hes1-protein--human and Liver-Neoplasms

Mesenchymal hamartoma of the liver (MHL) is an uncommon benign hepatic tumor typically affecting children under 2 years of age. Only 5% of MHL occur after 5 years and are very rarely observed in adults. According to age, MHL may differ in their morphologic features. We report a case of an 11-year-old boy with MHL, resembling a malignant lesion from a clinical point of view, characterized by unusual histologic features: a prominent myxoid stroma, with a minimal ductular component, and absent cystic spaces. The present case and others reported in older children or adults demonstrate that these lesions may represent a potential diagnostic pitfall when occurring outside their classic clinical context especially because of their peculiar histologic findings. Moreover, it may be hypothesized that variation in morphology might be related to different evolutive stages of the cell of origin. To support this hypothesis, we therefore studied the presence of components of the Notch pathway inside and outside the lesion. Their absence inside the tumor and, in contrast, the expression of Notch2 and HES1 evident in overrepresented bile ducts present at the periphery might explain not only the lack of bile ducts, but also indicate a more adult phenotype compared with classic pediatric MHL, which show more bile ducts and liver trabeculae embedded in the mesenchymal matrix.

Topics: Adolescent; Adult; Aged; Basic Helix-Loop-Helix Transcription Factors; Bile Ducts; Child; Child, Preschool; Female; Gene Expression Regulation, Neoplastic; Hamartoma; Homeodomain Proteins; Humans; Liver; Liver Neoplasms; Male; Middle Aged; Receptor, Notch2; Signal Transduction; Transcription Factor HES-1

Kita-Kyushu lung cancer antigen-1 (KK-LC-1) is a cancer/testis antigen reactivated in several human malignancies. So far, the major focus of studies on KK-LC-1 has been on its potential as diagnostic biomarker and immunotherapy target. However, its biological functions and molecular mechanisms in cancer progression remain unknown.. Expression of KK-LC-1 in HCC was analysed using RT-qPCR, Western blot and immunohistochemistry. The roles of KK-LC-1 on HCC progression were examined by loss-of-function and gain-of-function approaches. Pathway inhibitor DAPT was employed to confirm the regulatory effect of KK-LC-1 on the downstream Notch signalling. The interaction of KK-LC-1 with presenilin-1 was determined by co-immunoprecipitation. The association of CpG island methylation status with KK-LC-1 reactivation was evaluated by methylation-specific PCR, bisulphite sequencing PCR and 5-Aza-dC treatment.. We identified that HCC tissues exhibited increased levels of KK-LC-1. High KK-LC-1 level independently predicted poor survival outcome. KK-LC-1 promoted cell growth, migration, invasion and epithelial-mesenchymal transition in vitro and in vivo. KK-LC-1 modulated the Notch1/Hes1 pathway to exacerbate HCC progression through physically interacting with presenilin-1. Upregulation of KK-LC-1 in HCC was attributed to hypomethylated CpG islands.. This study identified that hypomethylation-induced KK-LC-1 overexpression played an important role in HCC progression and independently predicted poor survival. We defined the KK-LC-1/presenilin-1/Notch1/Hes1 as a novel signalling pathway that was involved in the growth and metastasis of HCC.

Topics: Animals; Antigens, Neoplasm; Carcinoma, Hepatocellular; Cell Line, Tumor; CpG Islands; Disease Progression; DNA Methylation; Epithelial-Mesenchymal Transition; Gene Knockdown Techniques; Hep G2 Cells; Heterografts; Humans; Liver Neoplasms; Male; Mice; Mice, Nude; Presenilin-1; Prognosis; Receptor, Notch1; RNA, Small Interfering; Signal Transduction; Transcription Factor HES-1; Up-Regulation

Accumulating studies have suggested that microRNA-760 (miR-760) plays an important role in chemoresistance of various cancer cells. However, whether miR-760 regulates the chemoresistance of hepatocellular carcinoma (HCC) remains unclear. In this study, we found that miR-760 was decreased in HCC cell lines, and doxorubicin (Dox) treatment significantly decreased miR-760 expression in HCC cells. Overexpression of miR-760 sensitized HCC cells to Dox-induced cytotoxicity and apoptosis, whereas miR-760 inhibition showed the opposite effects. Notch1 was predicted as a target gene of miR-760. miR-760 negatively regulated Notch1 expression and Notch1/Hes1 signaling. Overexpression of miR-760 increased PTEN expression and decreased the phosphorylation of Akt. Activation of Notch signaling significantly reversed the inhibitory effect of miR-760 on Dox-resistance and abrogated the effect of miR-760 on the PTEN/Akt signaling pathway in HCC cells. Overall, our results demonstrate that miR-760 inhibits Dox-resistance in HCC cells through inhibiting Notch1 and promoting PTEN expression.

