FOXM1 and CHD4 expression is associated with chemoresistance in hepatoblastoma.

Hepatoblastoma (HB) is the most common malignant liver tumor in childhood. Although pre-operative cisplatin (CDDP)-based chemotherapy is often used in cases of HB, about 20％ of HB patients exhibit resistance to CDDP. Forkhead box protein M1 (FOXM1) and chromo-domain-helicase-DNA-binding protein 4 (CHD4) have been associated with CDDP resistance in various tumors. We here analyzed the immunohistochemical expression of FOXM1 and CHD4 in HB specimens of 33 patients (mean age: 20 months) post-chemotherapy. The differentiation of specimens was assessed using the digital pathology software QuPath®, and then the relation between the FOXM1 or CHD4 expression and the differentiation and various other clinicopathological parameters was investigated. The histological type was epithelial in 19 cases (57.6%) and mixed epithelial and mesenchymal in 14 cases (42.4%). Nine cases had only a fetal component, 1 case had only an embryonal component, 22 cases had both fetal and embryonal components, and 1 case had no viable tumor. Both the FOXM1 and CHD4 immunoexpressions were found significantly more frequently in the embryonal than fetal components (p<0.0001 and p<0.0001, respectively). Regarding chemotherapy efficacy, the alpha-fetoprotein (AFP) level after chemotherapy was correlated with both the imaging shrinkage rate (R=-0.52) and histological residual rate (the percentage of the viable tumors of HB after chemotherapy)(R=0.62). High FOXM1 score was correlated with a high-postoperative AFP value (p<0.01) and a low AFP attenuation rate (p<0.05), but the FOXM1 score was not correlated with the imaging shrinkage rate (p=0.4418) or histological residual rate (p=0.4418). High CHD4 score showed a nonsignificant trend toward correlation with high postoperative AFP value (p=0.0849) and was not significantly correlated with the other parameters. Collectively, our results showed that FOXM1 expression may be useful in evaluating the response to CDDP-based chemotherapeutic regimens. Accurate measurement of FOXM1 expression by our scoring system using QuPath® is important in cases with mixed HB components of various differentiation levels.

Intratumoral CXCR4hi neutrophils display ferroptotic and immunosuppressive signatures in hepatoblastoma.

Pediatric hepatoblastoma (HB) is the most common primary liver malignancy in infants and children. With great diversity and plasticity, tumor-infiltrating neutrophils were one of the most determining factors for poor prognosis in many malignant tumors. In this study, through bulk RNA sequencing for sorted blood and tumor-infiltrated neutrophils and comparison of neutrophils in tumor and para-tumor tissue by single-cell sequencing, we found that intratumoral neutrophils were composed of heterogenous functional populations at different development stages. Our study showed that terminally differentiated neutrophils with active ferroptosis prevailed in tumor tissue, whereas, in para-tumor, pre-fate naïve neutrophils were dominant and ferroptotic neutrophils dispersed in a broad spectrum of cell maturation. Gene profiling and in vitro T-cell coculture experiment confirmed that one of main functional intratumoral neutrophils was mainly immunosuppressive, which relied on the activation of ferroptosis. Combining the bulk RNA-seq, scRNA-seq data, and immunochemistry staining of tumor samples, CXCL12/CXCR4 chemotaxis pathway was suggested to mediate the migration of neutrophils in tumors as CXCR4 highly expressed by intratumoral neutrophils and its ligand CXCL12 expressed much higher level in tumor than that in para-tumor. Moreover, our study pinpointed that infiltrated CXCR4hi neutrophils, regardless of their differential distribution of cell maturation status in HB tumor and para-tumor regions, were the genuine perpetrators for immune suppression. Our data characterized the ferroptosis-dependent immunosuppression energized by intratumoral CXCR4 expression neutrophils and suggest a potential cell target for cancer immunotherapies.

Cellular origin and molecular mechanisms of lung metastases in patients with aggressive hepatoblastoma.

