Deubiquitylase USP9X Suppresses Tumorigenesis Via Stabilization Of LATS2 In The Hippo Pathway

The Hippo pathway was initially identified in Drosophila by genetic mosaic screens for tumor suppressor genes.Researches indicated that the Hippo pathway plays an important role in cell proliferation,cell apoptosis,organ size control and progenitor cell/stem cell self-renewal.This pathway is quite conserved during evolution.In mammals,MST1/2 kinases together with the adaptor protein SAV phosphorylate LATS1/2 kinases.Together with the adaptor protein MOB,activated LATS1/2 in turn phosphorylate and inhibit Yes-associated protein(YAP).YAP is a key downstream effector of the Hippo pathway,and is a transcriptional co-activator that mainly interacts with TEAD family transcription factors to promote gene expression.Alteration of gene expression by YAP leads to cell proliferation,apoptosis evasion,and also stem cell amplification.The kinase activity of LATS1/2 is regulated by phosphorylation in response to extracellular signals.In addition,LATS2 protein level is negatively regulated by the ubiquitin-proteasome system in conditions such as hypoxia by E3 ligase SIAH2.However,the mechanism removing ubiquitin modification from LATS2 thus stabilizing the protein is not well understood.Here we report identification of the anaphase-promoting complex/cyclosome(APC/C),an ubiquitin ligase complex,and USP9X,a deubiquitylase,as specific LATS2-interacting proteins by tandem affinity purification(TAP).Although APC1 co-localizes with LATS2 to intracellular vesicle structures,it does not regulate the protein level and activity of LATS2.However,ablation of USP9X drastically diminishes the protein level of LATS2.We demonstrated that USP9X deubiquitinates LATS2 and thus prevents LATS2 degradation by the proteasome.In a genetic screen for tumor suppressors of pancreatic ductal adenocarcinoma(PDAC)carried out in mice,USP9X was found to be the most commonly mutated gene in over 50%of the tumors,indicating its strong tumor suppressive activity.Furthermore,in pancreatic cancer cells,loss of USP9X leads to YAP activation and enhanced oncogenic potential.In addition,tumorigenesis induced by USP9X ablation depends not only on LATS2 repression,but also on YAP/TAZ activity.Thus we identified USP9X as a deubiquitylase of the Hippo pathway kinase LATS2,and revealed the Hippo pathway as a downstream signaling mediating the tumor suppressive activity of USP9X.This work uncovers the regulation mechanism of ubiquitination in the Hippo pathway,and provides an advanced direction for therapic targeting.