Topics: Antibiotics, Antineoplastic; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Down-Regulation; Doxorubicin; Drug Resistance, Neoplasm; Humans; Liver Neoplasms; MicroRNAs; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Receptor, Notch1; Signal Transduction; Transcription Factor HES-1

Vasculogenic mimicry (VM) has been suggested to be present in various malignant tumors and associated with tumor nutrition supply and metastasis, leading to poor prognosis of patients. Notch1 has been demonstrated to contribute to VM formation in hepathocellular carcinoma (HCC). Celastrus orbiculatus extract (COE), a mixture of 11 terpenoids isolated from the Chinese Herb Celastrus orbiculatus Vine, has been suggested to be effective in cancer treatment.. In the current study, experiments were carried out to examine the effect of COE on VM formation and HCC tumor growth both in vitro and in vivo.. CCK-8 assay and Nikon live-work station were used to observe the viability of malignant cells treated with COE. Cell invasion was examined using Transwell. Matrigel was used to establish a 3-D culture condition for VM formation. Changes of mRNA and protein expression were examined by RT-PCR and Western Blot respectively. Tumor growth in vivo was monitored using in vivo fluorescence imaging device. PAS-CD34 dual staining and electron microscopy were used to observe VM formation. Immunohistochemical staining (IHC) was used to examine Notch1 and Hes1 expression in tumor tissues.. Results showed that COE can inhibit HCC cells proliferation and invasion in a concentration-dependent manner. VM formation induced by TGF-β1 was blocked by COE. In mouse xenograft model, COE inhibited tumor growth and VM formation. Both in vitro and in vivo studies showed that COE can downregulate expression of Notch1 and Hes1.. The current results indicate that COE can inhibit VM formation and HCC tumor growth by downregulating Notch1 signaling. This study demonstrates that COE is superior to other anti-angiogenesis agents and can be considered as a promising candidate in HCC treatment.

Topics: Angiogenesis Inhibitors; Animals; Carcinoma, Hepatocellular; Celastrus; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Humans; Liver Neoplasms; Male; Mice, Inbred BALB C; Neovascularization, Pathologic; Plant Extracts; Receptor, Notch1; Transcription Factor HES-1; Transforming Growth Factor beta1

Hairy and enhancer of split 1 (Hes-1) protein is a downstream target of Notch signaling and is a basic helix-loop-helix transcriptional repressor. However, definitive evidence for a role in hepatocellular carcinoma (HCC) cells has not been reported. Here, Hes-1 was revealed to an important component of the Notch signaling cascade in HCC cell lines possessing different potential for lung metastasis.. RNAi mediated by plasmid constructs was used to analyze the role of Hes-1 in MHCC-97L HCC cells by assessing proliferation, apoptosis, cell migration and matrigel invasion following transfection. Hes-1 protein expression analysis in HCC tissue was also conducted by immunohistochemistry.. Our studies revealed that Hes-1 was decreased in HCC cell lines with higher lung metastasis potential at both the mRNA and protein levels. Down-regulation of the Hes-1 gene in MHCC-97L cells resulted in increased cell proliferation, reduced apoptosis and increased migration and invasion.. Hes-1 has potential prognostic value in post-surgical HCC patients and may be an independent prognostic indicator for overall survival and tumor recurrence. These findings have important implications for understanding the mechanisms by which Hes-1 participates in tumor proliferation and invasion.

