Hepatoblastoma Samples Research Articles

Abstract 1100: Fascin-1 inhibitor decreases hepatoblastoma cells tumorigenicity via YAP 1

Abstract Introduction: Hepatoblastoma (HB) is a liver tumor that arises in children. It's a sporadic malignancy that is often overly aggressive. The current treatment consists of chemotherapy. However, chemotherapy in young patients has disastrous and long-term side effects such as ototoxicity, cardiomyopathy, and infertility. Thus, alternative strategies are needed. One hint is to target the most common mutations in HB. It has been demonstrated that 90% of HB tumors are mutated for the Wnt pathway effector ß-catenin. This mutation leads to an aberrant constitutive activation of Wnt/ß-catenin signaling. Here, we investigate one of ß-catenin transcriptional targets, Fascin-1 that is found up-regulated in many tumors. Fascin1 affects actin organization into bundles, and this leads to cell migration and invasion. Whereas Fascin-1 is absent from normal hepatocytes, we found its expression associated to the poor prognosis C2 subtype of HB. In both human and murine HB samples, Fascin-1 is associated to undifferentiated tumor cells. We further demonstrated that Fascin-1 expression modulates tumor hepatocyte differentiation status through gene expression. In this study, we investigate how Fascin-1 can regulate tumor cell plasticity and whether Fascin-1 is a druggable target in HB tumors. Methods: We use two classical HB model cells Huh6 and HepG2 and 3 Patient-Derived-Xenograft cell lines. We explore the effect of Fascin-1 actin-bundling activity impairment by using inhibitors, on invasion and migration using Trans-well and wound-healing assays. We follow proliferation and cell death by Flow cytometry and investigate gene expression by PCR and reporter assay. We investigate Fascin modulation of the kinome with PamGene technology. Results: We show that the inhibition of Fascin actin-binding activity decreases cell invasion and migration as well as proliferation. We show an increase of cell death in Huh6/HepG2 cells but not in the PDX models. A cell cycle arrest is induced, and the cells become senescent. Differentiation genes are overexpressed and EMT genes are repressed. Yap expression is downregulated; Yap promoter activity is downregulated, and Yap is found translocated into the cytoplasm upon Fascin-1 inhibition. These data suggest that Fascin inhibition decreases tumors cell invasion, induces cell senescence, and favors a switch towards a differentiated and these effects on the cells are mediated via the Hippo pathway. The kinome is also modulated. Conclusion: Fascin-1 is an interesting target in hepatoblastoma, commercialized phase-2 drugs are available, and this study will confirm the potential use of those drugs in HB treatment and elucidate by which mechanism Fascin-1 inhibition impacts tumors. Citation Format: Lydia DIF, Amandine Martin, Sara Basbous, Gregoire Manaud, Violaine Moreau. Fascin-1 inhibitor decreases hepatoblastoma cells tumorigenicity via YAP 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1100.

Abstract A003: A novel treatment strategy for high-risk and relapse/refractory hepatoblastoma

Abstract Background: Patients with metastatic, treatment-refractory, and relapsed hepatoblastoma (HB) have survival rates of less than 50% due to limited treatment options. To better understand these aggressive phenotypes, our lab has developed a preclinical testing pipeline consisting of patient-derived spheroids (PDSp) and orthotopic xenograft models for chemotherapy and targeted therapy testing. We hypothesized that with this preclinical pipeline we could find an effective combination treatment strategy utilizing pan-histone deacetylase (HDAC) inhibition. Methods: RNA sequencing, microarray, NanoString, and immunohistochemistry (IHC) data of patient HB samples were analyzed for expression of all HDAC classes. HB cell lines were used to screen HDAC inhibitors. PDSp were used to screen which combination of standard chemotherapy regimens with a HDAC inhibitor. HB patient derived xenograft (PDX) mice models were developed and treated based off the lowest viability of PDSp drug screen. Mice were started on study when tumor volume reached 0.1-0.4 cm3 and were sacked when tumors reached diameter of 1.5 cm or at the end of the 6-week study period. Tumors were sectioned and evaluated for Ki67% and necrosis. Results: HDAC RNA and protein expression was elevated in HB tumors compared to normal livers. Four unique HB PDX models, including high risk, relapse and treatment refractory cases, were developed and validated from treatment-refractory patient tumors. Panobinostat (IC50 of 0.013-0.059 mM) showed strong in vitro effects with the lowest cell viability compared to other HDAC inhibitors. PDSp demonstrated the highest level of cell death with combination treatment of vincristine/irinotecan/panobinostat (VIP). After 6 week of treatment[SV1] , all four models had a limited response to VI therapy; however, showed a much improved response with VIP. All four models responded to therapy with a decrease in tumor size compared to placebo and survival to 6 weeks. Two of our four models that had showed some initial response to VI therapy demonstrated necrotic cell death, lower Ki67%, decreased serum alpha fetoprotein (AFP) and reduced tumor burden compared to paired vincristine/irinotecan (VI) and placebo. Conclusions: Utilizing a preclinical pipeline for HB, we have shown that panobinostat in combination with VI chemotherapy can induce an effective tumor response in models developed from high risk, relapse, and treatment refractory HB patients. This clinically relevant methodology can be used to screen novel targeted combination therapies with the VI backbone in order to create novel treatment strategies for future clinical trials. Citation Format: Andres F Espinoza, Roma Patel, Sai Govindu, Pavan Kureti, Bryan Armbruster, Sarah Woodfield, Sanjeev A Vasudevan. A novel treatment strategy for high-risk and relapse/refractory hepatoblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A003.

