Distinct and Overlapping Roles of Hippo Effectors YAP and TAZ During Human and Mouse Hepatocarcinogenesis.

Yes-associated protein (YAP) and its paralog transcriptional co-activator with post synaptic density protein, drosophila disc large tumor suppressor and zonula occludens-1-binding motif (TAZ) are 2 co-activators downstream of Hippo tumor-suppressor cascade. Both have been implicated in the development of hepatocellular carcinoma (HCC). However, whether YAP and TAZ have distinct or overlapping functions during hepatocarcinogenesis remains unknown. Expression patterns of YAP and TAZ were analyzed in human HCC samples. The requirement of Yap and/or Taz in protein kinase B (Akt)/ neuroblastoma RAS viral oncogene homolog (NRas) -driven liver tumorigenesis was analyzed using conditional Yap, Taz, and Yap;Taz knockout mice. Transcriptional programs regulated by YAP and/or TAZ were identified via RNA sequencing. We found that in human HCC samples, an almost ubiquitous activation of YAP or TAZ occurs, underlying their role in this tumor type. Intriguingly, 70% of HCC samples showed only nuclear YAP or TAZ immunoreactivity. In the Akt/NRas liver tumor model, where nuclear Yap and Taz can be detected readily, deletion of Yap or Taz alone only mildly delayed liver tumor development, whereas their concomitant ablation strongly inhibited tumor cell proliferation and significantly suppressed Akt/NRas-driven hepatocarcinogenesis. In HCC cell lines, silencing of either YAP or TAZ led to decreased expression of both overlapping and distinct sets of genes, with the most prominent gene signatures related to cell-cycle progression and DNA replication. YAP and TAZ have overlapping and distinct roles in hepatocarcinogenesis. HCCs may display unique activation of YAP or TAZ, thus relying on either YAP or TAZ for their growth.

Wang H ，Wang J ，Zhang S ，Jia J ，Liu X ，Zhang J ，Wang P ，Song X ，Che L ，Liu K ，Ribback S ，Cigliano A ，Evert M ，Wu H ，Calvisi DF ，Zeng Y ，Chen X ... -  《Cellular and Molecular Gastroenterology and Hepatology》

TAZ is indispensable for c-MYC-induced hepatocarcinogenesis.

Mounting evidence implicates the Hippo downstream effectors Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) in hepatocellular carcinoma (HCC). We investigated the functional contribution of YAP and/or TAZ to c-MYC-induced liver tumor development. The requirement for YAP and/or TAZ in c-Myc-driven hepatocarcinogenesis was analyzed using conditional Yap, Taz, and Yap;Taz knockout (KO) mice. An hepatocyte-specific inducible TTR-CreERT2 KO system was applied to evaluate the role of YAP and TAZ during tumor progression. Expression patterns of YAP, TAZ, c-MYC, and BCL2L12 were analyzed in human HCC samples. We found that the Hippo cascade is inactivated in c-Myc-induced mouse HCC. Intriguingly, TAZ mRNA levels and activation status correlated with c-MYC activity in human and mouse HCC, but YAP mRNA levels did not. We demonstrated that TAZ is a direct transcriptional target of c-MYC. In c-Myc induced murine HCCs, ablation of Taz, but not Yap, completely prevented tumor development. Mechanistically, TAZ was required to avoid c-Myc-induced hepatocyte apoptosis during tumor initiation. The anti-apoptotic BCL2L12 gene was identified as a novel target regulated specifically by YAP/TAZ, whose silencing strongly suppressed c-Myc-driven murine hepatocarcinogenesis. In c-Myc murine HCC lesions, conditional knockout of Taz, but not Yap, led to tumor regression, supporting the requirement of TAZ for c-Myc-driven HCC progression. TAZ is a pivotal player at the crossroad between the c-MYC and Hippo pathways in HCC. Targeting TAZ might be beneficial for the treatment of patients with HCC and c-MYC activation. The identification of novel treatment targets and approaches for patients with hepatocellular carcinoma is crucial to improve survival outcomes. We identified TAZ as a transcriptional target of c-MYC which plays a critical role in c-MYC-dependent hepatocarcinogenesis. TAZ could potentially be targeted for the treatment of patients with c-MYC-driven hepatocellular carcinoma.

Wang H ，Zhang S ，Zhang Y ，Jia J ，Wang J ，Liu X ，Zhang J ，Song X ，Ribback S ，Cigliano A ，Evert M ，Liang B ，Wu H ，Calvisi DF ，Zeng Y ，Chen X ... -  《-》

The Clinicopathological Significance of YAP/TAZ Expression in Hepatocellular Carcinoma with Relation to Hypoxia and Stemness.