Topics: Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Down-Regulation; Female; Flow Cytometry; Follow-Up Studies; Homeodomain Proteins; Humans; Immunoenzyme Techniques; Liver Neoplasms; Lung Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Prognosis; Real-Time Polymerase Chain Reaction; Receptor, Notch1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Survival Rate; Transcription Factor HES-1; Tumor Cells, Cultured

Aberrant Notch signaling is observed in human hepatocellular carcinoma (HCC) and has been associated with the modulation of cell growth. However, the role of Notch signaling in HCC and its underlying mechanism remain elusive. RBP-J-interacting and tubulin-associated (RITA) mediates the nuclear export of RBP-J to tubulin fibers and downregulates Notch-mediated transcription. In this study, we found that RITA overexpression increased protein expression of p53 and Fbxw7 and downregulated the expression of cyclin D1, cyclin E, CDK2, Hes-1 and NF-κB p65. These changes led to growth inhibition and induced G0/G1 cell cycle arrest and apoptosis in SMMC7721 and HepG2 cells. Our findings indicate that RITA exerts tumor-suppressive effects in hepatocarcinogenesis through induction of G0/G1 cell cycle arrest and apoptosis and suggest a therapeutic application of RITA in HCC.

Topics: Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; DNA-Binding Proteins; Down-Regulation; F-Box Proteins; F-Box-WD Repeat-Containing Protein 7; Gene Knockdown Techniques; Hep G2 Cells; Homeodomain Proteins; Humans; Immunoglobulin J Recombination Signal Sequence-Binding Protein; Liver Neoplasms; Neoplasm Proteins; RNA, Small Interfering; Signal Transduction; Transcription Factor HES-1; Transcription Factor RelA; Tubulin; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Up-Regulation

Chemotherapeutic treatment of hepatocellular carcinoma often leads to chemoresistance during therapy or upon relapse of tumors. For the development of better treatments a better understanding of biochemical changes in the resistant tumors is needed. In this study, we focus on the characterization of in vivo chemoresistant human hepatocellular carcinoma HUH-REISO established from a metronomically cyclophosphamide (CPA) treated HUH7 xenograft model.. SCID mice bearing subcutaneous HUH7 tumors were treated i.p. with 75 mg/kg CPA every six days. Tumors were evaluated by immunohistochemistry, a functional blood-flow Hoechst dye assay, and qRT-PCR for ALDH-1, Notch-1, Notch-3, HES-1, Thy-1, Oct-4, Sox-2 and Nanog mRNA levels. Cell lines of these tumors were analyzed by qRT-PCR and in endothelial transdifferentiation studies on matrigel.. HUH-REISO cells, although slightly more sensitive against activated CPA in vitro than parental HUH-7 cells, fully retained their in vivo CPA chemoresistance upon xenografting into SCID mice. Histochemical analysis of HUH-REISO tumors in comparison to parental HUH-7 cells and passaged HUH-PAS cells (in vivo passaged without chemotherapeutic pressure) revealed significant changes in host vascularization of tumors and especially in expression of the tumor-derived human endothelial marker gene PECAM-1/CD31 in HUH-REISO. In transdifferentiation studies with limited oxygen and metabolite diffusion, followed by a matrigel assay, only the chemoresistant HUH-REISO cells exhibited tube formation potential and expression of human endothelial markers ICAM-2 and PECAM-1/CD31. A comparative study on stemness and plasticity markers revealed upregulation of Thy-1, Oct-4, Sox-2 and Nanog in resistant xenografts. Under therapeutic pressure by CPA, tumors of HUH-PAS and HUH-REISO displayed regulations in Notch-1 and Notch-3 expression.. Chemoresistance of HUH-REISO was not manifested under standard in vitro but under in vivo conditions. HUH-REISO cells showed increased pluripotent capacities and the ability of transdifferentiation to endothelial like cells in vitro and in vivo. These cells expressed typical endothelial surface marker and functionality. Although the mechanism behind chemoresistance of HUH-REISO and involvement of plasticity remains to be clarified, we hypothesize that the observed Notch regulations and upregulation of stemness genes in resistant xenografts are involved in the observed cell plasticity.

