cyclin-d1 and Hepatoblastoma

Hepatoblastoma is the most common malignant liver tumor in children. Since it is often unresectable and exhibits drug resistance, the treatment of advanced hepatoblastoma is challenging. The orphan nuclear receptor liver receptor homolog‑1 (LRH‑1) serves prominent roles in malignancy; however, to the best of our knowledge, the role of LRH‑1 in hepatoblastoma remains unknown. In the present study, human hepatoblastoma cell lines were analyzed; the mRNA and protein expression levels of LRH‑1 were significantly higher in HepG2 and HuH6 cells compared with those in HepT1 cells and control THLE‑2 cells. Knockdown of LRH‑1 resulted in decreased HepG2 and HuH6 cell proliferation via downregulation of cyclin D1 (CCND1) and c‑Myc. Furthermore, treatment with an LRH‑1 antagonist (LRA) inhibited the proliferation and colony formation of cell lines in a dose‑dependent manner, and induced cell cycle arrest at G1 phase through inhibition of CCND1 expression. Finally, LRA treatment enhanced the cytotoxic effects of doxorubicin on hepatoblastoma cells. Collectively, these findings suggested that LRH‑1 may have an important role in the progression of hepatoblastoma and implicated LRA as a novel, potential therapeutic agent for the treatment of hepatoblastoma.

Topics: Cell Line, Tumor; Cell Proliferation; Cell Survival; Child, Preschool; Cyclin D1; Doxorubicin; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Hep G2 Cells; Hepatoblastoma; Humans; Liver Neoplasms; Proto-Oncogene Proteins c-myc; Receptors, Cytoplasmic and Nuclear; Up-Regulation

Hepatoblastoma (HB) is a rare malignant liver tumour found in infants. Many heterogeneous histological tumour subtypes exist. Although survival rates have improved dramatically in recent years with the use of platinum-based chemotherapy, there still exists a subset of HB that does not respond to treatment. There are currently no tumour biomarkers in use and in this study we aim to evaluate potential biomarkers to aid identification of relapse cases that would otherwise be overlooked by current prognostication. This may identify patients that would benefit from more aggressive therapy and could improve overall survival rates. We used immunohistochemistry to analyse the expression of β-catenin, E-cadherin, Cyclin D1, Ki-67 and alpha-fetoprotein (AFP) protein in tumours from 91 patients prospectively enroled into the SIOPEL 3 clinical trial. The relationship between these biomarkers and clinicopathologic features and patient survival were statistically analysed. We identified one biomarker, Cyclin D1, which has a correlation with mixed epithelial/mesenchymal HB approaching significance (P = 0.07). Survival analysis using these markers has revealed two potential prognostic indicators; Cyclin D1 and Ki-67 (P = 0.01, 0.01).

Topics: alpha-Fetoproteins; beta Catenin; Biomarkers, Tumor; Cadherins; Cyclin D1; Hepatoblastoma; Humans; Immunohistochemistry; Ki-67 Antigen; Liver Neoplasms; Prognosis; Randomized Controlled Trials as Topic

Hepatitis C virus Core plays a vital role in the development of hepatocellular carcinoma; however, its action mechanism is still controversial. Here, we showed that Core down-regulated levels of p16, resulting in inactivation of Rb and subsequent activation of E2F1, which lead to growth stimulation of hepatocytes. For this effect, Core inhibited p16 expression by inducing promoter hypermethylation via up-regulation of DNA methyltransferase 1 (DNMT1) and DNMT3b. The growth stimulatory effect of Core was abolished when levels of p16 were restored by either exogenous complementation or treatment with 5-Aza-2'dC, indicating that the effect is critical for the stimulation of cell growth by Core.