BACKGROUND AND AIMS: Lung metastases are the most threatening signs for patients with aggressive hepatoblastoma (HBL). Despite intensive studies, the cellular origin and molecular mechanisms of lung metastases in patients with aggressive HBL are not known. The aims of these studies were to identify metastasis-initiating cells in primary liver tumors and to determine if these cells are secreted in the blood, reach the lung, and form lung metastases. APPROACH: We have examined mechanisms of activation of key oncogenes in primary liver tumors and lung metastases and the role of these mechanisms in the appearance of metastasis-initiating cells in patients with aggressive HBL by RNA-Seq, immunostaining, chromatin immunoprecipitation, Real-Time Quantitative Reverse Transcription PCR and western blot approaches. Using a protocol that mimics the exit of metastasis-initiating cells from tumors, we generated 16 cell lines from liver tumors and 2 lines from lung metastases of patients with HBL. RESULTS: We found that primary HBL liver tumors have a dramatic elevation of neuron-like cells and cancer-associated fibroblasts and that these cells are released into the bloodstream of patients with HBL and found in lung metastases. In the primary liver tumors, the ph-S675-ß-catenin pathway activates the expression of markers of cancer-associated fibroblasts; while the ZBTB3-SRCAP pathway activates the expression of markers of neurons via cancer-enhancing genomic regions/aggressive liver cancer domains leading to a dramatic increase of cancer-associated fibroblasts and neuron-like cells. Studies of generated metastasis-initiating cells showed that these cells proliferate rapidly, engage in intense cell-cell interactions, and form tumor clusters. The inhibition of ß-catenin in HBL/lung metastases-released cells suppresses the formation of tumor clusters. CONCLUSIONS: The inhibition of the ß-catenin-cancer-enhancing genomic regions/aggressive liver cancer domains axis could be considered as a therapeutic approach to treat/prevent lung metastases in patients with HBL.

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

BACKGROUND AND AIMS: 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. APPROACH AND RESULTS: 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-ß cancer-associated fibroblast and an immunosuppressive microenvironment induced by the upregulated TGF-ß 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-ß. Furthermore, clinical prognostic analysis showed that serum AFB1-ALB is a potential indicator of HB chemoresistance and prognosis. CONCLUSIONS: Our studies offer new insights into the relationship between aflatoxin and HB chemoresistance and provide important implications for its diagnosis and treatment.

Identification and experimental validation of druggable epigenetic targets in hepatoblastoma.

BACKGROUND & AIMS: Hepatoblastoma (HB) is the most frequent childhood liver cancer. Patients with aggressive tumors have limited therapeutic options; therefore, a better understanding of HB pathogenesis is needed to improve treatment. HBs have a very low mutational burden; however, epigenetic alterations are increasingly recognized. We aimed to identify epigenetic regulators consistently dysregulated in HB and to evaluate the therapeutic efficacy of their targeting in clinically relevant models. METHODS: We performed a comprehensive transcriptomic analysis of 180 epigenetic genes. Data from fetal, pediatric, adult, peritumoral (n = 72) and tumoral (n = 91) tissues were integrated. Selected epigenetic drugs were tested in HB cells. The most relevant epigenetic target identified was validated in primary HB cells, HB organoids, a patient-derived xenograft model, and a genetic mouse model. Transcriptomic, proteomic and metabolomic mechanistic analyses were performed. RESULTS: Altered expression of genes regulating DNA methylation and histone modifications was consistently observed in association with molecular and clinical features of poor prognosis. The histone methyltransferase G9a was markedly upregulated in tumors with epigenetic and transcriptomic traits of increased malignancy. Pharmacological targeting of G9a significantly inhibited growth of HB cells, organoids and patient-derived xenografts. Development of HB induced by oncogenic forms of ß-catenin and YAP1 was ablated in mice with hepatocyte-specific deletion of G9a. We observed that HBs undergo significant transcriptional rewiring in genes involved in amino acid metabolism and ribosomal biogenesis. G9a inhibition counteracted these pro-tumorigenic adaptations. Mechanistically, G9a targeting potently repressed the expression of c-MYC and ATF4, master regulators of HB metabolic reprogramming. CONCLUSIONS: HBs display a profound dysregulation of the epigenetic machinery. Pharmacological targeting of key epigenetic effectors exposes metabolic vulnerabilities that can be leveraged to improve the treatment of these patients. IMPACT AND IMPLICATIONS: In spite of recent advances in the management of hepatoblastoma (HB), treatment resistance and drug toxicity are still major concerns. This systematic study reveals the remarkable dysregulation in the expression of epigenetic genes in HB tissues. Through pharmacological and genetic experimental approaches, we demonstrate that the histone-lysine-methyltransferase G9a is an excellent drug target in HB, which can also be harnessed to enhance the efficacy of chemotherapy. Furthermore, our study highlights the profound pro-tumorigenic metabolic rewiring of HB cells orchestrated by G9a in coordination with the c-MYC oncogene. From a broader perspective, our findings suggest that anti-G9a therapies may also be effective in other c-MYC-dependent tumors.

