Fibronectin 1 derived from tumor-associated macrophages and fibroblasts promotes metastasis through the JUN pathway in hepatocellular carcinoma.

OBJECTIVE: Intercellular communication in the tumor microenvironment is a potential regulator of metastasis. To explore the specific mechanism, we performed a multi-omics analysis of hepatocellular carcinoma. MATERIALS AND METHODS: Multiple omics data including scRNA-seq, ATAC-seq, RNA-seq, and methylation data were obtained from GEO and TCGA databases. SCENIC was used to identify key transcription factors and their Regulatory networks. ScMLnet was used to explore the mechanism of intercellular communication in the microenvironment. Multiple omics studies based on RNA-seq, ATAC-seq, and methylation data were used to explore downstream mechanisms of key transcription factors. Based on the analysis of cell differentiation trajectory and transcription subtypes, the regulation of cell communication on tumor subtypes was studied, and possible therapeutic compounds were explored. The universality of this mechanism was investigated by post-Pan-cancer analysis. RESULTS: JUN and its regulatory network play a key role in HCC, which was mainly positively correlated with tumor-associated macrophages and fibroblasts. Intercellular communication analysis showed that macrophage and fibroblast-derived FN1 could increase JUN by TNFRSF11B/SMAD3. Multiomics analysis showed that KIF13A was a key downstream gene of JUN, which was involved in the activation of the hippo pathway. Analysis of cell differentiation trajectory, transcriptome subtypes, and neural network modeling showed that intercellular communication in the microenvironment can regulate the transcriptome characterization of HCC. Pan-cancer analysis indicates that this mechanism may be universal. CONCLUSION: FN1 derived from tumor-associated macrophages and fibroblasts promotes metastasis and alters transcriptome subtypes through the JUN-Hippo signaling pathway in HCC, which may be universal in cancers.

Down-regulation of EVA1A by miR-103a-3p promotes hepatocellular carcinoma cells proliferation and migration.

BACKGROUND: EVA1A (Eva-1 homolog A), a novel protein involved in autophagy and apoptosis, functions as a tumor suppressor in some human primary cancers, including hepatocellular carcinoma (HCC). While it is consistently downregulated in several cancers, its involvement in hepatocarcinogenesis is still largely unknown. METHODS: We first detected the expression of EVA1A in HCC tissues and cell lines using RT‒qPCR, immunohistochemistry and western blotting and detected the expression of miR-103a-3p by RT‒qPCR. Then, bioinformatics prediction, dual-luciferase reporter gene assays and western blotting were used to screen and identify the upstream microRNA of EVA1A. After manipulating the expression of miR-103a-3p or EVA1A, wound healing, invasion, proliferation, colony formation, apoptosis, autophagy, mitosis and mitochondrial function assays, including mitochondrial membrane potential, ROS and ATP production assays, were performed to investigate the functions of miR-103a-3p targeting EVA1A in HCC cells. Apoptosis-related proteins were assessed by RT‒qPCR (TP53) or western blotting (TP53, BAX, Bcl-2 and caspase-3). Autophagy level was evaluated by observing LC3 puncta and examining the protein levels of p62, Beclin1 and LC3-II/I. RESULTS: We found that EVA1A expression was decreased while miR-103a-3p expression was increased in HCC tissues and cell lines and that their expression was inversely correlated in HCC patients. The expression of miR-103a-3p was associated with HCC tumor stage and poor prognosis. miR-103a-3p could target EVA1A through direct binding to its 3'-UTR and suppress its expression. Overexpression of miR-103a-3p significantly downregulated the expression of EVA1A, TP53 and BAX, upregulated the JAK2/STAT3 pathway and promoted HCC cell migration, invasion and proliferation, while repression of miR-103a-3p dramatically upregulated the expression of EVA1A, TP53, BAX and cleaved-caspase-3, inhibited HCC cell migration, invasion and proliferation, and caused mitochondrial dysfunction and apoptosis. Overexpression of EVA1A significantly attenuated the cancer-promoting effects of miR-103a-3p in HCC cells, while knockdown of EVA1A alleviated the mitochondrial dysfunction and apoptosis caused by miR-103a-3p inhibition. Overexpression of EVA1A did not induce significant changes in autophagy levels, nor did it affect G2/M transition or mitosis. CONCLUSION: These findings indicate that the downregulation of the tumor suppressor EVA1A by miR-103a-3p potentially acts as a key mediator in HCC progression, mainly by inhibiting apoptosis and promoting metastasis. The miR-103a/EVA1A/TP53 axis provides a new potential diagnostic and therapeutic target for HCC treatment.