Topics: Amino Acid Sequence; Blotting, Western; Cell Cycle; Cell Line; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3B; Down-Regulation; G1 Phase; Hep G2 Cells; Hepatoblastoma; Host-Pathogen Interactions; Humans; Liver; Liver Neoplasms; Molecular Sequence Data; Promoter Regions, Genetic; RNA Interference; S Phase; Transfection; Viral Core Proteins

Hepatoblastoma (HBL), a major childhood malignant neoplasm, represents the most frequent malignant liver tumor in childhood. Recent reports have shown the CTNNB1 coding beta-catenin protein to be frequently mutated or deleted at hot-spot regions involving exon 3 in HBL. We investigated the genetic alterations of the CTNNB1 coding beta-catenin protein and expression of several genes downstream of Wnt signals in 4 benign and 17 malignant pediatric liver tumors (PLTs) consisting of 15 HBL, 1 hepatocellular carcinoma, and 1 hepatic immature sarcoma. Of 17 malignant PLTs, 10 (56%) revealed pathogenic alterations of the CTNNB1 gene, including 4 with missense mutations at codons 28, 32, 34 or 44, and 6 with interstitial deletions that partially or totally affected exon 3. All 6 deletions were in-frame deletions without a frame shift. The high frequency without any correlation to histological type indicates that the CTNNB1 gene alteration is a crucial event in the tumorigenesis of malignant PLTs. The immunohistochemical studies in 17 malignant PLTs demonstrated the nuclear/cytoplasmic accumulation of beta-catenin to be positive in all tumor specimens except for one hepatic sarcoma. A histological examination revealed all HBL cases involving tumors without detectable CTNNB1 gene alterations to show high expression of beta-catenin, thus indicating the accumulation of beta-catenin to be a common event in malignant PLTs, including HBL and hepatocellular carcinoma. Among the Wnt signal genes downstream of beta-catenin, E-cadherin is expressed in all malignant PLTs, while cyclin D1 expression was significantly detected in malignant PLTs with an advanced stage of disease. An immunohistological examination of nuclear accumulation of beta-catenin may thus be useful for diagnosing malignant PLTs. On the other hand, the expression of cyclin D1, a gene downstream of beta-catenin, might play a role in tumor progression.

Topics: beta Catenin; Cadherins; Carcinoma, Hepatocellular; Child; Child, Preschool; Cyclin D1; DNA Mutational Analysis; DNA, Neoplasm; Female; Hepatoblastoma; Humans; Immunohistochemistry; Infant; Liver Neoplasms; Male; Mutation; Prognosis

Hepatoblastomas are the most frequent malignant liver tumors of childhood. A high frequency of activating beta-catenin mutations in hepatoblastomas indicates that the Wnt signaling pathway plays an important role in the development of this embryonic neoplasm. Stabilization of beta-catenin leads to an increased formation of nuclear beta-catenin-T-cell factor complexes and altered expression of Wnt-inducible target genes. In this study, we analyzed the mRNA expression levels of nine Wnt genes, including c-JUN, c-MYC, CYCLIN D1, FRA-1, NKD-1, ITF-2, MMP-7, uPAR, and beta-TRCP, by competitive reverse transcription-PCR. We analyzed 23 hepatoblastoma biopsies for which matching liver tissue was available, 6 hepatoblastoma cell lines, and 3 human fetal liver samples. beta-TRCP and NKD-1 were highly expressed in all hepatoblastoma samples, independent of the beta-catenin mutational status, in comparison with their nontumorous counterparts. beta-TRCP mRNA overexpression was associated with accumulation of intracytoplasmic and nuclear beta-TrCP protein. In human liver tumor cells without beta-catenin mutations, Nkd-1 inhibited the Wnt-3a-activated Tcf-responsive-luciferase reporter activity, whereas Nkd-1 in hepatoblastomas with beta-catenin mutations had no antagonistic effect. Our data emphasize the inhibitory effect of beta-TrCP and Nkd-1 on the Wnt signaling pathway in a manner analogous to Conductin (AXIN2) and Dkk-1, inhibitors shown previously to be up-regulated in hepatoblastomas. Our findings indicate that overexpression of the Wnt antagonists Nkd-1 and beta-TrCP reveals an activation of the Wnt signaling pathway as a common event in hepatoblastomas. We propose that Nkd-1 and beta-TrCP may be used as possible diagnostic markers for the activated Wnt signaling pathway in hepatoblastomas.

