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Xenotropic and polytropic retrovirus receptor 1 (XPR1) is the only known transporter associated with Pi efflux in mammals, and its impact on tumor progression is gradually being revealed. However, the role of XPR1 in hepatocellular carcinoma (HCC) is unknown. A bioinformatics screen for the phosphate exporter XPR1 was performed in HCC patients. The expression of XPR1 in clinical specimens was analyzed using quantitative real-time PCR, Western blot analysis, and immunohistochemical assays. Knockdown of the phosphate exporter XPR1 was performed by shRNA transfection to investigate the cellular phenotype and phosphate-related cytotoxicity of the Huh7 and HLF cell lines. In vivo tests were conducted to investigate the tumorigenicity of HCC cells xenografted into immunocompromised mice after silencing XPR1. Compared with that in paracancerous tissue, XPR1 expression in HCC tissues was markedly upregulated. High XPR1 expression significantly correlated with poor patient survival. Silencing of XPR1 leads to decreased proliferation, migration, invasion, and colony formation in HCC cells. Mechanistically, knockdown of XPR1 causes an increase in intracellular phosphate levels; mitochondrial dysfunction characterized by reduced mitochondrial membrane potential and adenosine triphosphate levels; increased reactive oxygen species levels; abnormal mitochondrial morphology; and downregulation of key mitochondrial fusion, fission, and inner membrane genes. This ultimately results in mitochondria-dependent apoptosis. These findings reveal the prognostic value of XPR1 in HCC progression and, more importantly, suggest that XPR1 might be a potential therapeutic target., (© 2024 Wiley Periodicals LLC.)

Introduction: An optimal follow-up schedule for small (≤3-cm) hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA) remains unclear in clinical guidelines. We aimed to assess the cost-effectiveness of follow-up strategies in patients with small HCC after RFA., Methods: In total, 11,243 patients were collected from global institutions to calculate recurrence rates. Subsequently, a Markov model covering a 10-year period was developed to compare 25 surveillance strategies involving different surveillance techniques (computed tomography [CT], magnetic resonance imaging or ultrasonography [US], and α-fetoprotein [AFP]) and intervals (3 or 6 months). The study endpoint was incremental cost-effectiveness ratio (ICER), which represented additional cost per incremental quality-adjusted life year. Sensitivity analysis was conducted by varying the values of input parameters to observe the ICER., Results: In a base case analysis, the dominant strategy was CT every 3 months during an initial 2 years, followed by semiannual CT, and then switch to biannual the combination of US screening and AFP testing after 5 years (m3_CT-m6_CT-m6_USAFP), with an ICER of $68,570.92 compared with the "not followed" strategy. One-way sensitivity analysis showed the ICER consistently remained below the willingness-to-pay threshold of $100,000.00. In a probabilistic sensitivity analysis, m3_CT-m6_CT-m6_USAFP was the most cost-effective approach in 95.6% of simulated scenarios at a willingness-to-pay threshold., Discussion: For small HCC after RFA, the recommended follow-up strategy is CT, with scans scheduled every 3 months for the first 2 years, every 6 months thereafter, and transition to biannual the combination of US screening and AFP testing after 5 years., (Copyright © 2024 by The American College of Gastroenterology.)

Background: Liver cancer stem cells (CSCs) contribute to tumor initiation, progression, and recurrence in hepatocellular carcinoma (HCC). The Wnt/β-catenin pathway plays a crucial role in liver cancer stemness, progression, metastasis, and drug resistance, but no clinically approved drugs have targeted this pathway efficiently so far. We aimed to elucidate the role of COLEC10 in HCC stemness., Methods: The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) databases were employed to search for the association between COLEC10 expression and HCC stemness. Colony formation, sphere formation, side population, and limiting dilution tumor initiation assays were used to identify the regulatory role of COLEC10 overexpression in the stemness of HCC cell lines. Wnt/β-catenin reporter assay and immunoprecipitation were performed to explore the underlying mechanism., Results: COLEC10 level was negatively correlated with HCC stemness. Elevated COLEC10 led to decreased expressions of EpCAM and AFP (alpha-fetoprotein), two common markers of liver CSCs. Overexpression of COLEC10 inhibited HCC cells from forming colonies and spheres, and reduced the side population numbers in vitro, as well as the tumorigenic capacity in vivo. Mechanically, we demonstrated that overexpression of COLEC10 suppressed the activity of Wnt/β-catenin signaling by upregulating Wnt inhibitory factor WIF1 and reducing the level of cytoplasmic β-catenin. COLEC10 overexpression promoted the interaction of β-catenin with the component of destruction complex CK1α. In addition, KLHL22 (Kelch Like Family Member 22), a reported E3 ligase adaptor predicted to interact with CK1α, could facilitate COLEC10 monoubiquitination and degradation., Conclusion: COLEC10 inhibits HCC stemness by downregulating the Wnt/β-catenin pathway, which is a promising target for liver CSC therapy., (© 2024. Springer Nature Switzerland AG.)

Background: The role of circRNAs in hepatocellular carcinoma (HCC) progression remains unclear. CircPIAS1 (circBase ID: hsa_circ_0007088) was identified as overexpressed in HCC cases through bioinformatics analysis. This study aimed to investigate the oncogenic properties and mechanisms of circPIAS1 in HCC development., Methods: Functional analyses were conducted to assess circPIAS1's impact on HCC cell proliferation, migration, and ferroptosis. Xenograft mouse models were employed to evaluate circPIAS1's effects on tumor growth and pulmonary metastasis in vivo. Bioinformatics analysis, RNA immunoprecipitation, and luciferase reporter assays were utilized to elucidate the molecular pathways influenced by circPIAS1. Additional techniques, including RNA pulldown, fluorescence in situ hybridization (FISH), chromatin immunoprecipitation (ChIP), qPCR, and western blotting, were used to further explore the underlying mechanisms., Results: CircPIAS1 expression was elevated in HCC tissues and cells. Silencing circPIAS1 suppressed HCC cell proliferation and migration both in vitro and in vivo. Mechanically, circPIAS1 overexpression inhibited ferroptosis by competitively binding to miR-455-3p, leading to upregulation of Nuclear Protein 1 (NUPR1). Furthermore, NUPR1 promoted FTH1 transcription, enhancing iron storage in HCC cells and conferring resistance to ferroptosis. Treatment with ZZW-115, an NUPR1 inhibitor, reversed the tumor-promoting effects of circPIAS1 and sensitized HCC cells to lenvatinib., Conclusion: This study highlights the critical role of circPIAS1 in HCC progression through modulation of ferroptosis. Targeting the circPIAS1/miR-455-3p/NUPR1/FTH1 regulatory axis may represent a promising therapeutic strategy for HCC., (© 2024. The Author(s).)

