Hepatoblastoma Cell Line Research Articles

Gold Nanosystems Covered with Doxorubicin/DNA Complexes: A Therapeutic Target for Prostate and Liver Cancer.

Different gold nanosystems covered with DNA and doxorubicin (Doxo) were designed and synthesized for cancer therapy, starting from Au@16-Ph-16 cationic nanoparticles and DNA-Doxo complexes prepared under saturation conditions. For the preparation of stable, biocompatible, and small-sized compacted Au@16-Ph-16/DNA-Doxo nanotransporters, the conditions for the DNA-Doxo compaction process induced by gold nanoparticles were first explored using fluorescence spectroscopy, circular dichroism and atomic force microscopy techniques. The reverse process, which is fundamental for Doxo liberation at the site of action, was found to occur at higher CAu@16-Ph-16 concentrations using these techniques. Zeta potential, dynamic light scattering and UV-visible spectroscopy reveal that the prepared compacted nanosystems are stable, highly charged and of adequate size for the effective delivery of Doxo to the cell. This fact is verified by in vitro biocompatibility and internalization studies using two prostate cancer-derived cell lines (LNCaP and DU145) and one hepatocellular carcinoma-derived cell line (SNU-387), as well as a non-tumor prostate (PNT2) cell line and a non-hepatocarcinoma hepatoblastoma cell line (Hep-G2) model used as a control in liver cells. However, the most outstanding results of this work are derived from the use of the CI+NI combined treatments which present strong action in cancer-derived cell lines, while a protective effect is observed in non-tumor cell lines. Hence, novel therapeutic targets based on gold nanoparticles denote high selectivity compared to conventional treatment based on free Doxo at the same concentration. The results obtained show the viability of both the proposed methodology for internalization of compacted nanocomplexes inside the cell and the effectiveness of the possible treatment and minimization of side effects in prostate and liver cancer.

Hepatoblastoma Cancer Stem Cells Express PD-L1, Reveal Plasticity and Can Emerge upon Chemotherapy

The biology of cancer stem cells (CSCs) of pediatric cancers, such as hepatoblastoma, is sparsely explored. This is mainly due to the very immature nature of these tumors, which complicates the distinction of CSCs from the other tumor cells. Previously, we identified a CSC population in hepatoblastoma cell lines expressing the CSC markers CD34 and CD90, cell surface Vimentin (csVimentin) and binding of OV-6. In this study, we detected the co-expression of the immune escape factor PD-L1 in the CSC population, whereas the other tumor cells remained negative. FACS data revealed that non-CSCs give rise to CSCs, reflecting plasticity of CSCs and non-CSCs in hepatoblastoma as seen in other tumors. When we treated cells with cisplatin and decitabine, a new CD34+/lowOV-6lowCD90+ population emerged that lacked csVimentin and PD-L1 expression. Expression analyses showed that this new CSC subset shared similar pluripotency and EMT features with the already-known CSCs. FACS results further revealed that this subset is also generated from non-CSCs. In conclusion, we showed that hepatoblastoma CSCs express PD-L1 and that the biology of hepatoblastoma CSCs is of a plastic nature. Chemotherapeutic treatment leads to another CSC subset, which is highly chemoresistant and could be responsible for a poor prognosis after postoperative chemotherapy.

Preclinical Evidence Supporting Novel Protein Phosphatase 2A Activators as Therapeutics in Hepatoblastoma

