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Objective: This study aims to explore ADH4 expression in hepatocellular carcinoma (HCC), its prognostic impact, and its immune correlation to provide novel insights into HCC prognostication and treatment., Methods: HCC prognostic marker genes were rigorously selected using GEO database, Lasso regression, GEPIA, Kaplan-Meier and pROC analyses. The expression of interested markers (ADH4, DNASE1L3, RDH16, LCAT, HGFAC) in HCC and adjacent tissues was assessed by Immunohistochemistry (IHC). We observed that ADH4 exhibited low expression levels in liver cancer tissues and high expression levels in normal liver tissues. However, the remaining four genes did not manifest any statistically significant differences between hepatocellular carcinoma (HCC) tissue and adjacent non-cancerous tissue. Consequently, ADH4 became the primary focus of our research. ADH4 expression was validated by signed-rank tests and unpaired Wilcoxon rank sum tests across pan-cancer and HCC datasets. Clinical significance and associations with clinicopathological variables were determined using Kaplan-Meier, logistic regression and Cox analyses on TCGA data. The ADH4-related immune responses were explored by Spearman correlation analysis using TIMER2 data. CD68, CD4, and CD19 protein levels were confirmed by IHC in HCC and non-cancerous tissues., Results: ADH4 showed significant downregulation in various cancers, particularly in HCC. Moreover, low ADH4 expression was associated with clinicopathological variables and served as an independent prognostic marker for HCC patients. Additionally, ADH4 affects a variety of biochemical functions and may influence cancer development, prognosis, and treatment by binding to immune cells. Furthermore, at the immune level, the low expression pattern of ADH4 is TME-specific, indicating that ADH4 has the potential to be used as a target for cancer immunotherapy., Conclusion: This study highlights the diagnostic, prognostic and immunomodulatory roles of ADH4 in HCC. ADH4 could serve as a valuable biomarker for HCC diagnosis and prognosis, as well as a potential target for immunotherapeutic interventions., (© 2024. The Author(s).)

Chronic hepatitis C virus (HCV) infection is an important public health issue. However, knowledge on how the virus remodels the metabolic and immune response toward hepatic pathologic environment is limited. The transcriptomic and multiple evidences reveal that the HCV core protein-intestine-specific homeobox (ISX) axis promotes a spectrum of metabolic, fibrogenic, and immune modulators (e.g., kynurenine, PD-L1, and B7-2), regulating HCV-infection relevant pathogenic phenotype in vitro and in vivo. In a transgenic mice model, the HCV core protein-ISX axis enhance metabolic disturbance (particularly lipid and glucose metabolism) and immune suppression, and finally, chronic liver fibrosis in a high-fat diet (HFD)-induced disease model. Mechanistically, cells with HCV JFH-1 replicons upregulate ISX and, consequently, the expressions of metabolic, fibrosis progenitor, and immune modulators via core protein-induced nuclear factor-κB signaling. Conversely, cells with specific ISX shRNAi inhibit HCV core protein-induced metabolic disturbance and immune suppression. Clinically, the HCV core level is significantly correlated with ISX, IDOs, PD-L1, and B7-2 levels in HCC patients with HCV infection. Therefore, it highlights the significance of HCV core protein-ISX axis as an important mechanism in the development of HCV-induced chronic liver disease and can be a specific therapeutic target clinically., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Background and Aims: The study objective was to compare the effectiveness of microwave ablation (MWA) and laparoscopic liver resection (LLR) on solitary 3-5-cm HCC over time., Approach and Results: From 2008 to 2019, 1289 patients from 12 hospitals were enrolled in this retrospective study. Diagnosis of all lesions were based on histopathology. Propensity score matching was used to balance all baseline variables between the two groups in 2008-2019 (n = 335 in each group) and 2014-2019 (n = 257 in each group) cohorts, respectively. For cohort 2008-2019, during a median follow-up of 35.8 months, there were no differences in overall survival (OS) between MWA and LLR (HR: 0.88, 95% CI 0.65-1.19, p = 0.420), and MWA was inferior to LLR regarding disease-free survival (DFS) (HR 1.36, 95% CI 1.05-1.75, p = 0.017). For cohort 2014-2019, there was comparable OS (HR 0.85, 95% CI 0.56-1.30, p = 0.460) and approached statistical significance for DFS (HR 1.33, 95% CI 0.98-1.82, p = 0.071) between MWA and LLR. Subgroup analyses showed comparable OS in 3.1-4.0-cm HCCs (HR 0.88, 95% CI 0.53-1.47, p = 0.630) and 4.1-5.0-cm HCCs (HR 0.77, 95% CI 0.37-1.60, p = 0.483) between two modalities. For both cohorts, MWA shared comparable major complications (both p > 0.05), shorter hospitalization, and lower cost to LLR (all p < 0.001)., Conclusions: MWA might be a first-line alternative to LLR for solitary 3-5-cm HCC in selected patients with technical advances, especially for patients unsuitable for LLR., (© 2022 American Association for the Study of Liver Diseases.)

