Clinical Staging Of Hepatocellular Carcinoma Research Articles

Scutellaria baicalensis Induces Cell Apoptosis and Elicits Mesenchymal-Epithelial Transition to Alleviate Metastatic Hepatocellular Carcinoma via Modulating HSP90β.

Hepatocellular carcinoma is one of the most common malignant tumors in the world and shows strong metastatic potential. Current medicine for hepatocellular carcinoma therapy is invalid, while Scutellaria baicalensis Georgi exhibits the pharmaceutical potential to treat liver diseases and liver cancer. Herein, we verified the inhibitory properties and the pivotal molecules regimented by Scutellaria baicalensis on advanced hepatocellular carcinoma. At first, the viability of SK-Hep-1 cells was significantly reduced under treatment of Scutellaria baicalensis extract in a dose-dependent manner without affecting the growth of normal hepatocyte. Scutellaria baicalensis extract application could remarkably cause apoptosis of SK-Hep-1 cells through p53/cytochrome C/poly-ADP ribose polymerase cascades and arrest the cell cycle at the G1/S phase by downregulating cyclin-dependent kinases. Meanwhile, administration of Scutellaria baicalensis extract remarkably attenuated the migration capability as well as suppressed matrix metalloproteinase activity of advanced hepatocellular carcinoma cells. The proteome profiles and network analysis particularly implied that exposure to Scutellaria baicalensis extract downregulated the expression of HSP90β, and the clinical stage of hepatocellular carcinoma is also positively correlated with the HSP90β level. Combined treatment of Scutellaria baicalensis extract and HSP90β siRNAs could markedly enhance the ubiquitination activity and the degradation of vimentin to subsequently inhibit the metastatic property of SK-Hep-1 cells. Moreover, application of Scutellaria baicalensis extract and HSP90β siRNAs depleted phosphorylation of AKT, which stimulated the expression of p53 and consecutively triggered cell apoptosis. These findings suggest that HSP90β may be a prospective target for the effective therapy of advanced hepatocellular carcinoma via accelerating apoptosis of hepatocellular carcinoma cells and eliciting mesenchymal-epithelial transition with the administration of Scutellaria baicalensis extract.

Expression profile and prognostic values of SMC family members in HCC.

Objective:The structural maintenance of chromosome (SMC) gene family, including 6 proteins, is involved in a wide range of biological functions in different human cancers. Nevertheless, there is little research on the expression patterns, potential functions and prognostic value of SMC genes in hepatocellular carcinoma (HCC). Based on publicly available databases and integrative bioinformatics analysis, we tried to determine the value of SMC gene expression in predicting the risk of developing HCC.Methods:The expression and copy number variations data of SMC family members were obtained from TCGA (The Cancer Genome Atlas). We identified the prognostic values of SMC family members and their clinical features. GSEA (Gene Set Enrichment Analysis) was conducted to detect the mechanism underlying the involvement of SMC family members in liver cancer. We used Tumor Immune Estimation Resource database to explore the associations between TIICs (Tumor Immune Infiltrating Cells) and the SMC family members.Results:Our analysis proved that downregulation of SMC family members was common modification in HCC patients. In HCC, the expression of SMC1A, SMC2, SMC3, SMC4, SMC6 were upregulated. Upregulation of SMC2, SMC3, and SMC4, along with the clinical stage of HCC, were associated with a poor prognosis according to the results of univariate and multivariate Cox proportional hazards regression analysis. SMC2, SMC3, and SMC4 are also related to tumor purity and immune infiltration levels of HCC. The GSEA results proved that SMC family members take part in numerous biological processes underlying tumorigenesis.Conclusion:In this study, we comprehensively analyzed the expression of SMC family members in patients with HCC. This can provide insights for further investigation of the SMC members as potential therapeutic targets in HCC and suggest that the use of SMC inhibitor targeting SMC2, SMC3, and SMC4 can be a practical strategy for the therapy of HCC.

LOXL3-promoted hepatocellular carcinoma progression via promotion of Snail1/USP4-mediated epithelial-mesenchymal transition.

