ABSTRACT: The thyromimetic agent GC-1 induces hepatocyte proliferation via Wnt/?-catenin signaling and may promote regeneration in both acute and chronic liver insufficiencies. However, ?-catenin activation due to mutations in CTNNB1 is seen in a subset of hepatocellular carcinomas (HCC). Thus, it is critical to address any effect of GC-1 on HCC growth and development before its use can be advocated to stimulate regeneration in chronic liver diseases. In this study, we first examined the effect of GC-1 on ?-catenin-T cell factor 4 activity in HCC cell lines harboring wild-type or mutated-CTNNB1. Next, we assessed the effect of GC-1 on HCC in FVB mice generated by hydrodynamic tail vein injection of hMet-S45Y-?-catenin, using the sleeping beauty transposon-transposase. Four weeks following injection, mice were fed 5 mg/kg GC-1 or basal diet for 10 or 21 days. GC-1 treatment showed no effect on ?-catenin-T cell factor 4 activity in HCC cells, irrespective of CTNNB1 mutations. Treatment with GC-1 for 10 or 21 days led to a significant reduction in tumor burden, associated with decreased tumor cell proliferation and dramatic decreases in phospho-(p-)Met (Y1234/1235), p-extracellular signal-related kinase, and p-STAT3 without affecting ?-catenin and its downstream targets. GC-1 exerts a notable antitumoral effect on hMet-S45Y-?-catenin HCC by inactivating Met signaling. GC-1 does not promote ?-catenin activation in HCC. Thus, GC-1 may be safe for use in inducing regeneration during chronic hepatic insufficiency.
Project description:Hepatocellular cancer (HCC) remains a significant therapeutic challenge due to its poorly understood molecular basis. In the current study, we investigated two independent cohorts of 249 and 194 HCC cases for any combinatorial molecular aberrations. Specifically we assessed for simultaneous HMET expression or hMet activation and catenin ?1 gene (CTNNB1) mutations to address any concomitant Met and Wnt signaling. To investigate cooperation in tumorigenesis, we coexpressed hMet and ?-catenin point mutants (S33Y or S45Y) in hepatocytes using sleeping beauty transposon/transposase and hydrodynamic tail vein injection and characterized tumors for growth, signaling, gene signatures, and similarity to human HCC. Missense mutations in exon 3 of CTNNB1 were identified in subsets of HCC patients. Irrespective of amino acid affected, all exon 3 mutations induced similar changes in gene expression. Concomitant HMET overexpression or hMet activation and CTNNB1 mutations were evident in 9%-12.5% of HCCs. Coexpression of hMet and mutant-?-catenin led to notable HCC in mice. Tumors showed active Wnt and hMet signaling with evidence of glutamine synthetase and cyclin D1 positivity and mitogen-activated protein kinase/extracellular signal-regulated kinase, AKT/Ras/mammalian target of rapamycin activation. Introduction of dominant-negative T-cell factor 4 prevented tumorigenesis. The gene expression of mouse tumors in hMet-mutant ?-catenin showed high correlation, with subsets of human HCC displaying concomitant hMet activation signature and CTNNB1 mutations.We have identified cooperation of hMet and ?-catenin activation in a subset of HCC patients and modeled this human disease in mice with a significant transcriptomic intersection; this model will provide novel insight into the biology of this tumor and allow us to evaluate novel therapies as a step toward precision medicine. (Hepatology 2016;64:1587-1605).
