Proliferation Of Hepatoblastoma Cells Research Articles

IMPDH2 Positively Impacts the Proliferation Potential of Hepatoblastoma Cells by Activating JunB Signaling Pathway.

Amplification of inosine monophosphate dehydrogenase II, EC 1,1,1,205 (IMPDH2) has been reported in various cancers, which results in transformation and tumorigenicity. In our current work, we have explored the oncogenic properties and the underlying pathophysiology of IMPDH2 in hepatoblastoma (HB). To investigate IMPDH2 expression in HB tissues and prognostic significance in HB patients, gene expression profiling interactive analysis (GEPIA) has been adopted. Immunohistochemistry has also helped to validate the protein expression of IMPDH2 in HB tissues. The effect of IMPDH2 overexpression or depletion on the proliferation of Hepatoblastoma cells in vitro has been evaluated by CCK8 assays and colony formation assays. Xenograft tumor growth of mice has been detected. Luciferase reporter assays have been conducted to determine the interaction of IMPDH2 and JunB, which was further asserted by pharmacological inhibition of JunB. IMPDH2 was highly expressed in HB tissues. Experimentally, the proliferation and colony formation of HuH6 cells were increased by IMPDH2 overexpression. Conversely, genetic inactivation of IMPDH2 impaired the proliferative efficiency and colony-forming rate of HepG2 cells. Besides, the luciferase reporter assay validated IMPDH2 overexpression to be associated with enhanced JunB transcriptional activity, while its activity was diminished in the case of IMPDH2 depletion. JunB inhibitor neutralized the IMPDH2-mediated increased phosphorylation of JunB. Our findings, thus, suggest that IMPDH2 exhibits its oncogenic role in HB partially via JunB-dependent proliferation.

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.

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.

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. Invitro 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.

Propofol Suppresses Cell Progression by Inhibiting CCL18 Expression in Hepatoblastoma

Background Propofol is an anesthetic commonly used clinically and has been found to have antitumor activity in various cancers. The purpose of this study was to investigate the role of propofol in hepatoblastoma (HB). Methods CCK-8 and transwell were used to measure cell proliferation, migration, and invasion in HB cells. Cell apoptosis rate was measured by FCM. The expression of CCL18 in HB tissues and cells was detected by RT-qPCR. Western blotting was used to explore the protein expression of CCK18- and PI3K/AKT-related proteins. Results The expression of CCL18 in HB tissues and cells was overexpressed compared with control groups. CCL18 knockdown was found to notably block cell proliferation and progression, while enhancing cell apoptosis in HuH-6 and HepT1 cells. Furthermore, propofol suppressed the proliferation of HB cells in a dose-dependent manner. According to the results, we chose 5 μg/mL of propofol-treated cells for 48 hours as the subsequent experimental conditions. We found that propofol (5 μg/mL, 48 h) significantly blocked cell migration and invasion, but induced cell apoptosis in HuH-6 and HepT1 cells. In addition, CCK18 overexpression facilitated cell progression in HB cells, while propofol dramatically suppressed the effect of CCK18. Besides that, propofol suppressed the PI3K/AKT pathway. Conclusion Propofol suppressed the development of HB cells by inhibiting CCK18 expression and the PI3K/AKT pathway. Therefore, we infer that propofol plays a role in the treatment of HB.

CircRNA CDR1as promotes hepatoblastoma proliferation and stemness by acting as a miR-7-5p sponge to upregulate KLF4 expression.

Hepatoblastoma (HB) is a malignant embryonal tumor of the liver that consists of heterogenous populations of stem/progenitor cells. Although circular RNAs (circRNAs) play an essential role in tumor development, the effects of circRNA on the proliferation of HB cells, especially cancer stem cells (CSCs), remain unclear. We found that the circRNA, CDR1as, was highly expressed in CSC-enriched populations of HB cell lines. Results from flow cytometric and sphere-forming assays revealed that CDR1as knockdown in HB cell lines decreased the proportion of stem cells. The Cell Counting Kit-8 (CCK-8) assay, colony formation experiments, and EdU assay revealed that CDR1as knockdown in HB cell lines decreased cell growth and the colony-forming abilities. Biotin-coupled probe pull-down assays and biotin-coupled microRNA capture were conducted to evaluate the interaction between CDR1as and miR-7-5p. Dual-luciferase reporter assays demonstrated that Kruppel-like factor 4 (KLF4), expression of which is highly correlated with cancer stemness, was a target of miR-7-5p. Overall, the knockdown of CDR1as significantly inhibited the proliferation and stemness of HB cells by reducing the sponge activity on miR-7-5p and subsequently suppressing the interaction between miR-7-5p and KLF4. Results from this study suggest that CDR1as is an oncogene that effects the proliferation and stemness of HBs.