INTRODUCTION: The tumor suppressor, protein phosphatase 2A (PP2A), is downregulated in hepatoblastoma. Our laboratory has shown that PP2A activation using the sphingosine analog, FTY720, decreased hepatoblastoma viability in vitro and tumor growth in vivo. Because FTY720 has significant immunosuppressive properties, we wished to examine the effects 2 novel compounds, ATUX-3364 (3364) and ATUX-8385 (8385), designed to activate PP2A without causing immunosuppression in hepatoblastoma. METHODS: The established human hepatoblastoma cell line, HuH6, and a human hepatoblastoma patient-derived xenograft, COA67, were used. Cells were treated with increasing doses of 3364 or 8385 for 24 hours. Proliferation and viability were investigated using CellTiter96 and alamarBlue assays, respectively. The ability of the COA67 cells to form tumor spheres, a marker of cancer cell stemness, was assessed following treatment with 3364 or 8385 (2 μM). RESULTS: Viability was significantly decreased following doses as low as 5 μM treatment in HuH6 (Fig. 1A, upper panel) and 0.5 μM treatment in COA67 (Fig. 1A, lower panel). Proliferation was significantly decreased in HuH6 after treatment with 3364 (7.5 μM, 57 ± 0.1%; p ≤ 0.01) and 8385 (12.5 μM, 54 ± 0.1%; p ≤ 0.01) vs 0 μM and COA67 cells at 4 μM (3364: 64 ± 0.7%; 8385: 93 ± 0.3%, vs 0 μM; p ≤ 0.05) after treatment. Both compounds significantly affected the ability of COA67 cells to form tumor spheres (Fig. 1B).CONCLUSION: The novel PP2A activators, 3364 and 8385, decreased proliferation, viability, and stemness in hepatoblastoma. These findings provide evidence for further investigation of these novel PP2A activators as potential therapeutics for hepatoblastoma.

Multiomics characterization of mouse hepatoblastoma identifies yes-associated protein 1 target genes.

Hepatoblastoma (HB) is the most common primary liver malignancy in childhood and lacks targeted therapeutic options. We previously engineered, to our knowledge, the first yes-associated protein 1 (YAP1) S127A -inducible mouse model of HB, demonstrating tumor regression and redifferentiation after YAP1 withdrawal through genome-wide enhancer modulation. Probing accessibility, transcription, and YAP1 binding at regulatory elements in HB tumors may provide more insight into YAP1-driven tumorigenesis and expose exploitable vulnerabilities in HB. Using a multiomics approach, we integrated high-throughput transcriptome and chromatin profiling of our murine HB model to identify dynamic activity at candidate cis -regulatory elements (cCREs). We observed that 1301 of 305,596 cCREs exhibit "tumor-modified" (TM) accessibility in HB. We mapped 241 TM enhancers to corresponding genes using accessibility and histone H3K27Ac profiles. Anti-YAP1 cleavage under targets and tagmentation in tumors revealed 66 YAP1-bound TM cCRE/gene pairs, 31 of which decrease expression after YAP1 withdrawal. We validated the YAP1-dependent expression of a putative YAP1 target, Jun dimerization protein 2 (JDP2), in human HB cell lines using YAP1 and LATS1/2 small interfering RNA knockdown. We also confirmed YAP1-induced activity of the Jdp2 TM enhancer in vitro and discovered an analogous human enhancer in silico. Finally, we used transcription factor (TF) footprinting to identify putative YAP1 cofactors and characterize HB-specific TF activity genome wide. Our chromatin-profiling techniques define the regulatory frameworks underlying HB and identify YAP1-regulated gene/enhancer pairs. JDP2 is an extensively validated target with YAP1-dependent expression in human HB cell lines and hepatic malignancies.

Investigation of Radiotracer Metabolic Stability In Vitro with CYP-Overexpressing Hepatoma Cell Lines.

The characterization of novel radiotracers toward their metabolic stability is an essential part of their development. While in vitro methods such as liver microsome assays or ex vivo blood or tissue samples provide information on overall stability, little or no information is obtained on cytochrome P450 (CYP) enzyme and isoform-specific contribution to the metabolic fate of individual radiotracers. Herein, we investigated recently established CYP-overexpressing hepatoblastoma cell lines (HepG2) for their suitability to study the metabolic stability of radiotracers in general and to gain insight into CYP isoform specificity. Wildtype HepG2 and CYP1A2-, CYP2C19-, and CYP3A4-overexpressing HepG2 cells were incubated with radiotracers, and metabolic turnover was analyzed. The optimized protocol, covering cell seeding in 96-well plates and analysis of supernatant by radio thin-layer-chromatography for higher throughput, was transferred to the evaluation of three 18F-labeled celecoxib-derived cyclooxygenase-2 inhibitors (coxibs). These investigations revealed time-dependent degradation of the intact radiotracers, as well as CYP isoform- and substrate-specific differences in their metabolic profiles. HepG2 CYP2C19 proved to be the cell line showing the highest metabolic turnover for each radiotracer studied here. Comparison with human and murine liver microsome assays showed good agreement with the human metabolite profile obtained by the HepG2 cell lines. Therefore, CYP-overexpressing HepG2 cells provide a good complement for assessing the metabolic stability of radiotracers and allow the analysis of the CYP isoform-specific contribution to the overall radiotracer metabolism.

