 2) CHI3L1 promoted cancer cell invasion and metastasis; 3) CHI3L1 regulate liver cancer potentially by regulating the TGF-β signaling pathways; 4) CHI3L1 has direct kinase activities or activate kinase to phosphorylate SMAD2, SMAD3.

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. Cancer stem cells (CSC) represent a subpopulation of tumor cells endowed with the capacity for self-renewal and multilineage differentiation. Previous studies have indicated that CSC may participate in the formation of VM. With the advance of high-resolution microarrays and massively parallel sequencing technology, long noncoding RNAs (lncRNAs) are suggested to play a critical role in tumorigenesis and, in particular, the development of human hepatocellular carcinoma (HCC). Currently, no definitive relationship between lncRNA and VM formation has been described. In the current study, we demonstrated that expression of the lncRNA, n339260, is associated with CSC phenotype in HCC, and n339260 level correlated with VM, metastasis, and shorter survival time in an animal model. Overexpression of n339260 in HepG2 cells was associated with a significant increase in CSC. Additionally, the appearance of VM and vascular endothelial (VE)-cadherin, a molecular marker of VM, was also induced by n339260 overexpression. Using a short hairpin RNA approach, n339260 was silenced in tumor cells, and knockdown of n339260 was associated with reduced VM and CSC. The results of this study indicate that n339260 promotes VM, possibly by the development of CSC. The related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Chronic hepatitis B (HBV) and C virus (HCV) infection causes liver cancer. This protocol is to provide the methods used to assess the relationship between statins and HCC risk in hepatitis B or C patients., Methods: We will search comprehensively the PubMed (medline), Embase, Web of Science, Cochrane Library, and China National Knowledge Infrastructure, Wanfang Database from their inception to November 2017. We will include studies that evaluated and clearly defined exposure to statins, reported the HCC incidence in hepatitis B/C patients or HBV/HCV-related cirrhosis patients, provided effective comparison groups, and reported risk estimates, such as hazard ratios, relative risks, or odds ratios with corresponding 95% confidence intervals or sufficient data for their estimation. We will use Stata (version 15.0) to compute the data synthesis carefully when a meta-analysis is allowed., Results: This study will provide a high-quality synthesis of current evidence of statins on HCC risk in hepatitis B or C patients., Conclusion: The conclusion of our systematic review will provide evidence to judge whether statin is an effective intervention for hepatitis B or C patients.

Resistance to TGF-β-induced growth repression is prevalent in various cancer cells, but the underlying mechanisms remain unclear. In this study, we showed that activation of TGF-β signaling caused Sma- and Mad-related family (Smad) 2 and Smad3 to bind directly to the promoter region of miR-195, and then activated miR-195 transcription in normal hepatocytes. Conversely, miR-195 inhibited the expression of Smad7 by binding to its 3'-UTR, thereby strengthening TGF-β-Smad signaling. These data identify a novel TGF-β-miR-195 positive regulatory circuitry in normal hepatocytes. Further investigation revealed that HDAC1, a histone deacetylase that was abnormally overexpressed in hepatocellular carcinoma, could bind to the miR-195 promoter via Smad3 and cause hypoacetylation in the histones associated with the miR-195 promoter in hepatoma cells. This resulted in transcriptional repression of miR-195 and, subsequently, disruption of the TGF-β-miR-195 regulatory loop and evasion of TGF-β-mediated growth inhibition. Moreover, silencing HDAC1 in hepatoma cells restored TGF-β-mediated growth suppression, but this effect was attenuated if miR-195 expression decreased. These findings suggest that HDAC1-induced miR-195 down-regulation is an important mechanism for tumor cells to resist the cytostatic activity of TGF-β, and highlight the importance of TGF-β-Smad2/3-miR-195-Smad7 circuitry in preventing uncontrolled cell proliferation.-Wang, R., Fu, T., You, K., Li, S., Zhao, N., Yang, J., Zhuang, S.-M. Identification of a TGF-β-miR-195 positive feedback loop in hepatocytes and its deregulation in hepatoma cells.