Oncogenic ß-catenin stimulation of AKT2-CAD-mediated pyrimidine synthesis is targetable vulnerability in liver cancer.

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

DYRK1A suppression attenuates HIF­1α accumulation and enhances the anti­liver cancer effects of regorafenib and sorafenib under hypoxic conditions.

Hypoxia promotes drug resistance and induces the expression of hypoxia inducible factor (HIF)­1α in liver cancer cells. However, to date, no selective HIF­1α inhibitor has been clinically approved. The aim of this study is to investigate a drug­targetable molecule that can regulate HIF­1α under hypoxia. The present study demonstrated that hyperactivation of dual­specificity tyrosine­phosphorylation­regulated kinase 1A (DYRK1A)/HIF­1α signaling was associated with an increased risk of liver cancer. In addition, DYRK1A knockdown using small interfering RNA transfection or treatment with harmine, a natural alkaloid, significantly reduced the protein expression levels of HIF­1α in liver cancer cells under hypoxic conditions in vitro. Conversely, DYRK1A overexpression­vector transfection in liver cancer cell lines notably induced HIF­1α expression under the same conditions. Furthermore, DYRK1A was shown to interact and activate STAT3 under hypoxia to regulate HIF­1α expression. These findings indicated that DYRK1A may be a potential upstream activator of HIF­1α and positively regulate HIF­1α via the STAT3 signaling pathway in liver cancer cells. Additionally, treatment with harmine attenuated the proliferative ability of liver cancer cells under hypoxic conditions using sulforhodamine B and colony formation assay. Furthermore, DYRK1A knockdown could significantly enhance the anti­liver cancer effects of regorafenib and sorafenib under hypoxia. Co­treatment with harmine and either regorafenib or sorafenib also promoted cell death via the STAT3/HIF­1α/AKT signaling pathway under hypoxia using PI staining and western blotting. Overall, the results from the present study suggested that DYRK1A/HIF­1α signaling may be considered a novel pathway involved in chemoresistance, thus providing a potentially effective therapeutic regimen for treating liver cancer.

Propofol suppresses proliferation, migration, invasion, and tumor growth of liver cancer cells via suppressing cancer susceptibility candidate 9/phosphatase and tensin homolog/AKT serine/threonine kinase/mechanistic target of rapamycin kinase axis.

Propofol is a commonly used drug for sedation and general anesthesia during cancer surgery. Previous studies indicate that propofol exerts anti-tumor effect in various cancers. The aim of this study was to investigate the underlying molecular mechanism of propofol in liver cancer. The effects of propofol on liver cancer cells were evaluated by cell viability assay, colony formation assay, and tumor xenograft model. Dysregulated lncRNAs of propofol-treated liver cancer cells were evaluated by transcriptome RNA sequencing. The underlying molecular mechanisms of lncRNA cancer susceptibility candidate 9 (CASC9) in propofol-induced anti-tumor effects were evaluated by western blot, quantitative real-time polymerase chain reaction (qRT-PCR), wound scratch healing assay, transwell cell migration and invasion assay, TUNEL staining, fluorescence in situ hybridization, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP). We found that propofol suppressed proliferation, migration, invasion, and tumor xenograft growth of liver cancer cells in a dose-dependent manner. Exosomes transfer from propofol-treated cells inhibited proliferation, migration, and invasion and promoted apoptosis of liver cancer cells. Transcriptional profiling of propofol-treated liver cancer cells identified CASC9 as significantly downregulated lncRNA in cells and exosomes. Enforced CASC9 expression partially rescued the inhibitory effects of propofol on liver cancer cells. Furthermore, CASC9 was found to interact directly with EZH2 and epigenetically regulated PTEN expression. Restoration of CASC9 partially abrogated the inhibition of propofol on Akt/mTOR signaling. Our results indicated that propofol exerted anti-tumor effects by downregulating CASC9, and subsequently suppressed Akt/mTOR signaling. Our findings provided a novel insight into propofol-induced anti-tumor effects in liver cancer.