Inhibitory effects of LOXL2 knockdown on cellular functions of liver cancer stem cells.

BackgroundLysyl oxidase-like 2 (LOXL2) plays a role in tumor microenvironment formation and metastasis of hepatocellular carcinoma (HCC), which has a high mortality burden. Liver cancer stem cells (LCSCs) are related with the major malignant phenotypes of HCC. The function of LOXL2 in regulation of LCSCs remains unknown.MethodsCD133+HepG2 and CD133+Hep3B cells were sorted by fluorescence-activated cell sorting (FACS) from two human hepatoblastoma cell lines. Spheroid formation, apoptosis, cell cycle, as well as transwell assays were performed upon LOXL2 knockdown in CD133+HepG2 and CD133+Hep3B cells. Protein and mRNA levels were quantified by Western blotting, immunofluorescence and reverse transcription-PCR (RT-PCR).ResultsKnockdown of LOXL2 decreased spheroid formation, migration and invasion (P<0.05), also induced apoptosis (P<0.05) and cell cycle arrest (P<0.05) in CD133+HepG2 and CD133+Hep3B cells. Knockdown of LOXL2 effectively inhibited expression of the anti-apoptosis proteins baculoviral inhibitor of apoptosis protein (IAP) repeat-containing 3 (BIRC3) and murine double minute 2 (MDM2) (P<0.01), as well as autophagy marker microtubule-associated protein 1 light chain 3 B (LC3B) and autophagy gene ATG5 in CD133+HepG2 and CD133+Hep3B cells (P<0.01).ConclusionsThe results revealed that LOXL2 inhibition could reduce the proliferation and expansion of LCSCs, making LOXL2 inhibitors an attractive and novel therapeutic strategy of HCC.

Abstract 1825: Romidepsin therapy results in p53 mediated apoptosis in hepatoblastoma

Abstract Purpose: Relapsed and treatment refractory hepatoblastoma (HB) has a survival rate less than 50% due to limited treatment options. We have previously shown that HB and treatment refractory HB has upregulation of class I histone deacetylase (HDAC) genes (HDAC1-4) through microarray, confirmed by RT-PCR. The highest consistent upregulation noted was with HDAC 2 and 3. Given this upregulation, we tested established and treatment refractory HB cell lines with HDAC 2/3 inhibitor, Romidepsin. We sought to characterize Romidepsin efficacy and mechanism of action. Methods: Romidepsin was tested with cytotoxic assays (MTT). Tumor derived cell lines were treated with Romidepsin and evaluated for gene expression of p53 pathway via qRT-PCR. Immunoblotting assays were used to assess changes in expression of Acetyl-p53 at time points of 0, 4, 8, and 24 hours after 0.01 µM concentrated Romidepsin treatment. Evaluation of the specific cell death pathway involved was performed via cell viability assays using 0.01 µM concentration of Romidepsin in combination with cell death reversal agents including Z-VAD for apoptosis, 3-Methyladenine for autophagy, Necrostatin-1 for necroptosis, and Ferrostatin-1 for ferroptosis at 48 hours. Results: Romidepsin (IC50 of 0.001-0.02 µM) showed strong in vitro effects for viability. HB cell lines treated with HDAC inhibitor had increased p21 and PUMA expression on RT-PCR, most notably at the 24-hour mark (p = &amp;lt; 0.05). Acetyl p-53 was noted to increase in HUH-6 and Hep G2 throughout the 24 hours of treatment on western blots, while Hep T1 was noted to have no changes in expression. All cell lines showed the highest level of cell death reversal with ZVAD in cell viability assay after Romidepsin treatment. Hep T1 showed the highest cell death reversal with Romidepsin: relative cell viability of 52% (±5%) versus 95% (±2%) in ZVAD/Romidepsin. Conclusions: Romidepsin demonstrates strong in vitro efficacy and suggest HDAC 2/3 inhibition may act through p53 mediated mechanism, notably acetylated p53. Cell death via apoptosis is further supported by cell death reversal with ZVAD/Romidepsin treatment in HB cell lines. Citation Format: Andres F. Espinoza, Richard Whitlock, Rohit Srivastava, Roma Patel, Sai Govindu, Aayushi Shah, Samuel Larson, Sarah Woodfield, Sanjeev Vasudevan. Romidepsin therapy results in p53 mediated apoptosis in hepatoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1825.