Nuclear translocation regulated by phosphorylation is a key step in providing activated mitogen-activated protein kinases (MAPKs) access to their nuclear targets; however, the mechanisms linking MAPK-induced nuclear translocation and target gene expression mediating oncologic activity remain obscure. Here, we show that the MAPK extracellular signal-regulated kinase (ERK) 1, but not ERK2, phosphorylated intestine-specific homeobox (ISX), leading to its nuclear translocation and downstream oncogenic signaling. Mechanistically, ERK1 phosphorylated serine 183 of ISX, facilitating its nuclear translocation and downstream target gene expression. In contrast, dominant-negative ERK1 expression in hepatoma cells inhibited the nuclear translocation of ISX and the expression of downstream genes involved in cell proliferation, malignant transformation, and epithelial-mesenchymal transition in vitro and in vivo. An activating mutation in ISX (S183D) exhibited a constitutive nuclear localization and resistance to sorafenib. Additionally, in 576 paired clinical hepatocellular carcinoma (HCC) samples and adjacent normal tissues, ERK1 and ISX were co-expressed in a tumor-specific manner at mRNA and protein levels, while their mRNA levels showed significant correlation with survival duration, tumor size, number, and stage. These results highlight the significance of ERK1/ISX signaling in HCC progression and its potential as a prognostic and therapeutic target in HCC., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Epigenetic regulation is important for cancer tumor metastasis and progression, including lung and liver cancer. However, the mechanism of epigenetic regulation in liver cancer leaves much to be discussed. According to a previous study, p300/CBP-associated factor (PCAF) mediated epithelial-mesenchymal transition (EMT) and promotes cancer metastasis by recruiting intestine-specific homeobox (ISX) and bromodomain-containing protein 4 (BRD4) in lung cancer. To figure out whether the three genes are also expressed in patients with hepatocellular carcinoma (HCC) or not, and their correlation with patients' outcome, BRD4, PCAF, and ISX messenger RNA (mRNA) expression levels in 377 patients with HCC were investigated using quantitative polymerase chain reaction and confocal fluorescence imaging. The correlation of the gene expression (PCAF, ISX, and BRD4) in liver cancer is also being investigated. Here, we show that the mRNA expression of PCAF, BRD4, and ISX in 377 paired specimens from patients with HCC, and the adjacent normal tissues exhibited a tumor-specific expression pattern, highly correlated with disease pathogenesis, patient survival time, progression stage, and poor prognosis. The results show that ISX and BRD4 can potentially be a target for improving the survival rate., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Purpose: To build a novel predictive model for hepatocellular carcinoma (HCC) patients based on DNA methylation data., Methods: Four independent DNA methylation datasets for HCC were used to screen for common differentially methylated genes (CDMGs). Gene Ontology (GO) enrichment, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to explore the biological roles of CDMGs in HCC. Univariate Cox analysis and least absolute shrinkage and selection operator (LASSO) Cox analysis were performed to identify survival-related CDMGs (SR-CDMGs) and to build a predictive model. The importance of this model was assessed using Cox regression analysis, propensity score-matched (PSM) analysis and stratification analysis. A validation group from the Cancer Genome Atlas (TCGA) was constructed to further validate the model., Results: Four SR-CDMGs were identified and used to build the predictive model. The risk score of this model was calculated as follows: risk score = (0.01489826 × methylation level of WDR69) + (0.15868618 × methylation level of HOXB4) + (0.16674959 × methylation level of CDKL2) + (0.16689301 × methylation level of HOXA10). Kaplan-Meier analysis demonstrated that patients in the low-risk group had a significantly longer overall survival (OS; log-rank P-value =0.00071). The Cox model multivariate analysis and PSM analysis identified the risk score as an independent prognostic factor (P<0.05). Stratified analysis results further confirmed this model performed well. By analyzing the validation group, the results of receiver operating characteristic (ROC) curve analysis and survival analysis further validated this model., Conclusion: Our DNA methylation-based prognosis predictive model is effective and reliable in predicting prognosis for patients with HCC., (© 2021 The Author(s).)