AGA Clinical Practice Guideline on Systemic Therapy for Hepatocellular Carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC), the most common primary liver cancer, remains a deadly cancer, with an incidence that has tripled in the United States since 1980. In recent years, new systemic therapies for HCC have been approved and a critical assessment of the existing data is necessary to balance benefits and harms and inform the development of evidence-based guidelines. METHODS: The American Gastroenterological Association formed a multidisciplinary group consisting of a Technical Review Panel and a Guideline Panel. The Technical Review Panel prioritized clinical questions and outcomes according to their importance for clinicians and patients and conducted an evidence review of systemic therapies in patients with advanced-stage HCC. The Grading of Recommendations Assessment, Development and Evaluation framework was used to assess evidence. The Guideline Panel reviewed the evidence and used the Evidence-to-Decision Framework to develop recommendations. RESULTS: The Panel reviewed the evidence, summarized in the Technical Review, for the following medications approved by the US Food and Drug Administration for HCC: first-line therapies: bevacizumab+atezolizumab, sorafenib, and lenvatinib; second-line therapies: cabozantinib, pembrolizumab, ramucirumab, and regorafenib; and other agents: bevacizumab, nivolumab, and nivolumab+ipilimumab. CONCLUSIONS: The Panel agreed on 11 recommendations focused on systemic therapy for HCC in patients who are not eligible for locoregional therapy or resection, those with metastatic disease and preserved liver function, those with poor liver function, and those on systemic therapy as adjuvant therapy.

Independent Assessment of the Children's Hepatic Tumors International Collaboration Risk Stratification for Hepatoblastoma and the Association of Tumor Histological Characteristics With Prognosis.

Importance: Hepatoblastoma is the most common pediatric liver malignant neoplasm, and accurate risk stratification is essential for guiding treatment. Objective: To validate the Children's Hepatic Tumors International Collaboration-Hepatoblastoma Stratification (CHIC-HS) in an independent cohort of patients with hepatoblastoma and evaluate the association of pretreatment hepatoblastoma histological subtype with prognosis. Design, Setting, and Participants: This is a single-institution retrospective cohort study of 96 pediatric patients with hepatoblastoma diagnosed and treated between June 1, 2000, and December 31, 2016, with recent therapy and independent of the CHIC-HS discovery cohort. Each patient was assigned a risk group according to CHIC-HS. The histological characteristics of each tumor were assessed based on the International Pediatric Liver Tumor Consensus Classification. Data were analyzed from May 2018 to May 2019. Main Outcomes And Measures: The main outcomes were event-free survival (EFS) and overall survival (OS). Cox regression analysis was used to examine the associations of patient characteristics and tumor histological characteristics with survival. Results: A total of 96 patients (median [range] age, 1.9 [0.4-18] years; 36 [38%] girls and 60 [63%] boys) were assessed, including 15 with very low risk, 28 with low risk, 23 with intermediate risk, and 30 with high risk, according to CHIC-HS criteria. There were a total of 13 cancer-related deaths; median (range) follow-up was 3.5 (0.1-17.8) years for those alive at the last follow-up. The estimated 5-year OS rates were 100% in the very low-risk group, 94.7% (95% CI, 68.1%-99.2%) in the low-risk group, 89.2% (95% CI, 63.1%-97.2%) in the intermediate-risk group, and 57.9% (95% CI, 34.6%-75.5%) in the high-risk group. In a multivariable analysis, we confirmed that CHIC-HS significantly estimated EFS (high-risk group vs very low- and low-risk groups: hazard ratio [HR], 45.59; 95% CI, 9.39-209.5; P < .001) and OS (high-risk group vs very low- and low-risk groups: HR, 21.95; 95% CI, 2.76-174.29; P < .001). In the subcohort of 84 patients for whom pretreatment tumor histological data were available, tumor epithelial histological subtypes were found to be significantly associated with both EFS and OS. Patients in the CHIC-HS high-risk group and with embryonal-only histological subtype had the highest risk of relapse or disease progression (high-risk: HR, 42.62; 95% CI, 9.91-203.9; embryonal: HR, 3.28; 95% CI, 1.21-8.9) and death (high-risk: HR, 18.78; 95% CI, 2.31-152.84; embryonal: HR, 7.12; 95% CI, 1.51-33.52). Conclusions and Relevance: This cohort study found that CHIC-HS performed as expected in an independent cohort that was more recently treated. Incorporation of pretreatment tumor histological data into CHIC-HS may provide additional prognostic value.