Abstract 2492: Identification of RBMS1 in the amplified region 2q24 as a major driver of cellular growth in childhood hepatoblastoma

Abstract Hepatoblastoma represents the most common primary malignancy of the liver in childhood. Pathological activation of the WNT signaling pathway is characteristic for hepatoblastoma caused by mutations of CTNNB1 or other genes encoding components of the pathway. Genome-wide chromosomal analyses uncovered recurrent chromosomal alterations, in particular gain of chromosome 2q, and in some cases amplification of the region 2q24 that suggests the presence of a so far unidentified oncogene in this chromosomal region. The aim of this study was to identify and further characterize this oncogene. In the framework of the clinical hepatoblastoma study of the German Society for Pediatric Oncology and Hematology, we had the opportunity to study samples of 76 hepatoblastoma patients. Using molecular inversion probe (MIP) array technology, genome-wide, high-resolution, quantitative chromosomal copy number profiles were generated and the smallest overlapping amplified region was identified. RNA and protein expression in normal and diseased tissues as well as ascribed functions of the genes located in this area were evaluated in silico in available databases. RNA expression levels of these genes were analyzed by Nanostring method and Affymetrix arrays and compared between 2q24-amplified tumors versus non-amplified tumors and normal liver tissue. The most promising candidate gene regarding its expression pattern and described function was selected for further analysis. Its protein expression was studied in situ by immunohistochemistry in hepatoblastoma tissue microarrays. The biological function was analyzed in vitro by targeted siRNA-mediated knockdown experiments in 3 hepatoblastoma cell lines (HepT1, HepG2, HuH6) und subsequent cell growth assays (MTT) and WNT signaling pathway specific luciferase reporter assays. Gain of chromosome 2q was present in 44.7% of the cases. Amplification of the 2q24.2-24.3 region was detected in 11.8%. In the smallest overlapping amplified region of 5.6 Mbp, we could accurately map 20 protein coding genes, three genes encoding long noncoding RNAs and one gene encoding a micro-RNA. The RBMS1 gene encoding a single-stranded DNA/RNA binding protein showed significant RNA overexpression in 2q24 amplified tumors. This overexpression was validated by immunohistochemical studies at the protein level. RBMS1 knockdown by specific siRNA transfection resulted in a significantly reduced proliferation of hepatoblastoma cells. RBMS1 knockdown also resulted in a marked reduction of the WNT pathway activity in these cells. We identified RBMS1 located within the amplicon on chromosome 2q24 as a potential oncogenic driver in hepatoblastoma. Initial data suggest that RBMS1 may exert this function by interaction with the WNT signaling pathway that is pathologically activated in hepatoblastoma. Citation Format: Martin Rodemann, Verena Dreschmann, Evelyn Dörner, Dietrich von Schweinitz, Christian Vokuhl, Torsten Pietsch. Identification of RBMS1 in the amplified region 2q24 as a major driver of cellular growth in childhood hepatoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2492.