Lysyl-oxidase-like 3 (LOXL3) was reported to be essential in epithelial-mesenchymal transition (EMT) of cancers. However, the role of LOXL3 in hepatocellular carcinoma (HCC) remained unclear. In this study, we explored clinical significance, biological functions, and regulatory mechanisms of LOXL3 in HCC. Our study found that LOXL3 expression was markedly associated with the tumor size and clinical stage of HCC, and it was highly expressed in tumor tissues of metastatic HCC patients. High expression of LOXL3 predicted a poor prognosis of HCC. TGF-β1 treatment elevated LOXL3 protein expression and cell invasion, and reduced cell apoptosis in HCC cell lines (SMMC-7721 and Huh-7), while downregulation of LOXL3 reversed the promotive effects of TGF-β1 treatment on LOXL3 protein expression and cell invasion, and the inhibitory effect on cell apoptosis. Mechanistically, LOXL3 interacted with snail family transcriptional repressor 1 (Snail1) through STRING database and RIP assay, and Snail1 bound to ubiquitin-specific peptidase 4 (USP4) promoter by JASPAR database, luciferase reporter gene and Co-IP assays. Overexpression of USP4 reversed the inhibitory effect of LOXL3 silence on EMT in HCC cells through deubiquitinating and stabilizing the expression of Snail1. Moreover, LOXL3-promoted HCC EMT through Wnt/β-catenin/Snail1 signaling pathway. In vivo study revealed that silence of LOXL3-inhibited HCC tumor growth. In conclusion, LOXL3 silence inhibited HCC invasion and EMT through Snail1/USP4-mediated circulation loop and Wnt/β-catenin signaling pathway.

Construction of a Novel Clinical Stage-Related Gene Signature for Predicting Outcome and Immune Response in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) with high heterogeneity is one of the most frequent malignant tumors. However, there were no studies to create a clinical stage-related gene signature for HCC patients. Differentially expressed genes (DEGs) associated with clinical stage of HCC were analyzed based on TCGA datasets. Functional enrichment analysis was carried out by the use of stage-related DEGs. Then, the least absolute shrinkage and selection operator (LASSO) regression and univariate Cox regression were performed to reduce the overfit and the number of genes for further analysis. Next, survival and ROC assays were carried out to demonstrate the model using TCGA. Functional analysis and immune microenvironment analysis related to stage-related DEGs were performed. Reverse transcriptase polymerase chain reaction (RT-PCR) and Cell Counting Kit-8 (CCK-8) assays were applied to examine the expression and function of PNCK in HCC. In this research, there were 21 DEGs between HCC specimens with stage (I-II) and HCC specimens with stage (III-IV), including 20 increased genes and 1 decreased genes. A novel seven-gene signature (including PITX2, PNCK, GLIS1, SCNN1G, MMP1, ZNF488, and SHISA9) was created for the prediction of outcomes of HCC patients. The ROC curves confirmed the prognostic value of the new model. Cox assays demonstrated that the seven-gene signature can independently forecast overall survival. The immune analysis revealed that patients with low risk score exhibited more immune activities. Moreover, we confirmed that PNCK expressions were distinctly increased in HCC, and its silence suppressed the proliferation of HCC cells. Overall, our research offered a robust and reliable gene signature which displayed an important value in the prediction of overall survival of HCC patients and might deliver more effective personalized therapies.

Comprehensive Analyses of the Infiltrating Immune Cell Landscape and Its Clinical Significance in Hepatocellular Carcinoma.

BackgroundImmunotherapy has gradually played a significant role in treating cancer over the past 10 years. Meanwhile, significant connections have been found between infiltrating immune cells and prognosis, as well as the development of immunotherapy in hepatocellular carcinoma (HCC). Thus, analyses of the immune cell landscape are urgently needed for the future development of immunotherapy in HCC.MethodsExpression data of HCC and normal liver tissue were searched and downloaded from The Cancer Genome Atlas. The fraction of various immune cells was estimated by CIBERSORT, which is a computational analysis tool. Comparisons of the fractions of 22 types of immune cells were performed between HCC and normal tissues, as well as survival analyses and clinical significance. xCell was used to validate the results.ResultsThe fractions of 12 types of immune cells, including follicular helper T (Tfh) cells, monocytes, M0, and M2 macrophages, were significantly different between tissues. In survival analyses, higher fractions of Tfh cells (P=0.037), M0 (P=0.001), and M2 macrophages (P=0.045) were associated with a poorer prognosis, and monocytes with a better prognosis (P=0.040). Furthermore, a higher fraction of M0 macrophages (P=0.001), lower fraction of monocytes (P=0.031), and M2 macrophages (P=0.018) were found as risk factors of a poorer histological grade of HCC. Only M0 macrophages showed a significant association with the clinical stage of HCC. Meanwhile, xCell showed monocytes and M2 macrophages significantly reduced in tumor tissues, which validated the results.ConclusionTfh cells, monocytes, M0 and M2 macrophages may play an indicator role in carcinogenesis, progression, and clinical outcomes of HCC. Our research can serve as a reference contributing to future immunotherapy strategies of HCC.