The Effects of Protein Phosphatase 2A Activation with Novel Tricyclic Sulfonamides on Hepatoblastoma.

BACKGROUND: The tumor suppressor, protein phosphatase 2A (PP2A), is downregulated in hepatoblastoma. We aimed to examine the effects of two novel compounds of the tricyclic sulfonamide class, ATUX-3364 (3364) and ATUX-8385 (8385), designed to activate PP2A without causing immunosuppression, on human hepatoblastoma. METHODS: An established human hepatoblastoma cell line, HuH6, and a human hepatoblastoma patient-derived xenograft, COA67, were treated with increasing doses of 3364 or 8385, and viability, proliferation, cell cycle and motility were investigated. Cancer cell stemness was evaluated by real-time PCR and tumorsphere forming ability. Effects on tumor growth were examined using a murine model. RESULTS: Treatment with 3364 or 8385 significantly decreased viability, proliferation, cell cycle progression and motility in HuH6 and COA67 cells. Both compounds significantly decreased stemness as demonstrated by decreased abundance of OCT4, NANOG, and SOX2 mRNA. The ability of COA67 to form tumorspheres, another sign of cancer cell stemness, was significantly diminished by 3364 and 8385. Treatment with 3364 resulted in decreased tumor growth in vivo. CONCLUSION: Novel PP2A activators, 3364 and 8385, decreased hepatoblastoma proliferation, viability, and cancer cell stemness in vitro. Animals treated with 3364 had decreased tumor growth. These data provide evidence for further investigation of PP2A activating compounds as hepatoblastoma therapeutics.

KDM1A drives hepatoblastoma progression by activating the Wnt/ß-catenin pathway through inhibition of DKK3 transcription.

This study is to explore the mechanism of KDM1A-regulated hepatoblastoma (HB) development. Cancerous and paracancer tissues of 30 HB patients were collected for detection of KDM1A and DKK3 expression. HuH-6 and HepG2 cells were subjected to assays of cellular activities after treatment with sh-KDM1A, sh-DKK3, and/or XAV-939 (an inhibitor of the Wnt/ß-catenin pathway). Chromatin immunoprecipitation was used to determine the interaction of KDM1A with DKK3. Nude mice were injected with HuH-6 cells in which KDM1A was knocked down. KDM1A was highly expressed and DKK3 was lowly expressed in HB patients. Knockdown of KDM1A reduced the proliferative and invasive capabilities of HepG2 and HuH-6 cells and accelerated the cell apoptosis; these influences were nullified by knockdown of DKK3. KDM1A inhibited DKK3 transcription by reducing H3 methylation. XAV-939 treatment inhibited the development of HepG2 and HuH-6 cells in which KDM1A and DKK3 were both knocked down. Knockdown of KDM1A reduced the tumor mass, inactivated the Wnt/ß-catenin signaling, and increased the expression of DKK3 in nude mice. KDM1A stimulates HB development by activating the Wnt/ß-catenin pathway through inhibition of DKK3 transcription.

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

PURPOSE: The purpose of our study is to identify potential biomarkers of hepatoblastoma (HB) and further explore the pathogenesis of it. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Immunohistochemical expression of beta-catenin in hepatoblastoma and its clinical significance.

