Activation of β-catenin and Yap1 in human hepatoblastoma and induction of hepatocarcinogenesis in mice.

Aberrant activation of β-catenin and Yes-associated protein 1 (Yap1) signaling pathways have been associated with the development of multiple tumor types. Yap functions as a transcriptional coactivator by interacting with TEA domain DNA binding proteins. We investigated the interactions among these pathways during hepatic tumorigenesis. We used immunohistochemical analysis to determine expression of β-catenin and Yap1 in liver cancer specimens collected from patients in Europe and the United States, consisting of 104 hepatocellular carcinoma, 62 intrahepatic cholangiocarcinoma, and 94 hepatoblastoma samples. We assessed β-catenin 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 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 (ΔN90/β-catenin) and Yap1 (YapS127A) in livers of mice; tissues were collected, and histological and immunohistochemical analyses were performed. We observed nuclear localization of β-catenin and Yap1 in 79% of hepatoblastoma samples but not in most hepatocellular carcinoma or intrahepatic cholangiocarcinoma samples. Yap1 and β-catenin coprecipitated in hepatoblastoma but not hepatocellular carcinoma cells. Small interfering RNA-mediated knockdown of Yap1 or β-catenin in hepatoblastoma cells reduced proliferation in an additive manner. Knockdown of Yap1 reduced its ability to coactivate transcription with β-catenin; β-catenin inhibitors inactivated Yap1. Overexpression of constitutively active forms of Yap1 and β-catenin in mouse liver led to rapid tumorigenesis, with 100% mortality by 11 weeks. Tumor cells expressed both proteins, and human hepatoblastoma cells expressed common targets of their 2 signaling pathways. Yap1 binding of TEA domain factors was required for tumorigenesis in mice. 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.

Tao J ，Calvisi DF ，Ranganathan S ，Cigliano A ，Zhou L ，Singh S ，Jiang L ，Fan B ，Terracciano L ，Armeanu-Ebinger S ，Ribback S ，Dombrowski F ，Evert M ，Chen X ，Monga SPS ... -  《-》

β-Catenin and Yes-Associated Protein 1 Cooperate in Hepatoblastoma Pathogenesis.

Hepatoblastoma (HB), the most common pediatric primary liver neoplasm, shows nuclear localization of β-catenin and yes-associated protein 1 (YAP1) in almost 80% of the cases. Co-expression of constitutively active S127A-YAP1 and ΔN90 deletion-mutant β-catenin (YAP1-ΔN90-β-catenin) causes HB in mice. Because heterogeneity in downstream signaling is being identified owing to mutational differences even in the β-catenin gene alone, we investigated if co-expression of point mutants of β-catenin (S33Y or S45Y) with S127A-YAP1 led to similar tumors as YAP1-ΔN90-β-catenin. Co-expression of S33Y/S45Y-β-catenin and S127A-YAP1 led to activation of Yap and Wnt signaling and development of HB, with 100% mortality by 13 to 14 weeks. Co-expression with YAP1-S45Y/S33Y-β-catenin of the dominant-negative T-cell factor 4 or dominant-negative transcriptional enhanced associate domain 2, the respective surrogate transcription factors, prevented HB development. Although histologically similar, HB in YAP1-S45Y/S33Y-β-catenin, unlike YAP1-ΔN90-β-catenin HB, was glutamine synthetase (GS) positive. However, both ΔN90-β-catenin and point-mutant β-catenin comparably induced GS-luciferase reporter in vitro. Finally, using a previously reported 16-gene signature, it was shown that YAP1-ΔN90-β-catenin HB tumors exhibited genetic similarities with more proliferative, less differentiated, GS-negative HB patient tumors, whereas YAP1-S33Y/S45Y-β-catenin HB exhibited heterogeneity and clustered with both well-differentiated GS-positive and proliferative GS-negative patient tumors. Thus, we demonstrate that β-catenin point mutants can also collaborate with YAP1 in HB development, albeit with a distinct molecular profile from the deletion mutant, which may have implications in both biology and therapy.

Min Q ，Molina L ，Li J ，Adebayo Michael AO ，Russell JO ，Preziosi ME ，Singh S ，Poddar M ，Matz-Soja M ，Ranganathan S ，Bell AW ，Gebhardt R ，Gaunitz F ，Yu J ，Tao J ，Monga SP ... -  《-》

Induction of Chromosome Instability by Activation of Yes-Associated Protein and Forkhead Box M1 in Liver Cancer.