The morphology of nanoparticles influences their cellular uptake process, while rough surface-enhanced affinity renders rough nanoparticles desirable in related biomedical applications. In this work, rattle-structured rough nanocapsules (Au@HSN-PGEA, AHPs) composed of in-situ-formed gold nanorod (Au NR) cores and polycationic mesoporous silica shells were constructed for trimodal complementary cancer therapy. Taking advantage of surface roughness, near-infrared (NIR) responsiveness, and controlled release manner, AHPs were expected to realize the co-delivery of sorafenib (SF, a hydrophobic antiproliferative and antiangiogenic drug) and antioncogene p53 for malignant hepatocellular carcinoma treatment. The rough surface feature of AHP was investigated for cellular uptake and the subsequent gene transfection. The feasibility of photothermal Au NR cores for NIR-triggered SF release was also tested. Notably, synergistic effects based on photothemal therapy-enhanced chemotherapy were achieved. In addition, the good in vivo performance of the proposed multifunctional nanoparticles with rough surfaces was also demonstrated. The current work extends the biomedical applications of the intriguing rough nanoparticles and provides a facile strategy to construct flexible platforms for complementary gene/chemo/photothermal therapy.

Excessive triglyceride accumulation in hepatocytes is the hallmark of obesity-associated nonalcoholic fatty liver disease (NAFLD). Elevated levels of the saturated free fatty acid palmitate in obesity are a major contributor to excessive hepatic lipid accumulation. The nuclear orphan receptor Nur77 is a transcriptional regulator and a lipotoxicity sensor. Using human HepG2 hepatoma cells, this study aimed to investigate the functional role of Nur77 in palmitate-induced hepatic steatosis. The results revealed that palmitate significantly induced lipid accumulation and suppressed lipolysis in hepatocytes. In addition, palmitate significantly suppressed Nur77 expression and stimulated the expression of peroxisome proliferator-activated receptor γ (PPARγ) and its target genes. Nur77 overexpression significantly reduced palmitate-induced expression of PPARγ and its target genes. Moreover, Nur77 overexpression attenuated lipid accumulation and augmented lipolysis in palmitate-treated hepatocytes. Importantly, G0S2 knockdown significantly attenuated lipid accumulation and augmented lipolysis in palmitate-treated hepatocytes, whereas G0S2 knockdown had no effect on the palmitate-induced expression of Nur77, PPARγ, or PPARγ target genes. In summary, palmitate suppresses Nur77 expression in HepG2 cells, and Nur77 overexpression alleviates palmitate-induced hepatic fat accumulation by down-regulating G0S2. These results display a novel molecular mechanism linking Nur77-regulated G0S2 expression to palmitate-induced hepatic steatosis.

Combining near-infrared fluorescence (NIRF) and nuclear imaging techniques provides a novel approach for hepatocellular carcinoma (HCC) diagnosis. Here, we report the synthesis and characteristics of a dual-modality NIRF optical/positron emission tomography (PET) imaging probe using heptamethine carbocyanine dye and verify its feasibility in both nude mice and rabbits with orthotopic xenograft liver cancer. This dye, MHI-148, is an effective cancer-specific NIRF imaging agent and shows preferential uptake and retention in liver cancer. The corresponding NIRF imaging intensity reaches 10 9 /cm 2 tumor area at 24 h after injection in mice with HCC subcutaneous tumors. The dye can be further conjugated with radionuclide 68 Ga ( 68 Ga-MHI-148) for PET tracing. We applied the dual-modality methodology toward the detection of HCC in both patient-derived orthotopic xenograft (PDX) models and rabbit orthotopic transplantation models. NIRF/PET images showed clear tumor delineation after probe injection (MHI-148 and 68 Ga-MHI-148). The tumor-to-muscle (T/M) standardized uptake value (SUV) ratios were obtained from PET at 1 h after injection of 68 Ga-MHI-148, which was helpful for effectively capturing small tumors in mice (0.5 cm × 0.3 cm) and rabbits (1.2 cm × 1.8 cm). This cancer-targeting NIRF/PET dual-modality imaging probe provides a proof of principle for noninvasive detection of deep-tissue tumors in mouse and rabbit and is a promising technique for more accurate and early detection of HCC.

