Investigation of CTNNB1 gene mutations and expression in hepatocellular carcinoma and cirrhosis in association with hepatitis B virus infection.
ABSTRACT: Hepatitis B virus (HBV), along with Hepatitis C virus chronic infection, represents a major risk factor for hepatocellular carcinoma (HCC) development. However, molecular mechanisms involved in the development of HCC are not yet completely understood. Recent studies have indicated that mutations in CTNNB1 gene encoding for ?-catenin protein lead to aberrant activation of the Wnt/ ?-catenin pathway. The mutations in turn activate several downstream genes, including c-Myc, promoting the neoplastic process. The present study evaluated the mutational profile of the CTNNB1 gene and expression levels of CTNNB1 and c-Myc genes in HBV-related HCC, as well as in cirrhotic and control tissues. Mutational analysis of the ?-catenin gene and HBV genotyping were conducted by direct sequencing. Expression of ?-catenin and c-Myc genes was assessed using real-time PCR. Among the HCC cases, 18.1% showed missense point mutation in exon 3 of CTNNB1, more frequently in codons 32, 33, 38 and 45. The frequency of mutation in the hotspots of exon 3 was significantly higher in non-viral HCCs (29.4%) rather than HBV-related cases (12.7%, P?=?0.021). The expression of ?-catenin and c-Myc genes was found upregulated in cirrhotic tissues in association with HBV infection. Mutations at both phosphorylation and neighboring sites were associated with increased activity of the Wnt pathway. The results demonstrated that mutated ?-catenin caused activation of the Wnt pathway, but the rate of CTNNB1 gene mutations was not related to HBV infection. HBV factors may deregulate the Wnt pathway by causing epigenetic alterations in the HBV-related HCC.
Project description:HBx mutations (T1753V, A1762T, G1764A, and T1768A) are frequently observed in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Aberrant activation of the Wnt/?-catenin signaling pathway is involved in the development of HCC. However, activation of the Wnt/?-catenin signaling pathway by HBx mutants has not been studied in hepatoma cells or HBV-associated HCC samples. In this study, we examined the effects of HBx mutants on the migration and proliferation of HCC cells and evaluated the activation of Wnt/?-catenin signaling in HBx-transfected HCC cells and HBV-related HCC tissues. We found that HBx mutants (T, A, TA, and Combo) promoted the migration and proliferation of hepatoma cells. The HBx Combo mutant potentiated TOP-luc activity and increased nuclear translocation of ?-catenin. Moreover, the HBx Combo mutant increased and stabilized ?-catenin levels through inactivation of glycogen synthase kinase-3?, resulting in upregulation of downstream target genes such as c-Myc, CTGF, and WISP2. Enhanced activation of Wnt/?-catenin was found in HCC tissues with HBx TA and Combo mutations. Knockdown of ?-catenin effectively abrogated cell migration and proliferation stimulated by the HBx TA and Combo mutants. Our results indicate that HBx mutants, especially the Combo mutant, allow constitutive activation of the Wnt signaling pathway and may play a pivotal role in HBV-associated hepatocarcinogenesis.
Project description:The Wnt/?-catenin pathway plays a vital role in initiating and sustaining hepatocellular carcinoma (HCC). However, few studies have investigated polymorphisms in the Wnt/?-catenin signaling pathway genes in the Chinese Han population. The aim of the present retrospective study was to investigate the correlations between polymorphisms of the Wnt/?-catenin signaling pathway genes (CTNNB1 and WNT2) and HCC susceptibility, development, and progression.Twenty tagging single nucleotide polymorphisms were chosen from HapMap data and genotyped in 320 patients with HCC, 320 chronic hepatitis B virus (HBV)-infected patients without HCC (N-HCC, including 95 liver cirrhosis, 164 chronic hepatitis B, and 61 asymptomatic HBV carriers), and 320 healthy controls. Associations between polymorphisms in the 2 Wnt/?-catenin signaling pathway genes (CTNNB1 and WNT2) and HCC susceptibility, development, and progression were investigated.Genotype AA (P?=?0.002, odds ratio [OR]?=?2.524) and allele A (P?=?0.0003, OR?=?1.613) of the WNT2 rs4730775 polymorphism were associated with HCC susceptibility compared with healthy controls. Genotype GA (P?=?0.001, OR?=?0.567) and allele A (P?=?0.002, OR?=?0.652) of rs3864004, and genotype AG (P?=?0.0004, OR?=?0.495) and allele G (P?=?0.001, OR?=?0.596) of rs11564475 in the CTNNB1 gene were correlated with HCC compared with N-HCC patients. These findings were consistent in dominant and recessive models. Multidimensionality reduction analysis revealed that interactions among rs3864004, rs11564475, and rs4730775 were significantly associated with HCC compared with N-HCC patients. The polymorphism rs4135385 of CTNNB1 genotype GA was associated with a higher risk for Stage III + IV HCC (modified Union for International Cancer Control) (P?=?0.001, OR?=?2.238).Genetic polymorphisms in the WNT2 and CTNNB1 genes were closely associated with HCC risk and progression in a Chinese Han population.