Abstract 4005: Identification of IGF2 as genomic driver and actionable therapeutic target in hepatoblastoma

Abstract Background and Aims: Management of hepatoblastoma (HB), the most frequent pediatric liver cancer, is based on surgical resection and perioperative platin-based regimens, commonly cisplatin. Here, we aimed to identify actionable targets in HB and assess the efficacy of molecular therapies in HB cell lines, organoids and murine models. Methods: Paired tumor and adjacent tissues from 31 resected HBs and a validation set of 50 HBs were analyzed at the transcriptomic, genomic and epigenomic level using RNAseq, SNP and methylation arrays, respectively. The main targetable driver in HB was identified by gene co-expression network analysis (GCN) and its overexpression was confirmed by qRT-PCR. The anti-tumor effect of driver inhibition with molecular therapies alone or in combination with cisplatin was assessed in cell lines, patient-derived HB organoids and in a HB xenograft murine model. Results: IGF2 overexpression (FC&amp;gt; 4 vs adjacent tissue) was identified as the top targetable HB driver (71%, 22/31). IGF2high tumors displayed progenitor cell features and were significantly enriched in molecular classes with aggressive phenotypes and CTNNB1 mutations, while IGF2low tumors were enriched in inflammatory and TGF-β signaling. IGF2high tumors correlated with shorter recurrence-free survival after resection (median 34 months vs not reached for IGF2low; p = 0.02). IGF2 overexpression correlated in most cases (86%) with fetal promoter hypomethylation (50%), 11p15.5 loss of heterozygosity (LOH, 57%) or overexpression of miR483 (55%). Xentuzumab (anti-IGF1/2 mAb) alone or combined with cisplatin reduced proliferation and clonogenic capacity in IGF2high cell lines. The combination of xentuzumab and cisplatin exhibited synergistic effects in terms of cell viability in organoids derived from IGF2high human HBs. In mice, this combination induced a significant decrease in the viable tumor volume and extended survival compared to cisplatin alone (n = 13-14 per arm, p = 0.04). Conclusion: IGF2 is an actionable driver in HB and its overexpression was associated with fetal promoter hypomethylation, LOH or miR483 overexpression. The combination of a mAb against IGF1/2 (xentuzumab) with cisplatin led to remarkable anti-tumoral effects in pre-clinical models, providing the rationale for exploring this regimen in IGF2high HB patients. Citation Format: Jordi Abril-Fornaguera, Laura Torrens, Juan Carrillo-Reixach, Alexander Rialdi, Ugne Balaseviciute, Carla Montironi, Philipp Haber, Álvaro Del Río-Álvarez, Montserrat Domingo-Sàbat, Laura Royo, Nicholas Akers, Catherine E. Willoughby, Judit Peix, Miguel Torres-Martin, Marc Puigvehi, Roser Pinyol, Stefano Cairo, Margaret Childs, Rudolf Maibach, Rita Alaggio, Piotr Czauderna, Bruce Morland, Bojan Losic, Vincenzo Mazzaferro, Ernesto Guccione, Daniela Sia, Carolina Armengol, Josep M. Llovet. Identification of IGF2 as genomic driver and actionable therapeutic target in hepatoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4005.

Abstract 3891: Efficacy of dinaciclib in hepatoblastoma through inhibition of cyclin dependent kinases