Ubiquitin-specific protease 4 (USP4) is a member of the deubiquitinating enzyme family, which plays an important role in human tumor diseases. However, the mechanisms by which USP4 facilitates tumor development, especially in hepatocellular carcinoma (HCC), remain unclear. Clinically, we found that USP4 is overexpressed in human HCC tissues compared with adjacent non-tumoral tissues and is significantly correlated with malignant phenotype characteristics, including tumor size, tumor number, differentiation, serum alpha-fetoprotein level, and vascular invasion. Moreover, Kaplan-Meier survival analysis showed a poor overall survival rate in patients with USP4-overexpressing tumors. Analyses of univariate and multivariate Cox proportional hazard models indicated that USP4 is a prognostic biomarker for poor outcome. Using in vitro and in vivo assays, we demonstrated that USP4 overexpression enhanced HCC cell growth, migration, and invasion. Mechanistically, cyclophilin A (CypA) was identified as an important molecule for USP4-mediated oncogenic activity in HCC. We observed that USP4 interacted with CypA and inhibited CypA degradation via deubiquitination in HCC cells. Subsequently, the USP4/CypA complex activated the MAPK signaling pathway and prevented CrkII phosphorylation. These data suggest that USP4 acts as a novel prognostic marker, offering potential therapeutic opportunities for HCC.

HOX transcript antisense RNA (HOTAIR), a newly discovered long noncoding RNA (lncRNA), has been reported to be a poor prognostic marker in many types of cancers. The current study attempted to investigate the biological roles and clinicopathlogical implications of HOTAIR in hepatocellular carcinoma (HCC), as well as understand the molecular mechanisms of HOTAIR in HCC progression. HOTAIR expression in 95 HCC patients with paired HCC tissues and adjacent non‑cancer tissues were investigated using quantitative reverse transcription‑polymerase chain reaction. The association between HOTAIR expression and clinicopathological features was assessed. The effects of HOTAIR were examined in vitro assays by silencing the lncRNA. Pathway analyses were performed to illustrate the biological functions of the HOTAIR and coexpression genes. The expression level of HOTAIR was observed significantly higher in the HCC tissue than the adjacent non‑tumor tissue. HOTAIR expression levels were significantly higher in tumor samples from patients with distant metastasis, advanced stage, portal vein tumor embolus, vasoinvasion, tumor capsular infiltration or positive nm23 expression than those from patients without these conditions, correspondingly. The silencing of HOTAIR in liver cancer cells induced the inhibition of cell proliferation and promotion of apoptosis. Several pathways such as extracellular matrix‑receptor interaction, focal adhesion, pathways in cancer were annotated with the HOTAIR and coexpression genes. In summary, the present analysis indicates that HOTAIR might be an oncogene in HCC. It functions though promoting tumor cell growth and inhibiting apoptosis. HOTAIR may potentially be involved in HCC metastatic progression by several pathways correlated to cell adhesion, and may be a therapeutic target in future.