Piperlongumine inhibits diethylnitrosamine induced hepatocellular carcinoma in rats.

BACKGROUND: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Piperlongumine (PL) has been claimed to have cytotoxic and HCC inhibitory effects in various cancer cell lines and xenograft models, but the chemopreventive potential of PL has not been studied in experimentally induced HCC yet. RESEARCH DESIGN: Twenty-four Wistar male rats were divided into four groups of six each, Group A: untreated control; Group B: Diethylnitrosamine (DEN) control (200 mg/kg), Group C: DEN + PL 10 mg/kg; and Group D: DEN + PL 20 mg/kg. Rats from all groups were assessed for liver cancer progression or inhibition by evaluating biochemical, cytokines, tumor markers, lipid peroxidation, and histological profiles. RESULTS: The liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) levels, and lipid peroxidation were significantly decreased in Group C and Group D compared to Group B. Upregulation in the level of pro-inflammatory cytokines IL-1B, TNF-α, inflammatory mediator (NF-κB) and tumour marker alpha-fetoprotein (AFP) in Group B were brought down upon treatment with piperlongumine in a dose-dependent manner. Antitumor cytokine (IL-12) was upregulated in PL-treated rats compared to DEN control rats. DEN treated group (Group B) showed histological features of HCC, and in rats treated with PL (Groups C, D) partial to complete reversal to normal liver histoarchitecture was observed. CONCLUSIONS: The potential chemopreventive actions of piperlongumine may be due to its free radical scavenging and antiproliferative effect. Therefore, piperlongumine may serve as a novel therapeutic agent for the treatment of hepatocellular carcinoma.

Prognosis of patients with hepatocellular carcinoma treated with immunotherapy - development and validation of the CRAFITY score.