Project description:The mechanisms of Myc-driven liver tumorigenesis are inadequately understood. Herein we show that Myc-driven hepatocellular carcinoma (HCC) is dramatically aggravated in mice with hepatocyte-specific Ptpn11/Shp2 deletion. However, Myc-induced tumors develop selectively from the rare Shp2-positive hepatocytes in Shp2-deficent liver, and Myc-driven oncogenesis depends on an intact Ras-Erk signaling promoted by Shp2 to sustain Myc stability. Despite a stringent requirement of Shp2 cell autonomously, Shp2 deletion induces an immunosuppressive environment, resulting in defective clearance of tumor-initiating cells and aggressive tumor progression. The basal Wnt/β-catenin signaling is upregulated in Shp2-deficient liver, which is further augmented by Myc transfection. Ablating Ctnnb1 suppresses Myc-induced HCC in Shp2-deficient livers, revealing an essential role of β-catenin. Consistently, Myc overexpression and CTNNB1 mutations are frequently co-detected in HCC patients with poor prognosis. These data elucidate complex mechanisms of liver tumorigenesis driven by cell-intrinsic oncogenic signaling in cooperation with a tumor-promoting microenvironment generated by disrupting the specific oncogenic pathway.
Project description:<h4>Purpose</h4>Hepatocellular carcinoma (HCC) is the sixth most common solid tumor worldwide and the third leading cause of cancer-related death. HCC is a particularly serious threat to the Chinese population. Although many molecular alterations are known to be involved in the tumorigenesis of hepatocytes, no systemic survey has examined the somatic mutations in HCC samples from Chinese patients. Our goal was to elucidate somatic mutations in Chinese HCC patients and investigate the possible molecular mechanisms involved in tumorigenesis.<h4>Experimental design</h4>A total of 110 hepatitis B virus (HBV)-positive HCC samples and 46 HBV-negative HCC samples were genotyped for hot-spot mutations in the CSF1R, CTNNB1, KRAS, BRAF, NRAS, ERBB2, MET, PIK3CA, JAK1, and SMO genes. The transcriptomes of the CTNNB1 mutation-positive HCC samples from the HBV-positive patients (CB+ HCC) were compared to adjacent non-cancerous livers, and significantly altered genes were functionally validated in vitro.<h4>Results</h4>CTNNB1 mutations accounted for the majority of the mutations detected in our study. A slightly higher mutation rate was found in the HBV-positive patients than in their negative counterparts. A distinct pattern of CTNNB1 mutation was detected in these two populations, and drastic changes at the transcriptomic level were detected in the CB+ tumors compared to adjacent non-cancerous livers. Potential tumor suppressors (FoxA3 and Onecut1) and oncogenes (MAFG and SSX1) were functionally validated.<h4>Conclusions</h4>Our work is the first systemic characterization of oncogenic mutations in HCC samples from Chinese patients. Targeting the Wnt-?-catenin pathway may represent a valid treatment option for Chinese HCC patients. Our work also suggests that targeting ONECUT1, FOXA3, SSX1, and MAFG may be a valid treatment option for CTNNB1 mutation positive HCC patients.