Background: We aimed to redefine Immune checkpoint inhibitors (ICIs)-responsive "hot" TME and develop a corresponding stratification model to maximize ICIs-efficacy in Hepatocellular Carcinoma (HCC)., Methods: Hypoxic scores were designed, and the relevance to immunotherapy responses were validated in pan-cancers through single cell analysis. Multi-omics analysis using the hypoxic scores and immune infiltrate abundance was performed to redefine the ICIs-responsive TME subtype in HCC patients from TCGA (n = 363) and HCCDB database (n = 228). The immune hypoxic stress index (IHSI) was constructed to stratify the ICIs-responsive TME subtype, with exploring biological mechanism in vitro and in vivo. MRI-radiomics models were built for clinical applicability., Results: The hypoxic scores were lower in the dominant cell-subclusters of responders in pan-cancers. The higher immune infiltrate-normoxic (HIN) subtype was redefined as the ICIs-responsive TME. Stratification of the HIN subtype using IHSI effectively identified ICIs-responders in Melanoma (n = 122) and urological cancer (n = 22). TRAF3IP3, the constituent gene of IHSI, was implicated in ICIs-relevant "immune-hypoxic" crosstalk by stimulating MAVS/IFN-I pathway under normoxic condition. MRI-radiomics models assessing TRAF3IP3 with HIF1A expression (AUC > 0.80) screened ICIs-Responders in HCC cohort (n = 75)., Conclusion: The hypoxic-immune stratification redefined ICIs-responsive TME and provided MRI-Radiomics models for initial ICIs-responders screening, with IHSI facilitating further identification., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: To identify reliable magnetic resonance imaging (MRI) features that can differentiate confluent fibrosis (CF) from infiltrative hepatocellular carcinoma (HCC)., Methods: A retrospective analysis was conducted on Twenty CF patients and 28 infiltrative HCC patients who underwent upper abdomen MRI scans. The imaging features of lesions were analyzed, and the apparent diffusion coefficient (ADC) of lesions were measured. Accuracy, sensitivity and specificity for the diagnosis of CF were calculated for each category individually and combined., Results: Compared to infiltrative HCC, hepatic capsular retraction at the site of lesion, hepatic volume loss at the site of lesion and "nodular surround sign" were more common in patients with CF (all P < 0.001). Hepatic volume loss at the site of lesion, no or mild enhancement in arterial phase, and hyper-enhancing in delayed phase to the background parenchyma showed superior diagnostic accuracy (83.3%, 85.4%, 97.9%, respectively). When the lesion exhibited hepatic volume loss at the site of lesion or no or mild enhancement in arterial phase or hyper-enhancing in delayed phase, a sensitivity of 100.0% for the diagnosis of CF was achieved. When the lesion was positive for any two of three categories, or positive for all three categories, a specificity of 100.0% was achieved. The ADC values of CF were higher than those of infiltrative HCC (P < 0.001)., Conclusion: The combination of the hepatic volume loss at the site of lesion, no or mild enhancement in arterial phase, and hyper-enhancing in delayed phase to the background parenchyma can be considered reliable MR features for the diagnosis of CF, as they allow differentiation from infiltrative HCC., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: With increasing life expectancy, the number of elderly patients (≥ 65 years) with hepatocellular carcinoma (HCC) has steadily increased. Hepatectomy remains the first-line treatment for HCC patients. However, the prognosis of hepatectomy for elderly patients with HCC remains unclear., Methods: Clinical and follow-up data from 1331 HCC patients who underwent surgery between 2008 and 2020 were retrospectively retrieved from a multicentre database. Patients were divided into elderly (≥ 65 years) and non-elderly (< 65 years) groups, and PSM was used to balance differences in the baseline characteristics. The postoperative major morbidity and cancer-specific survival (CSS) of the two groups were compared and the independent factors that were associated with the two study endpoints were identified by multivariable regression analysis., Results: Of the 1331 HCC patients enrolled in this study, 363 (27.27%) were elderly, while 968 (72.73%) were not. After PSM, 334 matched samples were obtained. In the propensity score matching (PSM) cohort, a higher rate of major morbidity was found in elderly patients (P = 0.040) but the CSS was similar in the two groups (P = 0.087). Multivariate analysis revealed that elderly age was not an independent risk factor associated with high rates of major morbidity (P = 0.117) or poor CSS (P = 0.873). The 1-, 3- and 5-year CSS rates in the elderly and non-elderly groups were 91.0% versus 86.2%, 71.3% versus 68.8% and 55.9% versus 58.0%, respectively. Preoperative alpha fetoprotein (AFP) level, Child‒Pugh grade, intraoperative blood transfusion, extended hemi hepatectomy, and tumour diameter could affect the postoperative major morbidity and preoperative AFP level, cirrhosis, Child‒Pugh grade, macrovascular invasion, microvascular invasion (MVI), satellite nodules, and tumor diameter were independently and significantly associated with CSS., Conclusion: Age itself had no significant effect on the prognosis of elderly patients with HCC after hepatectomy. Hepatectomy can be safely performed in elderly patients after cautious perioperative management., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Collectin subfamily member 10 (COLEC10), a C-type lectin mainly expressed in the liver, is involved in the development of hepatocellular carcinoma (HCC). However, its underlying molecular mechanism in HCC progression remains unknown. In this study, reduced COLEC10 expression in tumor tissues was validated using various HCC cohorts and was associated with poor patient prognosis. COLEC10 overexpression attenuated HCC cell growth and migration abilities in vitro and in vivo. We identified that COLEC10 was a novel interactor of 78-kDa glucose-regulated protein (GRP78), a master modulator of the unfolded protein response in the endoplasmic reticulum (ER). COLEC10 overexpression potentiated ER stress in HCC cells, as demonstrated by elevated expression levels of phosphorylated protein kinase RNA-like ER kinase, phosphorylated inositol-requiring protein 1α, activating transcription factor 4, DNA damage-inducible transcript 3, and X-box-binding protein 1s. The ER in COLEC10-overexpressing cells also showed a dilated and fragmented pattern. Mechanistically, COLEC10 overexpression increases GRP78 occupancy through direct binding by the C-terminal carbohydrate recognition domain in the ER, which released and activated the ER stress transducers protein kinase RNA-like ER kinase and phosphorylated inositol-requiring protein 1α, triggering the unfolded protein response activity. COLEC10-overexpressing HCC cells generated a relatively high reactive oxygen species level and switched to apoptotic cell death under sorafenib-treated conditions. Our study provides the first novel view that COLEC10 inhibits HCC progression by regulating GRP78-mediated ER stress signaling and may serve as a promising therapeutic and prognostic biomarker., (Copyright © 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Metastasis is a landmark event for rapid postsurgical relapse and death of HCC patients. Although distinct genomic and transcriptomic profiling of HCC metastasis had been reported previously, the causal relationships of somatic mutants, mRNA levels and metastatic potentials were difficult to be established in clinic. Therefore, 11 human HCC cell lines and 7 monoclonal derivatives with definite metastatic potentials and tropisms were subjected to whole exome sequencing (WES) and whole transcriptome sequencing (WTS). TP53, MYO5A, ROS1 and ARID2 were the prominent mutants of metastatic drivers in HCC cells. During HCC clonal evaluation, TP53, MYO5A and ROS1 mutations occurred in the early stage, EXT2 and NIN in the late stage. NF1 mutant was unique in lung tropistic cell lines, RNF126 mutant in lymphatic tropistic ones. PER1, LMO2, GAS7, NR4A3 expression levels were positively associated with relapse-free survival (RFS) of HCC patients. The integrative analysis revealed 58 genes exhibited both somatic mutation and dysregulated mRNA levels in high metastatic cells. Altogether, metastatic drivers could accumulate gradually at different stages during HCC progression, some drivers might modulate HCC metastatic potentials and the others regulate metastatic tropisms., Competing Interests: Declaration of Competing Interest The authors report no conflicts of interest in this work., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: Ubiquitin conjugate enzyme E2O (UBE2O) is a ubiquitin-conjugating enzyme that has been reported to be involved in tumorigenesis. This study investigated the role of UBE2O in hepatocellular carcinoma (HCC)., Methods: The expression of UBE2O was detected using qRT-PCR, Western blotting, and immunohistochemical staining. Cell proliferation and Transwell assays were used to detect proliferation, migration, and invasion of HCC cells, respectively. Bioinformatic analysis was performed to analyze the relationship between UBE2O and the clinical features, prognosis, and immune cell infiltration of HCC., Results: UBE2O was significantly over-expressed in HCC tissues. High expression of UBE2O was associated with poor tumor grade and poor prognosis. Functional experiments showed that down-regulation of UBE2O inhibited HCC cell proliferation, migration, and invasion. Co-expression gene analysis and gene set enrichment analysis showed that UBE2O was associated with protein hydrolysis, cell cycle, and cancer-related pathways in HCC. The results of immune analysis revealed that the expression of UBE2O was positively correlated with the immune infiltration and expression of immune-related chemokines of HCC., Conclusions: UBE2O is significantly correlated with the prognosis of HCC and may be a valuable prognostic biomarker for HCC., (© 2022. Huazhong University of Science and Technology.)

For patients with inoperable huge hepatocellular carcinoma (H-HCC, tumor size ≥10 cm), treatment options are limited. This study aimed to evaluate the characteristics and outcomes of patients with H-HCC who use Chinese herbal medicine (CHM). Multi-institutional cohort data were obtained from the Chang Gung Research Database (CGRD) between 1 January 2002 and 31 December 2018. All patients were followed up for 3 years or until the occurrence of death. Characteristics of CHM users and risk of all-cause mortality were assessed, and core CHMs with potential pharmacologic pathways were explored. Among 1618 patients, clinical features of CHM users (88) and nonusers (1530) were similar except for lower serum α-fetoprotein (AFP) and higher serum albumin levels in CHM users. CHM users had significantly higher 3 year overall survival rates (15.0% vs. 9.7%) and 3 year liver-specific survival rates (13.4% vs. 10.7%), about 3 months longer median survival time, and lower risk of all-cause mortality. Core CHMs were discovered from the prescriptions, including Hedyotis diffusa Willd combined with Scutellaria barbata D.Don, Salvia miltiorrhiza Bunge., Curcuma longa L., Rheum palmatum L., and Astragalus mongholicus Bunge. CHM use appears safe and is possibly beneficial for inoperable H-HCC patients; however, further clinical trials are still required.