BACKGROUND & AIMS: Immunotherapy with atezolizumab plus bevacizumab represents the new standard of care in systemic front-line treatment of hepatocellular carcinoma (HCC). However, biomarkers that predict treatment success and survival remain an unmet need. METHODS: Patients with HCC put on PD-(L)1-based immunotherapy were included in a training set (n = 190; 6 European centers) and a validation set (n = 102; 8 European centers). We investigated the prognostic value of baseline variables on overall survival using a Cox model in the training set and developed the easily applicable CRAFITY (CRP and AFP in ImmunoTherapY) score. The score was validated in the independent, external cohort, and evaluated in a cohort of patients treated with sorafenib (n = 204). RESULTS: Baseline serum alpha-fetoprotein ≥100 ng/ml (hazard ratio [HR] 1.7; p = 0.007) and C-reactive protein ≥1 mg/dl (HR, 1.7; p = 0.007) were identified as independent prognostic factors in multivariable analysis and were used to develop the CRAFITY score. Patients who fulfilled no criterion (0 points; CRAFITY-low) had the longest median overall survival (27.6 (95% CI 19.5-35.8) months), followed by those fulfilling 1 criterion (1 point; CRAFITY-intermediate; 11.3 (95% CI 8.0-14.6) months), and patients meeting both criteria (2 points; CRAFITY-high; 6.4 (95% CI 4.8-8.1) months; p <0.001). Additionally, best radiological response (complete response/partial response/stable disease/progressive disease) was significantly better in patients with lower CRAFITY score (CRAFITY-low: 9%/20%/52%/20% vs. CRAFITY-intermediate: 3%/25%/36%/36% vs. CRAFITY-high: 2%/15%/22%/61%; p = 0.003). These results were confirmed in the independent validation set and in different subgroups, including Child-Pugh A and B, performance status 0 and ≥1, and first-line and later lines. In the sorafenib cohort, CRAFITY was associated with survival, but not radiological response. CONCLUSIONS: The CRAFITY score is associated with survival and radiological response in patients receiving PD-(L)1 immunotherapy. The score may help with patient counseling but requires prospective validation. LAY SUMMARY: The immunotherapy-based regimen of atezolizumab plus bevacizumab represents the new standard of care in systemic first-line therapy of hepatocellular carcinoma (HCC). Biomarkers to predict treatment outcome are an unmet need in patients undergoing immunotherapy for HCC. We developed and externally validated a score that predicts outcome in patients with HCC undergoing immunotherapy with immune checkpoint blockers.

Comparative performance of risk prediction models for hepatitis B-related hepatocellular carcinoma in the United States.

BACKGROUND & AIMS: Guidelines recommend hepatocellular carcinoma (HCC) surveillance in patients with chronic HBV infection. Several HCC risk prediction models are available to guide surveillance decisions, but their comparative performance remains unclear. METHODS: Using a retrospective cohort of patients with HBV treated with nucleos(t)ide analogues at 130 Veterans Administration facilities between 9/1/2008 and 12/31/2018, we calculated risk scores from 10 HCC risk prediction models (REACH-B, PAGE-B, m-PAGE-B, CU-HCC, HCC-RESCUE, CAMD, APA-B, REAL-B, AASL-HCC, RWS-HCC). We estimated the models' discrimination and calibration. We calculated HCC incidence in risk categories defined by the reported cut-offs for all models. RESULTS: Of 3,101 patients with HBV (32.2% with cirrhosis), 47.0% were treated with entecavir, 40.6% tenofovir, and 12.4% received both. During a median follow-up of 4.5 years, 113 patients developed HCC at an incidence of 0.75/100 person-years. AUC values for 3-year HCC risk were the highest for RWS-HCC, APA-B, REAL-B, and AASL-HCC (all >0.80). Of these, 3 (APA-B, RWS-HCC, REAL-B) incorporated alpha-fetoprotein. AUC values for the other models ranged from 0.73 for PAGE-B to 0.79 for CAMD and HCC-RESCUE. Of the 7 models with AUC >0.75, only APA-B was poorly calibrated. In total, 10-20% of the cohort was deemed low-risk based on the published cut-offs. None of the patients in the low-risk groups defined by PAGE-B, m-PAGE-B, AASL-HCC, and REAL-B developed HCC during the study timeframe. CONCLUSION: In this national cohort of US-based patients with HBV on antiviral treatment, most models performed well in predicting HCC risk. A low-risk group, in which no cases of HCC occurred within a 3-year timeframe, was identified by several models (PAGE-B, m-PAGE-B, CAMD, AASL-HCC, REAL-B). Further studies are warranted to examine whether these patients could be excluded from HCC surveillance. LAY SUMMARY: Risk prediction models for hepatocellular carcinoma (HCC) in patients infected with hepatitis B virus (HBV) could guide HCC surveillance decisions. In this large cohort of US-based patients receiving treatment for HBV, most published models discriminated between those who did or did not develop HCC, although the RWS-HCC, REAL-B, and AASL-HCC performed the best. If confirmed in future studies, these models could help identify a low-risk subset of patients on antiviral treatment who could be excluded from HCC surveillance.