Isoorientin triggers apoptosis of hepatoblastoma by inducing DNA double-strand breaks and suppressing homologous recombination repair

Hepatoblastoma (HB) is the most common malignant liver tumor in children. DNA and DNA-associated processes are one of the most important targets of chemotherapeutic agents. Isoorientin (Iso), a natural flavonoid compound, can be extracted from several plant species. The effects of Iso and its molecular mechanisms on hepatic malignancies remain unclear. Herein, the anti-tumor effects of Iso in HB and its underlying mechanisms were explored. We found that Iso significantly inhibited the proliferation of HB cells both in vitro and in vivo. Mechanistic studies showed that Iso triggered cell apoptosis by inducing DNA double-stranded breaks and blocking the initiation process of homologous recombination repair, which was related to the attenuation of ataxia telangiectasia mutated (ATM) activation and inhibiting the binding of phosphorylated ataxia telangiectasia mutated (pATM) and the MRE11-RAD50-NBS1 (MRN) complex. Furthermore, Iso markedly sensitized HB cells to the anti-proliferative effects of the poly ADP-ribose polymerase (PARP) inhibitor olaparib both in vivo and in vitro. Taken together, our study first showed that Iso was a DNA-damage agent, and the combination of Iso with a PARP inhibitor might be a promising strategy for treating HB patients.

Inhibitory effect of RNA interference targeting human telomerase reverse transcriptase against hepatoblastoma cells

Objective To construct a recombinant retrovirus vector expressing small interfering RNA (siRNA) targeting human telomerase reverse transcriptase (hTERT), and assess its effect on proliferation and apoptosis of human hepatoblastoma cells. Methods The sequences of the siRNA targeting hTERT, U6 promoter and enhanced green fluorescent protein (EGFP) gene were amplified by polymerase chain reaction (PCR) and inserted into the mammalian retroviral expression vector pLXSN. The PCR method was used to amplify hTERT-siRNA, EGFP, DNA fragment of U6+ 27. The retroviral expression vector pLXSN-EGFP-U6-siTERT was constructed and subjected to enzyme digestion identification. The recombinant retroviral vector pLXSN-EGFP-U6-siTERT was constructed. The vector was then used to infect human hepatoblastoma cell HepG2. The telomerase activity of the infected cells was detected by telomerase repeat amplification protocol-silver staining, and the cell apoptosis was examined using flow cytometry. The inhibition rate of HepG2 cell proliferation was analyzed by methyl thiazol tetrazolium (MTT) assay. Results hTERT-siRNA retroviral expression vectors were successfully prepared. In the control group, the telomerase activity was 2 143.06±198.69. At 24, 48 and 72 h after colony forming units (CFU) recombinant virus infection the telomerase activity was 1 632.02±116.28, 899.38±126.11 and 321.25±25.25 respectively. As compared with before infection, the telomerase activity was respectively decreased by 23.84%, 58.03% and 85.01%. There was significant difference among different time groups (P=0.046, 0.024 and 0.008). The results of flow cytometry showed that there was significant difference in the cell apoptosis rate between the experimental group and the control group with different titer of recombinant virus infection, and there was a concentration-dependent relationship. At 24 h after 1×105 CFU recombinant virus infection, the apoptosis rate was 29.05%. MTT results showed that apoptosis rate was 29.05% at 24 h after recombinant virus infection, the tumor cell death was significant, and with the increase in virus titer and prolongation of time, cell death also gradually increased. At 24, 48 and 72 h after virus infection of 6.0×105 CFU, the results of MTT assay were 0.29±0.14, 0.20±0.13 and 0.18±0.11 respectively. At 24, 48 and 72 h after virus infection of 3.0×105 CFU, MTT results were 0.32±0.11, 0.26±0.12 and 0.25±0.10. At 24, 48 and 72 h after virus infection of 1.0×105 CFU, results of MTT assay were 0.33±0.12, 0.26±0.13 and 0.26±0.12. At 24, 48 and 72 h after virus infection of 1.0×104 CFU, the results of MTT assay were 0.43±0.14, 0.35±0.10 and 0.33±0.15. At 24, 48 and 72 h after virus infection of 1.0×103 CFU, the results of MTT assay were 0.52±0.11, 0.44±0.13 and 0.44±0.10. At 24, 48 and 72 h after virus infection of 1.0×102 CFU, the results of MTT assay were 0.65±0.13, 0.61±0.15 and 0.60±0.16. In the negative control group, the results of MTT assay were 0.69±0.11, 1.01±0.14 and 2.98±0.16 respectively at 24, 48 and 72 h. There existed the concentration-effect and time-effect relationship (P=0.037, 0.034 and 0.028). Conclusion hTERT-siRNA can effectively silence hTERT gene and suppress the telomerase activity and proliferation of HepG2 cells. Key words: RNA interference; Human telomerase reverse transcriptase; Hepatoblastoma; Apoptosis