Abstract Introduction: Hepatoblastoma (HB) is the most frequent liver malignancy of childhood with an annual incidence of 1.5 per million and a survival rate less than 50%. Due to significant side effects and ineffectiveness of standard chemotherapy for relapse refractory disease, the treatment options are now shifting towards targeted therapies. Cyclin-dependent kinases (CDKs) are potential cancer therapeutic targets because of their critical role in promoting cell growth and regulating transcriptional processes. This study aims to assess the therapeutic efficacy of dinaciclib, cyclin-dependent kinase inhibitor, with HB in preclinical in vitroand in vivo models. Method: Dinaciclib was tested in patient derived cell lines (PDCL) from different patient derived xenograft (PDX) models using CellTiter-Glo Luminescent Viability assays. Dinaciclib was also assayed in vitro for cytotoxicity (MTT assay) and proliferation (CCK-8 assay). Differential expression of CDKs was checked by microarray analysis between normal liver and HB patients. Results were confirmed by immunoblotting assays. Immunoblotting assays were also used to see the effect of dinaciclib on CDK 1/2/5/9 and to assess the induction of apoptosis (PARP cleavage) in vitro. Dinaciclib was also tested in our orthotopic PDX models of high-risk HB. MRI and alpha-fetoprotein (AFP) ELISA were done to evaluate tumor growth. Tumor weights and volumes, as well as, immunohistochemistry were also used to assess effects of dinaciclib in vivo. Result: Dinaciclib suppressed cell proliferation and viability in a dose-dependent manner in all HB cell lines tested with IC50 in the low micromolar range (HepG2- 3.75; HepT1- 0.007; Huh6- 1.42; HB17- 0.72). Microarray analysis showed significant upregulation of CDK1 (p=0.0003), CDK2 (p=0.00023) and CDK9 (p=0.015) in HB patient samples, however, immunoblotting with dinaciclib-treated cells showed decreased expression of CDKs 1/2/5/9 as well as induction of apoptosis. Dinaciclib also showed in vivo inhibition of tumor growth compared to placebo in both PDX models (HB47: relative tumor volume p = 0.03, T/C ratio= 0.36, tumor weight p = 0.02; HB52: relative tumor volume p = 0.06, T/C ratio= 0.16, tumor weight p = 0.07). Conclusion: Dinaciclib treatment showed promising effects with preclinical HB cell lines and PDX models. Citation Format: Rohit K. Srivastava, Roma Patel, Andres F. Espinoza, Yang Yu, Richard S. Whitlock, Samuel Larson, Andrew Badachhape, Jianhua Yang, Sarah E. Woodfield, Sanjeev A. Vasudevan. Efficacy of dinaciclib in hepatoblastoma through inhibition of cyclin dependent kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3891.

Abstract 5674: A developmentally prometastatic niche to hepatoblastoma in neonatal liver mediated by the Cxcl1/Cxcr2 axis

Abstract Background and Rationale: Hepatoblastoma (HB) is the most common pediatric liver cancer. Its predominant occurrence in very young children led us to investigating whether the neonatal liver provides a protumorigenic niche to HB development. Methods: HB development was compared between orthotopic transplantation models established in postnatal day 5 and 60 mice (P5Tx and P60Tx models). Single-cell RNA-sequencing 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. Results: 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 which 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 in HepG2 cells abolished their metastatic potential in the P5Tx model. Lastly, we showed that in metastatic HB patient tumors there was a similar larger population of aHSCs in the tumor-surrounding liver than in localized tumors, and tumor hypoxia was uniquely associated with HB patient prognosis among pediatric cancers. Conclusion: We demonstrated that the neonatal liver provides a prometastatic niche to HB development via the Cxcl1/Cxcr2 axis. Citation Format: Li Fan, Qingfei Pan, Wentao Yang, Selene C. Koo, Cheng Tian, Liyuan Li, Meifen Lu, Anthony Brown, Bensheng Ju, John Easton, Sarangarajan Ranganathan, Soona Shin, Alexander Bondoc, Jun J. Yang, Jiyang Yu, Liqin Zhu. A developmentally prometastatic niche to hepatoblastoma in neonatal liver mediated by the Cxcl1/Cxcr2 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5674.

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.

Chemical investigations and cytotoxic effects of metabolites from Antrodia camphorata against human hepatocellular carcinoma cells

Antrodia camphorata is used as a medicinal fungus in Taiwan to treat fatigue, food intoxication, and enhance liver function. Here we identified fermented metabolic components from the mycelium of A. camphorata KH37 and explored their anti-hepatoma potentials with study models of human hepatoblastoma cell lines. Bioassay-guided fractionation of the solid fermentation powder of A. camphorata KH37 led to the isolation of one new quinonol, antroquinonol Z (1), and nine known compounds (2–10). Treatment with 10 μM antrocamols LT1 (2) or LT3 (3) reduced cell viability of HepG2 and Huh-7 cells to about 60% in 48 hours. Antroquinonol Z (1) exhibited mild cytotoxicity against Huh-7 cells in 48 and 72 hours. Interestingly, two fractions showed cytotoxicity in HepG2 and Huh-7 cells, even better than compounds isolated from these fractions. The significant cytotoxicity of partially purified samples from A. camphorata KH37 exhibited a potential for developing alternative or complementary therapeutics against hepatoma.