Immune checkpoint blockade (ICB) therapy is an important treatment option for individuals with cancer, but it has certain limitations. Identifying a better target that can overcome tumor immune escape and stimulate T cell activity is critical. This research aimed to delve into the molecular mechanism underlying the immunoregulatory function of metadherin (MTDH), which is a novel and potential therapeutic target in hepatocellular cancer (HCC). A small interfering RNA library was screened using the luciferase reporter assay and PD-L1 promoter. The Cancer Genome Atlas database and HCC tissues were used to investigate the relationship between MTDH and PD-L1. The association between MTDH and β-catenin/lymphoid enhancer binding factor (LEF-1) was discovered by co-immunoprecipitation. The chromatin immunoprecipitation assay was used to investigate the interaction of MTDH with the PD-L1 promoter when LEF-1 expression was silenced. Locked nucleic acid antisense oligonucleotides (ASOs) were used to inhibit MTDH. We utilized in vitro co-cultures and in vivo syngeneic tumor development experiments to confirm the effectiveness of MTDH ASO combined with PD-1 monoclonal antibody (mAb). MTDH was demonstrated to be a PD-L1 modulator. MTDH increased PD-L1 expression and upregulated PD-L1 transcriptional activity through β-catenin/LEF-1 signaling. More importantly, MTDH ASO improved the anti-PD-1 response and increased cytotoxic T-cell infiltration in PD-1 mAb-treated malignancies. MTDH effectively predicts the therapeutic efficacy of ICB therapy. Our results imply that combining MTDH ASO with PD-1 mAb could be a promising therapeutic strategy for HCC. In addition, MTDH is a potential novel biomarker for predicting the effectiveness of immune checkpoint inhibitor treatment., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background & Aims: Cancer stemness and immune evasion are closely associated and play critical roles in tumor development and resistance to immunotherapy. However, little is known about the underlying molecular mechanisms that coordinate this association., Methods: The expressions of heterogeneous nuclear ribonucleoprotein M (HNRNPM) in 240 hepatocellular carcinoma (HCC) samples, public databases, and liver development databases were analyzed. Chromatin immunoprecipitation assays were performed to explore the associations between stem-cell transcription factors and HNRNPM. HNRNPM-regulated alternative splicing (AS) and its binding motif were identified by RNA-seq and RIP-seq. HNRNPM-specific antisense oligonucleotides were developed to explore potential therapeutic targets in HCC. CD8+ T cells that were co-cultured with tumor cells were sorted by flow cytometry assays., Results: We identified an elevated oncofetal splicing factor in HCC, HNRNPM, that unifies and regulates the positive association between cancer stemness and immune evasion. HNRNPM knockdown abolished HCC tumorigenesis and diminished cancer stem cell properties in vitro and in vivo. Mechanistically, HNRNPM regulated the AS of MBD2 by binding its flanking introns, whose isoforms played opposing roles. Although MBD2a and MBD2c competitively bound to CpG islands in the FZD3 promoter, MBD2a preferentially increased FZD3 expression and then activated the WNT/β-catenin pathway. Interestingly, FZD3 and β-catenin further provided additional regulation by targeting OCT4 and SOX2. We found that HNRNPM inhibition significantly promoted CD8+ T cell activation and that HNRNPM- antisense oligonucleotides effectively inhibited WNT/β-catenin to enhance anti-programmed cell death protein-1 immunotherapy by promoting CD8+ T cell infiltration., Conclusions: HNRNPM has a tumor-intrinsic function in generating an immunosuppressive HCC environment through an AS-dependent mechanism and demonstrates proof of the concept of targeting HNRNPM in tailoring HCC immunotherapeutic approaches., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Immunotherapy with hepatitis B virus (HBV)-specific TCR redirected T (HBV-TCR-T) cells in HBV-related hepatocellular carcinoma (HBV-HCC) patients after liver transplantation was reported to be safe and had potential therapeutic efficacy. We aim to investigate the safety of HBV-TCR-T-cell immunotherapy in advanced HBV-HCC patients who had not met the criteria for liver transplantation., Methods: We enrolled eight patients with advanced HBV-HCC and adoptively transferred short-lived autologous T cells expressing HBV-specific TCR to perform an open-label, phase 1 dose-escalation study (NCT03899415). The primary endpoint was to evaluate the safety of HBV-TCR-T-cell therapy according to National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.03) during the dose-escalation process. The secondary endpoint was to assess the efficacy of HBV-TCR-T-cell therapy by evaluating the anti-tumor responses using RECIST criteria (version 1.1) and the overall survival., Results: Adverse events were observed in two participants among the 8 patients enrolled. Only one patient experienced a Grade 3 liver-related adverse event after receiving a dose of 1 × 10 5 HBV-TCR-T cells/kg, then normalized without interventions with immunosuppressive agents. Among the patients, one achieved a partial response lasting for 27.7 months. Importantly, most of the patients exhibited a reduction or stabilization of circulating HBsAg and HBV DNA levels after HBV-TCR-T-cell infusion, indicating the on-target effects., Conclusions: The adoptive transfer of HBV-TCR-T cells into advanced HBV-HCC patients were generally safe and well-tolerated. Observations of clinical efficacy support the continued development and eventual application of this treatment strategy in patients with advanced HBV-related HCC., Clinical Trials Registration: This study was registered at ClinicalTrials.gov (NCT03899415)., (© 2021. The Author(s).)

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide due to a high rate of tumour metastasis and disease recurrence. In physiological conditions, tetraspanins interact with specific partner proteins in tetraspanin-enriched microdomains and regulate their subcellular localization and function. However, the function of Tspan5 in pathological processes, particularly in cancer biology and its clinical significance, are still unclear. Here, we describe that a high expression of Tspan5 is significantly associated with some clinicopathological features including invasive length, vascular invasion, clinical stage and poor overall survival of HCC patients. Alterations of Tspan5 expression by lentivirus transductions in HCC cells demonstrated that Tspan5 promotes wound healing and cell migration in vitro and tumour metastasis of HCC cells in vivo. Mechanistic studies revealed that Tspan5 promoted cell migration and tumour metastasis by increasing the enzymatic maturation of ADAM10 and activating Notch signalling via the increase of the cleavage of the Notch1 receptor catalysed by the γ-secretase complex. Activation of Notch signalling by Tspan5 was shown further to enhance the epithelial-mesenchymal transition (EMT) and actin skeleton rearrangement of tumour cells. In clinical HCC samples, Tspan5 expression is strongly correlated with many key molecules acting in Notch signalling and EMT, highlighting the role of Tspan5 in the regulation of Notch signalling, EMT and tumour metastasis of HCC. Our findings provide new insights into the mechanism of tumour metastasis and disease progression of HCC and may facilitate the development of novel clinical intervention strategies against HCC., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Autophagy is upregulated in response to metabolic stress, a hypoxic tumor microenvironment, and therapeutic stress in various cancers and mediates tumor progression and resistance to cancer therapy. Herein, we identified a cinchona alkaloid derivative containing urea ( C1 ), which exhibited potential cytotoxicity and inhibited autophagy in hepatocellular carcinoma (HCC) cells. We showed that C1 not only induced apoptosis but also blocked autophagy in HCC cells, as indicated by the increased expression of LC3-II and p62, inhibition of autophagosome-lysosome fusion, and suppression of the Akt/mTOR/S6k pathway in the HCC cells. Finally, to improve its solubility and efficacy, we encapsulated C1 into PEGylated lipid-poly(lactic- co -glycolic acid) (PLGA) nanoscale drug carriers. Systemic administration of nanoscale C1 significantly suppressed primary tumor growth and prevented distant metastasis while maintaining a desirable safety profile. Our findings demonstrate that C1 combines autophagy modulation and apoptosis induction in a single molecule, making it a promising therapeutic option for HCC.

A persimmon tannin- Aloe vera composite powder (PT-A) was investigated for its capacity to protect against ionizing radiation. Human hepatic cells (L02 cells) and human hepatoma cells (HepG2 cells) were pretreated with different concentrations of PT-A or the single compounds (PT or Aloe vera ) and radiated with X-rays. After radiation and post-incubation for 12 h or 24 h, the cell viability, apoptosis, and reactive oxygen species (ROS) production were analyzed by Cell Counting Kit 8 (CCK-8), 2',7'-dichlorfluorescein diacetate (DCFH-DA) staining, and Hoechst 33258 staining/flow cytometry, respectively. CCK-8 results illustrated that the optimal radiation dose L02 cells was 8 Gy for L02 cells, and the cell activity was 71.72% (IC 50 = 412.1 μg/mL) after post-radiation incubation of 12 h. For HepG2 cells, the optimal radiation dose was 8 Gy, and the cell activity was 62.37% (IC 50 = 213.0 μg/mL). The cell apoptotic rate was the lowest at a PT-A concentration of 200 μg/mL in L02 cells (4.32%, P < 0.05), and at 100 μg/mL in HepG2 cells (9.80%, P < 0.05). ROS production induced by radiation could be effectively inhibited by 200 μg/mL of PT-A in L02 cells, and by 100 μg/mL of PT-A in HepG2 cells. The PT-A composite has good radioprotective effects on cell vitality and apoptosis of X-rays radiation exposure towards L02 cells and HepG2 cells compared to the persimmon tannin or Aloe vera . Therefore, PT-A composite might be useful as a natural, harmless anti-ionizing radiation agent, and has various clinical application prospects in future.