Abstract A17: Silencing of Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) impairs growth and proliferation of hepatoblastoma cells

Abstract Background: Hepatoblastoma (HB) is the most common primary, malignant liver tumor in children, occurring primarily in children under the age of 4 years. Although chemotherapy in conjunction with surgical resection has improved the prognosis for patients with HB, disease-free survival rates remain less than 50% for patients with advanced, metastatic or relapsed disease, highlighting the need for novel treatments. Additional subsets of tumors with a worse prognosis include those with histologic features of hepatocellular carcinoma (HCC) and tumors which develop in children older than 4 years. Nuclear factor erythroid-2–related factor 2 (NFE2L2, also known as Nrf2), a key transcriptional regulator for the cellular response to oxidative stress in normal cells, has been shown to contribute to malignant phenotypes of cancers. Recently, activating mutations in NFE2L2 have been reported in HCC and also in HB by our research team (Trevino et al1). These activating mutations of NFE2L2 and/or high expression of NFE2L2 have been observed notably in several pediatric liver tumors with high-risk features associated with a poor outcome. We hypothesize that NFE2L2 activation/overexpression may contribute to an aggressive tumor phenotype, and investigated its role in HB cell proliferation. Methods: Hep293TT cells, previously established using primary tumor tissues from a highly aggressive hepatoblastoma from a 5-year-old Caucasian female, were transfected with NFE2L2 small hairpin RNAs (shRNAs) to determine the effect of NFE2L2 suppression on cell proliferation and to identify potential NFE2L2-regulated targets. Analyses of Hep293TT cell proliferation and cell cycle kinetics were conducted using cell viability assays (CellTiter-Glo Luminescence), propidium iodide staining and FACS analysis. A comprehensive transcriptome analyses of Hep293TT cells was carried out by performing RNA sequencing (RNA-seq) using an Illumina HiSeq 2000. Results: RNA-seq analysis was performed on a set of pediatric cancer cell lines including Hep293TT, as well as reference liver cell lines HepG2 and Hep3B. After normalization, the analysis revealed high expression of NFE2L2 in Hep293TT cells with fold change of 2.01 relative to the mean expression level from the pediatric cancer cell line panel (14 cell lines). Compared to other liver cancer cell lines, fold change of 1.28 and 1.5 were measured from Hep3B and HepG2, respectively. Differential expression between NFE2L2 knockdown and parental cell will further demonstrate the molecular and functional influence of NFE2L2. Furthermore, knockdown of NFE2L2 inhibited cell proliferation in Hep293TT cells by inducing cycle arrest. Conclusions: Our results suggest that NFE2L2 may contribute to cell growth and proliferation in hepatoblastoma. Further studies are warranted to determine if NFE2L2 status can be used to stratify highly aggressive pediatric liver tumors and establish NFE2L2 as a potentially novel target for the treatment of HBs. 1Trevino et al, AACR Annual Meeting, 2012, Abstract #4592 This work was supported by grants from the Cancer Prevention and Research Institute of Texas RP101195 and NIH R21 CA122565. Citation Format: Hima Bansal, Tina Chen, Zhao Lai, Donald Parsons, Lisa Trevino, Dolores Lopez-Terrada, Milton Finegold, Dinesh Rakheja, Sarah Comerford, Robert Hammer, Bradley Pollock, Jaclyn Hung, Sanjay Bansal, Yidong Chen, Gail Tomlinson. Silencing of Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) impairs growth and proliferation of hepatoblastoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A17.

Hepatocyte growth factor protects hepatoblastoma cells from chemotherapy-induced apoptosis by AKT activation

Hepatocyte growth factor/scatter factor (HGF) is a ubiquitously expressed molecule that elicits pleiotropic functions on epithelial cells, including mitogenic, motogenic, differentiating, angiogenic and morphogenic effects. In hepatoblastoma (HB), post-operative residual tumor growth and tumor recurrences are often associated with markedly elevated serum levels of HGF, suggesting a link between this molecule and tumor malignancy. Here, we demonstrate that HGF has no impact on overall cell viability and proliferation of HB cells, although signal transduction occurs downstream of HGF, such as c-Met phosphorylation, activation of phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK)/ERK-1/2 signaling. Instead of being mitogenic, HGF confers anti-apoptotic properties upon serum starvation and moreover protects HB cells against strong apoptotic inducers such as cisplatin and camptothecin, thereby contributing to chemotherapeutic resistance. This effect is mainly dependent on the PI3K/AKT signaling pathway, since inhibition by wortmannin resulted in abrogation of HGF-mediated survival, whereas inhibition of the MAPK pathway had no effect. Together, these findings highlight the importance of HGF in tumor cell survival and suggest that HGF and its cognate receptor c-Met should be considered as a candidate for combined therapeutic strategies of advanced pediatric liver tumors.