Hippo signaling interactions with Wnt/β-catenin and Notch signaling repress liver tumorigenesis
Wantae Kim, Sanjoy Kumar Khan, Jelena Gvozdenovic-Jeremic, Youngeun Kim, Jason Dahlman, Hanjun Kim, Ogyi Park, Tohru Ishitani, Eek-hoon Jho, Bin Gao, Yingzi Yang
Wantae Kim, Sanjoy Kumar Khan, Jelena Gvozdenovic-Jeremic, Youngeun Kim, Jason Dahlman, Hanjun Kim, Ogyi Park, Tohru Ishitani, Eek-hoon Jho, Bin Gao, Yingzi Yang
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Research Article Genetics Hepatology

Hippo signaling interactions with Wnt/β-catenin and Notch signaling repress liver tumorigenesis

Abstract

Malignant tumors develop through multiple steps of initiation and progression, and tumor initiation is of singular importance in tumor prevention, diagnosis, and treatment. However, the molecular mechanism whereby a signaling network of interacting pathways restrains proliferation in normal cells and prevents tumor initiation is still poorly understood. Here, we have reported that the Hippo, Wnt/β-catenin, and Notch pathways form an interacting network to maintain liver size and suppress hepatocellular carcinoma (HCC). Ablation of the mammalian Hippo kinases Mst1 and Mst2 in liver led to rapid HCC formation and activated Yes-associated protein/WW domain containing transcription regulator 1 (YAP/TAZ), STAT3, Wnt/β-catenin, and Notch signaling. Previous work has shown that abnormal activation of these downstream pathways can lead to HCC. Rigorous genetic experiments revealed that Notch signaling forms a positive feedback loop with the Hippo signaling effector YAP/TAZ to promote severe hepatomegaly and rapid HCC initiation and progression. Surprisingly, we found that Wnt/β-catenin signaling activation suppressed HCC formation by inhibiting the positive feedback loop between YAP/TAZ and Notch signaling. Furthermore, we found that STAT3 in hepatocytes is dispensable for HCC formation when mammalian sterile 20–like kinase 1 and 2 (Mst1 and Mst2) were removed. The molecular network we have identified provides insights into HCC molecular classifications and therapeutic developments for the treatment of liver tumors caused by distinct genetic mutations.

Authors

Wantae Kim, Sanjoy Kumar Khan, Jelena Gvozdenovic-Jeremic, Youngeun Kim, Jason Dahlman, Hanjun Kim, Ogyi Park, Tohru Ishitani, Eek-hoon Jho, Bin Gao, Yingzi Yang

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Figure 1

Notch signaling activates and forms a positive feedback loop with YAP/TAZ.

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Notch signaling activates and forms a positive feedback loop with YAP/TA...
(A) Western blot analysis of control and DKO liver tissues with the indicated antibodies. (B) Notch reporter assay in primary hepatocytes derived from control and DKO mice (n = 3). (C) Western blot analysis of JAG1, β-catenin, YAP, and TAZ protein levels in primary hepatocytes derived from control and DKO mice. (D) qRT-PCR of Notch or YAP/TAZ response gene expression in liver tissues from control and DKO mice (n = 3). (E) JAG1 upregulation in DKO liver depended on the presence of YAP or YAP/TAZ. Western blot analysis of lysates from the indicated mice. (F) Western blot analysis of primary hepatocytes treated with neutralizing anti-JAG1 antibody (20 μg/ml). (G) YAP/TAZ-dependent reporter assay in primary hepatocytes isolated from control and DKO-treated mice with 15 μg/ml neutralizing anti-JAG1 antibody (n = 3). (H) Increase in YAP/TAZ reporter activity by NICD in Huh7 cells. (I) Western blot analysis of the indicated protein levels induced by NICD expression in Huh7 cells. (J) Western blot analysis of TAZ protein in Huh7 cells treated with 25 μg/ml CHX for the indicated durations. Line graphs show quantified TAZ levels at the indicated time points (n = 4). Data in J represent the mean ± SD. *P < 0.05 and **P < 0.01, by 2-tailed Student’s t test. (K) NICD associated with endogenous TAZ or YAP in Huh7 cells. IP was performed using anti-NICD antibody, and coprecipitated protein was analyzed by Western blotting. (L) Flag-tagged NICD was transfected into Huh7 cells, which were treated with 20 μM MG132 for 8 hours. IP with anti-TAZ antibody was performed to detect interaction with β-TrCP. (D, G, and H) Data are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01, by 2-tailed Student’s t test. Ctrl, control.