Topics: Administration, Metronomic; Aldehyde Dehydrogenase 1 Family; Animals; Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Transdifferentiation; Disease Models, Animal; Drug Resistance, Neoplasm; Endothelial Cells; Female; Gene Expression Profiling; Homeodomain Proteins; Humans; Isoenzymes; Liver Neoplasms; Male; Mice; Nanog Homeobox Protein; Neovascularization, Pathologic; Octamer Transcription Factor-3; Receptor, Notch1; Retinal Dehydrogenase; SOXB1 Transcription Factors; Spheroids, Cellular; Thy-1 Antigens; Transcription Factor HES-1; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

CCAAT/enhancer binding protein α is one of the key transcription factors of the hepatocyte nuclear factors family, which plays a critical role in liver cell proliferation and differentiation. However, the role of CCAAT/enhancer binding protein α in hepatocarcinogenesis remains to be defined.. A recombinant adenovirus carrying the C/EBPα gene was constructed to determine its effect on hepatocarcinogenesis in vitro and in vivo.. We demonstrated that overexpression of CCAAT/enhancer binding protein α inhibited the tumourigenicity of Huh7 cells, re-established the expression of certain liver-specific genes and induced G0/G1 arrest. Overexpression of CCAAT/enhancer binding protein α significantly suppressed the proliferation of primary hepatocarcinogenesis cells and tumour associated fibroblasts in vitro. Additionally, intratumoural injection of adenovirus carrying the C/EBPα reduced the growth of subcutaneous hepatocarcinogenesis xenografts in nude mice. Systemic administration of adenovirus carrying the C/EBPα resulted in the eradication of orthotopic liver hepatocarcinogenesis nodules in nude mice. Further, up-regulation of CCAAT/enhancer binding protein α reduced the expression of Notch3, thereby suppressing Hes1 transactivation activity and leading to decreased p27 expression. Overexpression of Hes1 partially abolished the anti-proliferation effect of CCAAT/enhancer binding protein α on Huh7 cells.. These results suggested that the effect of CCAAT/enhancer binding protein α on hepatocarcinogenesis is partially through by reducing Notch3/Hes1/p27 cascades and CCAAT/enhancer binding protein α may possess a novel therapeutic potential for human hepatocarcinogenesis.

Topics: Adenoviridae; Animals; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Hepatocellular; CCAAT-Enhancer-Binding Protein-alpha; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Genetic Vectors; Homeodomain Proteins; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Proliferating Cell Nuclear Antigen; Receptor, Notch3; Receptors, Notch; Signal Transduction; Transcription Factor HES-1; Transfection

This study was conducted to evaluate the effect of mesenchymal stem cells (MSCs) and a novel curcumin derivative (NCD) on HepG2 cells (hepatoma cell line) and to investigate their effect on Notch1 signaling pathway target genes. HepG2 cells were divided into HepG2 control group, HepG2 cells treated with MSC conditioned medium (MSCs CM), HepG2 cells treated with a NCD, HepG2 cells treated with MSCs CM and NCD, and HepG2 cells treated with MSCs CM (CM of MSCs pretreated with a NCD). Expression of Notch1, Hes1, VEGF, and cyclin D1 was assessed by real-time, reverse transcription-polymerase chain reaction (RT-PCR) in HepG2 cells. In addition, HepG2 proliferation assay was performed in all groups. Notch1 and its target genes (Hes1 and cyclin D1) were downregulated in all treated groups with more suppressive effect in the groups treated with both MSCs and NCD. Also, treated HepG2 cells showed significant decrease in cell proliferation rate. These data suggest that modulation of Notch1 signaling pathway by MSCs and/or NCD can be considered as a therapeutic target in HCC.

Topics: Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Hepatocellular; Cell Proliferation; Curcumin; Cyclin D1; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Homeodomain Proteins; Humans; Liver Neoplasms; Mesenchymal Stem Cells; Receptor, Notch1; Signal Transduction; Transcription Factor HES-1; Vascular Endothelial Growth Factor A

