Mechanistic Study Of USP5 In Promoting The Proliferation Of Non-small Cell Lung Cancer Cells By Stabilizing CCND1

Background and aims:Lung cancer is the most commonly diagnosed cancer and the leading cause of cancerrelated death worldwide.Non-small cell lung cancer(NSCLC)account for about 85%of all lung cancer cases.Last decade witnesses the advances of targeted therapy and immunotherapy for lung cancer patients,but the five-year survival rate of patients with the disease remains low.Therefore,gaining an understanding of the mechanisms of disease progression,screening biomarkers,and identifying potential therapeutic targets will be instructive and meaningful to treating and improving the prognosis of NSCLC.NSCLC has the characteristics of a high proliferation rate and rapid growth.Abnormal cell cycle regulation and uncontrolled proliferation are considered to be important mechanisms for the occurrence of NSCLC.Cell cycle is determined by a welltuned cascade signaling transduction,in which cyclin D1(CCND1)plays a critical role,which can interact with and activates cyclin-dependent kinases 4/6(CDK4/6).Once activated,CDK4/6 phosphorylates the retinoblastoma(Rb)protein and promotes G1/S phase cell cycle transition.In NSCLC,CCND1 has been found to be elevated and to promote carcinogenesis,cell cycle progression,metastasis,and drug resistance.CCND1 is a rapid responsive protein in cell cycle progress with a very short half-life.It has been reported that CCND1 could be degraded by Ubiquitin proteasome system(UPS).Moreover,CCND1 ubiquitination is a dynamic process and certain deubiquitinases can remove the conjugated ubiquitin molecules.Among them,some members of the ubiquitin specific protease(USP)family such as USP2a,USP10,and USP22 have been found to potentially deubiquitinate CCND1 protein and enhance its stability.Recent studies have reported that CCND1 can be upregulated by USP5,another member of USP family,in pancreatic cancer and NSCLC,however,the mechanism of action is still unclear.Given its deubiquitinating effect on multiple oncoproteins,the present study was performed to investigate whether USP5 upregulates the expression level of CCND1 protein in NSCLC via deubiquitination and stabilization of CCND1.In order to provide a therapeutic strategy moving from our mechanistic insights,we further evaluated the potential anticancer activity of USP5 inhibition.Methods:We first examined the effect of USP5 on CCND1 by cotransfecting the tool cell line HEK293T with Flag-CCND1 and Myc-USP5 plasmids.We next examined the half-life of CCND1 protein in the presence of USP5 and cycloheximide(CHX).We analyzed the effect of USP5 overexpression or knockdown on the protein levels of endogenous CCND1 in the NSCLC cell lines.Moreover,we evaluated the association of the endogenous expression of USP5 and CCND1 in a panel of NSCLC cell lines.The mechanism by which USP5 regulates the protein level of CCND1 was further explored by coimmunoprecipitation and the protein ubiquitination assay.Next,we constructed the domains of USP5 and detected the binding domains of USP5 and CCND1 by Western blotting(WB).Knockdown of USP5 with siRNA in A549 and H1299 cells,the effects of USP5 on CCND1-CDK4/6-Rb signaling were detected by WB and cell cycle was examined by flow cytometry.CCK-8 growth assay,colony formation assay and wound healing assay were performed to investigate the potential role of USP5 on the proliferation and migration in NSCLC cells.We analyzed the data on USP5 and CCND1 mRNA expression in lung adenocarcinoma tissues and normal lung tissues from The Cancer Genome Atlas(TCGA)database.Additionally,the prognostic value of USP5 and CCND1 in lung cancer tissues was evaluated by Immunohistochemistry(IHC)staining.We also analyzed the relationship of the mRNA and protein expression levels of USP5 and CCND1 with the overall survival(OS)of lung adenocarcinoma(LUAD)patients via TCGA(UALCAN)and The Human Protein Atlas(THPA)databases,respectively.We used CCK8 to determine A549 and H1299 cell viability following treatment with different concentrations USP5 inhibitors(WP1130 or EOAI3402143(G9))for 24h、48h and 72h.After 24 hours of