Background: Primary hepatic malignancies account for 0.5-2% of all solid tumours in childhood. Hepatoblastoma, a rare embryonic tumour in the general population, represents the most frequent primary hepatic malignancy in the paediatric age group, with an incidence of one new case per million under 15 years of age, median age at diagnosis being 1 year. Aberrant activation of the Wnt/beta-catenin signalling pathway is likely to result in tumorigenesis of hepatoblastoma. The nuclear and intra-cytoplasmic accumulations of beta-catenin correlate with the likely prognosis of the disease. Nuclear expression of beta catenin is associated with a shorter survival, higher stage, and seen in embryonal/undifferentiated types. Aim: To study the expression of beta-catenin in hepatoblastoma by immunohistochemistry and correlate it with the tumour histology and survival outcome. Materials & Methods: This is a retrospective study of 11 children over a period of 5 years with the diagnosis of hepatoblastoma. These children underwent partial hepatectomy or liver transplantation at the Department of Paediatric Surgery. The clinical, histological and survival data were collected. Immunohistochemical analysis with beta-catenin was done and analysed. Results: Mean birth weight of the children was 2.75kg.63.6% had an epithelial type of histology.Beta catenin expression by IHC was studied in 11 cases and found to be positive in 4 cases. Nuclear positivity was noted in 2/4 cases of embryonal type and Cytoplasmic and membranous positivity was seen in the other 2/4 cases. Normal liver showed a membranous pattern of positivity in one case. Negative staining was seen in 6 out of 11 cases. Conclusion: Beta catenin is considered to be an useful tool for assessing the prognosis of patients with hepatoblastoma and its expression is associated with a poor survival outcome. There are no validated biomarkers for prognosis so far. However, larger studies incorporating molecular profiling is warranted to establish prognostic factors for planning effective treatment strategies.

Hepatoblastomas with carcinoma features represent a biological spectrum of aggressive neoplasms in children and young adults.

BACKGROUND & AIMS: Hepatoblastoma (HB) and hepatocellular carcinoma (HCC) are the predominant liver cancers in children, though their respective treatment options and associated outcomes differ dramatically. Risk stratification using a combination of clinical, histological, and molecular parameters can improve treatment selection, but it is particularly challenging for tumors with mixed histological features, including those in the recently created hepatocellular neoplasm not otherwise specified (HCN NOS) provisional category. We aimed to perform the first molecular characterization of clinically annotated cases of HCN NOS. METHODS: We tested whether these histological features are associated with genetic alterations, cancer gene dysregulation, and outcomes. Namely, we compared the molecular features of HCN NOS, including copy number alterations, mutations, and gene expression profiles, with those in other pediatric hepatocellular neoplasms, including HBs and HCCs, as well as HBs demonstrating focal atypia or pleomorphism (HB FPAs), and HBs diagnosed in older children (>8). RESULTS: Molecular profiles of HCN NOS and HB FPAs revealed common underlying biological features that were previously observed in HCCs. Consequently, we designated these tumor types collectively as HBs with HCC features (HBCs). These tumors were associated with high mutation rates (∼3 somatic mutations/Mb) and were enriched with mutations and alterations in key cancer genes and pathways. In addition, recurrent large-scale chromosomal gains, including gains of chromosomal arms 2q (80%), 6p (70%), and 20p (70%), were observed. Overall, HBCs were associated with poor clinical outcomes. CONCLUSIONS: Our study indicates that histological features seen in HBCs are associated with combined molecular features of HB and HCC, that HBCs are associated with poor outcomes irrespective of patient age, and that transplanted patients are more likely to have good outcomes than those treated with chemotherapy and surgery alone. These findings highlight the importance of molecular testing and early therapeutic intervention for aggressive childhood hepatocellular neoplasms. LAY SUMMARY: We molecularly characterized a class of histologically aggressive childhood liver cancers and showed that these tumors are clinically aggressive and that their observed histological features are associated with underlying recurrent molecular features. We proposed a diagnostic algorithm to identify these cancers using a combination of histological and molecular features, and our analysis suggested that these cancers may benefit from specialized treatment strategies that may differ from treatment guidelines for other childhood liver cancers.

M-CSFR expression in the embryonal component of hepatoblastoma and cell-to-cell interaction between macrophages and hepatoblastoma.

Tumor-associated macrophages (TAMs) have protumor functions in various cancers. However, their significance in hepatoblastoma, the most common liver tumor in children, remains unclear. The aim of this study was to explore the potential roles of TAMs in hepatoblastoma. Immunohistochemical analysis revealed that the density of CD204-positive TAMs was significantly higher in the embryonal component than in other histological subtypes of hepatoblastoma. An in vitro co-culture study with Huh6 cells and human monocyte-derived macrophages (HMDMs) showed that macrophage-colony-stimulating factor receptor (M-CSFR) was strongly up-regulated in the Huh6 cells that were directly co-cultured with HMDMs. The expressions of M-CSFR ligands (interleukin-34 and M-CSF) were also increased by co-culture with HMDMs. The proliferation of HepG2 cells (another hepatoblastoma cell line expressing M-CSFR) was inhibited by an M-CSFR inhibitor. M-CSFR was found to be highly expressed in the embryonal component and in recurrent lesions. The number of CD204-positive macrophages was also higher in the M-CSFR-positive areas than in the M-CSFR-negative areas. Thus, M-CSFR expression appeared to be induced by cell-cell contact with macrophages in hepatoblastoma cells, and M-CSFR inhibitor is potentially effective against M-CSFR-positive hepatoblastoma, especially recurrent cases.