Project description:Recently, we have shown that coexpression of hMet and mutant-?-catenin using sleeping beauty transposon/transposase leads to hepatocellular carcinoma (HCC) in mice that corresponds to around 10% of human HCC. In the current study, we investigate whether Ras activation, which can occur downstream of Met signaling, is sufficient to cause HCC in association with mutant-?-catenin. We also tested therapeutic efficacy of targeting ?-catenin in an HCC model. We show that mutant-K-Ras (G12D), which leads to Ras activation, cooperates with ?-catenin mutants (S33Y, S45Y) to yield HCC in mice. Affymetrix microarray showed?>?90% similarity in gene expression in mutant-K-Ras-?-catenin and Met-?-catenin HCC. K-Ras-?-catenin tumors showed up-regulation of ?-catenin targets like glutamine synthetase (GS), leukocyte cell-derived chemotaxin 2, Regucalcin, and Cyclin-D1 and of K-Ras effectors, including phosphorylated extracellular signal-regulated kinase, phosphorylated protein kinase B, phosphorylated mammalian target of rapamycin, phosphorylated eukaryotic translation initiation factor 4E, phosphorylated 4E-binding protein 1, and p-S6 ribosomal protein. Inclusion of dominant-negative transcription factor 4 at the time of K-Ras-?-catenin injection prevented HCC and downstream ?-catenin and Ras signaling. To address whether targeting ?-catenin has any benefit postestablishment of HCC, we administered K-Ras-?-catenin mice with EnCore lipid nanoparticles (LNP) loaded with a Dicer substrate small interfering RNA targeting catenin beta 1 (CTNNB1; CTNNB1-LNP), scrambled sequence (Scr-LNP), or phosphate-buffered saline for multiple cycles. A significant decrease in tumor burden was evident in the CTNNB1-LNP group versus all controls, which was associated with dramatic decreases in ?-catenin targets and some K-Ras effectors, leading to reduced tumor cell proliferation and viability. Intriguingly, in relatively few mice, non-GS-positive tumors, which were evident as a small subset of overall tumor burden, were not affected by ?-catenin suppression.Ras activation downstream of c-Met is sufficient to induce clinically relevant HCC in cooperation with mutant ?-catenin. ?-catenin suppression by a clinically relevant modality is effective in treatment of ?-catenin-positive, GS-positive HCCs. (Hepatology 2017;65:1581-1599).
Project description:<h4>Background and aims</h4>4-phenylbutyric acid (4-PBA) is a low molecular weight fatty acid that is used in clinical practice to treat inherited urea cycle disorders. In previous reports, it acted as a chemical chaperone inhibiting endoplasmic reticulum (ER) stress and unfolded protein response signaling. A few studies have suggested its function against hepatic fibrosis in mice models. However, its role in hepatocarcinogenesis remained unknown.<h4>Methods</h4>4-PBA was administered alone or in combination with diethylnitrosamine to investigate its long-term effect on liver tumorigenesis. The role of 4-PBA in oncogene-induced hepatocellular carcinoma (HCC) mice model using sleeping beauty system co-expressed with hMet and β-catenin point mutation (S45Y) was also observed. RNA-seq and PCR array were used to screen the pathways and genes involved. In vitro and in vivo studies were conducted to explore the effect of 4-PBA on liver and validate the underlying mechanism.<h4>Results</h4>4-PBA alone didn't cause liver tumor in long term. However, it promoted liver tumorigenesis in HCC mice models via initiation of liver cancer stem cells (LCSCs) through Wnt5b-Fzd5 mediating β-catenin signaling. Peroxisome proliferator-activated receptors (PPAR)-α induced by 4-PBA was responsible for the activation of β-catenin signaling. Thus, intervention of PPAR-α reversed 4-PBA-induced initiation of LCSCs and HCC development in vivo. Further study revealed that 4-PBA could not only upregulate the expression of PPAR-α transcriptionally but also enhance its stabilization via protecting it from proteolysis. Moreover, high PPAR-α expression predicted poor prognosis in HCC patients.<h4>Conclusions</h4>4-PBA could upregulate PPAR-α to initiate LCSCs by activating β-catenin signaling pathway, promoting HCC at early stage. Therefore, more discretion should be taken to monitor the potential tumor-promoting effect of 4-PBA under HCC-inducing environment.