Diet-induced rewiring of the Wnt gene regulatory network connects aberrant splicing to fatty liver and liver cancer in DIAMOND mice

Several preclinical models have been recently developed for metabolic associated fatty liver disease (MAFLD) and associated hepatocellular carcinoma (HCC) but comprehensive analysis of the regulatory and transcriptional landscapes underlying disease in these models are still missing. We investigated the regulatory and transcriptional landscape in fatty livers and liver tumours from DIAMOND mice that faithfully mimic human HCC development in the context of MAFLD. RNA-sequencing and ChIP-sequencing revealed rewiring of the Wnt/β-catenin regulatory network in DIAMOND tumours, as manifested by chromatin remodelling and associated switching in the expression of the canonical TCF/LEF downstream effectors. We identified splicing as a major mechanism leading to constitutive oncogenic activation of β-catenin in a large subset of DIAMOND tumours, a mechanism that is independent on somatic mutations in the locus and that has not been previously shown. Similar splicing events were found in a fraction of human HCC and hepatoblastoma samples.

Characterisation of Aberrant Metabolic Pathways in Hepatoblastoma Using Liquid Chromatography and Tandem Mass Spectrometry (LC-MS/MS).

Hepatoblastoma (HB) is a rare childhood tumour with an evolving molecular landscape. We present the first comprehensive metabolomic analysis using untargeted and targeted liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS) of paired tumour and non-tumour surgical samples in HB patients (n = 8 pairs). This study demonstrates that the metabolomic landscape of HB is distinct from that of non-tumour (NT) liver tissue, with 35 differentially abundant metabolites mapping onto pathways such as fatty acid transport, glycolysis, the tricarboxylic acid (TCA) cycle, branched-chain amino acid degradation and glutathione synthesis. Targeted metabolomics demonstrated reduced short-chain acylcarnitines and a relative accumulation of branched-chain amino acids. Medium- and long-chain acylcarnitines in HB were similar to those in NT. The metabolomic changes reported are consistent with previously reported transcriptomic data from tumour and non-tumour samples (49 out of 54 targets) as well as metabolomic data obtained using other techniques. Gene set enrichment analysis (GSEA) from RNAseq data (n = 32 paired HB and NT samples) demonstrated a downregulation of the carnitine metabolome and immunohistochemistry showed a reduction in CPT1a (n = 15 pairs), which transports fatty acids into the mitochondria, suggesting a lack of utilisation of long-chain fatty acids in HB. Thus, our findings suggest a reduced metabolic flux in HB which is corroborated at the gene expression and protein levels. Further work could yield novel insights and new therapeutic targets.

A TGF-β-dominant chemoresistant phenotype of hepatoblastoma associated with aflatoxin exposure in children.