Purpose: The purpose of this study was to investigate the predictive capability of neutrophil-to-apolipoprotein A1 ratio (NAR) for predicting overall survival (OS) among patients with hepatocellular carcinoma (HCC) receiving transarterial chemoembolization (TACE)., Patients and Methods: We investigated the clinical features of 554 patients with HCC receiving TACE and assessed NAR's predictive value for OS with 222 patients (the discovery cohort) and 332 patients (the validation cohort). The association of NAR with circulation lectin-type oxidized low-density lipoprotein receptor-1-positive (LOX-1 + ) polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) was illustrated., Results: Multivariate Cox regression revealed that lymphocyte count; Tumor, Node, Metastasis (TNM) stage; and NAR were independent prognostic factors in the discovery cohort. The validation cohort confirmed the independent prognostic value of TNM stage and NAR. Patients with low NAR (<2.7) displayed significantly increased OS in the discovery cohort (59.8 months vs. 21 months), the validation group (38.0 months vs. 23.6 months), and the total cohort (44.1 months vs. 22.0 months). A Cox proportional hazards model was used to combine Cancer of the Liver Italian Program (CLIP) score with discretized NAR. C-index illustrated that NAR-integrated CLIP score was the best model compared with NAR and CLIP score. Furthermore, NAR-CLIP presented superior predictive capacity for 10-, 20-, 30-, 40-, 50-, and 60-month survival compared with CLIP score by survival receiver-operator characteristic analysis in the discovery cohort, validation cohort, and total cohort. NAR was significantly associated with LOX-1 + PMN-MDSCs by linear regression., Conclusion: This study identified NAR as an independent predictor for OS among patients with HCC receiving TACE. NAR reflected circulation LOX-1 + PMN-MDSC level., Implications for Practice: The present study identified neutrophil-to-apolipoprotein A1 ratio (NAR) as an independent predictor for overall survival among patients with hepatocellular carcinoma receiving transarterial chemoembolization. NAR reflected circulation level of lectin-type oxidized low-density lipoprotein receptor-1-positive polymorphonuclear myeloid-derived suppressor cells., (© 2021 AlphaMed Press.)

Snail family transcriptional repressor 1 (SNAIL1) is a master inducer of the epithelial‑to‑mesenchymal transition (EMT) process, contributing to tumor metastasis and recurrence. Our previous study reported that G2 and S phase‑expressed‑1 (GTSE1) served a role in regulating SNAIL1 expression in hepatocellular carcinoma (HCC). However, the underlying mechanism remains unknown. Therefore, the present study aimed to reveal the regulatory mechanism of GTSE1 on SNAIL1 expression using in vitro assays performed in HCC cell models. It was demonstrated that endogenous SNAIL1 expression was downregulated and upregulated by GTSE1 overexpression or small interfering RNA‑mediated knockdown, respectively. Via cycloheximide chase experiments, it was identified that GTSE1 overexpression increased the protein turnover of SNAIL1, while knockdown of GTSE1 reduced its degradation rate. Furthermore, it was demonstrated that GTSE1 overexpression induced the cytoplasmic expression of SNAIL1 using immunofluorescence and subcellular fractionation methods. The nuclear export inhibitor leptomycin B was able to decrease the cytoplasmic retention of SNAIL1 caused by GTSE1 overexpression. In addition, TGF‑βI treatment increased both the mRNA and protein expression levels of GTSE1, and decreased the protein expression level of SNAIL1 without affecting its mRNA transcription in Huh7 cells. It was also found that TGF‑β signaling could upregulate the transcription of GTSE1 expression by transactivating the Smad binding elements in the GTSE1 promoter. Moreover, the TGF‑βI‑induced decrease in SNAIL1 protein expression was GTSE1‑dependent in Huh7 cells. In conclusion, the current study provides a novel mechanism via which GTSE1 affects the stability of SNAIL1 by regulating its subcellular localization in HCC cells.

Background : Stem cells play an important role in hepatocellular carcinoma (HCC). However, their precise effect on HCC tumorigenesis and progression remains unclear. The present study aimed to characterize stem cell-related gene expression in HCC. Methods : The mRNA expression-based stemness index (mRNAsi) was used to analyze the clinical characteristics and prognosis of HCC patients. The weighted gene co-expression network analysis (WGCNA) was used to construct a gene co-expression network of 374 HCC patients. Finally, six genes were used to construct the prognosis signature. Results : HCC patients had a higher mRNAsi score than healthy people, suggesting poor prognosis. Two gene modules highly related to mRNAsi were identified. Multivariate Cox analysis was carried out to establish a Cox proportional risk regression model. The risk score for each patient was the sum of the product of each gene expression and its coefficient. Survival analysis suggested that the low-risk group had a significantly better prognosis. Conclusions : The established six-gene signature was able to predict patient prognosis accurately. This new signature should be verified in prospective studies in order to determine patient prognosis in clinical decision-making.

CD45 + CD71 + erythroid cells generated through splenic extramedullary erythropoiesis have recently been found to suppress anti-infection and tumor immunity in neonates and adults with malignances. However, their role in tumor microenvironment has not been investigated. In the present study, we found that the number of CD45 + CD71 + erythroid cells was significantly elevated in hepatocellular carcinoma (HCC) tissues compared to that in paratumor region and circulation. Additionally, they were more abundant in HCC tissues compared to some immune suppressive cells as well as CD45 - CD71 + erythroid cells. CD45 + CD71 + erythroid cells suppressed T cells through generation of reactive oxygen species, IL-10, and TGF-β in a paracrine and cell-cell contact manner, and their suppressive effect was stronger than that of myeloid-derived suppressor cells. The abundance of CD45 + CD71 + erythroid cells in tumor tissue, as illustrated via immunofluorescence, predicted disease-free survival and overall survival, and its prognostic value was better than that of Cancer of the Liver Italian Program score. This study demonstrated that accumulation of intratumoral CD45 + CD71 + erythroid cells in HCC tissues could play a superior immunosuppressive role in tumor microenvironment and may serve as a valuable biomarker to predict recurrence of HCC., (Copyright © 2020. Published by Elsevier B.V.)

The high expression of sonic hedgehog ligand (SHh) is closely correlated to the metastasis, drug resistance and poor prognosis of hepatocellular carcinoma (HCC). Therefore, sensitive, specific and efficient detection methods for SHh are needed for the early diagnosis and assessment of prognosis. Herein, an aptamer, AP32 that specifically binds to SHh (K D  = 25.7 ± 4.1 nM) was obtained by SELEX technology with further optimization. In vivo experiments confirmed that AP32 has the potential to be an imaging probe for Huh-7 cell-derived xenograft. The interaction mode in 3-dimensional configuration between the aptamer and SHh was established by molecular simulation and confirmed by mutations at key sites of the aptamer. An aptasensor-based assay was successfully developed by conjugating Texas-Red-labeled AP32 to microbeads, and was used to analyze SHh content in hepatoma cell lysates, serum and HCC specimens. The method exhibited a broad detection range from 0.07 to 62.5 nM with a low detection limit of 69 pM, and a recovery rate of 104.6 ± 3.9% in serum. When the assay was used to measure SHh content in tissue lysates, the results demonstrated that it possessed 57.1% positivity, 100% specificity in distinguishing 28 HCC specimens from normal tissues, and was compensatory for detection of HCC in AFP-negative cases. Moreover, elevated SHh levels are indicative of portal vein invasion at 77.8% positive rate. This novel aptasensor-based SHh assay may offer a reliable means in predicting early metastasis and poor prognosis in hepatocellular carcinoma., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ginkgo biloba extract (GBE) is used in traditional Chinese medicine as a herbal supplement for improving memory. Exposure of B6C3F1/N mice to GBE in a 2-year National Toxicology Program (NTP) bioassay resulted in a dose-dependent increase in hepatocellular carcinomas (HCC). To identify key microRNAs that modulate GBE-induced hepatocarcinogenesis, we compared the global miRNA expression profiles in GBE-exposed HCC (GBE-HCC) and spontaneous HCC (SPNT-HCC) with age-matched vehicle control normal livers (CNTL) from B6C3F1/N mice. The number of differentially altered miRNAs in GBE-HCC and SPNT-HCC was 74 (52 up and 22 down) and 33 (15 up and 18 down), respectively. Among the uniquely differentially altered miRNAs in GBE-HCC, miR-31 and one of its predicted targets, Cdk1 were selected for functional validation. A potential miRNA response element (MRE) in the 3'-untranslated regions (3'-UTR) of Cdk1 mRNA was revealed by in silico analysis and confirmed by luciferase assays. In mouse hepatoma cell line HEPA-1 cells, we demonstrated an inverse correlation between miR-31 and CDK1 protein levels, but no change in Cdk1 mRNA levels, suggesting a post-transcriptional effect. Additionally, a set of miRNAs (miRs-411, 300, 127, 134, 409-3p, and 433-3p) that were altered in the GBE-HCCs were also altered in non-tumor liver samples from the 90-day GBE-exposed group compared to the vehicle control group, suggesting that some of these miRNAs could serve as potential biomarkers for GBE exposure or hepatocellular carcinogenesis. These data increase our understanding of miRNA-mediated epigenetic regulation of GBE-mediated hepatocellular carcinogenesis in B6C3F1/N mice.

