Deubiquitinase PSMD14 Promotes Epithelial Ovarian Cancer Progression By Regulating Ubiquitination On PKM2

Objective:With a five-year survival rate less than 50%,ovarian cancer is the deadliest gynecological cancer,thereby seriously threatening women’s health.Ubiquitination is one of the most common types of post-translational modifications,and it is involved in multiple cellular biological processes,such as regulation of protein stability,transcription and cell cycle.Dysregulation of ubiquitination and deubiquitination was disclosed to be closely related to the carcinogenesis and progression of various human cancers,including ovarian cancer.Therefore,ubiquitination and deubiquitination need to be further explored to elucidate the mechanisms of ovarian cancer progression,with the aim of finding potential therapeutic targets for ovarian cancer.26S proteasome non-ATPase regulatory subunit 14(PSMD14)belongs to the JAB1/MPN/Mov34(JAMM)domain metalloprotease family of deubiquitylating enzyme,playing pro-cancer roles in the carcinogenesis and progression of multiple malignant tumors,such as prostate cancer,hepatocellular carcinoma and colon cancer.However,its function and the underlying mechanism in the progression of ovarian cancer still remains to be characterized.Metabolic reprogramming is a key feature of cancer,sustaining cancer cell survival and growth.In recent years,studies have disclosed that ubiquitination and deubiquitination play an important role on the regulation of metabolic reprogramming in cancer cells.Our study aims to explore the function of deubiquitylating enzyme PSMD14 in the progression of ovarian cancer,its roles in metabolic reprogramming in ovarian cancer cells,as well as the underlying molecular mechanisms.Methods:1.The expression levels of PSMD14 in ovarian cancer tissues and normal ovarian tissues were analyzed by using the GEPIA database.The relationship between the level of PSMD14 expression and overall survival in ovarian cancer patients was explored in the Kaplan-Meier plotter database.The expression of PSMD14 in normal ovarian tissues,ovarian benign epithelial tumor,ovarian borderline epithelial tumor and epithelial ovarian cancer was detected by immunohistochemistry.The relationship between level of PSMD14 expression and clinicopathological parameters of ovarian cancer patients was analyzed.2.The endogenous expression level of PSMD14 in ovarian cancer cell lines,including A2780,OVCAR-3 and HO-8910,was detected by Western Blot.We then conducted PSMD14 knockdown ovarian cancer cells,PSMD14 overexpressed ovarian cancer cells,and ovarian cancer cells overexpressing PSMD14 with mutated functional site.Western Blot was used to test the efficiency of knockdown and overexpression.CCK8 assay,colony formation assay and soft agarose assay were used to detect the effect of PSMD14 expression as well as its enzymatic activity on the proliferation of ovarian cancer cells.Transwell assay was used to analyze the effect of PSMD14 and its enzymatic activity on the invasion of ovarian cancer cells.The effect of PSMD14 and its enzymatic activity on migration of ovarian cancer cells was investigated by scratch assays.PSMD 14 knockdown ovarian cancer cells were subcutaneously injected into female nude mice for the observation of tumor growth rate,tumor size and weight.After treated with PSMD 14 specific inhibitor O-phenanthroline(OPA),ovarian cancer cells were detected by CCK8 assay,colony formation assay,soft agarose assay,Transwell assay and scratch assay for their proliferation,invasion and migration ability.3.Co-IP assay combined with mass spectrometry was used to identify the proteins interacting with PSMD 14 in ovarian cancer cells.Co-IP assay was utilized to verify the binding between PSMD 14 protein and pyruvate kinase M2 isoform(PKM2)protein in ovarian cancer cells.The interaction between PSMD 14 protein and PKM2 protein was further verified by exogenous Co-IP in HEK293T cells.The localization of the exogenous PSMD 14 and PKM2 protein in HEK293T cells was studied by immunofluorescence.IP assay was used to detect the effect of PSMD 14 knockdown or OPA on PKM2 ubiquitination in ovarian cancer cells.In HEK293T cells,exogenous IP assay was used to detect the effects of PSMD 14 overexpression on PKM2 ubiquitination.The effect of PSMD 14 on the level of PKM2 protein expression was detected by Western Blot.The influence of PSMD 14 on the stability of PKM2 protein was detected by Western Blot after cycloheximide(CHX)treatment.The effects of PSMD 14 on the levels of PKM2 monomers,dimers and tetramers were studied by cross-linking assay with glutaraldehyde.4.The effects of PSMD14 on PKM2 enzymatic activity as well as glucose metabolism in ovarian cancer cells were detected by PK Activity Colorimetric Assay Kit,Colorimetric Glucose Uptake Assay Kit and Lactate Colorimetric Assay kit.The effect of PSMD14 on PKM2 localization in ovarian cancer cells was investigated by immunofluorescence.The effects of PSMD 14 on cytoplasmic as well as nuclear PKM2 levels were detected by Western Blot with