Hepatoblastoma (HB) is the most common liver cancer in children, posing a serious threat to children's health. Chemoresistance is the leading cause of mortality in patients with HB. A more explicit definition of the features of chemotherapy resistance in HB represents a fundamental urgent need. We performed an integrative analysis including single-cell RNA sequencing, whole-exome sequencing, and bulk RNA sequencing in 180 HB samples, to reveal genomic features, transcriptomic profiles, and the immune microenvironment of HB. Multicolor immunohistochemistry staining and in vitro experiments were performed for validation. Here, we reported four HB transcriptional subtypes primarily defined by differential expression of transcription factors. Among them, the S2A subtype, characterized by strong expression of progenitor ( MYCN , MIXL1 ) and mesenchymal transcription factors ( TWIST1 , TBX5 ), was defined as a new chemoresistant subtype. The S2A subtype showed increased TGF-β cancer-associated fibroblast and an immunosuppressive microenvironment induced by the upregulated TGF-β of HB. Interestingly, the S2A subtype enriched SBS24 signature and significantly higher serum aflatoxin B1-albumin (AFB1-ALB) level in comparison with other subtypes. Functional assays indicated that aflatoxin promotes HB to upregulate TGF-β. Furthermore, clinical prognostic analysis showed that serum AFB1-ALB is a potential indicator of HB chemoresistance and prognosis. Our studies offer new insights into the relationship between aflatoxin and HB chemoresistance and provide important implications for its diagnosis and treatment.

High-Resolution Magic-Angle-Spinning NMR in Revealing Hepatoblastoma Hallmarks.

Cancer is one of the leading causes of death in children and adolescents worldwide; among the types of liver cancer, hepatoblastoma (HBL) is the most common in childhood. Although it affects only two to three individuals in a million, it is mostly asymptomatic at diagnosis, so by the time it is detected it has already advanced. There are specific recommendations regarding HBL treatment, and ongoing studies to stratify the risks of HBL, understand the pathology, and predict prognostics and survival rates. Although magnetic resonance imaging spectroscopy is frequently used in diagnostics of HBL, high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy of HBL tissues is scarce. Using this technique, we studied the alterations among tissue metabolites of ex vivo samples from (a) HBL and non-cancer liver tissues (NCL), (b) HBL and adjacent non-tumor samples, and (c) two regions of the same HBL samples, one more centralized and the other at the edge of the tumor. It was possible to identify metabolites in HBL, then metabolites from the HBL center and the border samples, and link them to altered metabolisms in tumor tissues, highlighting their potential as biochemical markers. Metabolites closely related to liver metabolisms such as some phospholipids, triacylglycerides, fatty acids, glucose, and amino acids showed differences between the tissues.

Construction of a combined random forest and artificial neural network diagnosis model to screening potential biomarker for hepatoblastoma

The purpose of our study is to identify potential biomarkers of hepatoblastoma (HB) and further explore the pathogenesis of it. Differentially expressed genes (DEGs) were incorporated into the combined random forest and artificial neural network diagnosis model to screen candidate genes for HB. Gene set enrichment analysis (GSEA) was used to analyze the ARHGEF2. Student's t test was performed to evaluate the difference of tumor-infiltrating immune cells (TIICs) between normal and HB samples. Spearson correlation analysis was used to calculate the correlation between ARHGEF2 and TIICs. ARHGEF2, TCF3, TMED3, STMN1 and RAVER2 were screened by the new model. The GSEA of ARHGEF2 included cell cycle pathway and antigen processing presenting pathway. There were significant differences in the composition of partial TIICs between HB and normal samples (p < 0.05). ARHGEF2 was significantly correlated with memory B cells (Cor = 0.509, p < 0.05). These 5 candidate genes contribute to the molecular diagnosis and targeted therapy of HB. And we found "ARHGEF2-RhoA-Cyclin D1/CDK4/CDK6-EF2" is a key mechanism regulating cell cycle pathway in HB. This will be helpful in the treatment of HB. The occurrence of HB is related to abnormal TIICs. We speculated that memory B cells play an important role in HB.

Exploration of Potential Biomarkers and Immune Landscape for Hepatoblastoma: Evidence from Machine Learning Algorithm.