The aryl hydrocarbon receptor (AHR), a major chemical sensor, is thought to play a role in various biological contexts, including cell cycle regulation and tumorigenesis. However, its regulatory mechanisms remain unclear. We propose herein a novel mechanism through which AHR promotes tumorigenesis by targeting expression of the oncogene intestine-specific homeobox (ISX) in hepatocellular carcinoma (HCC). Compared to paired tumor-adjacent tissues and non-HCC tumors, HCCs exhibited an increased and hierarchical pattern of AHR expression. Patients exhibiting high AHR expression had a significantly shorter survival duration, compared to those with low and medium expression. Functionally, AHR was found to target the newly discovered proto-oncogene, ISX, resulting in the increased expression of this gene and its downstream targets, CCND1 and E2F1. Ablation of AHR or ISX in hepatoma cells suppressed cell growth, whereas overexpression promoted cell proliferation and led to enhanced tumorigenic activity in vitro and in vivo. These results provide evidence to support a critical role for the AHR/ISX axis in HCC tumorigenesis and suggest its potential utility as a new therapeutic and prognostic target for HCC., (© 2017 Wiley Periodicals, Inc.)

The epithelial-mesenchymal transition (EMT) is an important process in the progression of cancer. However, its occurrence and mechanism of regulation are not fully understood. We propose a regulatory pathway in which spermatogenic leucine zipper 1 (SPZ1) promotes EMT through its transactivating ability in increasing TWIST1 expression. We compared the expression of SPZ1 and TWIST1 in specimens of hepatocarcinoma cells (HCCs) and non-HCCs. Expression of SPZ1 exhibited a tumor-specific expression pattern and a high correlation with patients' survival time, tumor size, tumor number and progression stage. Moreover, forced expression and knockdown of SPZ1 in hepatoma cells showed that SPZ1 was able to regulate the cellular proliferation, invasion, and tumorigenic activity in a TWIST1-dependent manner in vitro and in vivo. These data demonstrate that SPZ1, a newly dscribed molecule, transactivates TWIST1 promoters, and that this SPZ1-TWIST axis mediates EMT signaling and exerts significant regulatory effects on tumor oncogenesis.