Multidrug resistance is one of the reasons for low survival of advanced hepatocellular carcinoma (HCC). Our previous studies indicate that the hedgehog signalling is involved in hepatic carcinogenesis, metastasis and chemo-resistance. The present study aims to uncover molecular mechanisms underlying hepatoma chemo-resistance. TAP1 and GLI1/2 gene expression was assessed in both poorly differentiated hepatoma cells and HCC specimens. Potential GLI-binding site in the TAP1 promoter sequence was validated by molecular assays. Approximately 75% HCC specimens exhibited an elevated expression of hedgehog GLI1 transcription factor compared with adjacent liver tissue. Both GLI1/2 and TAP1 protein levels were significantly elevated in poorly differentiated hepatoma cells. Both Huh-7-trans and Huh-7-DN displayed more karyotypic abnormalities and differential gene expression profiles than their native Huh-7 cells. Sensitivity to Sorafenib, doxorubicin and cisplatin was remarkably improved after either GLI1 or TAP1 gene was inhibited by an RNAi approach or by a specific GLI1/2 inhibitor, GANT61. Further experiments confirmed that hedgehog transcription factor GLI1/2 binds to the TAP1 promoter, indicating that TAP1 is one of GLI1/2 target genes. In conclusion, TAP1 is under direct transcriptional control of the hedgehog signalling. Targeting hedgehog signalling confers a novel insight into alleviating drug resistance in the treatment of refractory HCC., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Daphne genkwa , a Chinese medicinal herb, is used frequently in Southeast Asian countries to treat diseases; the flavonoid hydroxygenkwanin (HGK) is extracted from its flower buds. The bioactivity of HGK, particularly as an anti-liver cancer agent, has not been explored. In this study, human hepatocellular carcinoma (HCC) cell lines and an animal xenograft model were employed to investigate both the activity of HGK against liver cancer and its cellular signaling mechanisms. HCC cells treated with HGK were subjected to cell function assays. Whole transcriptome sequencing was used to identify genes whose expression was influenced by HGK, and the flavonoid's cancer suppression mechanisms were further investigated through gain- and loss-of-function assays. Finally, in vitro findings were tested in a mouse xenograft model. The data showed that HGK induced the expression of the microRNA miR-320a, which in turn inhibited the expression of the transcription factor 'forkhead box protein M1' (FOXM1) and downstream FOXM1-regulated proteins related to epithelial-mesenchymal transition, thereby leading to the suppression of liver cancer cell growth and invasion. Significant inhibition of tumor growth was also observed in HGK-treated mice. Hence, the present study demonstrated the activity of HGK against liver cancer and validated its potential use as a therapeutic agent.

Background: The tumor-targeting ability and pH-sensitive properties of intelligent drug delivery systems are crucial for effective drug delivery and anti-tumor therapy., Methods: In this study, sHA-DOX/HA-GA mixed micelles were designed with the following properties: sulfated hyaluronic acid (sHA) was synthesized to block cell migration by inhibiting HAase; sHA-DOX conjugates were synthesized via pH-sensitive hydrazone bond to realize DOX-sensitive release. The introduction of HA-GA conjugate could improve active-targeting ability and cellular uptake., Results: The results showed that the mixed micelles possessed a nearly spherical shape, nanoscale particle size (217.70±0.89 nm), narrow size distribution (PDI=0.07±0.04), negative zeta potential (-31.87±0.61 mV) and pH-dependent DOX release. In addition, the sHA-DOX/HA-GA micelles exhibited concentration-dependent cytotoxicities against liver carcinoma cells (HepG2) and HeLa cells, and were shown to be effectively taken up by HepG2 cells by confocal microscopy analysis. Furthermore, the in vivo anti-tumor study showed that mixed micelles had a superior anti-tumor effect compared to that of free DOX. Further evidence obtained from the hematoxylin-eosin staining and immunohistochemistry analysis also demonstrated that sHA-DOX/HA-GA exhibited stronger tumor inhibition and lower systemic toxicity than free DOX., Conclusion: The sHA-DOX/HA-GA mixed micelles could be a potential drug delivery system for anti-hepatoma therapy., Competing Interests: The authors report no conflicts of interest in this work., (© 2019 Li et al.)

Hepatocellular carcinoma (HCC) is one of the most common types of liver cancer and is the second leading cause of cancer mortality with an estimated 745 500 deaths annually (Jemal et al., 2011). Although new therapeutic modalities including novel chemotherapeutic interventions and targeted therapy have been applied, the prognosis of HCC patients remains unsatisfactory due to the high incidence of intrahepatic and distal metastases (Siegel et al., 2018).

Calcyclin-binding protein (CACYBP) is a multi-ligand protein implicated in the progression of various human cancers. However, its function in hepatocellular carcinoma (HCC) remains unknown. Methods : The expression of CACYBP and RNF41 (RING finger protein 41) in HCC cancer and adjacent non-tumor tissues was detected by immunohistochemistry. CCK-8 assays, colony formation assays, flow cytometry detection and xenograft models were used to evaluate the impact of CACYBP expression on HCC cell growth, apoptosis and cell cycle regulation. Immunoprecipitation and ubiquitination assays were performed to determine how RNF41 regulates CACYBP. The regulatory mechanism of RNF41-CACYBP signaling axis on P27 Kip1 was investigated by western blotting and immunofluorescence. Results : CACYBP was highly expressed and associated with poor prognosis in HCC. CACYBP expression was required for HCC cell growth in vitro and in vivo . Moreover, we identified RNF41 as a specific binding partner of CACYBP at exogenous and endogenous levels. RNF41 recruited CACYBP by its C-terminal substrate binding domain, subsequently ubiquitinating CACYBP and promoting its degradation in both proteasome- and lysosome-dependent pathways. In HCC tissues, RNF41 expression was reduced and conferred a negative correlation with CACYBP expression. Mechanistically, CACYBP overexpression stimulated the Ser10, Thr157 and Thr198 phosphorylation of P27 Kip1 and its cytoplasmic retention, and RNF41 co-expression attenuated this phenomenon. CACYBP depletion led to decreased levels of cyclin D1, cyclin A2, CDK2 and CDK4, causing a typical cell cycle arrest at G1/S phase and increasing apoptosis in HCC cells. P27 Kip1 -S10D but not P27 Kip1 -S10A reconstitution rescued partially the cell cycle function and apoptotic feature after CACYBP depletion. Conclusion : Our findings provide novel insights into the functional role and regulatory mechanism of CACYBP in HCC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Hepatocellular carcinoma (HCC) has become a great threat for people's health. Many long noncoding RNAs are involved in the pathogenesis of HCC. SNHG15, as a tissue specific long noncoding RNAs, has been studied in many human cancers, except HCC., Aim: To explore the regulatory mechanism of SNHG15 in HCC., Methods: In the present research, 101 HCC patient samples, two HCC cell lines and one normal liver cell line were used. RT-qPCR and Western blot analysis were applied to detect SNHG15, miR-490-3p and histone deacetylase 2 (HDAC2) expression. The regulatory mechanism of SNHG15 was investigated using CCK-8, Transwell and luciferase reporter assays., Results: Our research showed that up-regulation of SNHG15 was found in HCC and was related to aggressive behaviors in HCC patients. Moreover, knockdown of SNHG15 restrained HCC cell proliferation, migration and invasion. In addition, SNHG15 served as a molecular sponge for miR-490-3p. Further, miR-490-3p directly targets HDAC2. HDAC2 was involved in HCC progression by interacting with the SNHG15/miR-490-3p axis., Conclusion: In conclusion, long noncoding RNA SNHG15 promotes HCC progression by mediating the miR-490-3p/HDAC2 axis in HCC., Competing Interests: Conflict-of-interest statement: The authors declare no conflict of interest., (©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.)

Background: Sorafenib is the only approved first line systemic therapy for advanced hepatocellular carcinoma (HCC) in the last decade. Tumour resistance to sorafenib has been of major obstacles to improve HCC patient survival., Methods: We polarised THP-1 cells to M1 and M2 macrophages, performed various in vitro assays and developed sorafenib-resistant xenograft models to investigate the role of tumour-associated macrophages (TAM)-secreted molecules in HCC resistance to the targeted therapy., Results: We demonstrated M2, but not M1, macrophages not only promote proliferation, colony formation and migration of hepatoma cells but also significantly confer tumour resistance to sorafenib via sustaining tumour growth and metastasis by secreting hepatocyte growth factor (HGF). HGF activates HGF/c-Met, ERK1/2/MAPK and PI3K/AKT pathways in tumour cells. Tumour-associated M2 macrophages were accumulated in sorafenib-resistance tumours more than in sorafenib-sensitive tumours in vivo and produced abundant HGF. HGF chemoattracts more macrophages migrated from surrounding area, regulates the distribution of M2 macrophages and increases hepatoma resistance to sorafenib in a feed-forward manner., Conclusions: Our results provide new insights into the mechanisms of sorafenib resistance in HCC and rationale for developing new trials by combining sorafenib with a potent HGF inhibitor such as cabozantinib to improve the first line systemic therapeutic efficacy.