Embryologically, intrahepatic small bile ducts arise from hepatic progenitor cells via ductal plates, whereas the pancreato-extrahepatic biliary progenitor cells expressing the transcription factors PDX1 and HES1 are reportedly involved in the development of the extrahepatic biliary tract and ventral pancreas. The expression of cellular markers characteristic of the different anatomical levels of the biliary tree and pancreas, as well as PDX1 and HES1, was examined in cholangiocarcinoma components of combined hepatocellular cholangiocarcinoma (12 cases), intrahepatic cholangiocarcinoma (21 cases), hilar cholangiocarcinoma (25 cases), and pancreatic ductal adenocarcinoma (18 cases). Anterior gradient protein-2 and S100P were frequently expressed in hilar cholangiocarcinoma and pancreatic ductal adenocarcinoma, whereas neural cell adhesion molecule and luminal expression of epithelial membrane antigen were common in cholangiocarcinoma components of combined hepatocellular cholangiocarcinoma. PDX1 and HES1 were frequently and markedly expressed in pancreatic ductal adenocarcinoma and, to a lesser degree, in hilar cholangiocarcinoma, although their expression was rare and mild in cholangiocarcinoma components in combined hepatocellular cholangiocarcinoma. The expression patterns of these molecules in intrahepatic cholangiocarcinoma were intermediate between those in hilar cholangiocarcinoma and cholangiocarcinoma components of combined hepatocellular cholangiocarcinoma. Pancreatic ductal adenocarcinoma and hilar cholangiocarcinoma had a similar expression of mucin, immunophenotypes, as well as transcription factors. Pancreatic ductal adenocarcinoma and hilar cholangiocarcinoma showed similar postoperative prognosis. In conclusion, the similar expression of phenotypes related to pancreatobiliary anatomy and embryology may in part explain why these 2 types of carcinoma present similar clinicopathologic features. Further studies on the carcinogenesis of these carcinomas based on their similarities are warranted.

Topics: Aged; Basic Helix-Loop-Helix Transcription Factors; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Carcinoma, Pancreatic Ductal; Cholangiocarcinoma; Female; Homeodomain Proteins; Humans; Immunohistochemistry; Klatskin Tumor; Liver Neoplasms; Male; Middle Aged; Mucins; Pancreatic Neoplasms; Trans-Activators; Transcription Factor HES-1

Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma (HCC), however, little is known about the mechanism. Here, we investigated the relationship between HBx and Notch signaling in HepG2 cells after they were transfected with the HBx gene. It was found that HBx upregulated the expression of Notch-1, Jagged-1 and Hes-1 at the transcriptional level by binding to the Notch-1 intracellular domain, which is congruent with the observations of enhanced malignant biological activities of HBx-transfected HepG2 cells compared with normal HepG2 cells. However, while Notch signaling was blocked, the HBx-induced abnormalities were partially reversed. These findings suggest that HBx may promote the progression of HCC via the activated Notch pathway.

Topics: Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Calcium-Binding Proteins; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Proliferation; Dipeptides; Hep G2 Cells; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Liver Neoplasms; Membrane Proteins; Receptor, Notch1; Serrate-Jagged Proteins; Signal Transduction; Time Factors; Trans-Activators; Transcription Factor HES-1; Transfection; Viral Regulatory and Accessory Proteins

Current studies are ongoing to find new drugs for the treatment of hepatocellular carcinoma (HCC). The discovery of drugs depends on the identification of molecules that can play essential roles in the development of liver cancer, for example, Notch pathway molecules. γ-Secretase inhibitors (GSIs) can inhibit the cleavage of intramembranous substrates of all Notch receptors and subsequently suppress Notch signaling. However, whether the inhibition of the Notch pathway can suppress or promote HCC growth is still under debate. In this study, we examined the expression of Notch pathway molecules in 20 pairs of HCC tissue with their normal counterparts and a panel of eight HCC cell lines. We also determined the effects of different types of GSI treatments on the cell growth of those HCC cell lines. Our results showed that the molecules of the Notch pathway were expressed in six of the eight HCC cell lines. Those six HCC cell lines were more sensitive to GSI-I treatment than the nonexpression ones. Among the four inhibitors, GSI-X and GSI-XXI exerted no effect on HCC cells growth at all. GSI-IX inhibited the growth of four HCC cell lines at 40 μmol/l. In contrast, most of these HCC cell lines were susceptible to a low concentration of GSI-I (1.2 μmol/l) treatment. The suppressive effect of GSI-I on cell growth was because of the inhibition of C-Myc, a Notch target gene. In addition, 80% (16/20) of the specimens showed either an increased expression of at least one Notch receptor or an augmented expression of Jagged1 compared with their normal counterparts. Our study reports for the first time that different kinds of GSIs can block the growth of several HCC cell lines. Our finding suggests that GSI-I is a potential chemical reagent and warrants additional testing in liver cancer therapeutics.