TAZ is indispensable for c-MYC-induced hepatocarcinogenesis.

BACKGROUND & AIMS: Mounting evidence implicates the Hippo downstream effectors Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) in hepatocellular carcinoma (HCC). We investigated the functional contribution of YAP and/or TAZ to c-MYC-induced liver tumor development. METHODS: The requirement for YAP and/or TAZ in c-Myc-driven hepatocarcinogenesis was analyzed using conditional Yap, Taz, and Yap;Taz knockout (KO) mice. An hepatocyte-specific inducible TTR-CreERT2 KO system was applied to evaluate the role of YAP and TAZ during tumor progression. Expression patterns of YAP, TAZ, c-MYC, and BCL2L12 were analyzed in human HCC samples. RESULTS: We found that the Hippo cascade is inactivated in c-Myc-induced mouse HCC. Intriguingly, TAZ mRNA levels and activation status correlated with c-MYC activity in human and mouse HCC, but YAP mRNA levels did not. We demonstrated that TAZ is a direct transcriptional target of c-MYC. In c-Myc induced murine HCCs, ablation of Taz, but not Yap, completely prevented tumor development. Mechanistically, TAZ was required to avoid c-Myc-induced hepatocyte apoptosis during tumor initiation. The anti-apoptotic BCL2L12 gene was identified as a novel target regulated specifically by YAP/TAZ, whose silencing strongly suppressed c-Myc-driven murine hepatocarcinogenesis. In c-Myc murine HCC lesions, conditional knockout of Taz, but not Yap, led to tumor regression, supporting the requirement of TAZ for c-Myc-driven HCC progression. CONCLUSIONS: TAZ is a pivotal player at the crossroad between the c-MYC and Hippo pathways in HCC. Targeting TAZ might be beneficial for the treatment of patients with HCC and c-MYC activation. LAY SUMMARY: The identification of novel treatment targets and approaches for patients with hepatocellular carcinoma is crucial to improve survival outcomes. We identified TAZ as a transcriptional target of c-MYC which plays a critical role in c-MYC-dependent hepatocarcinogenesis. TAZ could potentially be targeted for the treatment of patients with c-MYC-driven hepatocellular carcinoma.

Downregulation of METTL6 mitigates cell progression, migration, invasion and adhesion in hepatocellular carcinoma by inhibiting cell adhesion molecules.

RNA modifications have attracted increasing interest in recent years because they have been frequently implicated in various human diseases, including cancer, highlighting the importance of dynamic post­transcriptional modifications. Methyltransferase­like 6 (METTL6) is a member of the RNA methyltransferase family that has been identified in many cancers; however, little is known about its specific role or mechanism of action. In the present study, we aimed to study the expression levels and functional role of METTL6 in hepatocellular carcinoma (HCC), and further investigate the relevant pathways. To this end, we systematically conducted bioinformatics analysis of METTL6 in HCC using gene expression data and clinical information from a publicly available dataset. The mRNA expression levels of METTL6 were significantly upregulated in HCC tumor tissues compared to that in adjacent non­tumor tissues and strongly associated with poorer survival outcomes in patients with HCC. CRISPR/Cas9­mediated knockout of METTL6 in HCC cell lines remarkably inhibited colony formation, cell proliferation, cell migration, cell invasion and cell attachment ability. RNA sequencing analysis demonstrated that knockout of METTL6 significantly suppressed the expression of cell adhesion­related genes. However, chromatin immunoprecipitation sequencing results revealed no significant differences in enhancer activities between cells, which suggests that METTL6 may regulate genes of interest post­transcriptionally. In addition, it was demonstrated for the first time that METTL6 was localized in the cytosol as detected by immunofluorescence analysis, which indicates the plausible location of RNA modification mediated by METTL6. Our findings provide further insight into the function of RNA modifications in cancer and suggest a possible role of METTL6 as a therapeutic target in HCC.

