The Role And Mechanism Of Deubiquitinase OTUD6A In Regulating CDC6 And Bladder Tumorigenesis

The incidence and mortality of bladder cancer(BCa)are increasing year-on-year globally.BCa ranks 12th in incidence and 14th in mortality,according to the latest published global cancer statistics.Ninety percent of BCa is urothelial carcinoma.BCa can be divided into non-muscle-invasive bladder cancer(NMIBC)and muscle-invasive bladder cancer(MIBC),accounting for approximately 75%and 25%,respectively.Distal metastasis occurs in about 5%MIBC patients.NMIBC patients are mainly treated with transurethral resection of bladder tumor(TURBT),adjuvant with intravesical chemotherapy or Bacillus Calmette-Guerin(BCG)instillation.MIBC patients mainly undergo radical cystectomy,adjuvant with chemotherapy,immunotherapy or targeted therapy.Despite many drugs and therapies,patients with BCa still suffer from chemotherapy resistance,low response rate to immunotherapy and strong side effects of targeted therapy.Therefore,it is important to investigate the molecular mechanism of BCa tumorigenesis for improving BCa diagnosis and treatment.Cell division cycle 6(CDC6),which belongs to the AAA+family of ATPase,has been identified as a major replication licensing regulatory factor during the initiation of DNA replication.CDC6 was not merely essential for the initiation of DNA replication,but functions beyond the G1 phase.Due to its integral role in cell cycle progression,aberration of CDC6 expression leads to various pathological changes.Aberrant upregulated CDC6 expression has been found in a broad range of human cancers and correlates with poor prognosis,including lung cancer,colon cancer,breast cancer,and so on.Increased CDC6 expression was found in the tumor tissues and urine of BCa patients,suggesting the great significance of CDC6 in BCa.Ubiquitination plays an important role in posttranslational regulation in various physical and pathological processes,including tumorigenesis,apoptosis,and DNA damage response.Ubiquitination can be reversed by a group of proteins known as deubiquitinases(DUBs).Nearly 100 DUBs in six different protein families are encoded by the human genome.CDC6 is degraded by several E3 ligase complexes through the ubiquitination pathway.However,whether CDC6 is regulated by DUBs is unknown.Here,we focus on the role and mechanism of OTUD6A in CDC6 stabilizing and OTUD6A-CDC6 axis in BCa,and the results were obtained below,Part Ⅰ OTUD6A deubiquitinates CDC6In order to clarify the molecular mechanism of deubiquitinating CDC6,we used the DUB plasmid library for screening,and achieved the following results:1.OTUD6A upregulates CDC6 protein expression.We transfected the DUBs plasmids into HEK293 cells,and the effect of different DUBs on CDC6 protein expression was detected by Western blot.The results showed that overexpression of OTUD6A could significantly upregulate CDC6 protein level,whereas there was no significant change in CDC6 mRNA expression,indicating OTUD6A upregulates CDC6 expression through posttranslational pathway.2.OTUD6A interacts with and promotes stability of CDC6.Immunofluorescence staining assays showed that both OTUD6A and CDC6 proteins were mainly expressed in the nucleus;co-immunoprecipitation(co-IP)and LC-MS/MS assays confirmed the association between OTUD6A and CDC6;CHX half-life assays showed that overexpression of OTUD6A prolonged the half-life of CDC6,and OTUD6A knockdown significantly shortened half-life of CDC6.Importantly,treatment of proteasome inhibitor MG 132 recovered the reduced CDC6 expression caused by OTUD6A knockdown.In addition,OTUD6A promoted the stability of CDC6 independent of CDC6 phosphorylation.These results suggest that OTUD6A stabilizes CDC6 protein by inhibiting a proteasome-dependent degradation of CDC6.3.OTUD6A deubiquitinates CDC6.Site-directed mutagenesis experiments confirmed OTUD6A reduced CDC6 polyubiquitination levels by its DUB activity.Further findings showed OTUD6A deubiquitinates K6,K33 and K48 ubiquitin chains on CDC6 protein;OTUD6A promoted CDC6 protein expression through rescuing CDC6 protein expression decreased by overexpression of CCNF and CDH1,and downregulated CDC6 polyubiquitination levels by its DUB activity.Part Ⅱ OTUD6A