This study aimed to investigate the immune landscape in hepatoblastoma (HB) based on deconvolution methods and identify a biomarkers panel for diagnosis based on a machine learning algorithm. Firstly, we identified 277 differentially expressed genes (DEGs) and differentiated and functionally identified the modules in DEGs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and GO (gene ontology) were used to annotate these DEGs, and the results suggested that the occurrence of HB was related to DNA adducts, bile secretion, and metabolism of xenobiotics by cytochrome P450. We selected the top 10 genes for our final diagnostic panel based on the random forest tree method. Interestingly, TNFRSF19 and TOP2A were significantly down-regulated in normal samples, while other genes (TRIB1, MAT1A, SAA2-SAA4, NAT2, HABP2, CYP2CB, APOF, and CFHR3) were significantly down-regulated in HB samples. Finally, we constructed a neural network model based on the above hub genes for diagnosis. After cross-validation, the area under the ROC curve was close to 1 (AUC = 0.972), and the AUC of the validation set was 0.870. In addition, the results of single-sample gene-set enrichment analysis (ssGSEA) and deconvolution methods revealed a more active immune responses in the HB tissue. In conclusion, we have developed a robust biomarkers panel for HB patients.

Molecular networks of hepatoblastoma predisposition and oncogenesis in Beckwith-Wiedemann syndrome.

Beckwith‐Wiedemann Syndrome (BWS) is the most common human overgrowth disorder caused by structural and epigenetic changes to chromosome 11p15. Patients with BWS are predisposed to developing hepatoblastoma (HB). To better understand the mechanism of HB oncogenesis in this cancer predisposition background, we performed the first multi‐dimensional study of HB samples collected from patients diagnosed with BWS. This multi‐omic investigation of seven BWS HB and five matched nontumor BWS liver samples from 7 unique patients included examination of whole exome sequences, messenger RNA/microRNA expression, and methylation levels to elucidate the genomic, transcriptomic, and epigenomic landscape of BWS‐associated HB. We compared the transcriptional profiles of the BWS samples, both HB and nontumor, to that of control livers. Genes differentially expressed across BWS tissues were identified as BWS HB predisposition factors; this gene group included cell cycle regulators, chromatin organizers, and WNT, mitogen‐activated protein kinase (MAPK), and phosphoinositide 3‐kinase (PI3K)/AKT members. We also compared transcriptional changes associated with non‐syndromic HB carrying BWS‐like 11p15 alterations compared to those without, as well as to BWS HB. Through this analysis, we identified factors specific to 11p15‐altered HB oncogenesis, termed the BWS oncogenesis network. We propose that 11p15 alterations drive HB oncogenesis by initially dysregulating cell‐cycle regulators and chromatin organizers, including histone deacetylase 1 (HDAC1), ATP‐dependent helicase X, and F‐Box and WD repeat domain containing 7. Furthermore, we found oncogenic factors such as dickkopf WNT signaling pathway inhibitor 1 and 4, WNT16, forkhead box O3 (FOXO3), and MAPK10 are differentially expressed in 11p15‐altered HB in both the BWS and non‐syndromic backgrounds. These genes warrant further investigation as diagnostic or therapeutic targets.

Molecular signatures of aggressive pediatric liver cancer

Liver masses account for 5 to 6% of pediatric cancer, which includes hepatoblastoma (HBL) along with rare cases of hepatocellular carcinoma (HCC). The most dangerous form of pediatric liver cancer is aggressive HBL, which can be characterized by chemo-resistance and multiple nodules or metastases at diagnosis, all correlating with worse clinical prognosis. Despite intensive studies and a significant improvement in overall outcomes, very little is known about the key molecular pathways which determine the aggressiveness of pediatric liver cancer. Although genetic mutations have been reported in aggressive HBL, they represent a low level (1.9% per case) and are found mainly in two genes CTNNB1 and NRF2. Over the past 5 years, our liver biology and tumor group at Cincinnati Children's Hospital Medical Center has investigated molecular signatures of aggressive HBL by examination of fresh tissue specimens, which were studied immediately after surgery to preserve the integrity of key biochemical pathways. Summarization of these high quality HBL samples discovered several critical pathways that are specific for aggressive pediatric liver cancer. These pathways include three characteristics: Conversion of tumor suppressor proteins (TSPs) by posttranslational modifications into oncogenesActivation of specific chromosomal regions, i.e., Aggressive Liver Cancer Domains (ALCDs) within many oncogenes, resulting in increased expression of oncogenesPotential epigenetic mechanisms that open chromatin structure of oncogenes via ALCDs. This commentary summarizes our key findings and discusses development of potential ALCD-based therapeutic approaches.