The GINS complex is one of the core components of the eukaryotic replicative helicase CMG (Cdc45-MCM helicase-GINS) complex that serves as the replicative helicase unwinding duplex DNA ahead of moving replication fork during chromosome duplication. Many studies have highlighted the important functions amongst GINS subunits in various cancers. Nevertheless, the functions and prognostic roles of distinct GINS subunits in hepatocellular carcinoma (HCC) were largely unexplored. In the present study, we reported the prognostic values of GINS subunits in HCC patients through analysis of several databases, including Oncomine, (TCGA), and Kaplan-Meier Plotter (KMPlotter). We found that mRNA expressions of all GINS subunits were significantly up-regulated in HCC tumor than in non-tumor liver tissues. Survival analysis revealed that elevated expression of individual GINS subunit predicts a poor overall survival (OS) in all HCC patients. When sorting the patients by gender, the correlation between elevated expression of individual GINS subunit and poor OS remains significant in male patient subgroup, but not in female patient subgroup. Additionally, we found that co-overexpression of all GINS subunits was significantly associated with a higher hazard ratio, suggesting the GINS complex may co-operate to promote HCC progression. Indeed, their expressions were highly correlated with each other in the same cohort and TRANSFAC analysis revealed that four transcription factors including C/EBPα, Oct-1, Sp1, and USF may serve as common transcription factors binding to the promoters of all four GINS subunits. Therefore, we propose that individual GINS subunit or GINS complex as a whole could be potential prognostic biomarkers for HCC., (© 2018 The Author(s).)

Melatonin is the main pineal hormone that relays light/dark-cycle information to the circadian system. Recent studies have examined the intrinsic antitumor activity of melatonin in various cancers, including hepatocellular carcinoma (HCC), the primary life-threatening malignancy in both sexes in Taiwan. However, the detailed regulatory mechanisms underlying melatonin's anti-HCC activity remain incompletely understood. Here, we investigated the mechanisms by which the anti-HCC activity of melatonin is regulated. Human hepatoma cell lines were treated with 1 and 2 mM melatonin, and functional assays were used to dissect melatonin's antitumor effect in HCC; small-RNA sequencing was performed to identify the microRNAs (miRNAs) involved in the anti-HCC activity of melatonin; and quantitative RT-PCR and Western blotting were used to elucidate how miRNAs regulate melatonin-mediated HCC suppression. Melatonin treatment at both doses strongly inhibited the proliferation, migration and invasion capacities of Huh7 and HepG2 cell lines, and melatonin treatment markedly induced the expression of the miRNA let7i-3p in cells. Notably, transfection of cells with a let7i-3p mimic drastically reduced RAF1 expression and activation of mitogen-activated protein kinase signaling downstream from RAF1, and rescue-assay results demonstrated that melatonin inhibited HCC progression by modulating let7i-3p-mediated RAF1 suppression. Our findings support the view that melatonin treatment holds considerable promise as a therapy for HCC.

Anillin (ANLN) is an actin-binding protein essential for assembly of cleavage furrow during cytokinesis. Although reportedly overexpressed in various human cancers, its role in hepatocellular carcinoma (HCC) is unclear. To address this issue, we confirmed that in 436 liver samples obtained from surgically removed HCC tissues, higher ANLN expression was detected in tumor tissues than in adjacent non-tumor tissues of HCC as measured by immunohistochemistry, quantitative real-time PCR and western blotting. Correlation and Kaplan-Meier analysis revealed that patients with higher ANLN expression were associated with worse clinical outcomes and a shorter survival time, respectively. Moreover, ANLN inhibition resulted in growth restraint, reduced colony formation, and a lower sphere number in suspension culture. Mechanistically, ANLN deficiency induced an increasing number of multinucleated cells along with the activation of apoptosis signaling and DNA damage checkpoints. Furthermore, HBV infection increased ANLN expression by inhibiting the expression of microRNA (miR)-15a and miR-16-1, both of which were identified as ANLN upstream repressors by targeting its 3' untranslated region. Thus, we conclude that ANLN promotes tumor growth by ways of decreased apoptosis and DNA damage. Expression level of ANLN significantly influences the survival probability of HCC patients and may represent a promising prognostic biomarker.

Autophagy is an intracellular "self-eating" process that is closely related to inflammation and cellular immunity. New studies indicate that autophagy is also involved in tumor suppression. The anti-inflammatory cytokine interleukin-37 (IL-37) has been shown to have tumor-suppressive abilities in hepatocellular carcinoma (HCC). Notably, autophagy appears to play a dual role in the development of HCC and may be involved in both tumorigenesis and tumor suppression. However, the potential role of IL-37 in autophagy is currently unknown. In this study, we investigated the effect of IL-37 on autophagy in multiple HCC cell lines. In doing so, we found that IL-37 inhibits proliferation in HCC cells and also induces autophagy and apoptosis in the SMMC-7721 and Huh-7 cell lines. Further experiments revealed that IL-37 treatment reduced the levels of phosphorylated protein kinase B (p-AKT), phosphorylated mammalian target of rapamycin (p-mTOR), phosphorylated p70 ribosomal protein s6 kinase (p-p70S6K) and phosphorylated 4E-binding protein 1 (4E-BP1). Moreover, treatment with an AKT agonist, insulin-like growth factor 1 (IGF-1), reversed these IL-37-mediated effects on autophagy, and treatment with an phosphoinositide-3-kinase (PI3K)/AKT inhibitor, LY294002, mimicked the effects of IL-37. Taken together, these results indicate that IL-37 regulates autophagy in SMMC-7721 and Huh-7 cells via inhibition of the PI3K/AKT/mTOR signaling pathway., (Copyright © 2017. Published by Elsevier Ltd.)

The aim of this study was to test the causal association between circulating transforming growth factor beta 1 (protein: TGF-β1 and coding gene: TGFB1) and hepatocellular carcinoma by choosing TGFB1 gene C-509T polymorphism as an instrument in a Mendelian randomization (MR) meta-analysis. Ten English articles were identified for analysis. Two authors independently assessed each article and abstracted relevant data. Odds ratio (OR) and weighted mean difference (WMD) with 95% confidence interval (CI) were synthesized under a random-effects model. Overall, the association of C-509T polymorphism with hepatocellular carcinoma was negative, but its association with circulating TGF-β1 was statistically significant, with a higher concentration observed in carriers of the -509TT genotype (WMD, 95% CI, P: 1.72, 0.67-2.78, 0.001) and -509TT/-509TC genotypes (WMD, 95% CI, P: 0.98, 0.43-1.53, < 0.001). In subgroup analysis, C-509T polymorphism was significantly associated with hepatocellular carcinoma in population-based studies under homozygous-genotype (OR, 95% CI, P: 1.74, 1.08-2.80, 0.023) and dominant (OR, 95% CI, P: 1.48, 1.01-2.17, 0.047) models. Further MR analysis indicated that per unit increase in circulating TGF-β1 was significantly associated with a 38% (95% CI: 1.03-4.65) and 49% (95% CI: 1.01-6.06) increased risk of hepatocellular carcinoma under homozygous-genotype and dominant models, respectively. Conclusively, based on a MR meta-analysis, our findings suggest that enhanced circulating TGF-β1 is causally associated with an increased risk of hepatocellular carcinoma.

Angiopoietin-like protein 1 (ANGPTL1) has been shown to act as a tumor suppressor by inhibiting angiogenesis, cancer invasion, and metastasis. However, little is known about the effects of ANGPTL1 on sorafenib resistance and cancer stem cell properties in hepatocellular carcinoma (HCC) and the mechanism underlying these effects. Here, we show that ANGPTL1 expression positively correlates with sorafenib sensitivity in HCC cells and human HCC tissues. ANGPTL1 significantly decreases epithelial-mesenchymal transition (EMT)-driven sorafenib resistance, cancer stemness, and tumor growth of HCC cells by repressing Slug expression. ANGPTL1 directly interacts with and inactivates MET receptor, which contributes to Slug suppression through inhibition of the extracellular receptor kinase/protein kinase B (ERK/AKT)-dependent early growth response protein 1 (Egr-1) pathway. ANGPTL1 expression inversely correlates with Slug expression, poor sorafenib responsiveness, and poor clinical outcomes in HCC patients., Conclusion: ANGPTL1 inhibits sorafenib resistance and cancer stemness in HCC cells by repressing EMT through inhibition of the MET receptor-AKT/ERK-Egr-1-Slug signaling cascade. ANGPTL1 may serve as a novel MET receptor inhibitor for advanced HCC therapy. (Hepatology 2016;64:1637-1651)., (© 2016 by the American Association for the Study of Liver Diseases.)

