Fbxw7 Induces Apoptosis And Growth Arrest By Targeting YAP For Ubiquitination And Proteasomal Degradation In Hepatocellular Carcinoma Cells

Introduction:Primary liver cancer(PLC),more than ninety percent are hepatocellular carcinoma(HCC),is one of the most common malignancies and the second leading cause of cancer-related death in China.The rising incidence of HCC coupled with emerging evidence for efficacy of screening in high-risk patients,liver transplantation as a curative option in select patients,ability to make definitive diagnosis using high-resolution imaging of the liver,and proven efficacy of transarterial chemoembolization(TACE),radiofrequency ablation(RFA)and sorafenib as palliative therapy have improved the outlook for HCC patients,but it has not yet achieved satisfactory curative effect.The clinical application of sorafenib has made up the gap of treatment for patients with advanced HCC.Therefore,further investigating the correlation between functional change of new genes and HCC development,malignant characteristics as well,play an important role in revealing the precise molecular mechanisms of HCC development,designing reasonable molecular targeted drugs,predicting the prognosis and improving the treatment of HCC in China.The ubiquitin proteasome system-mediated protein degradation is the main regulator and terminator of gene and protein function,and controls the majority of protein degradation in eukaryotic cells.Therefore,regulation of this pathway has become an innovative method in the treatment of disease.F-box and WD repeat domain-containing 7(Fbxw7)is a key factor,which determines the substrate specificity of the Skpl-Cullinl-F-box(SCF)-type E3 complex.Fbxw7 targets for ubiquitination and proteasomal degradation various oncoproteins,such as Cyclin E,c-Myc,c-Jun,MCL1,Notch and so on,and inhibits proliferation,induces apoptosis and increases chemotherapeutic sensitization of cancer cells.Lack of Fbxw7 expression could be found in a wide range of human cancers and low-expression of Fbxw7 was correlated with poor prognosis,indicating that Fbxw7 was a general tumor suppressor in human cancer.p53 regulated Fbxw7 gene expression at transcriptional level,the double heterozygous p53+/-Fbxw+/-mice and irradiated Fbxw+/-mice developed a range of tumors including HCC,these indicated that Fbxw7 was correlated with HCC development.Several proteins targeted by Fbxw7,such as Cyclin E,c-Myc,c-Jun,Notch and so on,were deregulated and correlated with proliferation,apoptosis,chemotherapy resistance and poor prognosis in HCC.The Hippo-YAP signaling pathway is a well-conserved potent regulator of cell growth and apoptosis in mammals.The core downstream effector of Hippo pathway is Yes-associated preotein(YAP).Aberrant of this pathway has been found in several types of human cancer.Using integrative oncogenomic and proteomic approaches,YAP was identified as a driving oncogene of the 11q22 amplicon in HCC and breast cancer.In addition,transgenic(Tg)mice with liver-targeted YAP overexpression demonstrated a dramatic increase in liver size and eventuallydeveloped tumors.In clinical studies,YAP was an independent predictor associated with poor diseasefreesurvival(DFS)and overall survival(OS)in HCC.These results indicate that Hippo-YAP signaling pathway plays an important role in the initiation and development of HCC.Previous study reported a quantitative atlas of mitotic phosphorylation by using liquid chromatograph-mass spectrometer/mass spectrometer(LC-MS/MS)and 17 phosphopeptides and 16 phosphorylation sites for YAP protein were found.we carefully examined the sequence around S131 and T141 and found a[(L)-V-pS-P-(P)-G-pT]motif,similar to the canonical phosphodegron[(L)-X-pT/pS-P-(P)-X-pS/pT/E/D]recognized by Fbxw7,suggesting YAP may be a potential target protein of Fbxw7.However,the expression of Fbxw7 and its clinical significance,the effect of Fbxw7 on proliferation and apoptosis and its precise molecular mechanisms remain unclear.Objectives:In this work,we intend to investigate the expression of Fbxw7 and its effect on proliferation and apoptosis in HCC,explore the role of Fbxw7 in HCC development and analysis the possible mechanisms