The Prognostic Value of Combined Preoperative and Postoperative Inflammation Indicators in Hepatocellular Carcinoma after Partial Hepatectomy

Purpose: The current clinical stage of hepatocellular carcinoma (HCC) is insufficient to predict the prognosis. Previous studies have shown that various preoperative inflammation indicators can predict the prognosis of HCC, but the role of postoperative inflammation indicators remains unclear. The purpose of this study was to explore the prognostic value of postoperative inflammation indicators and establish a clinical prognostic model combining preoperative and postoperative inflammation indicators. Methods: 88 patients with primary HCC from Beijing Tsinghua ChangGung Hospital in the National Hepatobiliary Standard Database were included in the study. Preoperative and postoperative prognostic models were established based on preoperative clinicopathological features and combined with postoperative inflammation indicators. The prognostic value of the four models was evaluated by the area under the curve of time-dependent receiver operating characteristic curves (td-AUC). Results: Multivariate analysis of preoperative clinicopathological indicators found that preoperative alpha-fetoprotein (AFP), platelet-lymphocyte ratio (PLR), lymphocyte-monocyte ratio (LMR), Prognostic Nutritional Index (PNI), tumor number and microvascular invasion were independent prognostic factors for disease-free survival (DFS). Introducing the postoperative inflammatory indicators for multivariate analysis found that preoperative AFP, PLR, LMR, PNI and postoperative neutrophil-lymphocyte ratio (NLR) and PLR were independent prognostic factors for DFS and establish a postoperative model. The postoperative model had higher td-AUC values than the preoperative model, TNM stage, Barcelona Clinic Liver Cancer (BCLC) stage in 1-year (0.849 VS 0.826, 0.514 and 0.532), 2-year (0.809 VS 0.799, 0.513 and 0.527), and 3-year (0.784 VS 0.783, 0.509 and 0.529) after surgery. Conclusions: Postoperative inflammation indicators are valuable prognostic indicators, and the predictive performance of the combined model is better than preoperative stage, TNM stage and BCLC stage.

Identifying 13 Hub Genes Associated with Progression and Prognosis of Hepatocellular Carcinoma with Weighted Gene Co-Expression Network Analysis

Hepatocellular carcinoma (HCC) is a type of common cancer, often accompanied by tumor recurrence and metastasis after surgery with poor prognosis. Therefore, searching into potential biomarkers that can effectively predict the prognosis and progression of HCC is crucial. In this study, we identified 1,981 differentially expressed genes (DEGs) using mRNA expression profiles from the TCGA-LIHC dataset. Subsequently, weighted gene co-expression network analysis found that the turquoise module closely associated with the pathological grade and clinical stage of HCC was identified. Then, from the key genes in the turquoise module and protein-protein interaction network analysis, 13 hub genes significantly related to the prognosis of HCC were screened. Through co-expression and functional enrichment analyses, these 13 hub genes were found to play an important role in mitosis. Finally, we evaluated the relationship between these hub genes and overall survival and disease-free survival through survival analysis. The result indicated that HCC patients with high hub gene expression had a poorer prognosis than HCC patients with low expression. Receiver operating characteristic curves showed that each hub gene could predict the prognosis of HCC patients. In summary, a total of 13 hub genes were identified that play an important role in the progression of HCC, which can be used as potential biomarkers for HCC patients.

High expression of WISP1 promotes metastasis and predicts poor prognosis in hepatocellular carcinoma.