LIM Homeobox-2 Suppresses Hallmarks of Adult and Pediatric Liver Cancers by Inactivating MAPK/ERK and Wnt/Beta-Catenin Pathways

Introduction: Hepatocellular carcinoma and hepatoblastoma are two liver cancers characterized by gene deregulations, chromosomal rearrangements, and mutations in Wnt/beta-catenin (Wnt) pathway-related genes. LHX2, a transcriptional factor member of the LIM homeobox gene family, has important functions in embryogenesis and liver development. LHX2 is oncogenic in many solid tumors and leukemia, but its role in liver cancer is unknown. Methods: We analyzed the expression of LHX2 in hepatocellular carcinoma and hepatoblastoma samples using various transcriptomic datasets and biological samples. The role of LHX2 was studied using lentiviral transduction, in vitro cell-based assays (growth, migration, senescence, and apoptosis), molecular approaches (phosphokinase arrays and RNA-seq), bioinformatics, and two in vivo models in chicken and Xenopus embryos. Results: We found a strong connection between LHX2 downregulation and Wnt activation in these two liver cancers. In hepatoblastoma, LHX2 downregulation correlated with multiple poor outcome parameters including higher patient age, intermediate- and high-risk tumors, and low patient survival. Forced expression of LHX2 reduced the proliferation, migration, and survival of liver cancer cells in vitro through the inactivation of MAPK/ERK and Wnt signals. In vivo, LHX2 impeded the development of tumors in chick embryos and repressed the Wnt pathway in Xenopus embryos. RNA-sequencing data and bioinformatic analyses confirmed the deregulation of many biological functions and molecular processes associated with cell migration, cell survival, and liver carcinogenesis in LHX2-expressing hepatoma cells. At a mechanistic level, LHX2 mediated the disassembling of beta-catenin/T-cell factor 4 complex and induced expression of multiple inhibitors of Wnt (e.g., TLE/Groucho) and MAPK/ERK (e.g., DUSPs) pathways. Conclusion: Collectively, our findings demonstrate a tumor suppressive function of LHX2 in adult and pediatric liver cancers.

Integrative Analysis of DNA Methylation and Gene Expression Profiling Data Reveals Candidate Methylation-Regulated Genes in Hepatoblastoma

PurposeThis study aimed to identify novel methylation-regulated genes as diagnostic biomarkers and therapeutic targets for hepatoblastoma (HB).Materials and MethodsThe DNA methylation data of 19 HB tumor samples and 10 normal liver samples from the GSE78732 dataset and gene expression profiling data of 53 HB tumor samples and 14 normal liver samples from the GSE131329 dataset and 31 HB tumor samples and 32 normal liver samples from the GSE133039 dataset were downloaded form the Gene Expression Omnibus database. Next, differentially methylated genes (DMGs) and differentially expressed genes (DEGs) were identified. Venn diagrams were used to identify methylation-regulated genes. The VarElect online tool was selected to identify key methylation-regulated genes, and a protein–protein interaction (PPI) network was constructed to show the interactions among key methylation-regulated genes and DEGs. Finally, Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed to investigate the potential regulatory mechanisms of key methylation-regulated genes.ResultsA total of 457 DMGs and 1597 DEGs were identified between the HB and normal liver samples. After DMGs and DEGs overlapping, 22 hypomethylated and upregulated genes and 19 hypermethylated and downregulated genes in HB were screened. Survival analysis revealed that 13 methylation-regulated genes were associated with the prognosis of liver cancer. Moreover, SPP1, UHRF1, and HEY1 were selected as the key DNA methylation-regulated genes. The PPI network revealed that all of them could affect TP53, while both UHRF1 and HEY1 could influence BMP4. Enrichment analysis suggested that the DEGs were involved in TP53-related pathways, including the cell cycle and p53 signaling pathway. Finally, SPP1, UHRF1, and HEY1 were hypomethylated and upregulated in the HB samples compared with those in the normal liver samples.ConclusionSPP1, UHRE1, and HEY1 may play important roles in HB and be used as biomarkers for its diagnosis and treatment.

The differential expression of perilipin-2 in hepatoblastoma and its association with prognosis.