P-029 - Enabling insights into the cytochrome P450 isoform specific metabolism of radiotracers by CYP overexpressing hepatoblastoma cell lines

P-029 - Enabling insights into the cytochrome P450 isoform specific metabolism of radiotracers by CYP overexpressing hepatoblastoma cell lines

Chicken Feather Waste Valorization Into Nutritive Protein Hydrolysate: Role of Novel Thermostable Keratinase From Bacillus pacificus RSA27

Microbial keratinases exhibit a momentous role in converting keratin biowastes into exceedingly valuable protein supplements. This study reports a novel, highly stable keratinase from Bacillus pacificus RSA27 for the production of pure peptides rich in essential amino acids from chicken feathers. Purified keratinase showed a specific activity of 38.73 U/mg, 2.58-fold purification, and molecular weight of 36 kDa. Kinetic studies using a chicken feather as substrate report Km and Vmax values of 5.69 mg/ml and 142.40 μg/ml/min, respectively, suggesting significant enzyme-substrate affinity/biocatalysis. Identification and in silico structural-functional analysis of keratinase discovered the presence of distinct amino acid residues and their positions. Besides, keratinase possesses a high-affinity calcium-binding site (Asp128, Leu162, Asn164, Ile166, and Val168) and a catalytic triad of Asp119, His151, and Ser308, known attributes of serine protease (subtilisin family). Furthermore, a scale-up to 5 L fermenter revealed complete feather hydrolysis (94.5%) within 24 h with high activity (789 U/ml) and total amino acid of 153.97 μmol/ml. Finally, cytotoxicity evaluation of protein hydrolysate resulted in negligible cytotoxic effects (1.02%) on the mammalian hepatoblastoma cell line, signifying its potential biotechnological applications.

Circular RNA Circrna_0072387 Promotes Hepatoblastoma Progression by Regulating Hsa-Mir-490/Histone Deacetylase 2 Axis

Circular RNAs have been reported to be associated with the occurrence and progress of various cancers. However, its biological roles in hepatoblastoma (HB) remain undefined. This study intended to explain a possible mechanism of hsa_circ_0072387 (circ72387) in hepatoblastoma progression. The expression of circ72387, hsa-miR-490 (miR490) and histone deacetylase 2 (HDAC2) in HB tissues and cell lines (HepG2/Huh6) were determined by quantitative realtime polymerase chain reaction (qPCR) or Western blot. Cell Counting Kit-8 (CCK-8) and colony formation assay were used to detect the proliferation of HB cell lines. The interaction between miR490 with circ72387 was predicted by CircInteractome and was verified by Dual-Luciferase reporter assay while the interaction between miR490 with HDAC2 was predicted by Targetscan and was verified by western blotting assay. Results showed that circ72387 was upregulated in both HB tissues and cells, and miR490 was downregulated in HB cells. Furthermore, the silence of circ72387 suppressed the proliferation of hepatoblastoma cells. Circ72387 promoted the proliferation of HB cells through competitively binding to HDAC2 against miR490 acting as a Sponge of miR490 in HB. Circ72387 played an essential role in HB development through the circ72387-miR490-HDAC2 regulatory axis, which may pave the way for potential diagnostic markers and therapeutic targets research in HB.

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

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. 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. 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.

Inhibition of cIAP1 in the effective suppression of chemotherapy‑resistant hepatoblastoma.