The aim of this study was to explore the expression level of Wnt1-inducible signaling pathway protein 1 (WISP1) and its clinical significance in hepatocellular carcinoma (HCC). Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to detect the expression level of WISP1 in HCC tissues and cells. Kaplan-Meier curves and Cox proportional hazard regression model were chosen for single and multiple factor analysis of survival analysis, respectively. Furthermore, wound healing assay and transwell assay were used to verify the effect of WISP1 on HCC cell metastasis in vitro. The expression level of WISP1 in HCC tissues was significantly higher than that in para-cancer tissues (p<0.05). WISP1 expression was positively correlated with lymph node metastasis and clinical stage of HCC. Kaplan-Meier curve showed that HCC patients with higher WISP1 expression exhibited significantly worse progression free survival (PFS) time and overall survival (OS) time. Both univariate and multivariate analysis indicated that high expression of WISP1 was an independent predictor of poor prognosis in HCC. In addition, WISP1 significantly promoted the invasion and migration of HCC cells in vitro. WISP1 might contribute to the development of HCC, serving as a clinical biomarker and therapeutic target for HCC patients.

Construction prognosis model based on autophagy-related gene signatures in hepatocellular carcinoma.

Aim: To develop robust and accurate prognostic biomarkers to help clinicians optimize therapeutic strategies. Materials & methods: Differentially prognosis-related autophagy genes were identified by bioinformatics analysis method. Results: Seven prognosis-related autophagy genes were more significantly related to the prognosis of hepatocellular carcinoma (HCC). Functional enrichment analysis demonstrated that these genes were mainly enriched in the autophagy pathway. BIRC5, HSPB8 and TMEM74 exhibited significant prognostic value for HCC. Besides, the risk score and BIRC5 have significant significance with clinicopathological significance of HCC. Conclusion: The research has identified a number of prognosis-related autophagy genes that associated with the survival and clinical stage of HCC. In addition, the prognostic model can be used to calculate the patient's risk score and these prognosis-related autophagy genes might serve as therapeutic targets.

Does interferon-free direct-acting antiviral therapy for hepatitis C after curative treatment for hepatocellular carcinoma lead to unexpected recurrences of HCC? A multicenter study by the Japanese Red Cross Hospital Liver Study Group.

Background and aimThis study aimed to elucidate whether interferon (IFN)-free direct-acting antiviral (DAA) therapy for hepatitis C after curative treatment of hepatocellular carcinoma (HCC) promotes HCC recurrence in a real-world large-scale cohort.MethodsThis multicenter study was conducted by the Japanese Red Cross Hospital Liver Study Group. This retrospective study analyzed 516 patients who underwent antiviral treatment for hepatitis C with either IFN (n = 148) or IFN-free DAA (n = 368) after curative HCC treatment; 78 IFN-treated patients and 347 IFN-free DAA-treated patients achieved sustained virological response (SVR). The recurrence rate of HCC was compared between the antiviral therapies. Logistic analysis and Cox proportional hazards analysis identified factors associated with early recurrence of HCC within 24 weeks of antiviral therapy and recurrence throughout the observation period, respectively.ResultsAFP at the completion of antiviral therapy, clinical stage of HCC, and non-SVR were independent factors associated with early recurrence of HCC. Among patients who had achieved SVR, the clinical stage of HCC and the level of AFP at completion of antiviral therapy were independent factors associated with early recurrence of HCC. For recurrence throughout the observation period in SVR patients, AFP at completion of antiviral therapy, duration between last HCC treatment to antiviral therapy, and the number of treatments were independent factors. There was no significant difference in the rate of early recurrence of HCC or recurrence throughout the observation period between IFN and IFN-free DAA treated patients.ConclusionsThere were no differences in the early recurrence rate of HCC between patients who underwent IFN and those who underwent IFN-free DAA as antiviral therapies.

Overexpression of RHEB is associated with metastasis and poor prognosis in hepatocellular carcinoma.