affects cell cycle and cell proliferation by regulating CDC6Given the important role of CDC6 in cell cycle and cell proliferation,we used in vivo and in vitro experiments to analyze the role of OTUD6A in cell cycle progression,cell proliferation and organismal development,and the results showed below:1.OTUD6A expression is cell cycle-dependent.Released from cell cycle synchronization,flow cytometry,Western blot and qRT-PCR showed OTUD6A expression was highest at M phase,downregulated after entering G1 phase,and lowest during the S phase of cell cycle.Immunofluorescence assays showed OTUD6A protein mainly located in the nucleus during M and G1 phase,and cytoplasm during S phase,indicating OTUD6A protein expression and subcellular localization are cell cycle-dependent.2.Inhibition of OTUD6A expression slows cell proliferation.The proteomics results showed that OTUD6A regulated the cell cycle-related proteins;Otud6a conditional knockout mice were crossed with Dppa3-Cre mice to obtain whole-body Otud6a knockout mice(CKO).The results revealed that Otud6a did not affect embryo development,and the weight of newborn homozygous CKO mice had no significant difference from wild type mice.However,homozygous CKO male mice showed growth retardation after 18 days of post-natal age,and the weight of the heart,lung,liver,spleen,bladder,and testis significantly lower than that of wild type male mice.CCK8 and EdU incorporation assays showed a slower proliferation rate in mouse embryonic fibroblasts(MEFs)of CKO mice.3.OTUD6A affects cell cycle progression by regulating CDC6.The results of flow cytometry showed that OTUD6A and CDC6 knockdown arrested U20S and HeLa cells in G2/M phase.Exogenous CDC6 overexpression could partially rescue G2/M phase arrest caused by endogenous OTUD6A knockdown,indicating CDC6 is one of the important targets of OTUD6A in regulating cell cycle.Part Ⅲ The role and mechanism of OTUD6A-CDC6 axis in BCaTo further clarify the role of OTUD6A-CDC6 axis in pathological state,we analyzed this pathway in BCa development and the results showed below:1.The expression levels of OTUD6A and CDC6 increased during bladder tumorigenesis.The protein levels of OTUD6A and CDC6 were consistently increased during bladder tumorigenesis in BBN-induced BCa mice model.2.OTUD6A promotes the proliferation of BCa cells through upregulating CDC6 expression.Knockout of OTUD6A significantly inhibited BBN-induced BCa.CCK8,colony formation,EdU incorporation assays and subcutaneous tumor xenograft models suggested that knockdown of OTUD6A inhibited the proliferation of BCa cells,and OTUD6A overexpression promoted the proliferation of BCa cells.Exogenous CDC6 overexpression could rescue the inhibited proliferation of T24 cells caused by endogenous OTUD6A knockdown,indicating OTUD6A promotes BCa cells proliferation via enhancing the expression of CDC6.3.OTUD6A promotes chemotherapy resistance and DNA damage repair response of BCa cells by upregulating CDC6 expression.OTUD6A knockdown BCa cells had significantly lower IC50 of gemcitabine and methotrexate.The overexpressed exogenous CDC6 could partially reverse the reduction of IC50 of chemotherapy drugs caused by OTUD6A.TUNEL,comet,Western blot and y-H2A.X fluorescence staining assays showed that OTUD6A knockdown increased DNA damage caused by chemotherapy agents and hydroxyurea and inhibited ATR-Chk1 pathway in BCa cells,whereas overexpressed exogenous CDC6 could significantly reduce the increased DNA damage caused by endogenous OTUD6A knockdown.4.OTUD6A and CDC6 expressions correlate with prognosis of patients with BCa.The positive correlation between OTUD6A protein and CDC6 protein was found in tissue specimens;and patients with high expression of OTUD6A and CDC6 had poor prognosis,confirmed by immunohistochemical staining and Kaplan-Meier survival curve analysis.In conclusion:1.OTUD6A promotes stability of CDC6 via interacting with and deubiquitinating CDC6.2.OTUD6A knockdown decreases CDC6 protein expression and induces G2/M arrest.3.OTUD6A knockout causes growth retardation in mice.4.OTUD6A knockdown inhibits the proliferation of BCa cells and enhances sensitivity of chemotherapy drugs.5.OTUD6A and CDC6 protein expression levels show prognostic value in BCa.