Many different types of cancer cells have chromosome instability. The hippo pathway leads to phosphorylation of the transcriptional activator yes-associated protein 1 (YAP1, YAP), which regulates proliferation and has been associated with the development of liver cancer. We investigated the effects of hippo signaling via YAP on chromosome stability and hepatocarcinogenesis in humans and mice. We analyzed transcriptome data from 242 patients with hepatocellular carcinoma (HCC) to search for gene signatures associated with chromosomal instability (CIN); we investigated associations with overall survival time and cancer recurrence using Kaplan-Meier curves. We analyzed changes in expression of these signature genes, at mRNA and protein levels, after small interfering RNA-mediated silencing of YAP in Sk-Hep1, SNU182, HepG2, or pancreatic cancer cells, as well as incubation with thiostrepton (an inhibitor of forkhead box M1 [FOXM1]) or verteporfin (inhibitor of the interaction between YAP and TEA domain transcription factor 4 [TEAD4]). We performed co-immunoprecipitation and chromatin immunoprecipitation experiments. We collected liver tissues from mice that express a constitutively active form of YAP (YAPS127A) and analyzed gene expression signatures and histomorphologic parameters associated with chromosomal instability. Mice were given injections of thiostrepton and livers were collected and analyzed by immunoblotting, immunohistochemistry, histology, and real-time polymerase chain reaction. We performed immunohistochemical analyses on tissue microarrays of 105 HCCs and 7 nontumor liver tissues. Gene expression patterns associated with chromosome instability, called CIN25 and CIN70, were detected in HCCs from patients with shorter survival time or early cancer recurrence. TEAD4 and YAP were required for CIN25 and CIN70 signature expression via induction and binding of FOXM1. Disrupting the interaction between YAP and TEAD4 with verteporfin, or inhibiting FOXM1 with thiostrepton, reduced the chromosome instability gene expression patterns. Hyperplastic livers and tumors from YAPS127A mice had increased CIN25 and CIN70 gene expression patterns, aneuploidy, and defects in mitosis. Injection of YAPS127A mice with thiostrepton reduced liver overgrowth and signs of chromosomal instability. In human HCC tissues, high levels of nuclear YAP correlated with increased chromosome instability gene expression patterns and aneuploidy. By analyzing cell lines, genetically modified mice, and HCC tissues, we found that YAP cooperates with FOXM1 to contribute to chromosome instability. Agents that disrupt this pathway might be developed as treatments for liver cancer. Transcriptome data are available in the Gene Expression Omnibus public database (accession numbers: GSE32597 and GSE73396).

Weiler SME ，Pinna F ，Wolf T ，Lutz T ，Geldiyev A ，Sticht C ，Knaub M ，Thomann S ，Bissinger M ，Wan S ，Rössler S ，Becker D ，Gretz N ，Lang H ，Bergmann F ，Ustiyan V ，Kalin TV ，Singer S ，Lee JS ，Marquardt JU ，Schirmacher P ，Kalinichenko VV ，Breuhahn K ... -  《-》

β-Catenin Sustains and Is Required for YES-associated Protein Oncogenic Activity in Cholangiocarcinoma.

YES-associated protein (YAP) aberrant activation is implicated in intrahepatic cholangiocarcinoma (iCCA). Transcriptional enhanced associate domain (TEAD)-mediated transcriptional regulation is the primary signaling event downstream of YAP. The role of Wnt/β-Catenin signaling in cholangiocarcinogenesis remains undetermined. Here, we investigated the possible molecular interplay between YAP and β-Catenin cascades in iCCA. Activated AKT (Myr-Akt) was coexpressed with YAP (YapS127A) or Tead2VP16 via hydrodynamic tail vein injection into mouse livers. Tumor growth was monitored, and liver tissues were collected and analyzed using histopathologic and molecular analysis. YAP, β-Catenin, and TEAD interaction in iCCAs was investigated through coimmunoprecipitation. Conditional Ctnnb1 knockout mice were used to determine β-Catenin function in murine iCCA models. RNA sequencing was performed to analyze the genes regulated by YAP and/or β-Catenin. Immunostaining of total and nonphosphorylated/activated β-Catenin staining was performed in mouse and human iCCAs. We discovered that TEAD factors are required for YAP-dependent iCCA development. However, transcriptional activation of TEADs did not fully recapitulate YAP's activities in promoting cholangiocarcinogenesis. Notably, β-Catenin physically interacted with YAP in human and mouse iCCA. Ctnnb1 ablation strongly suppressed human iCCA cell growth and Yap-dependent cholangiocarcinogenesis. Furthermore, RNA-sequencing analysis revealed that YAP/ transcriptional coactivator with PDZ-binding motif (TAZ) regulate a set of genes significantly overlapping with those controlled by β-Catenin. Importantly, activated/nonphosphorylated β-Catenin was detected in more than 80% of human iCCAs. YAP induces cholangiocarcinogenesis via TEAD-dependent transcriptional activation and interaction with β-Catenin. β-Catenin binds to YAP in iCCA and is required for YAP full transcriptional activity, revealing the functional crosstalk between YAP and β-Catenin pathways in cholangiocarcinogenesis.

Zhang Y ，Xu H ，Cui G ，Liang B ，Chen X ，Ko S ，Affo S ，Song X ，Liao Y ，Feng J ，Wang P ，Wang H ，Xu M ，Wang J ，Pes GM ，Ribback S ，Zeng Y ，Singhi A ，Schwabe RF ，Monga SP ，Evert M ，Tang L ，Calvisi DF ，Chen X ... -  《-》

Co-activation of PIK3CA and Yap promotes development of hepatocellular and cholangiocellular tumors in mouse and human liver.

Li X ，Tao J ，Cigliano A ，Sini M ，Calderaro J ，Azoulay D ，Wang C ，Liu Y ，Jiang L ，Evert K ，Demartis MI ，Ribback S ，Utpatel K ，Dombrowski F ，Evert M ，Calvisi DF ，Chen X ... -  《Oncotarget》