Background/aims: Accumulated evidence indicates that lncRNA NEAT1 has important roles in various malignant tumors. In this study, we conducted a comprehensive analysis to explore the exact role of NEAT1 in hepatocellular carcinoma (HCC)., Methods: The effects of NEAT1 on cell proliferation, apoptosis, migration, and invasion were measured by in vitro experiments. The expression level and clinical value of NEAT1 in HCC was evaluated based on data from The Cancer Genome Atlas (TCGA), Oncomine, and in-house real-time quantitative (RT-qPCR). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein-protein interaction (PPI) network analyses were conducted to investigate the potential molecular mechanisms of NEAT1., Results: NEAT1 siRNA not only inhibited proliferation, migration, and invasion of HCC cells but also induced HCC cell apoptosis. A total of four records from TCGA, Oncomine, and RT-qPCR analysis were combined to assess the expression level of NEAT1 in HCC. The pooled standard mean deviation (SMD) indicated that NEAT1 was up-regulated in HCC (SMD = 0.54; 95% CI, 0.36-0.73; P < 0.0001). The area under the curve value of the summary receiver operating characteristic curve was 0.71. NEAT1 expression was also related to race (P = 0.025) and distant metastasis (P = 0.002). Additionally, the results of GO, KEGG pathway, and PPI network analyses suggest that NEAT1 may promote the progression of HCC by interacting with several tumor-related genes (SP1, MDM4, CREBBP, TRAF5, CASP8, TRAF1, KAT2A, and HIST4H4)., Conclusions: NEAT1 contributes to the deterioration of HCC and provides a potential biomarker for the diagnosis and therapy of HCC., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Decreased level of miR-204-5p has been documented in various malignancies. However, the expression and clinical significance of miR-204-5p in hepatocellular carcinoma has not been investigated. The aim of this study is to examine the relationship between miR-204-5p expression and clinicopathological features in hepatocellular carcinoma (HCC) as well as to predict the relevant signaling pathways. The miR-204-5p expression level was detected in HCC and in matched paraneoplastic liver from 95 formalin-fixed paraffin-embedded tissues by the real-time reverse transcription polymerized chain reaction (qRT-PCR). The association of miR-204-5p expression with clinicopathological features as well as the prognosis of HCC was examined. Public data portals including the Gene Expression Omnibus and The Cancer Genome Atlas were used to retrieve the HCC-related data in order to perform a comprehensive meta-analysis. Meanwhile, protein-protein interaction (PPI) and enrichment analyses were performed using predicted target genes. The relative expression of miR-204-5p was remarkably reduced in HCC than that in paraneoplastic hepatic tissues. In HCC, the miR-204-5p expression was downregulated in the metastasis, vasoinvasion, and advanced stage (III and IV) subgroups compared with their counterparts. Furthermore, the meta-analysis based on qRT-PCR data demonstrated that miR-204-5p was markedly downregulated in HCC with a standardized mean difference of -5.19 (P < .001). However, no significant association was observed between miR-204-5p and survival outcomes. The potential target genes of miR-204-5p were significantly enriched in several pathways which might be associated with HCC, such as "cell proliferation" from GO terms and "pathways in cancer" from the KEGG analysis. A PPI network of miR-204-5p potential target genes identified prospective core genes potentially involved in the regulation of HCC oncogenesis and progression. Our findings suggested that miR-204-5p might act as a tumor-suppressive gene in the tumorigenesis and progression of HCC via vital signaling pathways and that miR-204-5p could be regarded as a protective factor in HCC.

Near infrared fluorescence (NIRF) imaging has strong potential for widespread use in noninvasive tumor imaging. Indocyanine green (ICG) is the only Food and Drug Administration (FDA) -approved NIRF dye for clinical diagnosis; however, it is unstable and poorly targets tumors. DZ-1 is a novel heptamethine cyanine NIRF dye, suitable for imaging and tumor targeting. Here, we compared the fluorescence intensity and metabolism of DZ-1 and ICG. Additionally, we assayed their specificities and abilities to target tumor cells, using cultured hepatocellular carcinoma (HCC) cell lines, a nude mouse subcutaneous xenograft model of liver cancer, and a rabbit orthotopic transplantation model. We found that DZ-1 accumulates in tumor tissue and specifically recognizes HCC in subcutaneous and orthotopic models. The NIRF intensity of DZ-1 was one order of magnitude stronger than that of ICG, and DZ-1 showed excellent intraoperative tumor targeting in the rabbit model. Importantly, ICG accumulated at tumor sites, as well as in the liver and kidney. Furthermore, DZ-1 analog-gemcitabine conjugate (NIRG) exhibited similar tumor-specific targeting and imaging properties, including inhibition of tumor growth, in HCC patient-derived xenograft (PDX) mice. DZ-1 and NIRG demonstrated superior tumor-targeting specificity, compared to ICG. We show that DZ-1 is an effective molecular probe for specific imaging, targeting, and therapy in HCC., Competing Interests: The authors declare no conflict of interest.