HepT1-derived murine models of high-risk hepatoblastoma display vascular invasion, metastasis, and circulating tumor cells.

Hepatoblastoma (HB) is the most common pediatric primary liver malignancy, and survival for high-risk disease approaches 50%. Mouse models of HB fail to recapitulate hallmarks of high-risk disease. The aim of this work was to generate murine models that show high-risk features including multifocal tumors, vascular invasion, metastasis, and circulating tumor cells (CTCs). HepT1 cells were injected into the livers or tail veins of mice, and tumor growth was monitored with magnetic resonance and bioluminescent imaging. Blood was analyzed with fluorescence-activated cell sorting to identify CTCs. Intra- and extra-hepatic tumor samples were harvested for immunohistochemistry and RNA and DNA sequencing. Cell lines were grown from tumor samples and profiled with RNA sequencing. With intrahepatic injection of HepT1 cells, 100% of animals grew liver tumors and showed vascular invasion, metastasis, and CTCs. Mutation profiling revealed genetic alterations in seven cancer-related genes, while transcriptomic analyses showed changes in gene expression with cells that invade vessels. Tail vein injection of HepT1 cells resulted in multifocal, metastatic disease. These unique models will facilitate further meaningful studies of high-risk HB. This article has an associated First Person interview with the first author of the paper.

Deleting the ß-catenin degradation domain in mouse hepatocytes drives hepatocellular carcinoma or hepatoblastoma-like tumor growth.

BACKGROUND & AIMS: One-third of hepatocellular carcinomas (HCCs) harbor mutations activating the ß-catenin pathway, predominantly via mutations in the CTNNB1 gene itself. Mouse models of Apc loss-of-function are widely used to mimic ß-catenin-dependent tumorigenesis. Given the low prevalence of APC mutations in human HCCs, we aimed to generate liver tumors through CTNNB1 exon 3 deletion (ßcatΔex3). We then compared ßcatΔex3 liver tumors with liver tumors generated via frameshift in exon 15 of Apc (Apcfs-ex15). METHODS: We used hepatocyte-specific and inducible mouse models generated through either a Cre-Lox or a CRISPR/Cas9 approach using adeno-associated virus vectors. Tumors generated by the Cre-Lox models were phenotypically analyzed using immunohistochemistry and were selected for transcriptomic analysis by RNA-sequencing (RNAseq). Mouse RNAseq data were compared to human RNAseq data (8 normal tissues, 48 HCCs, 9 hepatoblastomas) in an integrative analysis. Tumors generated via CRISPR were analyzed using DNA sequencing and immuno-histochemistry. RESULTS: Mice with CTNNB1 exon 3 deletion in hepatocytes developed liver tumors indistinguishable from Apcfs-ex15 liver tumors. Both Apcfs-ex15 and ßcatΔex3 mouse models induced growth of phenotypically distinct tumors (differentiated or undifferentiated). Integrative analysis of human and mouse tumors showed that differentiated mouse tumors cluster with well-differentiated human CTNNB1-mutated tumors. Conversely, undifferentiated mouse tumors cluster with human mesenchymal hepatoblastomas and harbor activated YAP signaling. CONCLUSION: Apcfs-ex15 and ßcatΔex3 mouse models both induce growth of tumors that are transcriptionally similar to either well-differentiated and ß-catenin-activated human HCCs or mesenchymal hepatoblastomas. LAY SUMMARY: New and easy-to-use transgenic mouse models of primary liver cancers have been generated, with mutations in the gene encoding beta-catenin, which are frequent in both adult and pediatric primary liver cancers. The mice develop both types of cancer, constituting a strong preclinical model.

A developmentally prometastatic niche to hepatoblastoma in neonatal liver mediated by the Cxcl1/Cxcr2 axis.