Topics: Amyloid Precursor Protein Secretases; Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Benzodiazepinones; Calcium-Binding Proteins; Carbamates; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dipeptides; Gene Expression Regulation, Neoplastic; Genes, myc; Hep G2 Cells; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Liver Neoplasms; Membrane Proteins; Oligopeptides; Receptors, Notch; Serrate-Jagged Proteins; Transcription Factor HES-1

CDKN1C/P57 is a cyclin-dependent kinase inhibitor implicated in different human cancers, including hepatocellular carcinoma (HCC); however, little is known regarding the role of CDKN1C/P57 and its regulation in HCC. In this study, we show that the down-regulation of Notch1 and Notch3 in two HCC cell lines resulted in Hes1 down-regulation, CDKN1C/P57 up-regulation, and reduced cell growth. In line with these data, we report that CDKN1C/P57 is a target of transcriptional repression by the Notch effector, Hes1. We found that the up-regulation of CDKN1C/P57 by cDNA transfection decreased tumor growth, as determined by growth curve, flow cytometry analysis, and cyclin D1 down-regulation, without affecting the apoptosis machinery. Indeed, the expression of Bax, Noxa, PUMA, BNIP(3), and cleaved caspase-3 was not affected by CDKN1C/P57 induction. Morphologically CDKN1C/p57-induced HCC cells became flat and lengthened in shape, accumulated the senescence-associated β-galactosidase marker, and increased P16 protein expression. Evaluation of senescence in cells depleted both for Hes1 and CDKN1C/P57 revealed that the senescent state really depends on the accumulation of CDKN1C/p57. Finally, we validated our in vitro results in primary HCCs, showing that Hes1 protein expression inversely correlates with CDKN1C/P57 mRNA levels. In addition, reduced Hes1 protein expression is accompanied by a shorter time to recurrence after curative resection, suggesting that Hes1 may represent a biomarker for prediction of patients with poor prognosis.

Topics: Aged; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Chromatin Immunoprecipitation; Cyclin-Dependent Kinase Inhibitor p57; Female; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Gene Silencing; Homeodomain Proteins; Humans; Liver Neoplasms; Male; Middle Aged; Receptor, Notch1; Receptor, Notch3; Receptors, Notch; Recurrence; Signal Transduction; Time Factors; Transcription Factor HES-1

RUNX3 takes a strong suppressive effect in many tumors including hepatocellular carcinoma (HCC). HES-1, a downstream target of Notch signaling, is shown to be decreased in human HCC cell line SMMC7721 with RUNX3 gene transfection. Since Notch signaling is oncogenic in HCC, RUNX3 might exert its inhibitory effect in HCC partly through the suppression on Notch signaling. To investigate the possible mechanism of the down-regulation of HES-1 by RUNX3, we performed Western blot and reporter assay and found that RUNX3 suppressed intracellular domain of Notch1 (ICN1)-mediated transactivation of Notch signaling while it did not alter the expression of ICN1 and recombination signal binding protein-J kappa (RBP-J) in SMMC7721 cells. Besides, confocal microscopy, co-immunoprecipitation and GST pull-down assays showed that RUNX3 could co-localize with ICN1 and RBP-J, forming a complex with these two molecules in nucleus of SMMC7721 cells by its direct interaction with ICN1. Furthermore, RUNX3 was recruited to RBP-J recognition motif of HES-1 promoter, which was identified by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Taken together, these findings indicate that RUNX3 suppresses Notch signaling in HCC SMMC7721 cells by its interaction with ICN1 and thus recruitment to the RBP-J recognition motif of downstream genes of Notch signaling.