Perilipin-2, a lipid droplet (LD) coating protein, has been found to be involved in cancer progression. However, its role in hepatoblastoma (HB) is undefined. We collected 87 HB samples and the corresponding clinical data. Immunohistochemistry (IHC) staining was performed to detect perilipin-2 and the association of the perilipin-2 expression with clinical characteristics and prognosis was analyzed. The expression of perilipin-2 was increased in fetal HB components in comparison to embryonal HB components. The predominant staining pattern was vesicular in fetal HB cells, while it was granular in embryonal HB cells. Furthermore, strong expression of perilipin-2 was associated with the histopathological type of fetal predominant HB. Although event-free survival (EFS) did not differ to a statistically significant extent between the strong and weak expression groups in a univariate survival analysis, a multivariate survival analysis revealed that EFS was significantly improved in the strong perilipin-2 expression group. In conclusion, perilipin-2 is differentially expressed in HB and the strong expression of perilipin-2 predicts a better prognosis.

A Comprehensive Genomic Analysis Constructs miRNA-mRNA Interaction Network in Hepatoblastoma.

Hepatoblastoma (HB) is a rare disease but nevertheless the most common hepatic tumor in the pediatric population. For patients with advanced HB, the prognosis is dismal and there are limited therapeutic options. Multiple microRNAs (miRNAs) were reported to be involved in HB development, but the miRNA–mRNA interaction network in HB remains elusive. Through a comparison between HB and normal liver samples in the GSE131329 dataset, we detected 580 upregulated differentially expressed mRNAs (DE-mRNAs) and 790 downregulated DE-mRNAs. As for the GSE153089 dataset, the first cluster of differentially expressed miRNAs (DE-miRNAs) were detected between fetal-type tumor and normal liver groups, while the second cluster of DE-miRNAs were detected between embryonal-type tumor and normal liver groups. Through the intersection of these two clusters of DE-miRNAs, 33 upregulated hub miRNAs, and 12 downregulated hub miRNAs were obtained. Based on the respective hub miRNAs, the upstream transcription factors (TFs) were detected via TransmiR v2.0, while the downstream target genes were predicted via miRNet database. The intersection of target genes of respective hub miRNAs and corresponding DE-mRNAs contributed to 250 downregulated candidate genes and 202 upregulated candidate genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses demonstrated the upregulated candidate genes mainly enriched in the terms and pathways relating to the cell cycle. We constructed protein–protein interaction (PPI) network, and obtained 211 node pairs for the downregulated candidate genes and 157 node pairs for the upregulated candidate genes. Cytoscape software was applied for visualizing the PPI network and respective top 10 hub genes were identified using CytoHubba. The expression values of hub genes in the PPI network were subsequently validated through Oncopression database followed by quantitative real-time polymerase chain reaction (qRT-PCR) in HB and matched normal liver tissues, resulting in six significant downregulated genes and seven significant upregulated genes. The miRNA–mRNA interaction network was finally constructed. In conclusion, we uncover various miRNAs, TFs, and hub genes as potential regulators in HB pathogenesis. Additionally, the miRNA–mRNA interaction network, PPI modules, and pathways may provide potential biomarkers for future HB theranostics.

Hepatoblastoma: glutamine depletion hinders cell viability in the embryonal subtype but high GLUL expression is associated with better overall survival

PurposeGlutamine plays an important role in cell viability and growth of various tumors. For the fetal subtype of hepatoblastoma, growth inhibition through glutamine depletion was shown. We studied glutamine depletion in embryonal cell lines of hepatoblastoma carrying different mutations. Since asparagine synthetase was identified as a prognostic factor and potential therapeutic target in adult hepatocellular carcinoma, we investigated the expression of its gene ASNS and of the gene GLUL, encoding for glutamine synthetase, in hepatoblastoma specimens and cell lines and investigated the correlation with overall survival.MethodsWe correlated GLUL and ASNS expression with overall survival using publicly available microarray and clinical data. We examined GLUL and ASNS expression by RT-qPCR and by Western blot analysis in the embryonal cell lines Huh-6 and HepT1, and in five hepatoblastoma specimens. In the same cell lines, we investigated the effects of glutamine depletion. Hepatoblastoma biopsies were examined for histology and CTNNB1 mutations.ResultsHigh GLUL expression was associated with a higher median survival time. Independent of mutations and histology, hepatoblastoma samples showed strong GLUL expression and glutamine synthesis. Glutamine depletion resulted in the inhibition of proliferation and of cell viability in both embryonal hepatoblastoma cell lines. ASNS expression did not correlate with overall survival.ConclusionGrowth inhibition resulting from glutamine depletion, as described for the hepatoblastoma fetal subtype, is also detected in established embryonal hepatoblastoma cell lines carrying different mutations. At variance with adult hepatocellular carcinoma, in hepatoblastoma asparagine synthetase has no prognostic significance.