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:Hepatocellular carcinoma (HCC) is a heterogeneous cancer with active Wnt signaling. Underlying biologic mechanisms remain unclear and no drug targeting this pathway has been approved to date. We aimed to characterize Wnt-pathway aberrations in HCC patients, and to investigate sorafenib as a potential Wnt modulator in experimental models of liver cancer.The Wnt-pathway was assessed using mRNA (642 HCCs and 21 liver cancer cell lines) and miRNA expression data (89 HCCs), immunohistochemistry (108 HCCs), and CTNNB1-mutation data (91 HCCs). Effects of sorafenib on Wnt signaling were evaluated in four liver cancer cell lines with active Wnt signaling and a tumor xenograft model.Evidence for Wnt activation was observed for 315 (49.1%) cases, and was further classified as CTNNB1 class (138 cases [21.5%]) or Wnt-TGF? class (177 cases [27.6%]). CTNNB1 class was characterized by upregulation of liver-specific Wnt-targets, nuclear ?-catenin and glutamine-synthetase immunostaining, and enrichment of CTNNB1-mutation-signature, whereas Wnt-TGF? class was characterized by dysregulation of classical Wnt-targets and the absence of nuclear ?-catenin. Sorafenib decreased Wnt signaling and ?-catenin protein in HepG2 (CTNNB1 class), SNU387 (Wnt-TGF? class), SNU398 (CTNNB1-mutation), and Huh7 (lithium-chloride-pathway activation) cell lines. In addition, sorafenib attenuated expression of liver-related Wnt-targets GLUL, LGR5, and TBX3. The suppressive effect on CTNNB1 class-specific Wnt-pathway activation was validated in vivo using HepG2 xenografts in nude mice, accompanied by decreased tumor volume and increased survival of treated animals.Distinct dysregulation of Wnt-pathway constituents characterize two different Wnt-related molecular classes (CTNNB1 and Wnt-TGF?), accounting for half of all HCC patients. Sorafenib modulates ?-catenin/Wnt signaling in experimental models that harbor the CTNNB1 class signature.
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.
Project description:PD-1 immune checkpoint inhibitors have produced encouraging results in patients with hepatocellular carcinoma (HCC). However, what determines resistance to anti-PD-1 therapies is unclear. We created a novel genetically engineered mouse model of HCC that enables interrogation of how different genetic alterations affect immune surveillance and response to immunotherapies. Expression of exogenous antigens in MYC;Trp53 -/- HCCs led to T cell-mediated immune surveillance, which was accompanied by decreased tumor formation and increased survival. Some antigen-expressing MYC;Trp53 -/- HCCs escaped the immune system by upregulating the ?-catenin (CTNNB1) pathway. Accordingly, expression of exogenous antigens in MYC;CTNNB1 HCCs had no effect, demonstrating that ?-catenin promoted immune escape, which involved defective recruitment of dendritic cells and consequently impaired T-cell activity. Expression of chemokine CCL5 in antigen-expressing MYC;CTNNB1 HCCs restored immune surveillance. Finally, ?-catenin-driven tumors were resistant to anti-PD-1. In summary, ?-catenin activation promotes immune escape and resistance to anti-PD-1 and could represent a novel biomarker for HCC patient exclusion. SIGNIFICANCE: Determinants of response to anti-PD-1 immunotherapies in HCC are poorly understood. Using a novel mouse model of HCC, we show that ?-catenin activation promotes immune evasion and resistance to anti-PD-1 therapy and could potentially represent a novel biomarker for HCC patient exclusion.See related commentary by Berraondo et al., p. 1003.This article is highlighted in the In This Issue feature, p. 983.
Project description:Hepatocellular carcinoma (HCC) represents a serious public health challenge with few therapeutic options available to cancer patients.Wnt/β-catenin pathway is thought to play a significant role in HCC pathogenesis. In this study, we confirmed high frequency of CTNNB1 (β-catenin) mutations in two independent cohorts of HCC patients and demonstrated significant upregulation of β-catenin protein in the overwhelming majority of HCC patient samples, patient-derived xenografts (PDX) and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we went on to investigate oncogenic dependency on β-catenin in an extensive collection of human HCC cells lines. Our results demonstrate that dependency on β-catenin generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1 or displayed elevated levels of non-phosphorylated (active) β-catenin were significantly more sensitive to β-catenin siRNA treatment than cell lines with wild-type CTNNB1 and lower active β-catenin. Finally, significant therapeutic benefit of β-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. β-catenin downregulation and tumor growth inhibition was associated with reduction in AXIN2, direct transcriptional target of β-catenin, and decreased cancer cell proliferation as measured by Ki67 staining. Taken together, our data highlight fundamental importance of aberrant β-catenin signaling in the maintenance of oncogenic phenotype in HCC.
Project description: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: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.