Yap-Sox9 signaling determines hepatocyte plasticity and lineage-specific hepatocarcinogenesis.

BACKGROUND & AIMS: Primary liver tumors comprise distinct subtypes. A subset of intrahepatic cholangiocarcinoma (iCCA) can arise from cell fate reprogramming of mature hepatocytes in mouse models. However, the underpinning of cell fate plasticity during hepatocarcinogenesis is still poorly understood, hampering therapeutic development for primary liver cancer. As YAP activation induces liver tumor formation and cell fate plasticity, we investigated the role of Sox9, a transcription factor downstream of Yap activation that is expressed in biliary epithelial cells (BECs), in Yap-induced cell fate plasticity during hepatocarcinogenesis. METHODS: To evaluate the function of Sox9 in YAP-induced hepatocarcinogenesis in vivo, we used several genetic mouse models of inducible hepatocyte-specific YAP activation with simultaneous Sox9 removal. Cell fate reprogramming was determined by lineage tracing and immunohistochemistry. The molecular mechanism underlying Yap and Sox9 function in hepatocyte plasticity was investigated by transcription and transcriptomic analyses of mouse and human liver tumors. RESULTS: Sox9, a marker of liver progenitor cells (LPCs) and BECs, is differentially required in YAP-induced stepwise hepatocyte programming. While Sox9 has a limited role in hepatocyte dedifferentiation to LPCs, it is required for BEC differentiation from LPCs. YAP activation in Sox9-deficient hepatocytes resulted in more aggressive HCC with enhanced Yap activity at the expense of iCCA-like tumors. Furthermore, we showed that 20% of primary human liver tumors were associated with a YAP activation signature, and tumor plasticity is highly correlated with YAP activation and SOX9 expression. CONCLUSION: Our data demonstrated that Yap-Sox9 signaling determines hepatocyte plasticity and tumor heterogeneity in hepatocarcinogenesis in both mouse and human liver tumors. We identified Sox9 as a critical transcription factor required for Yap-induced hepatocyte cell fate reprogramming during hepatocarcinogenesis. LAY SUMMARY: Sox9, a marker of liver progenitor cells and bile duct lining cells, is a downstream target of YAP protein activation. Herein, we found that YAP activation in hepatocytes leads to a transition from mature hepatocytes to liver progenitor cells and then to bile duct lining cells. Sox9 is required in the second step during mouse hepatocarcinogenesis. We also found that human YAP and SOX9 may play similar roles in liver cancers.

An artificial intelligence model to predict hepatocellular carcinoma risk in Korean and Caucasian patients with chronic hepatitis B.