Topics: Adaptor Proteins, Signal Transducing; Adolescent; Adult; beta Catenin; beta-Transducin Repeat-Containing Proteins; Calcium-Binding Proteins; Carrier Proteins; Cell Line, Tumor; Child; Cyclin D1; Cytoskeletal Proteins; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Hepatoblastoma; Humans; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Male; Matrix Metalloproteinase 7; Middle Aged; Mutation; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-myc; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; TCF Transcription Factors; Trans-Activators; Transcription Factor 7-Like 2 Protein; Transcription Factors; Wnt Proteins

Cyclin D1, encoded by the gene CCND1, is a major regulator of the cell cycle transition from G1 phase to S phase. A CCND1 polymorphism (G to A) at codon 242, the boundary of exon 4 and intron 4, affects splicing such that exon 5 is not expressed in the A allele. Since exon 5 is involved in rapid turnover, the variant cyclin D1 corresponding to the A allele may have a longer half-life. A previous study demonstrated that in families with hereditary nonpolyposis colorectal cancer, the age of onset of colorectal cancer varied according to variation at this polymorphic site. We examined this CCND1polymorphism in a series hepatoblastoma, a childhood liver cancer that shares other molecular features with colon cancer. We determined in an analysis of 84 children with hepatoblastoma that the G/A exon 4 polymorphism in CCND1 is correlated with the age of onset of hepatoblastomas. The A/A genotype is associated with an earlier age of onset compared to the G/A or G/G genotype. The median age of patients with the G/G genotype was 22 months, compared to 17 months in patients with the G/A genotype and 11 months for the A/A genotype. These findings suggest that the CCND1 A polymorphism may contribute to tumor development in children with hepatoblastoma.

Topics: Age Distribution; Age of Onset; Black People; Child; Child, Preschool; Confidence Intervals; Cyclin D1; DNA; Female; Gene Frequency; Hepatoblastoma; Hispanic or Latino; Humans; Infant; Liver Neoplasms; Male; Odds Ratio; Polymorphism, Genetic; Polymorphism, Single-Stranded Conformational; White People

In this study we used liver neoplasms induced by several chemical carcinogens to investigate potential nuclear targets associated with beta-catenin/Wnt signaling and potential membrane-associated beta-catenin binding partners. Strong expression of cyclin D1, in a pattern similar to that observed previously for beta-catenin, was observed by Western analysis for all five hepatoblastomas examined regardless of treatment. Increased expression of cyclin D1 was also detected in 12 of 35 (34%) hepatocellular neoplasms. Ten of 15 tumors (67%) that had mutations in the Catnb gene had upregulation of cyclin D1, while only 2 of 20 tumors (10%) without Catnb mutations had increased cyclin D1 expression. Immunohistochemical analysis confirmed strong expression of cyclin D1 in most nuclei of hepatoblastomas and scattered nuclear staining in hepatocellular tumors that had Catnb mutations. Increased c-Jun expression was observed in 19 of 30 (63%) hepatocellular tumors and all hepatoblastomas, although upregulation was not completely correlated with Catnb mutation. C-Myc expression was not increased in the tumors. Reduced expression of E-cadherin, which interacts with beta-catenin at the membrane, was observed in some tumors, but this did not correlate with Catnb mutation. Expression of the epidermal growth factor receptor, which may have a role in beta-catenin tyrosine phosphorylation, was lower in some tumors than in normal tissue depending on chemical treatment. The results provide evidence that increased expression of cyclin D1 and c-Jun may provide an advantage during tumor progression and in the transition from hepatocellular neoplasms to hepatoblastomas. Moreover, it is likely increased cyclin D1 expression results at least in part from Catnb mutation, beta-catenin accumulation, and increased Wnt signaling.