Previous studies have determined that activated hepatic stellate cells (aHSCs) promote the progression of hepatocellular carcinoma (HCC) by increasing angiogenesis in cancerous tissues. In addition, angiopoietin 1 (Ang‑1) has been reported to be involved in tumor growth and metastasis via the promotion of angiogenesis. It remains unclear whether aHSCs and Ang‑1 are involved in the angiogenesis in HCC. A total of 25 HCC and tumor‑adjacent tissues, and 21 normal liver tissues were used in the present study. Immunohistochemistry (IHC) was used to detect the expression of Ang‑1 and α smooth muscle actin (α‑SMA). The expression of CD34 was also analyzed using IHC to evaluate the microvessel density (MVD). The protein expression levels of Ang‑1 were evaluated using western blot analysis. The association between aHSC, Ang‑1 and angiogenesis was determined using Spearman's rank correlation coefficient. The present study determined that the expression of α‑SMA, Ang‑1 and MVD (CD34) was significantly higher in the HCC tissues when compared with tumor‑adjacent tissues and normal liver tissues. Spearman's rank analysis identified a positive correlation between the expression of α‑SMA, Ang‑1 and CD34. This suggests that α‑SMA‑positive aHSCs promoted angiogenesis by expressing Ang‑1, resulting in the proliferation and metastasis of HCC.

As the most predominant tumour-infiltrating immune cells, tumour-associated macrophages (TAMs) are significant for fostering tumour growth, progression and metastasis. CD68-positive TAMs display dissimilarly polarized programmes comprising CD11c-positive pro-inflammatory macrophages (M1) and CD206-positive immunosuppressive macrophages (M2). The aim of this study is to determine the prognostic significance of diametrically polarized TAMs in hepatocellular carcinoma (HCC) and their application to risk stratification of patients according to their specific prognostic values. This study included 80 consecutive patients with HCC, and we evaluated diametrically polarized functional status of macrophages by immunohistochemical staining of CD68, CD11c and CD206. Prognostic values and clinicopathologic features were assessed in these patients. High versus low CD11c-positive TAM density (P = 0.005) and low versus high CD206-positive TAM density (P = 0.002) were associated with better overall survival, whereas CD68-positive TAM density had no prognostic significance (low versus high, P = 0.065). Furthermore, the presence of these positive staining macrophages did not show any prognostic significance for recurrence-free survival (all P > 0.05). Multivariate Cox regression analysis identified CD11c-positive and CD206-positive TAMs as an independent prognostic factor (P < 0.001, P = 0.031, respectively). Intratumoural infiltration of diametrically polarized TAMs, a novel identified independent prognostic factor for survival in patients with HCC, could be combined with the TNM stage and the Barcelona Clinic Liver Cancer stage to improve a risk stratification system., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Objective: Liver transplantation for intrahepatic cholangiocarcinoma is notorious for rapid recurrence with poor survival rate postoperatively and has therefore been discontinued in most centers. The purpose of this study is to distinguish hepatocellular carcinoma (HCC) from cholangiocarcinoma in pretransplantation imaging evaluation by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI)., Materials and Methods: From January 2014 to September 2015, 19 patients were included in the study, with a mean age of 62.8 years. All subjects underwent pretransplantation DCE-MRI and surgical excision or core biopsy. The DCE-MRI parameters were measured using the Tofts model 1999. Statistical analysis included nonparametric tests and area under the curve for the receiver operating characteristic., Results: Fourteen HCCs and 5 cholangiocarcinomas were diagnosed by surgical pathology. The mean size of tumor was 6.4 cm (range, 1.5 cm to 13.7 cm). All DCE-MRI parameters were calculated as the ratio between the tumor and normal liver parenchyma and K(trans) (1/min) was used as a distinguishing parameter between the two tumors. K(trans) was higher in the cholangiocarcinoma group (1.89 ± 1.13) than in the HCC group (0.46 ± 0.35). Univariate analysis revealed that K(trans) has a high significant difference (P = .001). The optimal K(trans) value cutoffs were 1 or more (area under the curve = 0.971) for detection of HCCs or cholangiocarcinomas., Conclusion: The analysis of DCE-MRI with the kinetic model (Tofts, 1999) presents a new and practical approach indiscrimination of HCC from cholangiocarcinoma for pretransplantation imaging evaluation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: This study was undertaken to assess the long-term outcome of transcatheter arterial chemoembolization (TACE) after radiofrequency ablation (RFA) combined with a combined therapy for Chinese patients with intermediate (stage B) hepatocellular carcinoma (HCC) of single block type, and evaluate the survival rate for 1, 3, 5, and 7 years., Research Design and Methods: This prospective, single-center study consisted of patients with solitary massive intermediate (stage B) HCC treated by RFA combined with TACE from October 1999 to December 2013., Main Outcome Measures: The survival rate of the patients for 1, 3, 5, and 7 years, and safety of the RFA treatment in the interim, total RFA for each case, and number of TACE cycles were evaluated., Results: Ninety-three patients (aged 54.4 ± 8.0 years) underwent RFA combined with TACE as a combined therapy, and they were analyzed and followed up until December 2013. The mean time for the initial ablation was 1.5-3 h, and, on average, each patient received 1.39 RFA and 1.43 TACE therapies. Overall, complete ablation was achieved in nine patients, and the majority of ablation was seen in 84 patients. The longest survival time was 102 months and, among the survivors the 1, 3, 5, and 7 year survival rate was 94.4%, 52.3%, 26.1%, and 14.1%, respectively. The median survival time was 36 months (95% confidence interval = 32.7-39.3). Serum alpha-fetoprotein (AFP) levels showed significant correlation with tumor size in patients with HCC (r = 0.323, p = 0.0001). There were no major complications related to this therapy., Conclusion: This was the first study that performed RFA combined with TACE in Chinese patients with intermediate (stage B) HCC. RFA combined with TACE, as a combined therapy for intermediate (stage B) HCC, seems to be a promising regimen that showed a satisfactory clinical effect, which may become a new therapy mode for HCC. However, a larger cohort and control group(s) reflecting usual standards of care are needed to assess the external validity of these results in a wider population.

Kindlin-1 is a member of the Kindlin family of focal adhesion proteins and is implicated in cell adhesion, proliferation, polarity, and motility. Although expression of Kindlin-1 has recently been reported in a variety of human cancers, studies on its expression in human hepatocellular carcinoma (HCC) are currently lacking. This study aimed to determine the clinicopathological parameters and prognostic value of Kindlin-1 in HCC patients after surgical resection. The messenger RNA (mRNA) and protein levels of Kindlin-1 in 22 matched HCC specimens were assessed by quantitative real-time PCR (qRT-PCR) and Western blotting assays. The clinical and prognostic significance of Kindlin-1 in 68 cases of HCC was determined by immunohistochemistry. Kindlin-1 expression was higher in HCC tumor tissues relative to that in adjacent normal tissue at the both mRNA and protein levels (p < 0.05). Immunohistochemical results revealed that overexpression of Kindlin-1 was detected in 37 of 68 (54.4 %) tumor tissues and in seven of 68 (10.3 %) adjacent non-tumor tissues (p < 0.05). Positive Kindlin-1 expression was significantly correlated with tumor size, tumor capsula, status of metastasis, and tumor-node-metastasis (TNM) stage. Additionally, Kaplan-Meier survival analysis showed that positive Kindlin-1 expression was associated with unfavorable overall survival (OS) and disease-free survival (DFS). Multivariate analysis identified Kindlin-1 as an independent prognostic predictor for OS and DFS in HCC patients (p = 0.041 and 0.027, respectively). Taken together, our data suggest that Kindlin-1 could play an important role in HCC and might serve as a promising prognostic marker and potential target for HCC therapy.

The association of aberrant expression of Kindlin-2 with tumor progression has been reported in recent years. The purpose of this study was to investigate the expression of Kindlin-2 in hepatocellular carcinoma (HCC), and to evaluate its clinical and prognostic significance. The mRNA and protein levels of Kindlin-2 in HCC and adjacent non-cancerous tissues were examined by real-time PCR and western blotting. The relationships between Kindlin-2 expression, clinicopathological features and postoperative survival of HCC patients were also evaluated. Kindlin-2 expression was higher in HCC tissues as compared to adjacent non-cancerous tissues at both mRNA and protein levels (P<0.05, respectively). Positive expression of Kindlin-2 was significantly correlated with larger tumor size (P=0.034), capsular invasion (P=0.009), microvascular invasion (P=0.028) and poor prognosis of HCC patients (P<0.001). Moreover, multivariate survival analysis identified Kindlin-2 as an independent prognostic factor for overall and disease-free survival of HCC patients (P=0.018 and 0.001, respectively). Taken together, our findings suggested that Kindlin-2 was highly expressed in HCC tissues and was closely related to clinical progression. Therefore, Kindlin-2 protein could be a potential biomarker for predicting poor prognosis of HCC patients after surgery., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Objectives: Intrahepatic recurrence is the major cause of death among patients with hepatitis B virus (HBV)- related hepatocellular carcinoma (HCC) after curative surgical resection. Several approaches have been reported to decrease the recurrence rate. The objective of our study was to compare the clinical effects of transcatheter arterial chemoembolization (TACE) combined with interferon-alpha (IFN-α) therapy on recurrence after hepatic resection in patients with HBV-related HCC with that of TACE chemotherapy alone., Methods: We retrospectively analyzed the data from 228 patients who were diagnosed with HBV-related HCC and underwent curative resection between January 2001 to December 2008. The patients were divided into TACE (n = 126) and TACE-IFN-α (n = 102) groups for postoperative chemotherapy. The TACE regimen consisted of 5-fluorouracil (5-FU), cisplatin (DDP) , and the emulsion mixed with mitomycin C (MMC) and lipiodol. The recurrence rates, disease-free survival (DFS), overall survival (OS), and risk of recurrence were evaluated., Results: The clinicopathological parameters and adverse effects were similar between the 2 groups (P > 0.05). The median OS for the TACE- IFN-α group (36.3 months) was significantly longer than that of the TACE group (24.5 months, P < 0.05). The 3-and 5-year OS for the TACE-IFN-α group were significantly longer than those of the TACE group (P < 0.05) and the recurrence rate was significantly lower (P < 0.05). The TACE and IFN-α combination therapy, active hepatitis HBV infection, the number of tumor nodules, microvascular invasion, liver cirrhosis, and the BCLC stage were independent predictors of OS and DFS., Conclusions: The use of the TACE and IFN-α combination chemotherapy after curative hepatic resection safely and effectively improves OS and decreases recurrence in patients with HBV-related HCC who are at high risk. Our findings can serve as a guide for the selection of postoperative adjuvant chemotherapy for patients with HBV-related HCC who are at high risk of recurrence.