Hepatocellular carcinoma (HCC) has become the third most deadly disease worldwide and HBV is the major factor in Asia and Africa. We conducted 9 WGS (whole genome sequencing) analyses for matched samples of tumor, adjacent non-tumor tissues and normal blood samples of HCC patients from three HBV positive patients. We then validated the mutations identified in a larger cohort of 177 HCC patients. We found that the number of the unique somatic mutations (average of 59,136) in tumor samples is significantly less than that in adjacent non-tumor tissues (average 83, 633). We discovered that the TP53 R249S mutation occurred in 7.7% of the HCC patients, and it was significantly associated with poor diagnosis. In addition, we found that the L104P mutation in the VCX gene (Variable charge, X-linked) was absent in white blood cell samples, but present at 11.1% frequency in the adjacent tissues and increased to 14.6% in HCC tissues, suggesting that this mutation might be a tumor driver gene driving HCC carcinogenesis. Finally, we identified a TK1-RNU7 fusion, which would result in a deletion of 103 amino acids from its C-terminal. The frequencies of this fusion event decreased from the adjacent tissues (29.2%) to the tumors (16.7%), suggesting that a truncated thymidine Kinase1 (TK1) caused by the fusion event might be deleterious and be selected against during tumor progression. The three-way comparisons allow the identification of potential driver mutations of carcinogenesis. Furthermore, our dataset provides the research community a valuable dataset for identifying dynamic changes of mutation profiles and driver mutations for HCC.

The transcription factor Runx2 has been reported to promote epithelial-mesenchymal transition (EMT) in many tumors. Vasculogenic mimicry (VM) is described as the mimicry of endothelial cells by tumor cells to form microvascular tubes in aggressive tumors. Galectin-3 has been reported to regulate cell invasion, migration, and VM formation; it could be regulated by Runx2. However, the relationship between Runx2, Galectin-3, EMT, and VM has not been studied in hepatocellular carcinoma (HCC). We examined Runx2 expression in 89 human HCC samples and found Runx2 expression was associated with VM. Clinical-pathological data analysis revealed that Runx2 expression was associated with a shorter survival period. Overexpression of Runx2 promoted EMT and enhanced cell migration, invasion, and VM formation in HepG2 cells. Conversely, the downregulation of Runx2 inhibited EMT and reduced cell invasion, migration, and VM formation in SMMC7721. Galectin-3 expression declined following the downregulation of Runx2 in HepG2 cells, and increased in SMMC7721 cells after Runx2 knockdown. We consistently demonstrated that the downregulation of LGALS3 in HepG2-Runx2 cells reduced cell migration; invasion and VM formation; while upregulation of LGALS3 in SMMC7721-shRunx2 cells enhanced cell migration, invasion, and VM formation. The results indicate that Runx2 could promote EMT and VM formation in HCC and Galectin-3 might have some function in this process.

MicroRNAs (miRNAs) play vital roles in cell proliferation, differentiation and apoptosis in hepatocellular carcinoma (HCC). miR-26b has been confirmed as an important regulator in carcinogenesis and other pathological processes. miR-26b-5p is one member of the mature miR-26 family, and its functional role in proliferation, angiogenesis and apoptosis in HCC remains unknown. Here, we demonstrate that miR-26b-5p expression was significantly decreased in HCC tissues and HCC cell lines compared with normal liver tissues and liver cells by quantitative real-time polymerase chain reaction (qRT-PCR). The relationships between miR-26b-5p and the clinical characteristics of HCC patients were further analysed, and miR-26b-5p was positively correlated with the differentiation of HCC cells. Computational searches were further used to identify the downstream targets and signalling pathways of miR-26b-5p in HCC cells. Cell viability, proliferation and tube formation abilities were assessed by scrape, 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and three-dimensional culture assays to confirm that miR-26b-5p inhibited HCC cell growth and impaired the tube formation ability of the HCC cells. Both in vitro and in vivo studies showed that miR-26b-5p could suppress vascular mimicry (VM) and angiogenesis by down-regulating the expression of VE-cadherin, Snail and MMP2 and could inhibit the apoptosis of HCC cells. Using mouse models, we revealed that tumours derived from miR-26b-5p-expressing HCC cells displayed a significant decrease in microvessel density compared with those derived from control cells. Therefore, our data provide further insight into the role of miR-26b-5p as a negative regulator of proliferation, angiogenesis, and apoptosis in HCC.