The intestine-specific homeobox transcription factor intestine-specific homeobox (ISX) is an IL6-inducible proto-oncogene implicated in the development of hepatocellular carcinoma, but its mechanistic contributions to this process are undefined. In this study, we provide evidence that ISX mediates a positive feedback loop integrating inflammation, tryptophan catabolism, and immune suppression. We found that ISX-mediated IL6-induced expression of the tryptophan catabolic enzymes Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase in hepatocellular carcinoma cells, resulting in an ISX-dependent increase in the tryptophan catabolite kynurenine and its receptor aryl hydrocarbon receptor (AHR). Activation of this kynurenine/AHR signaling axis acted through a positive feedback mechanism to increase ISX expression and enhance cellular proliferation and tumorigenic potential. RNAi-mediated attenuation of ISX or AHR reversed these effects. In an IDO1-dependent manner, ectopic expression of ISX induced expression of genes encoding the critical immune modulators CD86 (B7-2) and programmed death ligand-1 (PD-L1), through which ISX conferred a significant suppressive effect on the CD8 + T-cell response. In hepatocellular carcinoma specimens, expression of IDO1, kynurenine, AHR, and PD-L1 correlated negatively with survival. Overall, our results identified a feed-forward mechanism of immune suppression in hepatocellular carcinoma organized by ISX, which involves kynurenine-AHR signaling and PD-L1, offering insights into immune escape by hepatocellular carcinoma, which may improve its therapeutic management. Cancer Res; 77(15); 4065-77. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Histone deacetylase 8 (HDAC8), a unique member of class I histone deacetylases, shows remarkable correlation with advanced disease stage and multiple malignant tumors However, little is known about the contribution of HDAC8 to the tumorigenesis of hepatocellular carcinoma (HCC). The present study investigated the expression of HDAC8 regulated by the aryl hydrocarbon receptor (AHR) in HCC cell lines and tissues, and the roles of HDAC8 overexpression in cell proliferation, including potentially underlying mechanisms. We assessed the correlation between the clinic-pathological parameters and the expression of AHR and HDAC8. Further, we analyzed the AHR siRNA transfection and HDAC8 inhibitors to explore the functions of HDAC8 in HCC progression in vitro and in vivo. In a panel of 289 HCC patients, HDAC8 was shown to be highly correlated with AHR expression at both mRNA and protein levels. HCC patients with high AHR expression showed a shorter survival time than that with low AHR expression. We then found that the expression of both AHR and HDAC8 was significantly upregulated in both HCC cell lines and tumor tissues compared to human normal hepatocytes and matched non-tumor tissues. Furthermore, HDAC8 inhibition remarkably inhibited hepatoma cell proliferation and transformation activity via upregulation of RB1 in vitro and in vivo. Our data revealed an important role of the AHR-HDAC8 axis in promoting HCC tumorigenesis, thus identifying HDAC8 as a potential therapeutic target for HCC treatment.

Aim: To determine whether diabetes mellitus (DM) affects prognosis/recurrence after liver transplantation (LT) for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC)., Methods: A retrospective study was conducted between January 2000 and August 2013 on 1631 patients with HBV-related HCC who underwent LT with antiviral prophylaxis. Patient data were obtained from the China Liver Transplant Registry (https://www.cltr.org/). To compare the outcomes and tumor recurrence in the HBV-related HCC patients with or without DM, statistical analyses were conducted using χ 2 tests, Mann-Whitney tests, the Kaplan-Meier method, log-rank tests and multivariate step-wise Cox regression analysis., Results: Univariate analysis of 1631 patients who underwent LT found overall 1-, 3- and 5-year survival rates of 79%, 73% and 71% respectively in the DM patients, and 84%, 78% and 76% in the non-DM patients respectively. Overall survival rate differences after LT between the two groups were significant ( P = 0.041), but recurrence-free survival rates were not ( P = 0.096). By stratified analysis, the overall survival rates in DM patients for age > 50 years ( P = 0.002), the presence of vascular invasion ( P = 0.096), tumors ≤ 3 cm ( P = 0.047), two to three tumor nodules ( P = 0.007), Child-Pugh grade B ( P = 0.018), and pre-LT alanine aminotransferase levels between 40 and 80 IU/L ( P = 0.017) were significantly lower than in non-DM patients. Additionally, serum α-fetoprotein level > 2000 ng/mL ( P = 0.052) was associated with a significant survival difference trend between DM and non-DM patients. Multivariate analysis showed that the presence of DM ( P < 0.001, HR = 1.591; 95%CI: 1.239-2.041) was an independent predictor associated with poor survival after LT., Conclusion: HBV-related HCC patients with DM have decreased long-term overall survival and poor LT outcomes. Prevention strategies for HCC patients with DM are recommended., Competing Interests: Conflict-of-interest statement: The authors declare that they have no conflicts of interest.