BACKGROUND & AIMS: Several models have recently been developed to predict risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB). Our aims were to develop and validate an artificial intelligence-assisted prediction model of HCC risk. METHODS: Using a gradient-boosting machine (GBM) algorithm, a model was developed using 6,051 patients with CHB who received entecavir or tenofovir therapy from 4 hospitals in Korea. Two external validation cohorts were independently established: Korean (5,817 patients from 14 Korean centers) and Caucasian (1,640 from 11 Western centers) PAGE-B cohorts. The primary outcome was HCC development. RESULTS: In the derivation cohort and the 2 validation cohorts, cirrhosis was present in 26.9%-50.2% of patients at baseline. A model using 10 parameters at baseline was derived and showed good predictive performance (c-index 0.79). This model showed significantly better discrimination than previous models (PAGE-B, modified PAGE-B, REACH-B, and CU-HCC) in both the Korean (c-index 0.79 vs. 0.64-0.74; all p <0.001) and Caucasian validation cohorts (c-index 0.81 vs. 0.57-0.79; all p <0.05 except modified PAGE-B, p = 0.42). A calibration plot showed a satisfactory calibration function. When the patients were grouped into 4 risk groups, the minimal-risk group (11.2% of the Korean cohort and 8.8% of the Caucasian cohort) had a less than 0.5% risk of HCC during 8 years of follow-up. CONCLUSIONS: This GBM-based model provides the best predictive power for HCC risk in Korean and Caucasian patients with CHB treated with entecavir or tenofovir. LAY SUMMARY: Risk scores have been developed to predict the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B. We developed and validated a new risk prediction model using machine learning algorithms in 13,508 antiviral-treated patients with chronic hepatitis B. Our new model, based on 10 common baseline characteristics, demonstrated superior performance in risk stratification compared with previous risk scores. This model also identified a group of patients at minimal risk of developing HCC, who could be indicated for less intensive HCC surveillance.

Validation of a Lab-on-Chip Assay for Measuring Sorafenib Effectiveness on HCC Cell Proliferation.

Hepatocellular carcinoma (HCC) is a highly lethal cancer, and although a few drugs are available for treatment, therapeutic effectiveness is still unsatisfactory. New drugs are urgently needed for hepatocellular carcinoma (HCC) patients. In this context, reliable preclinical assays are of paramount importance to screen the effectiveness of new drugs and, in particular, measure their effects on HCC cell proliferation. However, cell proliferation measurement is a time-consuming and operator-dependent procedure. The aim of this study was to validate an engineered miniaturized on-chip platform for real-time, non-destructive cell proliferation assays and drug screening. The effectiveness of Sorafenib, the first-line drug mainly used for patients with advanced HCC, was tested in parallel, comparing the gold standard 96-well-plate assay and our new lab-on-chip platform. Results from the lab-on-chip are consistent in intra-assay replicates and comparable to the output of standard crystal violet proliferation assays for assessing Sorafenib effectiveness on HCC cell proliferation. The miniaturized platform presents several advantages in terms of lesser reagents consumption, operator time, and costs, as well as overcoming a number of technical and operator-dependent pitfalls. Moreover, the number of cells required is lower, a relevant issue when primary cell cultures are used. In conclusion, the availability of inexpensive on-chip assays can speed up drug development, especially by using patient-derived samples to take into account disease heterogeneity and patient-specific characteristics.

The transcatheter arterial chemoembolization combined with targeted nanoparticle delivering sorafenib system for the treatment of microvascular invasion of hepatocellular carcinoma.

to explore the value of transcatheter arterial chemoembolization (TACE) combined with targeted nanoparticle delivery system for sorafenib (SFB) to treat hepatocellular carcinoma (HCC) with microvascular invasion. 42 HCC patients with microvascular invasion after liver cancer surgery were selected from our hospital from December 2020 and February 2021. Patients were divided into experimental group and control group based on their willingness. Patients in experimental group (18 cases) were treated with combination therapy of TACE and Ab-SFB-NP system; while patients in control group (24 cases) took TACE and non-nano drug delivery system. There was no obvious difference in liver function and blood test results between two groups of patients before treatment and one month after treatment (P > 0.05). Three months after treatment, differences of alanine aminotransferase (ALT) were statistically significant (P < 0.05); while differences of other test results were not (P > 0.05). The disease control rate (DCR) of patients in experimental group was higher slightly (P > 0.05). The incidence of adverse reactions of patients in experimental group was lower than the control group and the differences were statistically significant (P < 0.05). After three months of TACE, the DCR in the experimental group was significantly higher compared to control group. The toxic reactions of taking SFB with Ab-SFB-NP nano-drug delivery system mainly included hand-foot syndrome, diarrhea, and bleeding, the toxic reactions were mainly at level 1 ~ 2. After symptomatic treatment, the toxicity was effectively controlled, so the security was high.