The transcriptional regulator YAP induces Notch-pathway activity in hepatocytes and hepatocellular carcinoma cells

Aims: The Hippo-pathway regulates organ size and homeostasis. Studies revealed that deletion of Hippo-pathway kinases (e.g. Mst1/2) or overexpression of the transcriptional co-activator YAP lead to liver cancer; however, the underlying molecular mechanism have not identified so far. We therefore aimed to define possible effector mechanisms mediating YAP-dependent oncogenic activity in liver cancer. Methods: SiRNA-knockdown, expression profiling, and functional assays were performed using HCC cell lines. Gene expression and protein interaction were analyzed by immunoblotting or RT-PCR and co-immunoprecipitation, respectively. YAP transgenic (YAPLAP) and Mst1/2 knock-out mice as well as human HCCs were examined by IHC, immunoblotting, and RT-PCR. Results: Expression profiling after YAP inhibition in HCC cells and of YAPLAP liver tissues identified the Notch-pathway ligand Jag1 as potential target gene for the Hippo pathway. Expression of cleaved Notch receptor (NICD) and Notch target gene Hes1 were reduced in HCC cells after YAP inhibition. SiRNA-knockdown of Jag1 but not of Jag2 as well as inhibition of Notch signalling by γ-secretase phenocopied the biological effects detected after YAP inhibition (e.g., decreased viability). Overexpression of YAP induced tumor cell proliferation, whereas additional Jag1 inhibition partly compensated the effects. Expression of YAP/Jag1 and YAP/Hes1 significantly correlated in human HCCs. YAPLAP-mice developed dysplastic foci with increased Jag1 expression and elevated proliferation. Furthermore, time-resolved analysis showed a simultaneous increase of Jag1 and Hes1 levels after doxycylin-dependent YAP expression in isolated primary hepatocytes. Accordingly, Mst1/2-/- mice-derived HCCs showed high YAP, Jag1, and Hes1 levels. Conclusion: These data demonstrate that activation of YAP induces Notch-pathway activity by Jag1 expression and that Notch-signalling partly facilitates the oncogenic effects of YAP in vitro and in vivo.