Aberrant expression of Ras homolog enriched in brain (RHEB) has been observed in a variety of cancer tissues and is closely associated with clinicopathological features. However, the expression profile of RHEB in patients with hepatocellular carcinoma (HCC) and its clinical signature with underlying mechanisms have not been explored thus far. To analyze the association between RHEB expression and clinicopathological features, the RHEB expression levels were determined in the present study using gene microarrays, immunohistochemistry and western blotting in 60 liver cancer tissues and 35 normal liver tissues. Downregulation of RHEB expression in liver cancer cell lines was achieved by RNA interfering technology to explore its biological function in HCC. RHEB expression was high in liver cancer tissues, with an increase of 2.00±0.19-fold compared with normal tissues and of 2.00±0.27-fold compared with adjacent non-cancer tissues. RHEB expression increased along with the clinical staging of HCC, and the overall survival and mortality of patients were closely correlated to RHEB levels, micro-vascular invasion, hepatitis B virus-DNA titer, tumor differentiation and pathological satellites (P<0.05). After knocking down RHEB in SMMC-7721 cells, the growth of liver cancer cells was significantly reduced. The majority of cells were blocked in S-phase, and their colony-forming and proliferating abilities significantly decreased (P<0.05). In vivo, upon downregulation of RHEB expression, the tumorigenic ability of HCC significantly decreased (P<0.05). These data suggest that RHEB expression is a significant prognostic factor and may be important in HCC cell growth. The present study highlights the importance of RHEB as a novel prognostic marker of HCC.

Effects of ADAMTS14 genetic polymorphism and cigarette smoking on the clinicopathologic development of hepatocellular carcinoma.

BackgroundADAMTS14 is a member of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), which are proteolytic enzymes with a variety of further ancillary domain in the C-terminal region for substrate specificity and enzyme localization via extracellular matrix association. However, whether ADAMTS14 genetic variants play a role in hepatocellular carcinoma (HCC) susceptibility remains unknown.Methodology/Principal findingsFour non-synonymous single-nucleotide polymorphisms (nsSNPs) of the ADAMTS14 gene were examined from 680 controls and 340 patients with HCC. Among 141 HCC patients with smoking behaviour, we found significant associations of the rs12774070 (CC+AA vs CC) and rs61573157 (CT+TT vs CC) variants with a clinical stage of HCC (OR: 2.500 and 2.767; 95% CI: 1.148–5.446 and 1.096–6.483; P = 0.019 and 0.026, respectively) and tumour size (OR: 2.387 and 2.659; 95% CI: 1.098–5.188 and 1.055–6.704; P = 0.026 and 0.034, respectively), but not with lymph node metastasis or other clinical statuses. Moreover, an additional integrated in silico analysis proposed that rs12774070 and rs61573157 affected essential post-translation O-glycosylation site within the 3rd thrombospondin type 1 repeat and a novel proline-rich region embedded within the C-terminal extension, respectively.ConclusionsTaken together, our results suggest an involvement of ADAMTS14 SNP rs12774070 and rs61573157 in the liver tumorigenesis and implicate the ADAMTS14 gene polymorphism as a predict factor during the progression of HCC.

The expression and the diagnostic value of NOD2 in hepatocellular carcinoma

Objective To investigate the expression of nucleotide-binding oligomerization domain protein 2 (NOD2) in serum of patients with hepatocellular carcinoma (HCC), and to analyse the roles of NOD2 in HCC development and its clinical diagnostic value. Methods This study including 66 patients with HCC in the hospital from March 1, 2013 to December 31, 2014 and 61 healthy controls. Serum NOD2 levels were determined by enzyme-linked immunosorbent assay (ELISA). Analysis of significance was performed with rank sum test using SPSS statistical 16.0 software. Results Serum levels of NOD2 in HCC patients were 171 pg/ml, significantly higher than that of healthy controls(95 pg/ml, Z=-5.00, P=0.00), and the serum NOD2 levels were correlated with clinical stage of HCC (H=56.26, P=0.00). Compared with the serum NOD2 levels in stage Ⅰ, Ⅱ patients (106 pg/ml) and healthy controls (95 pg/ml), the serum NOD2 level in stage Ⅲ and Ⅳ (220 pg/ml) were significantly increased (χ2=31.24, P=0.00; χ2=47.23, P=0.00), but the expression of NOD2 in stage Ⅰ and Ⅱ were nearly equal to that of the healthy controls (χ2=0.36, P=0.83). The ROC analysis revealed that the best diagnostic cutoff-point of serum NOD2 levels for predicting the Ⅲ and Ⅳ stages of HCC was 148.78 pg/ml, meanwhile corresponding sensitivity was 89.1% and specificity was 77.0%. Additionally, correlation analysis demonstrated that there was no significant correlation between NOD2 and alpha-fetal protein (r=0.44, P=0.14). Survival curves obtained that the survival time of HCC patients with NOD2 serum concentrations ≥ 200 pg/ml was significantly less than that < 200 pg/ml (χ2=15.32, P<0.05). Conclusion NOD2 is highly expressed in the serum of HCC patients, especially in advanced patients, which is possibly involved in the development of HCC and has the potential to become an effective marker used for HCC diagnosis. Key words: Liver neoplasms; Diagnosis; Alpha fetoproteins; Nucleotide-binding oligomerization domain protein 2