Intestine-specific homeobox (ISX), a newly identified proto-oncogene, is involved in cell proliferation and progression of hepatocellular carcinoma (HCC). However, the underlying mechanisms linking gene expression and tumor formation remain unclear. In this study, we found that ISX transcriptionally activated E2F transcription factor 1 (E2F1) and associated oncogenic activity by directly binding to the E2 site of its promoter. Forced expression of ISX increased the expression of and phosphorylated the serine residue at position 332 of E2F1, which may be translocated into the nucleus to form the E2F1-DP-1 complex, suggesting that the promotion of oncogenic activities of the ISX-E2F1 axis plays a critical role in hepatoma cells. Coexpression of ISX and E2F1 significantly promoted p53 and RB-mediated cell proliferation and anti-apoptosis, and repressed apoptosis and autophagy. In contrast, short hairpin RNAi-mediated attenuation of ISX and E2F1 decreased cell proliferation and malignant transformation, respectively, in hepatoma cells in vitro and in vivo. The mRNA expression of E2F1 and ISX in 238 paired specimens from human HCC patients, and the adjacent, normal tissues exhibited a tumor-specific expression pattern which was highly correlated with disease pathogenesis, patient survival time, progression stage, and poor prognosis. Therefore, our results indicate that E2F1 is an important downstream gene of ISX in hepatoma progression., Competing Interests: The authors have no conflicts of interest.

The role of epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) inhibiting migration in hepatocellular carcinoma (HCC) remains unknown. Expression of EFEMP1 in HCC cell lines were quantified by western blotting and real-time PCR. The role of EFEMP1 in HCC cell migration was explored in vitro via siRNA and adding purified EFEMP1 protein. The associated molecule expression was detected by western blotting after downregulation of EFEMP1 and also tested by immunohistochemistry. Eight pairs of HCC non-HCC liver samples and 215 HCC samples were subjected to immunohistochemistry. EFEMP1 was highly expressed in 7,721 and HepG2 HCC cell lines while HuH7 HCC cell line expressed the lowest level of EFEMP1 compared with the others. Downregulating EFEMP1 by siRNA markedly increased the migration ability of HCC cells while adding purified EFEMP1 protein inhibited HCC cell migration. Downregulation of EFEMP1 increased the expression of ERK1/2, MMP2 and MMP9. Furthermore, U0126 (a highly selective and potent inhibitor of pERK1/2) could abrogate the migration ability enhanced by siRNA. Accordingly, MMP2 and MMP9 were inversely expressed with EFEMP1 expression by immunohistochemistry. EFEMP1 downregulated in HCC tissues, and lower EFEMP1 expression was significantly associated with HCC patients with ascites (P=0.050), vascular invasion (P=0.044), poorer differentiation (P=0.002) and higher clinical stage (P=0.003).

Chronic inflammation drives initiation of hepatocellular carcinoma (HCC), but the underlying mechanisms linking inflammation and tumor formation remain obscure. In this study, we compared the expression of interleukin (IL)-6 and cyclin D1 (CCND1) with the IL-6-induced homeobox gene ISX (intestine-specific homeobox) in 119 paired specimens of HCCs and adjacent normal tissues and also in paired specimens from 11 patients with non-HCCs. In pathologic analysis, ISX exhibited a tumor-specific expression pattern and a high correlation to patient survival time, tumor size, tumor number, and progression stage. Enforced expression of ISX accelerated cell proliferation and tumorigenic activity in hepatoma cells through CCND1 induction. In contrast, short hairpin RNA-mediated attenuation of ISX in hepatoma cells decreased cell proliferation and malignant transformation in vitro and in vivo. A high positive correlation existed in human hepatoma tumors between ISX and CCND1 expression. Together, our results highlight ISX as an important regulator in hepatoma progression with significant potential as a prognostic and therapeutic target in HCCs.