Topics: Adenoma, Liver Cell; Animals; beta Catenin; Biomarkers, Tumor; Cadherins; Carcinoma, Hepatocellular; Cyclin D1; Cytoskeletal Proteins; ErbB Receptors; Genes, ras; Hepatoblastoma; Immunoenzyme Techniques; Liver Neoplasms, Experimental; Mice; Mice, Inbred Strains; Mutation; Neoplasm Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-myc; Trans-Activators; Wnt Proteins; Zebrafish Proteins

Hepatoblastoma (HBL) is the most common malignant liver tumor in young children. Recent reports have shown that the beta-catenin gene was frequently mutated or deleted in HBLS: To elucidate the role of beta-catenin abnormalities in HBLs, we searched for mutations of beta-catenin and APC as well as expression of the target genes, cyclin D1, c-myc, and fibronectin, in 68 primary HBLS: The mutation analysis revealed that 44 (65%) tumors carried missense mutations or deletions of beta-catenin, all of which were somatic and targeted to the exon 3 encoding the amino acid residues involved in its degradation. However, no loss of function mutation of the APC gene was detected by the yeast functional assay. Of interest, beta-catenin mutation was significantly correlated with overexpression of the target genes, cyclin D1 and fibronectin, but not with that of c-myc in HBLs as measured by quantitative real-time reverse transcription-PCR. The immunohistochemical studies in 15 HBLs demonstrated that the nuclear/cytoplasmic accumulation of beta-catenin was positive in 13 tumors, 9 of which had the deletion or mutation of the gene. The significant correlation between the beta-catenin gene abnormality and the positive staining of cyclin D1 was also confirmed. Furthermore, the nuclear accumulation of beta-catenin was strongly associated with the poorly differentiated tumor cell components as well as with the positive staining of cyclin D1 within the tumor. Thus, our present results suggested that the gain of function mutation of beta-catenin played a crucial role in the malignant progression of HBL in vivo.

Topics: Adenomatous Polyposis Coli Protein; beta Catenin; Cell Differentiation; Child; Child, Preschool; Cyclin D1; Cytoskeletal Proteins; Fibronectins; Gene Deletion; Hepatoblastoma; Humans; Immunohistochemistry; Infant; Statistics as Topic; Trans-Activators

Abnormality of the cyclin D1/cdk4/p16INK4a/pRb pathway during tumorigenesis has recently been reported. Hepatoblastoma is a rare malignant liver tumor of childhood, but underlying abnormalities of cell-cycle regulating protein remain to be elucidated. The expression of cyclin D1, cdk4, p16 and retinoblastoma gene product (pRb) was studied by immunohistochemistry in 17 paraffin-embedded tissues consisting of both tumor and corresponding non-neoplastic tissues. Tumor tissues showed overexpression of cyclin D1 (13/17, 76%) and cdk4 (15/17, 88%). Eleven cases showed co-overexpression of both cyclin D1 and cdk4. No abnormal p16 or pRb expression was noted. In the group with a high score (+4) for cyclin D1 expression, a positive correlation with tumor recurrence was noted (P = 0.043). These data suggest that overexpressed cyclin D1 and cdk4 protein might play an important role in the tumorigenesis of hepatoblastoma and that in the group with high cyclin D1 expression, tumor recurrence may be more frequent.

Topics: Adolescent; Cell Transformation, Neoplastic; Child; Child, Preschool; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Female; Hepatoblastoma; Humans; Immunohistochemistry; Infant; Liver Neoplasms; Male; Proto-Oncogene Proteins; Retinoblastoma Protein

A rare case of hepatoblastoma in a 61-year-old Japanese housewife is described. This liver tumor mainly consisted of two tissue components: embryonal hepatocytes and primitive mesenchymal tissue. Fetal hepatocytes with alpha-fetoprotein production, gland formation, cartilage and osteoid were also found in a small portion. Molecular analysis by slot blot method revealed increased copy numbers of c-met and K-sam proto-oncogenes and cyclin D1 genes. These findings suggest that alterations of these oncogenes might play a role in the development of adult hepatoblastoma.

Topics: Cyclin D1; Cyclins; Female; Hepatoblastoma; Hepatocyte Growth Factor; Humans; Immunoblotting; Immunohistochemistry; Liver Neoplasms; Middle Aged; Oncogene Proteins; Proto-Oncogene Mas; Proto-Oncogene Proteins c-met; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 2; Receptors, Fibroblast Growth Factor