treatment,the protein levels of USP5 and CCND1 were detected by WB.Moreover,the effect of G9 on NSCLC xenograft tumor growth in vivo was determined in athymic BALB/c nude mice.At the endpoint of the experiment,animal body weight were measured and blood samples were analyzed.All tumor tissues were subjected to IHC assay,and each staining was given a score.Results:The immunoblotting(IB)assay demonstrated that USP5 increased the protein levels of CCND1 in a concentration dependent and time-dependent manner.The CHX chase assay showed that USP5 markedly prolonged the half-life of CCND1 protein.USP5 overexpression remarkably increased the levels of CCND1 protein in NSCLC cells,whereas knockdown of USP5 using its specific siRNA reduced the protein levels of CCND1.Western blotting results showed that the protein level of CCND1 was positively correlated with the USP5 expression in NSCLC cells.The co-immunoprecipitation results showed that USP5 was able to interact with CCND1 in HEK293T cells and NSCLC cells.Western blotting analyses suggested that the UBA1-UBA2 and H box domains in USP5 partly maintains CCND1 stability.Furthermore,our gain-and loss-of-function experiments demonstrated that overexpression of USP5 prevented endogenous CCND1 from polyubiquitinating in NSCLC cells and increased CCND1 protein level,whereas knockdown of USP5 produced the opposite effect.Ubiquitination experiment further revealed that knockdown of USP5 promoted the formation of K48-linked polyubiquitin chains on CCND1 and thus accelerated its proteasomal degradation.Flow cytometry experiment showed that knockdown of USP5 impeded cell cycle transition of NSCLC cells.And WB results demonstrated that USP5 knockdown did not change the expression level of CDK4 and CDK6 but suppressed the phosphorylation level of Rb(Ser780),and was accompanied by the downregulation of CCND1.As shown by the CCK-8 assay,A549,H1299 and H460 cell proliferation were promoted by the overexpression of USP5 but impaired by its knockdown.Meanwhile,the colony formation assay revealed that knockdown of USP5 also markedly reduced the number of colonies in NSCLC cells.Moreover,knockdown of USP5 attenuated the invasion migratory ability of A549 and H1299 cells.Analysis of public dataset showed that the level of USP5 mRNA was highly expressed in lung adenocarcinoma tissues.The mRNA correlation analysis of USP5 with CCND1 in TCGA lung adenocarcinoma tissues revealed that the expression of USP5 was not related to CCND1.Our IHC analysis in lung adenocarcinoma tissues and normal lung tissues showed that the levels of USP5 and CCND1 proteins were also upregulated in lung adenocarcinoma tissues.Moreover,clinical data confirmed that the expression level of USP5 was positively correlated with the protein level of CCND1 in lung adenocarcinoma tissues.UALCAN and THPA database demonstrates that the expression levels of USP5(either mRNA or protein)and CCND1(only protein)are positively correlated with poor prognosis of LUAD patients.Both WP1130 and G9 displayed very comparative activity in terms of suppressing the proliferation viability of A549 and H1299 NSCLC cells in vitro.And chemical inhibition of USP5 activity using WP1130 and G9 also showed similar ability to downregulate the protein level of CCND1 in NSCLC cells.The results of xenograft mouse model showed that G9 also effectively suppressed the NSCLC tumor growth in vivo with no significant toxicity.And the IHC staining of tumor tissues revealed that the expression levels of CCND1 were significantly reduced in the G9-treated mice whereas USP5 was not markedly affected.Conclusion:In conclusion,this study identified that CCND1 is a novel substrate of USP5.By decreasing CCND1 ubiquitination,USP5 stabilizes and upregulates its protein level and consequently promotes the proliferation,migration and colony formation of NSCLC cells.In contrast,genetic or chemical inhibition of USP5 leads to CCND1 degradation,thereby resulting in decreased NSCLC cell proliferation and tumor growth.Our findings indicates that the USP5-CCND1 axis may be a potential and novel target for the treatment of NSCLC,which may pave a new way for the study of NSCLC.