Project description:Recurrent somatic mutations in the promoter region of telomerase reverse transcriptase (TERT) gene and in the exon 3 of CTNNB1 gene have been recognized as common events in hepatocellular carcinoma (HCC) with variable frequencies depending on etiology and geographical region. We have analyzed TERT promoter and CTNNB1 gene mutations in 122 cases of hepatitis B (HBV) and hepatitis C (HCV) related HCCs, in 7 cases of cholangiocarcinoma (CC) and hepatocholangiocarcinoma (HCC-CC) as well as in autologous cirrhotic tissues. Overall, 50.4% and 26% of HCC as well as 14.3% and none of CC and HCC-CC were mutated in TERT promoter and in CTNNB1 exon 3, respectively. TERT and CTNNB1 mutations were found more frequently in HCV related (53.6% and 26.4%, respectively) than HBV related (41.7% and 16.7%, respectively) HCCs and coexisted in 57.6% of CTNNB1 mutated tumors. Mutations in TERT and CTNNB1 were not associated with the functional promoter polymorphism rs2853669. No mutations were detected in the 129 non-HCC cirrhotic tissues. In conclusion, mutations in TERT promoter and in CTNNB1 gene represent specific cancer signatures in the pathogenesis of viral related HCC and could be promising early biomarkers as well as targets for tailored therapies.
Project description:Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Here, we performed high-resolution copy-number analysis on 125 HCC tumors and whole-exome sequencing on 24 of these tumors. We identified 135 homozygous deletions and 994 somatic mutations of genes with predicted functional consequences. We found new recurrent alterations in four genes (ARID1A, RPS6KA3, NFE2L2 and IRF2) not previously described in HCC. Functional analyses showed tumor suppressor properties for IRF2, whose inactivation, exclusively found in hepatitis B virus (HBV)-related tumors, led to impaired TP53 function. In contrast, inactivation of chromatin remodelers was frequent and predominant in alcohol-related tumors. Moreover, association of mutations in specific genes (RPS6KA3-AXIN1 and NFE2L2-CTNNB1) suggested that Wnt/?-catenin signaling might cooperate in liver carcinogenesis with both oxidative stress metabolism and Ras/mitogen-activated protein kinase (MAPK) pathways. This study provides insight into the somatic mutational landscape in HCC and identifies interactions between mutations in oncogene and tumor suppressor gene mutations related to specific risk factors.
Project description:Hepatocellular cancer (HCC) remains a disease of poor prognosis, highlighting the relevance of elucidating key molecular aberrations that may be targeted for novel therapies. Wnt signalling activation, chiefly due to mutations in CTNNB1, have been identified in a major subset of HCC patients. While several in vitro proof of concept studies show the relevance of suppressing Wnt/?-catenin signalling in HCC cells or tumour xenograft models, no study has addressed the impact of ?-catenin inhibition in a relevant murine HCC model driven by Ctnnb1 mutations.We studied the in vivo impact of ?-catenin suppression by locked nucleic acid (LNA) antisense treatment, after establishing Ctnnb1 mutation-driven HCC by diethylnitrosamine and phenobarbital (DEN/PB) administration.The efficacy of LNA directed against ?-catenin vs. scrambled on Wnt signalling was demonstrated in vitro in HCC cells and in vivo in normal mice. The DEN/PB model leads to HCC with Ctnnb1 mutations. A complete therapeutic response in the form of abrogation of HCC was observed after ten treatments of tumour-bearing mice with ?-catenin LNA every 48h as compared to the scrambled control. A decrease in ?-catenin activity, cell proliferation and increased cell death was evident after ?-catenin suppression. No effect of ?-catenin suppression was evident in non-Ctnnb1 mutated HCC, observed after DEN-only administration.Thus, we provide the in vivo proof of concept that ?-catenin suppression in HCC will be of significant therapeutic benefit, provided the tumours display Wnt activation via mechanisms like CTNNB1 mutations.
