Cyclopeptide RA-V Inhibits Organ Enlargement And Tumorigenesis Induced By Defects Of The Hippo Pathway

The Hippo pathway restricts organ size during development and its inactivation plays a crucial role in cancer.Central to the Hippo pathway is a kinase cascade formed by the STE-20 family kinases MST1/2 and the downstream AGC family kinase LATS1/2.MST1/2 activate LATS1/2 by directly phosphorylating LATS1/2 and by phosphorylating adaptor proteins SAV1 and MOB1A/B.LATS 1/2 then phosphorylate YAP and TAZ,which are transcription co-activators and major effectors of the Hippo pathway.Phosphorylation of YAP/TAZ leads to cytoplasmic retention of YAP/TAZ through binding to scaffold protein 14-3-3,or degradation by the ubiquitin-proteasome pathway.When the Hippo pathway is inactive,dephosphorylated YAP/TAZ translocate to cell nucleus and interact with transcription factors such as the TEAD family proteins to induce expression of genes related to inducing cell proliferation and inhibiting apoptosis.Liver-specific knockout of Nf2,Sav1,Mst1/2 or overexpression of active Yap in mice induces liver enlargement and tumorigenesis.Further study demonstrated that activation of YAP/TAZ promotes liver tumorigenesis not only through cell-autonomous induction of hepatocyte dedifferentiation and proliferation,but also through non-cell-autonomous recruitment of type 2 macrophages to protect tumor-initiating cells from immune clearance.In human cancers,mutations of Hippo pathway upstream components lead to YAP activation.For instance,mutation of neurofibromin 2(NF2)is the major genetic driving force of neurofibromatosis type 2.Furthermore,activating mutations of GNAQ or GNA11 encoding G protein alpha subunits G?q and G?11 have been found in about 80%of uveal melanomas.In addition,the YAP gene locus was found amplified in many human cancers,including hepatocellular carcinoma(HCC).Importantly,YAP not only promotes tumor initiation and growth,but also plays key roles in metastasis and cancer relapse.These findings raise the attention on the Hippo pathway and YAP as targets for cancer therapy.Although progress has been made in the identification of YAP inhibitors,more effective and safe YAP inhibitors are still in need.In order to identify pharmacological inhibitors of YAP,we screened a library of 52,683 compounds using a YAP-specific reporter assay.In this screen we identified cyclopeptide RA-V,a natural product mainly purified from the roots and rhizomes of Rubia plants as a specific inhibitor of YAP and TAZ but not other reporters.Unexpectedly,later experiments demonstrated that RA-V represses the protein but not mRNA levels of YAP target genes such as CTGF and CYR61.It could be due to a protein synthesis inhibitory activity reported for a close derivative of RA-V.Since proteins encoded by YAP target genes are the ultimate executors of Hippo pathway functions,we further investigated whether RA-V could inhibit liver enlargement and tumorigenesis induced by YAP activation.Interestingly,RA-V strongly blocks liver enlargement induced by Mstl/2 knockout.Furthermore,RA-V not only inhibits liver tumorigenesis induced by YAP expression,but also induces regression of established tumors.We found that RA-V inhibits dedifferentiation and proliferation,while inducing apoptosis of hepatocytes.Furthermore,RA-V also induces apoptosis and inhibits proliferation of immune cells in the microenvironment,which are essential for YAP-induced tumorigenesis.RA-V is thus a potential candidate for development of drugs against cancers involving YAP/TAZ activation.