Aberrant expression of genes in de novo lipogenesis (DNL) pathway were associated with various cancers, including hepatocellular carcinoma (HCC). Single nucleotide polymorphisms (SNPs) of DNL genes have been reported to be associated with prognosis of some malignancies. However, the effects of SNPs in DNL genes on overall survival of HCC patients receiving transarterial chemoembolization (TACE) treatment are still unknown. In present study, nine SNPs in three genes (ACLY, ACACA and FASN) in DNL pathway were genotyped using the Sequenom iPLEX genotyping system in a hospital-based cohort with 419 HCC patients treated with TACE, and their associations with HCC overall survival were evaluated by Cox proportional hazard regression analysis under three genetic models (additive, dominant and recessive). Although we did not find any significant results in total analysis (all p>0.05), our stratified data showed that SNP rs9912300 in ACLY gene was significantly associated with overall survival of HCC patients with lower AFP level and SNP rs11871275 in ACACA gene was significantly associated with overall survival of HCC patients with higher AFP level. We further identified the significant interactions between AFP level and SNP rs9912300 or rs11871275 in the joint analysis. Conclusively, our data suggest that genetic variations in genes of DNL pathway may be a potential biomarker for predicting clinical outcome of HCC patients treated with TACE.

Background: MicroRNA-106b (miR-106b) is a member of the miR-106b ~ 25 cluster. It has been reported that miR-106b acts as an oncogene and is upregulated in many human cancers. However, the prognostic value of miR-106b in hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to investigate the clinical significance of miR-106b expression in HCC., Methods: We determined the expression level of miR-106b in 104 cases of paired HCC and adjacent non-tumor tissues by quantitative real-time PCR (qRT-PCR). The correlation between miR-106b expression and prognosis of HCC was studied by univariate and multivariate analysis. Multivariate analysis of the prognostic factors was performed with Cox proportional hazards model., Results: MiR-106b expression was significantly upregulated in as high as 76.0% of HCC tissues, compared with their non-tumor counterparts (P < 0.001). High miR-106b expression was significantly associated with large tumor size (P = 0.019) and vascular invasion (P = 0.016). Kaplan-Meier analysis showed that patients with high miR-106b expression had a worse overall survival than patients with low miR-106b expression (log-rank P = 0.004). The multivariate Cox regression analysis indicated that miR-106b expression was an independent prognostic factor for overall survival (HR, 2.002; 95% CI, 1.130-6.977; P = 0.027)., Conclusion: Our data indicated that miR-106b expression was significantly upregulated in HCC and could serve as a potential unfavorable prognostic biomarker., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000&#95;2014&#95;226.

HOXA13 is a member of homeobox genes that encode transcription factors regulating embryonic development and cell fate. Abnormal HOXA13 expression was reported in hepatocellular carcinoma (HCC), but its correlation with tumor angiogenesis and prognosis still remain unclear. This study was aimed to uncover the expression, diagnostic and prognostic significance of HOXA13 in HCC. Immunohistochemistry was performed to detect HOXA13 expression in HCC and corresponding paracarcinomatous tissues from 90 patients. Enzyme-linked immunosorbent assay was used to detect serum HOXA13 in 90 HCC patients and 20 healthy volunteers. Receiver operating characteristics was analyzed to calculate diagnostic accuracy of serum HOXA13, alpha-fetoprotein (AFP) and their combination. Immunoreactivity of HOXA13 was detected in 72.2% of HCC, and 12.2% of adjacent non-cancerous samples. HOXA13 expression was significantly associated with tumor size, microvascular invasion, pathological grade, tumor capsula status, AFP level, tumor-node-metastasis stage and positively correlated with VEGF (p < 0.001) and microvessel density (p < 0.001). The combination of serum HOXA13 and AFP had a markedly higher area under the curve than HOXA13 alone. HOXA13 expression was associated with unfavorable overall survival (OS) (p < 0.001) and disease-free survival (DFS) (p < 0.001). Multivariate analysis indicated that patients with HOXA13-expressing tumors had a significantly shorter OS (p = 0.030) and DFS (p = 0.005) than those with HOXA13-negative tumors. Thus, HOXA13 expression possibly plays an important role in tumor angiogenesis, progression and prognosis of HCC. Moreover, we demonstrate that serum HOXA13 may serve as a biomarker for early HCC diagnosing and predicting outcome.

Objective: Clinical trials suggest that combining transcatheter arterial chemoembolization with sorafenib in patients with advanced hepatocellular carcinoma shows a superior safety and tolerability profile. Our study aimed to retrospectively analyze the utility and prognostic factors of this combined therapy in these patients., Methods: Patients with advanced hepatocellular carcinoma, treated by transcatheter arterial chemoembolization and sorafenib subsequently, between February 2010 and September 2012 in our hospital, were retrospectively analyzed. After sorafenib treatment for 12 weeks, abdominal enhanced computed tomography or magnetic resonance imaging was used to evaluate short-term outcomes and clinical benefit rate. Overall survival and adverse events were recorded during follow-up. Univariate and multivariate analyses were used to identify relationships between baseline characteristics and overall survival., Results: Fifty-one advanced hepatocellular carcinoma patients were included. Common adverse events for sorafenib were hand-foot skin reaction, alopecia, diarrhea, anorexia and fatigue. The clinical benefit rate was 64% and the median survival time was 7.5 months. Median survival of patients with and without portal vein tumor thrombi was 6.0 months and 10.3 months (P < 0.001), respectively. Median survival of patients with cholinesterase ≥5000 U/l and < 5000 U/l was 10.6 months and 6.1 months (P < 0.001), respectively. Multivariate analysis identified the presence of portal vein tumor thrombi and low cholinesterase level as independent negative predictors of survival., Conclusions: Combining sorafenib and transcatheter arterial chemoembolization was safe and effective for advanced hepatocellular carcinoma patients with extrahepatic spread but without portal vein tumor thrombi. Portal vein tumor thrombi and cholinesterase level are independent predictors of prognosis following this combined therapy., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Previous research showed that microRNA-612 (miR-612) has inhibitory effects on cell proliferation, migration, invasion, and metastasis of hepatocellular carcinoma (HCC). AKT2 was confirmed to be a direct target of miR-612, through which the epithelial-mesenchymal transition (EMT) and metastasis of HCC were inhibited. Our present findings reveal that miR-612 is able to suppress the stemness of HCC by reducing the number and size of tumorspheres as well as clone formation in soft agar, and to relieve drug resistance to cisplatin and 5-fluorouracil. In addition, miR-612 hampered the capacity of tumorigenesis in NOD/SCID mice and redistributed the tumor invasive frontier of miR-612-modulating cells. Finally, our findings suggest that Wnt/β-catenin signaling is required in the regulation of EMT-associated stem cell-like traits by miR-612., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Unlabelled: Leukocyte cell-derived chemotoxin 2 (LECT2) has been shown to act as a tumor suppressor in hepatocellular carcinoma (HCC). However, the underlying mechanism has not yet been completely defined. Here, we employ a LECT2-affinity column plus liquid chromatography coupled with tandem mass spectrometry to identify LECT2-binding proteins and found that MET receptor strongly interacted with LECT2 protein. Despite the presence of hepatocyte growth factor, the LECT2 binding causes an antagonistic effect to MET receptor activation through recruitment of protein tyrosine phosphatase 1B. The antagonistic effect of LECT2 on MET activation also mainly contributes to the blockage of vascular invasion and metastasis of HCC. Furthermore, serial deletions and mutations of LECT2 showed that the HxGxD motif is primarily responsible for MET receptor binding and its antagonistic effects., Conclusion: These findings reveal a novel, specific inhibitory function of LECT2 in HCC by the direct binding and inactivation of MET, opening a potential avenue for treating MET-related liver cancer., (© 2014 by the American Association for the Study of Liver Diseases.)