Topics: Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Nucleus; Core Binding Factor Alpha 3 Subunit; Dipeptides; DNA; Gene Expression; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Immunoglobulin J Recombination Signal Sequence-Binding Protein; Liver Neoplasms; Peptide Fragments; Promoter Regions, Genetic; Protease Inhibitors; Protein Binding; Protein Interaction Domains and Motifs; Receptor, Notch1; Recombinant Fusion Proteins; Signal Transduction; Transcription Factor HES-1; Transcriptional Activation; Transfection

Hepatoblastoma is characterized by a diversity of differentiation patterns, some resembling stages of liver development, and occasionally associated with clinical behavior. Our hypothesis is that histologic microheterogeneity in hepatoblastoma correlates with molecular heterogeneity and reflects different stages of developmental arrest. We studied the activation status of the Wnt and Notch pathways and the differential expression of hepatocyte nuclear factor 4alpha, EGFR, and IGF2 genes, relevant to liver development and malignant transformation in histologic variants of hepatoblastoma. Eighty-seven percent of 32 hepatoblastoma cases studied carried CTNNB1 mutations within the ubiquitination domain. Large deletions were seen only in pure fetal cases, also characterized by CCND1 and GLUL (GS) overexpression. Hepatoblastomas with small-cell type appeared clearly distinct and were the only ones with negative GLUL expression. HES1 expression and HES1/AXIN2 used to measure Notch versus Wnt activation ratio were particularly elevated in pure fetal cases and were lowest in hepatoblastomas with small-cell component. Hepatocyte nuclear factor 4alpha was relatively elevated only in embryonal hepatoblastomas. DLK1, DKK, AXIN2, IGF2, and EGFR were increased in all subtypes. Our results support the hypothesis that hepatoblastoma microheterogeneity correlates with molecular heterogeneity. DLK1, a marker of bipotential oval cells, is consistently up-regulated in hepatoblastoma. Therefore, we speculate that hepatoblastomas may arise from a proliferating bipotential precursor. Wnt activation is prevalent in hepatoblastomas, most significantly in predominantly embryonal and mixed types, whereas Notch activation, needed for cholangiocytic differentiation at a more differentiated state, is highest in pure fetal hepatoblastomas. The relative Wnt versus Notch activation appears useful in stratifying different subtypes.

Topics: Basic Helix-Loop-Helix Transcription Factors; beta Catenin; Calcium-Binding Proteins; Child; Child, Preschool; ErbB Receptors; Female; Hepatoblastoma; Hepatocyte Nuclear Factor 4; Homeodomain Proteins; Humans; Immunohistochemistry; Infant; Infant, Newborn; Insulin-Like Growth Factor II; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Male; Membrane Proteins; Transcription Factor HES-1; Wnt Proteins

Notch signalling is altered in several solid tumours and it plays a role in growth inhibition and apoptosis of hepatocellular carcinoma (HCC)-derived cell lines, bile duct development and hepatocyte regeneration.. This study aims to analyse the expression of Notch3, Notch4 and HES1 and HES6 as Notch-target genes in HCC, matched non-neoplastic tissue and HEPG2 cells.. Notch3 and Notch4 are not expressed in normal liver and in chronic hepatitis surrounding HCC. Cirrhotic tissue stains negative for Notch3, while Notch4 is expressed by hepatocytes at the edge of regenerative nodules and in cell planes adjacent to fibrous septa. HCC tissue displays Notch3 and Notch4 abnormal accumulation, respectively, in 78% and 68% of the cases. The endothelium of hepatic veins with neoplastic permeation is frequently Notch4 positive. An upregulation of Notch3 mRNA was found in 95% of HCCs vs cirrhosis (P=0.0001), while Notch4 mRNA was downregulated in 80% of HCCs. HES6 mRNA expression was higher in HCC tissue when compared with cirrhosis (P=0.007), paralleling Notch3 mRNA expression. The HEPG2 cell line displays high Notch3 and low Notch4 protein and mRNA levels.. These descriptive findings suggest an aberrant expression of Notch3 and Notch4 in HCC and allow the hypothesis of an activation of Notch signalling by Notch3.

Topics: Aged; Aged, 80 and over; Basic Helix-Loop-Helix Transcription Factors; Blotting, Far-Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Hepatitis B, Chronic; Hepatitis C, Chronic; Homeodomain Proteins; Humans; Immunohistochemistry; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Proto-Oncogene Proteins; Receptor, Notch3; Receptor, Notch4; Receptors, Notch; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transcription Factor HES-1