MiR-195 expression is frequently reduced in various cancers, but its underlying mechanisms remain unknown. To explore whether abnormal transcription contributed to miR-195 downregulation in hepatocellular carcinoma (HCC), we characterized the -2165-bp site upstream of mature miR-195 as transcription start site and the -2.4 to -2.0-kb fragment as the promoter of miR-195 gene. Subsequent investigation showed that deletion of the predicted Sp1 binding site decreased the miR-195 promoter activity; Sp1 silencing significantly reduced the miR-195 promoter activity and the endogenous miR-195 level; Sp1 directly interacted with the miR-195 promoter in vitro and in vivo. These data suggest Sp1 as a transactivator for miR-195 transcription. Interestingly, miR-195 expression was also subjected to epigenetic regulation. Histone deacetylase 3 (HDAC3) could anchor to the miR-195 promoter via interacting with Sp1 and consequently repress the Sp1-mediated miR-195 transactivation by deacetylating histone in HCC cells. Consistently, substantial increase of HDAC3 protein was detected in human HCC tissues and HDAC3 upregulation was significantly correlated with miR-195 downregulation, suggesting that HDAC3 elevation may represent an important cause for miR-195 reduction in HCC. Our findings uncover the mechanisms underlying the transcriptional regulation and expression deregulation of miR-195 in HCC cells and provide new insight into microRNA biogenesis in cancer cells., (Copyright © 2016. Published by Elsevier B.V.)

Unlabelled: Twist1 is well known to induce epithelial-mesenchymal transition (EMT) and promote tumor metastasis. MicroRNAs (miRNAs) are involved in the EMT process and are associated with metastasis in hepatocellular carcinoma (HCC). In the present study, microRNA-26b-5p (miR-26b-5p) expression was consistently and significantly downregulated in HepG2-Twist1 HCC cell lines compared with HepG2-vector cell lines using microarrays (the HepG2-Twist1 cell line can stably express Twist1). miR-26b- 5p downregulation was directly mediated by Twist1 through binding to the promoter region of miR-26b-5p in HepG2-Twist1 cells by ChIP-seq technology. Both gain- and loss-of-function studies showed that miR-26b-5p dramatically suppressed EMT and the invasion ability of HCC cells in vitro. Using mouse models, tumors derived from miR- 26b-5p-overexpressed HCC cells exhibited a significant reduction in tumorigenicity compared with the control group. Subsequent investigation revealed that miR-26b-5p directly inhibited SMAD family member 1 (SMAD1) expression. miR-26b-5p repressed BMP4/Smad1 signaling following SMAD1 inhibition. Overexpression of SMAD1 reversed the function of miR-26b-5p. In human HCC tissues and mouse xenograft tumors, miR-26b-5p levels were inversely correlated with SMAD1 expression as well as metastasis., Conclusion: miR-26b-5p suppresses Twist1-induced EMT, invasion, and metastasis of HCC cells by targeting SMAD1 and BMP4/Smad1 signaling. This suggests a promising application for miR-26b-5p in anti-HCC therapy., Competing Interests: The authors declare no Conflicts of Interest.

