Deubiquitinase CYLD Inhibits Metastasis Of Hepatocellular Carcinoma Through Hippo Signaling Pathway

Liver cancer is a leading cause of cancer deaths worldwide,and the incidence of liver cancer has been increasing in many countries in recent years.There are two types of liver cancer: primary and metastatic.Primary liver cancer refers to malignant tumors originating from hepatocytes and intrahepatic bile duct epithelial cells.It is caused by various factors,such as hepatitis B virus infection,aflatoxin contamination,etc.Metastatic liver cancer is cancerous tumor that spreads to liver from another part of human body.Currently,the pathogenesis of liver cancer is not very clear.Therefore,the research on the molecular mechanisms of the occurrence and development of liver cancer will help to discover potential therapeutic targets.Ubiquitination is a reversible post-translational modification of proteins that play key roles in signal transduction and regulation of protein stability.Hundreds of E3 ubiquitin ligases facilitate the ubiquitination of specific proteins.All known deubiquitinases are grouped into seven families.This study focuses on cylindromatosis protein（CYLD）.CYLD belongs to the USP（ubiquitin-specific proteases）family,which recognizes and interacts with substrate proteins.CYLD specifically removes of K63-linked ubiquitin chains to regulate various signaling pathways,such as NF-κB（nuclear factor-κB）,TGF-β（transforming growth factor β）and JNK（c-Jun N-terminal kinase）.CYLD is a tumor suppressor that regulate the occurrence and development of various tumors such as cylindromas,breast cancer and cervical cancer.The previous research of our laboratory found that CYLD interacts with molecules of Hippo signaling pathway through a proximity labelling technology（Proximity-dependent biotin identification,Bio ID）.We assumed that CYLD might control the activation of Hippo signaling pathway.Hippo signaling pathway plays important roles in regulating tissue growth,organ size control and cell metabolism.The activation of Hippo signaling pathway involves a series of kinase cascades,including multiple transcriptional coactivators and transcription factors.YAP（yes associated protein）/TAZ（transcriptional coactivator with PDZ-binding motif）are the core transcriptional regulators of the Hippo signaling pathway.Once Hippo signaling pathway is activated,the phosphorylated protein kinase LATS1/2（large tumor suppressor kinase1/2）will phosphorylate YAP and TAZ,and the phosphorylated YAP/TAZ bind to the 14-3-3 protein and are retained in cytoplasm.Then YAP/TAZ are ubiquitinated and degraded.On the other hand,when Hippo signaling pathway is off,YAP/TAZ are dephosphorylated and transported to the nucleus,where they bind to downstream TEAD transcription factors and promote the activation target genes.The abnormal activation of the Hippo pathway leads to tumorigenesis.This study is based on the previous research of our laboratory,the molecular mechanisms of CYLD regulating the activation of Hippo signaling pathway was further confirmed and investigated,and the mechanisms of CYLD regulating the occurrence and development of liver cancer was further explored.In this paper,we first analyzed the RNA-seq data of TCGA-LIHC in TCGA（The Cancer Genome Atlas）database,including CYLD and the main members of Hippo signaling pathway: MST1/2,LATS1/2,YAP,TAZ and TEAD.We found that CYLD might be a tumor suppressor in the occurrence and development of liver cancer.At the same time,we found that the molecules of Hippo signaling pathway are differentially expressed in liver cancer.The molecules other than MST1 were highly expressed in hepatocellular carcinoma tissues.The protein kinase,MST1,is higher expressed in adjacent tissue than tumor,which is a upstream regulator of YAP.YAP/TAZ and the downstream transcriptional regualtors are higher expressed in tumor than adjacent tissue,which suggests that these proteins might promote the development of liver cancer.To confirm the inference,we performed cell proliferation,cell migration and cell invasion experiments in Hep G2 and MHCC-97 H cell lines.We showed that the knockout of CYLD had no effect on cell proliferation,but significantly promoted cell migration and invasion.Furthermore,we investigated whether CYLD regulates the development of hepatocellular carcinoma through LIMD1（LIM domain-containing protein）and Hippo signaling pathway.The results of phosphorylation experiments and nuclear protein extraction experiments.The results showed that CYLD partly depends on LIMD1 to regulate the activation of Hippo signaling pathway through YAP phosphorylation and localization assays.Finally,we performed cell proliferation and cell migration experiments in two liver cancer cell lines.The results showed that CYLD depends on LIMD1 to regulate Hippo signaling pathway and control the migration and invasion of liver cancer.