Cellular inhibitor of apoptosis protein‑1 (cIAP1) is a key regulator of programmed cell death and is known to be associated with chemotherapeutic resistance. The present study aimed to investigate the antitumor efficacy of birinapant, a novel selective inhibitor of cIAP1, against cisplatin (CDDP)‑resistant hepatoblastoma (HB) cells. Western blot analysis was used to investigate the antitumor effect of birinapant on cIAP1 expression in Huh6 cells at the protein level. A WST‑8 assay was performed to evaluate the tumor growth inhibitory effect of birinapant on the human HB cell lines, Huh6 and HepG2. Huh6 cells were exposed to CDDP and/or birinapant in order to confirm tumor growth inhibition. The antitumor efficacy of birinapant plus CDDP combination therapy was significantly higher than that of CDDP monotherapy in a dose‑dependent manner (P=0.035). The study also investigated the antitumor efficacy of birinapant plus CDDP combination therapy in an established xenograft model of SCID mice. Compared with CDDP monotherapy, birinapant combined with CDDP showed better inhibition of tumor growth (P=0.121). It was observed that the mRNA expression of cIAP1 in tumors was significantly enriched in the CDDP monotherapy group compared with that in the untreated group. Furthermore, immunohistochemical staining was performed to compare cIAP1 expression in pre‑ and post‑chemotherapy specimens in patients with HB, and a significant increase was observed in the post‑chemotherapy specimens (P<0.001). CDDP‑resistant Huh6 (Huh6‑CDDPR) cells were also established following repeated exposure to CDDP. Birinapant was substantially more effective against the Huh6‑CDDPR cells than against the Huh6 wild‑type cells. Taken together, these findings suggest that repeated exposure to CDDP enhances cIAP1 expression in HB cells and that birinapant is a promising therapeutic drug for CDDP‑resistant HB.

Long non-coding RNA Linc00205 promotes hepatoblastoma progression through regulating microRNA-154-3p/Rho-associated coiled-coil Kinase 1 axis via mitogen-activated protein kinase signaling

Hepatoblastoma (HB) is the most common pediatric liver tumor. The significant tumor heterogeneity of HB leads to varied prognoses among children with the disease. Recent studies have suggested that long non-coding RNAs (lncRNAs) can serve as novel therapies for HB treatment. Thus, in this study, we aimed to reveal the function and mechanism of the lncRNA Linc00205 in HB. Our results exhibited that, in both HB tissues and cell lines, levels of Linc00205 were significantly increased. In addition, knockdown of Linc00205 led to suppression of HB development. Moreover, we identified that Linc00205 was able to directly bind to miR-154-3p, thus isolating miR-154-3p from its target Rho-associated coiled-coil Kinase 1 (ROCK1). Further cellular behavioral experiments elucidated that the miR-154-3p inhibitor and ROCK1 overexpression were able to reverse the effect of downregulated Linc00205 on proliferation, migration, invasion, and apoptosis of HB cells by rescue assays via mitogen-activated protein kinase (MAPK) signaling. Our results demonstrated that Linc00205 enhanced HB progression by regulating ROCK1 expression via sponging miR-154-3p through MAPK signaling, which suggests a novel potential therapeutic target for HB.

Metastatic human hepatoblastoma cells exhibit enhanced tumorigenicity, invasiveness and a stem cell-like phenotype

Background/PurposeMetastatic hepatoblastoma continues to pose a significant treatment challenge, primarily because the precise mechanisms involved in metastasis are not fully understood, making cell lines and preclinical models that depict the progression of disease and metastasis-related biology paramount. We aimed to generate and characterize a metastatic hepatoblastoma cell line to create a model for investigation of the molecular mechanisms associated with metastasis. Materials/MethodsUsing a murine model of serial tail vein injections of the human hepatoblastoma HuH6 cell line, non-invasive bioluminescence imaging, and dissociation of metastatic pulmonary lesions, we successfully established and characterized the metastatic human hepatoblastoma cell line, HLM_3. ResultsThe HLM_3 cells exhibited enhanced tumorigenicity and invasiveness, both in vitro and in vivo compared to the parent HuH6 cell line. Moreover, HLM_3 metastatic hepatoblastoma cells exhibited a stem cell-like phenotype and were more resistant to the standard chemotherapeutic cisplatin. ConclusionThis newly described metastatic hepatoblastoma cell line offers a novel tool to study mechanisms of tumor metastasis and evaluate new therapeutic strategies for metastatic hepatoblastoma.