Discovery and validation of urinary metabotypes for the diagnosis of hepatocellular carcinoma in West Africans.

There is no clinically applicable biomarker for surveillance of hepatocellular carcinoma (HCC), because the sensitivity of serum alpha-fetoprotein (AFP) is too low for this purpose. Here, we determined the diagnostic performance of a panel of urinary metabolites of HCC patients from West Africa. Urine samples were collected from Nigerian and Gambian patients recruited on the case-control platform of the Prevention of Liver Fibrosis and Cancer in Africa (PROLIFICA) program. Urinary proton nuclear magnetic resonance ((1) H-NMR) spectroscopy was used to metabolically phenotype 290 subjects: 63 with HCC; 32 with cirrhosis (Cir); 107 with noncirrhotic liver disease (DC); and 88 normal control (NC) healthy volunteers. Urine samples from a further cohort of 463 subjects (141 HCC, 56 Cir, 178 DC, and 88 NC) were analyzed, the results of which validated the initial cohort. The urinary metabotype of patients with HCC was distinct from those with Cir, DC, and NC with areas under the receiver operating characteristic (AUROC) curves of 0.86 (0.78-0.94), 0.93 (0.89-0.97), and 0.89 (0.80-0.98) in the training set and 0.81 (0.73-0.89), 0.96 (0.94-0.99), and 0.90 (0.85-0.96), respectively, in the validation cohort. A urinary metabolite panel, comprising inosine, indole-3-acetate, galactose, and an N-acetylated amino acid (NAA), showed a high sensitivity (86.9% [75.8-94.2]) and specificity (90.3% [74.2-98.0]) in the discrimination of HCC from cirrhosis, a finding that was corroborated in a validation cohort (AUROC: urinary panel = 0.72; AFP = 0.58). Metabolites that were significantly increased in urine of HCC patients, and which correlated with clinical stage of HCC, were NAA, dimethylglycine, 1-methylnicotinamide, methionine, acetylcarnitine, 2-oxoglutarate, choline, and creatine. The urinary metabotyping of this West African cohort identified and validated a metabolite panel that diagnostically outperforms serum AFP.

Expression of LGR8 and Related Biomarkers in Hepatocellular Carcinoma: Correlation with Clinicopathological Parameters

This study aimed to evaluate the relationship between expression of LGR8, VEGF, MMP-2, MMP-9, fascin-1 and cortactin with clinicopathological parameters in hepatocellular carcinoma (HCC). The six biomarkers were investigated immunohistochemically using tissue microarrays of 93 HCC specimens. The tumor cells showed significant expression of LGR8, VEGF, MMP-9, fascin-1 and cortactin, but not of MMP-2. In addition, higher immunostaining scores for LGR8 in HCC showed negative correlation with T and AJCC clinical stages and upregulation of MMP-9, but no correlation with poorer survival rate; cortactin expression is correlated with poorer tumor differentiation in HCC. Thus, our data suggest that higher expression of LGR8 may facilitate tumor invasiveness in the early clinical stage of hepatocellular carcinoma, and synergic effects of cortactin also play a crucial role in the intrahepatic metastasis. Although tumor biological evidence implicates the relaxin-like hormone family as endocrine mediators of critical cellular action in cancer, characterization of target molecules and signaling pathways specific for LGR8 in defined tumor entities and crosstalk of the relaxin receptors with other receptor systems relevant to carcinogenesis will be of significant clinical relevance and may contribute to novel therapeutic strategies against hepatocellular carcinoma.

Relationship between clinical staging of hepatocellular carcinoma and serum transforming growth factor β1

Relationship between clinical staging of hepatocellular carcinoma and serum transforming growth factor β1