BACKGROUND AND AIMS: Hepatoblastoma (HB) is the most common pediatric liver cancer. Its predominant occurrence in very young children led us to investigate whether the neonatal liver provides a protumorigenic niche to HB development. APPROACH AND RESULTS: HB development was compared between orthotopic transplantation models established in postnatal day 5 (P5) and 60 (P60) mice (P5Tx and P60Tx models). Single-cell RNA-sequencing (sc-RNAseq) was performed using tumor and liver tissues from both models and the top candidate cell types and genes identified are investigated for their roles in HB cell growth, migration, and survival. CONCLUSIONS: We found that various HB cell lines including HepG2 cells were consistently and considerably more tumorigenic and metastatic in the P5Tx model than in the P60Tx models. Sc-RNAseq of the P5Tx and P60Tx HepG2 models revealed that the P5Tx tumor was more hypoxic and had a larger number of activated hepatic stellate cells (aHSCs) in the tumor-surrounding liver that express significantly higher levels of Cxcl1 than those from the P60Tx model. We found these differences were developmentally present in normal P5 and P60 liver. We showed that the Cxcl1/Cxcr2 axis mediated HB cell migration and was critical to HB cell survival under hypoxia. Treating HepG2 P60Tx model with recombinant CXCL1 protein induced intrahepatic and pulmonary metastasis and CXCR2 knockout (KO) in HepG2 cells abolished their metastatic potential in the P5Tx model. Lastly, we showed that in tumors from patients with metastatic HB, there was a similar larger population of aHSCs in the tumor-surrounding liver than in localized tumors, and tumor hypoxia was uniquely associated with prognosis of patients with HB among pediatric cancers. We demonstrated that the neonatal liver provides a prometastatic niche to HB development through the Cxcl1/Cxcr2 axis.

Mitochondrial fragmentation is crucial for c-Myc-driven hepatoblastoma-like liver tumors.

Hepatoblastoma is the most common liver cancer in children, and the aggressive subtype often has a poor prognosis and lacks effective targeted therapy. Although aggressive hepatoblastoma (HB) is often accompanied by abnormally high expression of the transcription factor c-Myc, the underlying mechanism remains unclear. In this study, we found that mitochondrial fragmentation was enhanced by c-Myc overexpression in human aggressive HB tissues and was associated with poor prognosis. Then, a mouse model resembling human HB was established via hydrodynamic injection of c-Myc plasmids. We observed that liver-specific knockout of the mitochondrial fusion molecule MFN1 or overexpression of mitochondrial fission molecule DRP1 promoted the occurrence of c-Myc-driven liver cancer. In contrast, when MFN1 was overexpressed in the liver, tumor formation was delayed. In vitro experiments showed that c-Myc transcriptionally upregulated the expression of DRP1 and decreased MFN1 expression through upregulation of miR-373-3p. Moreover, enhanced mitochondrial fragmentation significantly promoted aerobic glycolysis and the proliferation of HB cells by significantly increasing reactive oxygen species (ROS) production and activating the RAC-alpha serine/threonine-protein kinase (AKT)/mammalian target of rapamycin (mTOR) and nuclear factor κB (NF-κB) pathways. Taken together, our results indicate that c-Myc-mediated mitochondrial fragmentation promotes the malignant transformation and progression of HB by activating ROS-mediated multi-oncogenic signaling.

The Mutual Inhibition of FoxO1 and SREBP-1c Regulated the Progression of Hepatoblastoma by Regulating Fatty Acid Metabolism.

