The Molecular Mechanism Of WDR79 In Promoting The Proliferation Of Non-small Cell Lung Cancer

Lung cancer is one of the most common malignant tumors and the leading cause of cancer-related deaths worldwide.Small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC)are the two major pathological types of lung cancer,where about 85%of lung cancers accounting for NSCLC.Although combination of surgery,radiation and chemotherapy is being used for treating NSCLC,the five year survival rate remains discouragingly low under 15%.Therefore,exploring the molecular mechanism,and identification of new target biomarkers in carcinogenesis,will provide new strategies and breakthrough points for NSCLC therapy.WD-repeat protein always contains 4-16 WD-repeat domains which participate in various cellular activities,including signal transduction,cell division,cytoskeleton assembly,and chromatin assembly,RNA metabolism,apoptosis,and cell cycle regulation.WD-repeat domain is initiated by a glycine-histidine(GH)dipeptide from the N terminal and ended with a tryptophan-aspartic acid(WD)dipeptide at the C terminal,and it can mediate many protein-protein interactions.WD-repeat protein 79,also known as TCAB1,or WRAP53,contains six WD-repeat domains,is a member of WD-repeat protein family and highly conserved from yeast to human.Researches have shown that WDR79 involves in telomerase assembly and Cajal body formation and DNA double-strand break repair.In addition,the WDR79 gene encodes an antisense transcript of p53(Wrap53a)that can stabilize p53 mRNA and protein.WDR79 has been reported to participate in many diseases including congenital dyskeratosis syndrome,nasopharyngeal carcinoma,oesophageal squamous cell carcinoma,colon cancer,ER-negative breast cancer and ovarian cancer.But the mechanism of action of WDR79 in NSCLC is still not clarified.In this study we found that WDR79 is overexpressed in NSCLC tissues and cell lines and the expression difference is statistically significant comparing with that in the normal,those results suggest the WDR79 expression may be related with NSCLC.To explore the biological functions of WDR79 in NSCLC,we constructed WDR79 expression vector and shRNA lentiviral vectors,then performed MTT assay and clone formation assay.Knockdown of WDR79 in H1299 decreased the cell proliferation and its overexpression in A549 increased the cell proliferation.Tumorigenicity test confirmed that knockdown of WDR79 can inhibit the NSCLC cell proliferation.Those results suggested WDR79 is closely related NSCLC cell proliferation.Considering cell proliferation is dependented on the orderly cell cycle regulation,we investigated the function of WDR79 in cell cycle.Results showed that knockdown of WDR79 can cause G0/G1 phase arrest and further study showed the arrest is related with cyclins-CDKs(cyclin dependent kinase)complexes.Apoptosis is generally induced following cell cycle arrest.To ensure whether WDR79 knockdown induces cell apoptosis,we knockdowned WDR79 in A549 and H1299,and checked karyomorphism changes and the eversion of phosphatidylserine in the membrane,which revealed that WDR79 knockdown induces cell apoptosis in NSCLC through mitochondria pathway,first WDR79 knockdown changed the ratio of Bcl-2/Bax and then induced the release of cytochrome C from mitochondria,finally cytochrome C improved the activities of caspase-9 and caspase-3.The xenograft assay showed that WDR79 knockdown attenuates the tumorigenicity of NSCLC cell.By the protein sequence and structure analysis we found several USP7 binding domains in WDR79,this suggested that there may have an interaction between WDR79 and USP7.To confirm that hypothesis,we performed indirect immunofluorescence and Co-immunoprecipitation assay,and found that WDR79 interacted with USP7 in NSCLC cells.USP7 is involved in Mdm2-p53 pathway as a deubiquitinase.Since WDR79 can interact with USP7,we checked the effects of WDR79 on Mdm2 and p53 level.We found that overexpression of WDR79 upregulated Mdm2 and p53 protein levels and knockdown of WDR79 downregulated them,meanwhile,no changes in mRNA levels of Mdm2 and p53 were detected using qPCR assay indicating WDR79 regulates Mdm2 and p53 through post-transcription mechanism.As ubiquitin-proteasome pathway is the principal mechanism of protein degradation in cells,we aimed to check whether WDR79 affects the degradations of Mdm2 and p53 through this mechanism,Using MG132 to inhibit proteasomes activities and cycloheximide(CHX)to inhibit protein synthesis,we found that WDR79 affected the ubiquitination and proteolysis of Mdm2 and p53,then regualted their half-lives in cell.As a scaffold protein,WDR79 possessed no enzymatic activities.We overexpressed WDR79 in the USP7-knockdown cell,and found that WDR79 affected Mdm2 and p53 through USP7.Further studies showed that WDR79 did not induce proliferation in cells USP7 knockdown,the result confirmed that WDR79 promoted NSCLC cell proliferation through USP7 regulating Mdm2 and p53.In order to comprehensively understand the function of WDR79 in NSCLC we performed MS assay with WDR79 knockdown cells to construct the signal pathways it may involved in.The results shown that WDR79 knockdown induced hundreds of protein level changes,which were involved in MAPK,Wnt,NF-?B,ubiquitin proteasome pathway,PI3K-Akt,cAMP signaling pathway,cell division,cell adhesion,cell cycle,apoptosis,cell migration and other cellular procession.These results suggested the importance of WDR79 in NSCLC pathgenesis and indicated that WDR79 could be a novel target for NSCLC therapy.In summary,we clarify the relationship between WDR79 and NSCLC proliferation for the first time.WDR79 regulates Mdm2-p53 signaling pathway through the interaction with usp7,and then mediates NSCLC cell proliferation.We systematically reveal the signaling pathway network and biological processes that WDR79 may affect.Our study provides a demonstration for the potential of WDR79 as a therapeutic target for NSCLC.