underlying this process.Methods:1.Immunohistochemistry,Western blot and qRT-PCR were used to detect the expression of Fbxw7 in HCC and matched normal tumor-adjacent tissues,the correlation between Fbxw7 and clinicopathological features of HCC patients were analyzed.2.qRT-PCR was used to examine the Fbxw7 mRNA expression in human immortal hepatic cell line L02 and HCC cell lines HepG2,Hep3B,SMMC-7721,Bel-7402 and Huh7.3.Retrovirus-mediated Flag-Fbxw7 and Fbxw7-shRNAs were transfected into HepG2 and Hep3B,respectively.The expression of Fbxw7 were identified by qRT-PCR and Western blot,cell proliferation and apoptosis were analyzed by flow cytometry,Caspase 3/7 activity assay,BrdU incorporation assay and MTT assay.4.Immunohistochemistry was used to detect the expression of YAP in HCC and matched normal tumor-adjacent tissues,the correlation between Fbxw7 and YAP protein was analyzed.5.Retrovirus-mediated Flag-Fbxw7 and Fbxw7-shRNAs were transfected into HepG2 and Hep3B,respectively.The expression of YAP were identified by qRT-PCR,Western blot and immunofluorescence.6.Flag-Fbxw7 and HA-YAP were co-transfected into HEK293 cells,coimmunoprecipitation was used to determine the interaction between Fbxw7 and YAP;HA-YAP overexpression HepG2 cells were transfected with Flag-Fbxw7 and ubiquitination level of YAP protein was identified by immunoprecipitation,CHX chase assay was used to determine the half life of YAP protein with or without Fbxw7 overexpression.7.HA-YAP expressing Hep3B cells were transfected with Flag-Fbxw7,qRT-PCR and Western blot were used to determine the expression of Fbxw7 and YAP,cell proliferation and apoptosis were identified by flow cytometry,Caspase 3/7 activity assay,BrdU incorporation assay and MTT assay.8.Hep3B cells stablely overexpressing EV,Flag-Fbxw7 or Flag-Fbxw7+HA-YAP were used to construct nude mice subcutaneous tumor models,growth curve in all three groups were constructed by GraphPad Prism 5,immunohistochemistry and TUNEL assay were used to determine the expression of YAP and Ki-67 and apoptosis in xenografts.Results:1.The expression of Fbxw7 was significantly lower in the HCC tumor tissues compared to matched normal tumor-adjacent tissues(P<0.01).Fbxw7 protein was expressed at significantly lower level in patients with large tumor size(?5cm),venous infiltration,high histological grade and advanced tumor-node-metastasis(TNM)stage(P<0.0 5,respectively).2.Fbxw7 mRNA was expressed in all of the tested cells,L02 cell line expressing the highest Fbxw7 mRNA level and SMMC-7721 cell line expressing the lowest Fbxw7 mRNA level.3.Fbxw7 overexpression induced cell apoptosis and growth arrest in HepG2 cells(P<0.01,respectively),Fbxw7 knockdown led to enhanced cell proliferation and decreased apoptosis in Hep3B cells(P<0.01,respectively).4.YAP expression was significantly higher in cancer tissues than noncancerous tissues(P<0.01).In HCC tissues,Fbxw7 was negatively correlated with YAP protein(r=-0.572,P=0.003).5.Fbxw7 overexpression downregulated YAP protein expression but not YAP mRNA level in HepG2 cells(P<0.01).Fbxw7 knockdown led to YAP protein accumulation but not YAP mRNA in Hep3B cells(P<0.01).6.Fbxw7 interacted with YAP protein.Fbxw7 overexpression increased the ubiquitination level of YAP protein and shortened the half life of YAP in HepG2 cells.7.YAP protein partially abolished Fbxw7 induced apoptosis and growth arrest of Hep3B cells in vitro(P<0.01,respectively).8.Fbxw7 significantly inhibited Hep3B cell growth in vivo(P<0.01),Fbxw7 induced apoptosis and growth arrest of Hep3B cells were partially inverted by YAP protein in vivo(P<0.01,respectively).Conclusions:1.Reduced Fbxw7 expression closely correlates with malignant clinicopathological characteristics and inversely correlates with YAP protein,suggesting Fbxw7 may be a potential marker for predicting prognosis in HCC patients.2.Fbxw7 negatively regulates the stability of YAP protein through ubiquitin-proteasome proteolysis,YAP may be a novel target protein of Fbxw7 in HCC.3.YAP protein can partially abolish Fbxw7 induced apoptosis and growth arrest of HCC cells in vitro and in vivo,Fbxw7 may function as a tumor suppressor through Hippo-YAP signaling pathway.