BACKGROUND: Hepatoblastoma (HB) is the most common liver malignancy in pediatrics, but the treatment for this disease is minimal. This study is aimed at exploring the effect of FoxO1 and SREBP-1c on HB and their mechanism. METHODS: FoxO1, SREBP-1c, FASN, ACLY, ACC, and MAGL expressions in tissue samples were detected by RT-qPCR and WB. IHC was utilized to measure FASN content. Overexpression and knockdown of FoxO1 and sSREBP-1c were performed on Huh-6 cells. Cell proliferation, migration, and invasion were examined by CCK8, scratch, and transwell assay. ELISA was performed to test the ATP, FAO, NEFA, and Acetyl-CoA contents. ChIP was used to detect the interaction between SREBP-1c protein and the FoxO1 gene. In vivo tumorigenesis was conducted on mice. The morphology of tumor tissue sections was observed by HE staining. RESULTS: FoxO1 expression was downregulated in HB tissue, while the expressions of SREBP-1c, FASN, ACLY, ACC, and MAGL were upregulated. In Huh-6 cells and mouse tumor tissues, FoxO1 knockdown resulted in increased cell proliferation, migration, and invasion and active fatty acid metabolism. On the contrary, after the knockdown of SREBP-1c, cell proliferation, migration, and invasion were weakened, and fatty acid metabolism was significantly reduced. SREBP-1c interacted with the promoter of the FoxO1 gene. When FoxO1 was knocked down, the tumor tissue was more closely packed. After the knockdown of the SREBP-1c gene, the structure of tumor cells was deformed. CONCLUSION: FoxO1 and SREBP-1c inhibited each other in HB, leading to the increase of intracellular fatty acid metabolism, and ultimately facilitated the development of HB.

Comparison of the Impact of Xanthohumol and Phenethyl Isothiocyanate and Their Combination on Nrf2 and NF-κB Pathways in HepG2 Cells In Vitro and Tumor Burden In Vivo.

Background: Increasing evidence suggests that combinations of phytochemicals are more efficient than single components in the modulation of signaling pathways involved in cancer development. In this study, the impact of phenethyl isothiocyanate (PEITC), indole-3-carbinol (I3C), xanthohumol, (X), and resveratrol (RES) and their combinations on the activation and expression of Nrf2 and NF-κB in human hepatocytes and HCC cells were evaluated. Methods: THLE-2 and HepG2 cells were exposed to single phytochemicals and their combinations for 24 h. The activation of Nrf2 and NF-κB, expression of their target genes, and effect on cells survival were assessed. The tumor burden was evaluated in mice carrying xenografts. Results: All phytochemicals enhanced the activation and expression of Nrf2 and its target genes SOD and NQO1 in HepG2 cells. The increased expression of NQO1 (~90%) was associated with increased ROS generation. X + PEITC downregulated NF-κB activation reducing binding of its active subunits to DNA resulting in diminished COX-2 expression. In contrast to single phytochemicals, X + PEITC induced apoptosis. Moderate reduction of tumor burden in mice carrying xenografts following X and PEITC or their combination was observed. Conclusions: Since Nrf2 is overexpressed in HCC its reduced activation together with diminished level of NF-κB by X + PEITC may be considered as a strategy to support conventional HCC therapy.

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

PURPOSE: Glutamine 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. METHODS: We 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. RESULTS: High 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. CONCLUSION: Growth 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.

Quercetin inhibited the proliferation and invasion of hepatoblastoma cells through facilitating SIRT6-medicated FZD4 silence.

Hepatoblastoma (HB) is a malignant liver tumor that occurs during childhood. The histone deacetylase SIRT6 functions as a tumor suppressor in diverse cancers. Quercetin, as activators and antioxidants of sirtuins, exhibits remarkable anticancer activity in many tumors. However, whether quercetin ameliorates HB is still unclear. In our study, we found that SIRT6 was downregulated in HB tissues and cell lines. Overexpression of SIRT6 observably suppressed cell proliferation and invasion, promoted cell apoptosis. Mechanistically, SIRT6 suppressed frizzled 4 (FZD4) transcription by deacetylating histone H3K9. Upregulation of SIRT6 reduced the protein levels of FZD4 and H3K9ac. Additionally, quercetin treatment could enhance the expression of SIRT6, repress FZD4 level, cell viability and invasion, and promote apoptosis. Overexpression of FZD4 signally reversed quercetin-treated the promotion effect on cell apoptosis, and the inhibition effects on FZD4 expression, cell viability, invasion and Wnt/ß-catenin pathway related proteins. In addition, LiCl, an agonist of Wnt/ß-catenin pathway, could recover the inhibition effects of quercetin on Wnt/ß-catenin pathway related proteins, cell viability and invasion, and promotion effect on cell apoptosis. In vivo mouse xenograft tumor growth assay revealed that quercetin markedly suppressed tumor growth. In conclusion, these results demonstrated that the molecular mechanism of quercetin suppressing HB cell proliferation and invasion, promoting apoptosis was to promote the deacetylation of SIRT6 on FZD4 and inhibit the activation of Wnt/ß-catenin pathway.