Project description:Central nervous system primitive neuroectodermal tumours (CNS PNET) are high-grade, predominantly paediatric, brain tumours. Previously they have been grouped with medulloblastomas owing to their histological similarities. The WNT/beta-catenin pathway has been implicated in many tumour types, including medulloblastoma. On pathway activation beta-catenin (CTNNB1) translocates to the nucleus, where it induces transcription of target genes. It is commonly upregulated in tumours by mutations in the key pathway components APC and CTNNB1. WNT/beta-catenin pathway status was investigated by immunohistochemical analysis of CTNNB1 and the pathway target cyclin D1 (CCND1) in 49 CNS PNETs and 46 medulloblastomas. The mutational status of APC and CTNNB1 (beta-catenin) was investigated in 33 CNS PNETs and 22 medulloblastomas. CTNNB1 nuclear localisation was seen in 36% of CNS PNETs and 27% of medulloblastomas. A significant correlation was found between CTNNB1 nuclear localisation and CCND1 levels. Mutations in CTNNB1 were identified in 4% of CNS PNETs and 20% of medulloblastomas. No mutations were identified in APC. A potential link between the level of nuclear staining and a better prognosis was identified in the CNS PNETs, suggesting that the extent of pathway activation is linked to outcome. The results suggest that the WNT/beta-catenin pathway plays an important role in the pathogenesis of CNS PNETs. However, activation is not caused by mutations in CTNNB1 or APC in the majority of CNS PNET cases.
Project description:Desmoid tumors are benign mesenchymal neoplasms with a locally aggressive nature. The mutational status of β-catenin gene (CTNNB1) is presumed to affect the tumorous activity of the cells. In this study, we isolated three kinds of desmoid cell with different CTNNB1 status, and compared their characteristics. Cells were isolated from three patients with abdominal wall desmoid during surgery, all of which were resistant to meloxicam treatment. The mutational status of the CTNNB1 exon 3 was determined for both parental tumor tissues and isolated cultured cells. β-catenin expression was determined with immunohistochemistry. Responsiveness to meloxicam was investigated with MTS assay together with COX-2 immunostaining. mRNA expressions of downstream molecules of Wnt/β-catenin pathway were determined with real-time RT-PCR. Three kinds of cell isolated from desmoid tumors harboring different CTNNB1 mutation status (wild type, T41A, and S45F), all exhibited a spindle shape. These isolated cells could be cultured until the 20th passage with unchanged proliferative activity. Nuclear accumulation of β-catenin was observed in all cultured cells, particularly in those with S45F. Proliferating activity was significantly suppressed by meloxicam (25 μmol/L, P < 0.007) in all three cell cultures, of which parental desmoid was resistant to meloxicam clinically. The mRNA expressions of Axin2, c-Myc, and Cyclin D1 differently increased in the three cultured cell types as compared with those in human skin fibroblast cells (HDF). Inhibitors of Wnt/β-catenin pathway downregulated Axin2, c-Myc, and Cyclin D1 significantly. Isolated and cultured desmoid tumor cells harboring any one of the CTNNB1 mutation status had unique characteristics, and could be useful to investigate desmoid tumors with different mutation status of CTNNB1.
Project description:Up to 41% of hepatocellular carcinomas (HCCs) result from activating mutations in the CTNNB1 gene encoding ?-catenin. HCC-associated CTNNB1 mutations stabilize the ?-catenin protein, leading to nuclear and/or cytoplasmic localization of ?-catenin and downstream activation of Wnt target genes. In patient HCC samples, ?-catenin nuclear and cytoplasmic localization are typically patchy, even among HCC with highly active CTNNB1 mutations. The functional and clinical relevance of this heterogeneity in ?-catenin activation are not well understood. To define mechanisms of ?-catenin-driven HCC initiation, we generated a Cre-lox system that enabled switching on activated ?-catenin in (1) a small number of hepatocytes in early development; or (2) the majority of hepatocytes in later development or adulthood. We discovered that switching on activated ?-catenin in a subset of larval hepatocytes was sufficient to drive HCC initiation. To determine the role of Wnt/?-catenin signaling heterogeneity later in hepatocarcinogenesis, we performed RNA-seq analysis of zebrafish ?-catenin-driven HCC. At the single-cell level, 2.9% to 15.2% of hepatocytes from zebrafish ?-catenin-driven HCC expressed two or more of the Wnt target genes axin2, mtor, glula, myca and wif1, indicating focal activation of Wnt signaling in established tumors. Thus, heterogeneous ?-catenin activation drives HCC initiation and persists throughout hepatocarcinogenesis.