Identification of Ten Core Hub Genes as Potential Biomarkers and Treatment Target for Hepatoblastoma.

BackgroundThis study aimed to systematically investigate gene signatures for hepatoblastoma (HB) and identify potential biomarkers for its diagnosis and treatment.Materials and MethodsGSE131329 and GSE81928 were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between hepatoblastoma and normal samples were identified using the Limma package in R. Then, the similarity of network traits between two sets of genes was analyzed by weighted gene correlation network analysis (WGCNA). Cytoscape was used to visualize and select hub genes. PPI network of hub genes was construed by Cytoscape. GO enrichment and KEGG pathway analyses of hub genes were carried out using ClueGO. The random forest classifier was constructed based on the hub genes using the GSE131329 dataset as the training set, and its reliability was validated using the GSE81928 dataset. The resulting core hub genes were combined with the InnateDB database to identify the innate core genes.ResultsA total of 4244 DEGs in HB were identified. WGCNA identified four modules that were significantly correlated with the disease status. A total of 114 hub genes were obtained within the top 20 genes of each node rank. 6982 relation pairs and 3700 nodes were contained in the PPI network of 114 hub genes. GO enrichment and KEGG pathway analyses of hub genes were focused on MAPK, cell cycle, p53, and other crucial pathways involved in HB. A random forest classifier was constructed using the 114 hub genes as feature genes, resulting in a 95.5% true positive rate when classifying HB and normal samples. A total of 35 core hub genes were obtained through the mean decrease in accuracy and mean decrease Gini of the random forest model. The classification efficiency of the random forest model was 81.4%. Finally, CDK1, TOP2A, ADRA1A, FANCI, XRCC1, TPX2, CCNB2, CDK4, GLYATL1, and CFHR3 were identified by cross-comparison with the InnateDB database.ConclusionOur study established a random forest classifier that identified 10 core genes in HB. These findings may be beneficial for the diagnosis, prediction, and targeted therapy of HB.

Small Molecule Cjoc42 Improves Chemo-Sensitivity and Increases Levels of Tumor Suppressor Proteins in Hepatoblastoma Cells and in Mice by Inhibiting Oncogene Gankyrin.

Objective: Relapsed hepatoblastoma (HBL) and upfront hepatocellular carcinoma (HCC) are notoriously chemoresistant tumors associated with poor outcomes. Gankyrin (Gank) is a known oncogene that is overexpressed in pediatric liver cancer and implicated in chemo-resistance. The goal of this study was to evaluate if the Gank-tumor suppressor axis is activated in chemoresistant hepatoblastoma patients and examine if an inhibitor of Gank, Cjoc42, might improve the chemosensitivity of cancer cells. Methods: Expression of Gank and its downstream targets were examined in fresh human HBL samples using immunostaining, QRT-PCR, and Western Blot. Cancer cells, Huh6 (human HBL) and Hepa1c1c7 (mouse HCC) were treated with Cjoc42 and with Cjoc42 in combination with cisplatin or doxorubicin. Cell proliferation, apoptosis, and chemoresistance were examined. To examine activities of Cjoc42 in vivo, mice were treated with different doses of Cjoc42, and biological activities of Gank and cytotoxicity of Cjoc42 were tested. Results: Elevation of Gank and Gank-mediated elimination of TSPs are observed in patients with minimal necrosis after chemotherapy and relapsed disease. The treatment of Huh6 and Hepa1c1c7 with Cjoc42 was not cytotoxic; however, in combination with cisplatin or doxorubicin, Cjoc42 caused a significant increase in cytotoxicity compared to chemotherapy alone with increased apoptosis. Examination of Cjoc42 in WT mice showed that Cjoc42 is well tolerated without systemic toxicity, and levels of tumor suppressors CUGBP1, Rb, p53, C/EBPα, and HNF4α are increased by blocking their Gank-dependent degradation. Conclusions: Our work shows that Cjoc42 might be a promising adjunct to chemotherapy for the treatment of severe pediatric liver cancer and presents mechanisms by which Cjoc42 increases chemo-sensitivity.