nuclear and cytoplasmic protein extraction kit.Real-time PCR was used to detect the effect of PSMD14 on mRNA expression level of PKM2-dependent genes.5.PKM2 expression was further downregulated in PSMD14 overexpressed ovarian cancer cells.The Colorimetric Glucose Uptake Assay Kit and Lactate Colorimetric Assay kit were then used to explore the glucose metabolism.CCK-8 assay,colony formation assay and soft agarose assay were used to detect the proliferation in these ovarian cancer cells.Transwell assay was used to detect the invasion,and scratch assay was used to explore the migration of these ovarian cancer cells.Results:1.In GEPIA database,expression level of PSMD14 was higher in ovarian cancer tissues compared to that in normal ovarian tissues.In the Kaplan-Meier plotter database,the level of PSMD14 expression was found to be negatively associated with overall survival.Immunohistochemical results showed higher PSMD14 level in primary epithelial ovarian cancer and epithelial ovarian borderline tumor compared to that in normal ovarian tissues.Statistical analyses revealed that upregulated PSMD14 expression was positively correlated with higher International Federation of Gynecology and Obstetrics(FIGO)stage.2.Knockdown of PSMD14 significantly inhibited the proliferation,invasion and migration in ovarian cancer cells.PSMD14 stable knockdown also contributed to impaired in vivo tumor growth in female BALB/c nude mice and significantly lower ovarian tumor weight.PSMD14 inhibitor OPA significantly inhibited the proliferation,invasion and migration of ovarian cancer cells in a concentration-dependent manner.Overexpression of PSMD14 significantly increased the proliferation,invasion and migration of ovarian cancer cells.However,overexpression of PSMD14 with mutated functional site had no significant effect on the proliferation,invasion or migration of ovarian cancer cells.3.Co-IP combined with mass spectrometry analysis identified PKM2 as a protein co-immunoprecipitated with PSMD14 in ovarian cancer cells.Endogenous Co-IP and reciprocal Co-IP verified the endogenous interaction between PSMD14 protein and PKM2 protein in ovarian cancer cells.Exogenous Co-IP further confirmed the interaction between PKM2 and PSMD14.Immunofluorescence assay showed that PSMD14 was colocalized with PKM2.After downregulation of PSMD14 expression in OVCAR-3 cells or treatment of OVCAR-3 cells with PSMD14 inhibitor OPA,the ubiquitination level of PKM2 was markedly increased.The level of ubiquitinated PKM2 was significantly reduced by wild-type PSMD14,but not by PSMD14 mutants.K63 ubiquitin was markedly reduced by PSMD14,while K11 or K48 ubiquitin on PKM2 didn’t change significantly after PSMD14 overexpression.Knockdown and overexpression of PSMD14 didn’t alter the expression level of PKM2 protein in OVCAR-3 cells.Results of cycloheximide(CHX)pulse-chase assay indicated that altered PSMD14 expression had no influence on the stability of PKM2 protein.In the cross-linking experiments,knockdown of PSMD14 was found to upregulate the level of PKM2 tetramers and decrease the ratio of its dimers and monomers in OVCAR-3 cells.Tetrameric PKM2 reduced and was replaced by dimers and monomers after overexpression of PSMD14.4.Knockdown of PSMD14 increased PK activity,reduced glucose uptake and lactate production.On the contrary,overexpression of PSMD14 decreased PK activity,enhanced glucose uptake and lactate production.Both immunofluorescence staining and subcellular fractionation followed by Western blot analyses showed that the signal and expression level of nuclear PKM2 was decreased after PSMD14 knockdown,while was enhanced after PSMD14 overexpression.Knockdown of PSMD14 expression decreased the mRNA level of downstream PKM2-dependent genes,including cyclin D,HK1,GLUT1,MEK5,LDHA,PDK1,MYC and VEGFA,while overexpression of PSMD14 increased the mRNA level of these genes.However,overexpression of PSMD14 mutants had no significant effects on PKM2 oligomers,enzyme activity,localization,mRNA levels of downstream target genes,glucose uptake or lactate production.5.Silencing PKM2 abolished the induction of aerobic glycolysis and the promotion of viability,proliferation,invasion and migration induced by PSMD14 overexpression.Conclusion:The expression level of PSMD14 was increased in ovarian cancer,and its increased level correlated with higher FIGO stage and poor overall survival in ovarian cancer patients.PSMD14 promotes malignant biological behavior of ovarian cancer cells by regulating PKM2 through its deubiquitinating activity.PSMD14 binds to PKM2 and decreases the level of K63 ubiquitination on PKM2,downregulating the ratio of PKM2 tetramers to dimers and monomers,but PSMD14 does not change the total protein level or protein stability of PKM2.PSMD14 reduces PK activity and promotes glucose uptake and lactate production in ovarian cancer cells.PSMD14 promotes PKM2 nuclear translocation and increases the mRNA expression level of PKM2-dependent genes.PSMD14 specific inhibitor OPA represses the malignant behavior of ovarian cancer cells.