Unlabelled: Twist-1 and miRNAs have been reported to be associated with tumor metastasis and angiogenesis. However, the relationship between Twist-1 and miRNAs and the function of miRNAs remain largely undefined. We aimed to reveal the Twist-1-related miRNA expression profile and to determine whether Twist-1 functions in tumor metastasis and vasculogenic mimicry (VM) by regulating miRNA expression in hepatocellular carcinoma (HCC). Results showed that the expression of miR-27a-3p was consistently down-regulated in HCC cell lines and tissue samples displaying high expression of Twist-1. Both loss- and gain-of-function assays revealed suppressive effects of miR-27a-3p. Low miR-27a-3p expression was significantly associated with early metastasis in HCC. Subsequent investigations revealed that miR-27a-3p mediated the inhibition of epithelial-mesenchymal transition (EMT). Additional experiments showed that VE-cadherin is a direct target of miR-27a-3p and further demonstrated the critical role of miR-27a-3p in suppressing tumor metastasis and VM., Conclusions: Twist-1 up-regulation in HepG2 cells resulted in the differential expression of 18 miRNAs. Among them, miR-27a-3p deregulation contributed to VM and metastasis. The miR-27a-3p-mediated down-regulation of VE-cadherin and inhibition of EMT may be essential for Twist-1 to induce tumor metastasis and VM. Our findings highlight the importance of miR-27a-3p and suggest a promising new strategy for anti-HCC therapy.

Hepatic metastasis is the leading cause of mortality of colon cancer, which is still lack of an effective therapy. A new delivery system, pluronic P85 block copolymers, conveying chemotherapeutic agent 5-fluorouracil (5-Fu) for inhibiting growth and metastasis of colon cancer was designed and developed. In this study, we demonstrated that 5-Fu produce strong pesticide effect at lower doses in the present of pluronic P85 compared with control groups. The migration and invasion of HCT116 cells and RKO cells were examined and the results showed that migration and invasion capacities of HCT116 cells and RKO cells were reduced by administering 5-Fu/P85 copolymer micelles in vitro and in vivo which indicating an effectively activity. Interestingly, the content of CD133 + CXCR4+ cells in HCT116 cancer cells and RKO cells treated by 5-Fu/P85 copolymer micelles was decreased. Importantly, the epithelial-mesenchymal transition (EMT) of CD133 + CXCR4+ cells, which was strongly associated with liver metastasis of colon cancer, was also suppressed by giving 5-Fu/P85 copolymer micelles. The results indicated that 5-Fu/P85 copolymer micelles could inhibit the growth and metastasis of colon cancer, which could be attributed to the decrease of the content of CD133 + CXCR4+ cells and suppression of EMT of CD133 + CXCR4+ cells.

Epithelial-mesenchymal transition (EMT) is regulated by multiple signal transduction pathways. Twist-1 is one of the most important transcription factors in these pathways. In a previous study, we found that Bcl-2 enhanced the role of Twist-1 in EMT. Coexpression of Twist-1 and Bcl-2 may play an import role in vasculogenic mimicry (VM) through regulation of EMT. Moreover, regulators of EMT and VM are known to be important targets for microRNAs (miRNAs). To better understand how these critical pathways are induced by coexpression of Twist-1 and Bcl-2, we performed a comprehensive comparative bioinformatics analysis using microarrays on HCCs that overexpressed Twist-1 and Bcl-2. Eleven miRNAs associated with coexpression of Twist-1 and Bcl-2 were selected from the comprehensive analysis of miRNA microarray and ChIP-seq analysis. Changes in miRNAs were associated with significant differences in the expression of genes involved in signal transduction pathways related to processes including tumor invasion, metastasis, angiogenesis, and tumor cell shape. We confirmed the role of Twist-1 and Bcl-2 coexpression in HCC cells using wound healing assays, invasion assays, and 3D Matrigel assays. Furthermore, the role of miR-27a as a crucial regulator of EMT and VM was confirmed in HCC cells by RT-PCR and western blot analysis. These findings provide evidence that Bcl-2 enhances the role of Twist-1 in VM and EMT through miRNAs., (Copyright © 2015 Elsevier Inc. All rights reserved.)