To identify the integration sites in the host genome for the hepatitis B virus (HBV)-encoded X protein (HBx) in hepatocellular carcinoma (HCC) biopsies that are positive for hepatitis B surface antigen (HBsAg). HCC biopsies were obtained from six patients that were HBV carriers, as demonstrated by the presence of HBsAg in their serum and sero-negativity for antibody to HBsAg. DNA was extracted from the tissue, fractionated, and circularized. Primers were designed according to the HBx sequence and used to amplify the circularized DNA templates by inverse polymerase chain reaction (IPCR). The amplified DNA fragments were checked by electrophoresis, cloned into the PMD18-T expression vector, and sequenced. Sequence alignment was performed by the Blast algorithms. Seven electrophoresis bands yielded 22 sequencing results, which represented a total of three HBx integration sites in the host genome: 19q12, 2q32.2, 22q12. The 19q12 integration site encompasses the CCNE1 gene, which encodes a G1/S-specific cyclin-E1. HBx-related integration sites exist in HBsAg-positive HCC biopsies. The CCNE1 gene may play a role in the development of HBx-related HCC.

Objective: To observe the effect of SSd on reversing the malignant phenotype of HepG2 cells and to investigate its mechanism in order to prove that SSd is a new choice to prevent and treat HCC., Methods: HepG2 cells were cultured and treated by different concentration (0 mg/L, 2.5 mg/L, 5.0 mg/L, 10.0 mg/L and 20.0 mg/L) of SSd for 24 h, and treated by 10 mg/L of SSd for 0 h, 6 h, 12 h, 24 h, 48 h and 72h respectively. The cell inhibition rates were measured by MTT assay. Then cells were treated by 10 mg/L SSd for 48 hr in experimental group and treated by no SSd as a control, their morphological changes were observed by contrast phase microscope. The concentrations of ALB and AFP in clear supernatant liquid of cells were detected by radio-immunity and chemiluminescence. The cell migration rates were observed by transwell method, the relative expression levels of p27 mRNA were measured by RT-PCR., Results: The inhibitive effect of 10 mg/L SSd was the most significant among different concentrations ( F = 265.06, P less than 0.01). The shape of HepG2 from experimental group turned into small and round, and their volume ratios of nucleus to plasma decreased. ALB in supernatant liquid of HepG2 was higher ( t = 7.83, P less than 0.05, and its AFP was lower ( t = -10.72, P less than 0.01) as compared to control group. Cells migrated were fewer and p27 mRNA expression of HepG2 was higher in experimental group than that in control group (t = 22.00, P less than 0.05)., Conclusion: SSd could reverse the malignant phenotype of HepG2 cells. It was suggested that the up-regulation of p27 mRNA expression play an important role in the differentiation of HepG2 cells treated by SSd.

Objective: To compare the performance of Inverse-PCR, Alu-PCR and Cassette-ligation-mediated PCR (CLM-PCR) in HBV DNA integration sites identification., Methods: One HCC biopsy was obtained from surgically resected sample. The patient was positive for serum hepatitis B surface antigen (HBsAg). The genomic DNA was purified by the standard phenol/chloroform extraction and ethanol precipitation method. Seperated set of primers were designed to amplify the HBV DNA integration region by means of 3 different PCR methods respectively. The PCR products were analyzed by electrophoresis, then cloned to PMD18-T vector for DNA sequencing. The sequence alignment was performed under Blast software., Results: 7 bands and 22 sequencing results was obtained from IPCR and 3 integration sites was identified. Alu-PCR provided 12 bands and 32 sequencing results, and CLM-PCR showed 12 bands and 4 sequencing results. No integration site was identified from the latter two., Conclusion: IPCR compared with another two methods showed a reliable capacity in HBV DNA integration site identification.