Background & Aims Aberrant activation of βcatenin and Yes-associated protein 1 (Yap1) signaling pathways have been associated with development of multiple tumor types. Yap functions as a transcriptional co-activator by interacting with TEAD DNA binding proteins. We investigated the interactions among these pathways during hepatic tumorigenesis. Methods We used immunohistochemical analysis to determine expression of β-catenin and Yap1 in liver cancer specimens collected from patients in Europe and the US, consisting of 104 hepatocellular carcinoma (HCC), 62 intrahepatic cholangiocarcinoma (ICC), and 94 hepatoblastoma samples. We assessed βcatenin and Yap1 signaling and interactions in hepatoblastoma cell lines ((HuH6, HepG2, HepT1, HC-AFW1, HepG2, and HC-AFW1); proteins were knocked down with small interfering (si)RNAs and effects on proliferation and cell death were measured. Sleeping beauty-mediated hydrodynamic transfection was used to overexpress constitutively active forms of β catenin ( N90-βcatenin) and Yap1 (YapS127A) in livers of mice; tissues were collected and histologic and immunohistochemical analyses were performed. Results We observed nuclear localization of βcatenin and Yap1 in 79% of hepatoblastoma samples, but not in most HCC or ICC tissues. Yap1 and β catenin co-precipitated in hepatoblastoma but not HCC cells. siRNA-mediated knockdown of Yap1 or β catenin in hepatoblastoma cells reduced proliferation in an additive manner. Knockdown of Yap1 reduced its ability to co-activate transcription with βcatenin; βcatenin inhibitors inactivated Yap1. Overexpression of constitutively active forms of Yap1 and βcatenin in mouse liver led to rapid tumorigenesis, with 100% mortality by 11 weeks. Tumors cells expressed both proteins, and human hepatoblastoma cells expressed common targets of their 2 signaling pathways. Yap1 binding of TEAD factors was required for tumorigenesis in mice. Conclusions β catenin and the transcriptional regulator Yap1 interact physically and are activated in most human hepatoblastoma tissues; overexpression of activated forms of these proteins in livers of mice leads to rapid tumor development. Further analysis of these mice will allow further studies of these pathways in hepatoblastoma pathogenesis and could lead to the identification of new therapeutic targets.

Edited by Eric FearonHepatoblastoma (HB) is associated with aberrant activation of the ␤-catenin and Hippo/YAP signaling pathways. Overexpression of mutant ␤-catenin and YAP in mice induces HBs that express high levels of c-Myc (Myc). In light of recent observations that Myc is unnecessary for long-term hepatocyte proliferation, we have now examined its role in HB pathogenesis using the above model. Although Myc was found to be dispensable for in vivo HB initiation, it was necessary to sustain rapid tumor growth. Gene expression profiling identified key molecular differences between myc ؉/؉ (WT) and myc ؊/؊ (KO) hepatocytes and HBs that explain these behaviors. In HBs, these included both Myc-dependent and Myc-independent increases in families of transcripts encoding ribosomal proteins, non-structural factors affecting ribosome assembly and function, and enzymes catalyzing glycolysis and lipid bio-synthesis. In contrast, transcripts encoding enzymes involved in fatty acid ␤-oxidation were mostly down-regulated. Myc-independent metabolic changes associated with HBs included dramatic reductions in mitochondrial mass and oxidative function, increases in ATP content and pyruvate dehydrogenase activity, and marked inhibition of fatty acid ␤-oxidation (FAO). Myc-dependent metabolic changes included higher levels of neutral lipid and acetylCoA in WT tumors. The latter correlated with higher histone H3 acetylation. Collectively, our results indicate that the role of Myc in HB pathogenesis is to impose mutually dependent changes in gene expression and metabolic reprogramming that are unattainable in non-transformed cells and that cooperate to maximize tumor growth.

Amplification and/or activation of the c-Myc protooncogene is one of the leading genetic events along hepatocarcinogenesis. The oncogenic potential of c-Myc has been proven experimentally by the finding that its overexpression in the mouse liver triggers tumor formation. However, the molecular mechanism whereby c-Myc exerts its oncogenic activity in the liver remains poorly understood. Here, we demonstrate that the mammalian target of rapamycin complex 1 (mTORC1) cascade is activated and necessary for c-Myc dependent hepatocarcinogenesis. Specifically, we found that ablation of Raptor, the unique member of the mTORC1 complex, strongly inhibits c-Myc liver tumor formation. Also, p70S6K/ribosomal protein S6 (RPS6) and eukaryotic translation initiation factor 4E-binding protein 1/eukaryotic translation initiation factor 4E (4EBP1/eIF4E) signaling cascades downstream of mTORC1 are required for c-Myc-driven tumorigenesis. Intriguingly, microarray expression analysis revealed the upregulation of multiple amino acid transporters, including SLC1A5 and SLC7A6, leading to robust uptake of amino acids, including glutamine, into c-Myc tumor cells. Subsequent functional studies showed that amino acids are critical for activation of mTORC1, as their inhibition suppressed mTORC1 in c-Myc tumor cells. In human HCC specimens, levels of c-Myc directly correlate with those of mTORC1 activation as well as of SLC1A5 and SLC7A6. Conclusion Our current study indicates that an intact mTORC1 axis is required for c-Myc-driven hepatocarcinogenesis. Thus, targeting mTOR pathway or amino acid transporters may be an effective and novel therapeutic option for the treatment of HCC with activated c-Myc signaling.