Loss of Apc Cooperates with Activated Oncogenes to Induce Liver Tumor Formation in Mice

Hepatocellular carcinoma (HCC) and hepatoblastoma are the major types of primary liver cancer in adulthood and childhood, respectively. Wnt/β-catenin signaling deregulation is one of the most frequent genetic events in hepatocarcinogenesis. APC regulator of WNT signaling pathway (APC) encodes an inhibitor of the Wnt cascade and acts as a tumor suppressor. Germline defects of the APC gene lead to familial adenomatous polyposis, and its somatic mutations occur in multiple tumor types. However, the contribution of APC in hepatocarcinogenesis remains unclear. Therefore, APC mutations and expression patterns were examined in human HCC and hepatoblastoma samples. Whether loss of Apc alone or in cooperation with other oncogenes triggers liver tumor development invivo was also investigated. sgApc alone could not drive liver tumor formation, but synergized with activated oncogenes (YapS127A, TazS89A, and c-Met) to induce hepatocarcinogenesis. Mechanistically, Apc deletion induced the activation of β-catenin and its downstream targets in mouse liver tumors. Furthermore, Ctnnb1 ablation or TCF4-mediated transcription blockade completely prevented liver tumor formation, indicating the requirement of a functional β-catenin pathway for loss of Apc-driven hepatocarcinogenesis. This study shows that a subset of HCC patients with loss-of-function APC mutations mightbenefit from therapeutic strategies targeting the Wnt/β-catenin pathway.

Integrated multiomics analysis of hepatoblastoma unravels its heterogeneity and provides novel druggable targets

Although hepatoblastoma is the most common pediatric liver cancer, its genetic heterogeneity and therapeutic targets are not well elucidated. Therefore, we conducted a multiomics analysis, including mutatome, DNA methylome, and transcriptome analyses, of 59 hepatoblastoma samples. Based on DNA methylation patterns, hepatoblastoma was classified into three clusters exhibiting remarkable correlation with clinical, histological, and genetic features. Cluster F was largely composed of cases with fetal histology and good outcomes, whereas clusters E1 and E2 corresponded primarily to embryonal/combined histology and poor outcomes. E1 and E2, albeit distinguishable by different patient age distributions, were genetically characterized by hypermethylation of the HNF4A/CEBPA-binding regions, fetal liver-like expression patterns, upregulation of the cell cycle pathway, and overexpression of NQO1 and ODC1. Inhibition of NQO1 and ODC1 in hepatoblastoma cells induced chemosensitization and growth suppression, respectively. Our results provide a comprehensive description of the molecular basis of hepatoblastoma and rational therapeutic strategies for high-risk cases.

Human endogenous retrovirus-K mRNA expression and genomic alignment data in hepatoblastoma.

Human Endogenous Retroviruses are a class of genomic elements that are the result of ancient retroviral infection of the human germline. Many are biologically active elements that have been implicated in multiple diseases including cancer. The most recent class to invade the human genome is the HERV-K(HML-2) (HERV-K) family. Approximately 90 HERV-K proviruses and many smaller elements have been identified to date in the human genome. Additional proviruses are continually being discovered with the rapid advancement of deep-sequencing and long-read sequencing technologies. HERV-K proviruses are poorly annotated in human transcriptome databases making their analysis in RNA-seq data difficult. To enable analysis, we compiled the sequences of 91 HERV-K proviruses identified in NCBI GenBank (ID JN675007-JN675097) and created a proviral alignment tool for visualizing RNA-seq reads aligned across individual proviruses. This allowed us to analyse publicly available RNA-seq data from 10 hepatoblastoma samples and 3 normal liver controls (GEO Accession ID: GSE89775). This data report includes the raw FASTA sequence files of the HERV-K proviruses from NCBI, a differential gene expression list between hepatoblastoma samples, and genomic alignment figures from 5 HERV-K proviruses identified as differentially expressed in the companion research article “Upregulation of Human Endogenous Retrovirus-K (HML-2) mRNAs in hepatoblastoma: Identification of potential new immunotherapeutic targets and biomarkers [1]. The data provided here are available for other research groups interested in evaluating individual HERV-K proviral expression using RNA-seq data. Furthermore, the data analysis is highly flexible and will accommodate the addition of other HERV-K proviruses.