Background/Aims: Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) activation has been implicated in hepatocarcinogenesis and hepatic progenitor cell differentiation, and hypoxia has been shown to induce nuclear translocation of YAP in cancer cells. Here, we aimed to investigate the relationship between hypoxia, YAP and TAZ expression and stemness-related marker expression in human hepatocellular carcinomas (HCCs) and its clinical implications. Methods: Immunohistochemical stains were performed on tissue microarrays from 305 surgically resected HCCs, and the expression status of YAP and TAZ were correlated with CAIX, stemness markers (K19, EpCAM) and epithelial-mesenchymal transition (EMT)-related markers (uPAR, ezrin). The clinicopathological significance of YAP/TAZ expression was analyzed with relation to CAIX expression status. Results: YAP and TAZ expression were seen in 13.4 and 4.3% of HCCs, respectively. YAP/TAZ-positive HCCs frequently demonstrated higher serum AFP levels, microvascular invasion, advanced tumor stage, increased proliferative activity and expression of stemness- and EMT-related markers, CAIX, p53 and Smad2/3 (p < 0.05, all). Interestingly, YAP/TAZ-positivity was associated with microvascular invasion, higher serum AFP levels, stemness and EMT-related marker expression only in tumors expressing CAIX (p < 0.05, all), while these associations were not seen in CAIX-negative HCCs. Conclusions: YAP/TAZ expression is associated with vascular invasion, stemness and EMT in HCCs with hypoxia marker expression. The effect of Hippo signaling pathway deregulation in HCC may depend on the presence or absence of a hypoxic microenvironment, and hypoxia marker expression status should be taken into account when considering the use of YAP/TAZ as markers of aggressive biologic behavior in HCC.

Park H ，Lee Y ，Lee K ，Lee H ，Yoo JE ，Ahn S ，Park YN ，Kim H ... -  《-》

Differential requirement of Hippo cascade during CTNNB1 or AXIN1 mutation-driven hepatocarcinogenesis.

Gain-of-function (GOF) mutations of CTNNB1 and loss-of-function (LOF) mutations of AXIN1 are recurrent genetic alterations in hepatocellular carcinoma (HCC). We aim to investigate the functional contribution of Hippo/YAP/TAZ in GOF CTNNB1 or LOF AXIN1 mutant HCCs. The requirement of YAP/TAZ in c-Met/β-Catenin and c-Met/sgAxin1-driven HCC was analyzed using conditional Yap , Taz , and Yap;Taz knockout (KO) mice. Mechanisms of AXIN1 in regulating YAP/TAZ were investigated using AXIN1 mutated HCC cells. Hepatocyte-specific inducible TTR-CreER T2KO system was applied to evaluate the role of Yap;Taz during tumor progression. Cabozantinib and G007-LK combinational treatment were tested in vitro and in vivo . Nuclear YAP/TAZ was strongly induced in c-Met/sgAxin1 mouse HCC cells. Activation of Hippo via overexpression of Lats2 or concomitant deletion of Yap and Taz significantly inhibited c-Met/sgAxin1 driven HCC development, whereas the same approaches had mild effects in c-Met/β-Catenin HCCs. YAP is the major Hippo effector in c-Met/β-Catenin HCCs, and both YAP and TAZ are required for c-Met/sgAxin1-dependent hepatocarcinogenesis. Mechanistically, AXIN1 binds to YAP/TAZ in human HCC cells and regulates YAP/TAZ stability. Genetic deletion of YAP/TAZ suppresses already formed c-Met/sgAxin1 liver tumors, supporting the requirement of YAP/TAZ during tumor progression. Importantly, tankyrase inhibitor G007-LK, which targets Hippo and Wnt pathways, synergizes with cabozantinib, a c-MET inhibitor, leading to tumor regression in the c-Met/sgAxin1 HCC model. Our studies demonstrate that YAP/TAZ are major signaling molecules downstream of LOF AXIN1 mutant HCCs, and targeting YAP/TAZ is an effective treatment against AXIN1 mutant human HCCs.

Liang B ，Wang H ，Qiao Y ，Wang X ，Qian M ，Song X ，Zhou Y ，Zhang Y ，Shang R ，Che L ，Chen Y ，Huang Z ，Wu H ，Monga SP ，Zeng Y ，Calvisi DF ，Chen X ，Chen X ... -  《-》

TAZ target gene ITGAV regulates invasion and feeds back positively on YAP and TAZ in liver cancer cells.

The Hippo pathway effectors yes-associated protein (YAP) and WW domain containing transcription regulator 1 (TAZ/WWTR1) support tumor initiation and progression in various cancer entities including hepatocellular carcinoma (HCC). However, to which extent YAP and TAZ contribute to liver tumorigenesis via common and exclusive molecular mechanisms is poorly understood. RNAinterference (RNAi) experiments illustrate that YAP and TAZ individually support HCC cell viability and migration, while for invasion additive effects were observed. Comprehensive expression profiling revealed partly overlapping YAP/TAZ target genes as well as exclusively regulated genes. Integrin-αV (ITGAV) is a novel TAZ-specific target gene, whose overexpression in human HCC patients correlates with poor clinical outcome, TAZ expression in HCCs, and the abundance of YAP/TAZ target genes. Functionally, ITGAV contributes to actin stress fiber assembly, tumor cell migration and invasion. Perturbation of ITGAV diminishes actin fiber formation and nuclear YAP/TAZ protein levels. We describe a novel Hippo downstream mechanism in HCC cells, which is regulated by TAZ and ITGAV and that feedbacks on YAP/TAZ activity. This mechanism may represent a therapeutic target structure since it contributes to signal amplification of oncogenic YAP/TAZ in hepatocarcinogenesis.

Weiler SME ，Lutz T ，Bissinger M ，Sticht C ，Knaub M ，Gretz N ，Schirmacher P ，Breuhahn K ... -  《-》