Hepatocellular cancer (HCC) remains a significant therapeutic challenge due to poorly understood molecular basis. In the current study, we investigate 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 CTNNB1 mutations to address any concomitant Met and Wnt signaling. To investigate cooperation in tumorigenesis, we co-expressed hMet and β-catenin point-mutants (S33Y or S45Y) in hepatocytes using sleeping beauty (SB) 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. Co-expression of hMet and mutant-β-catenin led to notable HCC in mice. Tumors showed active Wnt and hMet signaling with evidence of glutamine synthetase and cyclin-D1 positivity and MAPK/ERK, AKT/Ras/mTOR activation. Introduction of dominant-negative TCF4 prevented tumorigenesis. The gene expression of mouse tumors in hMet-mutant-β-catenin showed high correlation with subsets of human HCC displaying concomitant hMet activation signature and CTNNB1 mutations. In conclusion, we have identified co-operation of hMet and β-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 towards precision medicine.

Hepatoblastoma is the most common pediatric liver malignancy, typically striking children within the first 3 years of their young lives. While advances in chemotherapy and newer surgical techniques have improved survival in patients with localized disease, unfortunately, for the 25% of patients with metastasis, the overall survival remains poor. These tumors, which are thought to arise from hepatic progenitors or hepatoblasts, hence the name hepatoblastoma, can be categorized by histological subtyping based on their level of cell differentiation. Genomic and histological analysis of human tumor samples has shown exon-3 deletions or missense mutations in gene coding for β-catenin, a downstream effector of the Wnt signaling pathway, in up to 90% of hepatoblastoma cases. The current article will review key aberrations in molecular pathways that are implicated in various subtypes of hepatoblastoma with an emphasis on Wnt signaling. It will also discuss cooperation among components of pathways such as β-catenin and Yes-associated protein in cancer development. Understanding the complex network of molecular signaling in oncogenesis will undoubtedly aid in the discovery of new therapeutics to help combat hepatoblastoma.

Highlights d mTORC1 activation is seen basally in pericentral hepatocytes because of Wnt/b-catenin d CTNNB1-mutated liver tumors are positive for GS and p-mTOR-S2448 d CTNNB1-mutated hepatocellular cancers are addicted to mTORC1 for metabolism d Targeting b-catenin-GS-mTORC1 axis in liver tumors may enable precision medicine

Recently we have shown that co-expression of hMet and mutant-β-catenin using sleeping beauty transposon/transposase leads to HCC in mice that represents around 10% of human HCC. In the current study, we investigate if Ras activation, which can occur downstream of Met signaling, is sufficient to cause HCC in association with mutant-β-catenin. We also tested therapeutic efficacy of targeting β-catenin in HCC model. We show that mutant-K-Ras (G12D), which leads to Ras activation, cooperates with β-catenin mutants (S33Y, S45Y) to yield HCC in mice. Affymetrix microarray shows >90% similarity in gene expression in mutant-K-Ras-β-catenin and Met-β-catenin HCC. K-Ras-β-catenin tumors showed upregulation of β-catenin targets like Glutamine Synthetase (GS), Lect2, Regucalcin and Cyclin-D1 and of K-Ras effectors including p-ERK, p-AKT, p-mTOR, p-EIF4E, p-4E-BP1 and p-S6 Ribosomal protein. Inclusion of dominant-negative TCF4 at the time of K-Ras-β-catenin injection prevented HCC and downstream β-catenin and Ras signaling. To address if targeting β-catenin has any benefit post-establishment of HCC, we administered K-Ras-β-catenin mice with EnCore lipid nanoparticle (LNP) loaded with a Dicer substrate siRNA targeting CTNNB1 (CTNNB1-LNP), scrambled sequence (Scr-LNP) or PBS for multiple cycles. A significant decrease in tumor burden was evident in CTNNB1-LNP group versus all controls, which was associated with dramatic decreases in β-catenin targets and some K-Ras effectors, leading to reduced tumor cell proliferation and viability. Intriguingly, in few mice, non-GS-positive tumors, which were evident as a small subset of overall tumor burden, were not affected by β-catenin suppression. In conclusion, we show that Ras activation downstream of c-Met is sufficient to induce clinically relevant HCC in cooperation with mutant β-catenin. β-Catenin suppression by a clinically relevant modality is effective in treatment of β-catenin-positive, GS-positive HCCs.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide and therapeutic agents for this malignancy are lacking. MicroRNAs play critical roles in carcinogenesis and present tremendous therapeutic potential. Here we report that microRNA-206 is a robust tumor suppressor that plays important roles in the development of HCC by regulating cell cycle progression and cMet signaling pathway. MicroRNA-206 was under-expressed in livers of two HCC mouse models, human individuals bearing HCC, and human HCC cell lines. Combining bioinformatic prediction and molecular and cellular approaches, we identified cMET (Met proto-oncogene), CCND1, and CDK6 as functional targets of microRNA-206. By inhibiting expression of cMET, CCND1 and CDK6, microRNA-206 delayed cell cycle progression, induced apoptosis and impaired proliferation of three distinct human HCC cell lines. Systemic administration of microRNA-206 completely prevented HCC development in both cMyc and AKT/Ras HCC mice, while 100% of control mice died from lethal tumor burdens. Conversely, re-introduction of cMet or Cdk6 into livers of cMyc and AKT/Ras HCC mice recovered growth of HCC inhibited by microRNA-206. These results strongly suggested that cMet and Cdk6 were two functional targets that mediated the inhibitory effect of microRNA-206 on the development of HCC. MicroRNA-206 overexpression demonstrated a profound therapeutic effect on HCC in xenograft and cMyc HCC mice. In summary, this study defines a potentially critical role of microRNA-206 in preventing the growth of HCC, and suggests its use as a potential therapeutic strategy for this malignancy.