Effects Of SIRT1 Subcellular Localization On Cisplatin Sensitivity, Migration And Invasion In Ovarian Carcinoma

Purpose:Silencing information regulator 1?SIRT1?is an important regulator in oncogenesis and tumor progression.However,its role as a tumor promotor or suppressor remains controversial.The nucleocytoplasmic shuttling of SIRT1 has been discovered for more than 10 years,but little is known about the roles of SIRT1 subcellular localization in ovarian carcinoma.The aim of this study was to explore the roles of SIRT1 subcellular localization on cisplatin sensitivity,migration and invasion and the related mechanisms in ovarian carcinoma.Methods:1.Human ovarian carcinoma IGROV1 cells and HEY cells overexpressing wild-type SIRT1?SIRT1 cells?,SIRT1 with mutated nuclear export sequences/NESs?SIRT1^NESmtESmt cells?and SIRT1 with mutated nuclear localization sequences/NLSs(SIRT1^NLSmt cells)were obtained via transfection of lentiviral vectors carrying the corresponding coding sequences,respectively.2.The subcellular localizations of exogenous SIRT1 were observed by the fluorescence signals of SIRT1-GFP fusion proteins encoded by lentiviral vectors.The mRNA and protein expression levels of SIRT1 were analyzed by real-time PCR and Western blot assay.Both cytoplasmic and nuclear extractions were used to analyze the subcellular localization of exogenous SIRT1 by Western blot assay.In order to identify the deacetylase activity of exogenous SIRT1,Western blot assay was performed to compare the acetylation level of p53 K382 and quantitative proteomic and acetylomic analyses were introduced to compare the overall acetylation level of proteins and lysine residues in different cell groups.3.In order to explore the role of SIRT1 subcellular localization on cisplatin sensitivity and its mechanisms,CCK-8 cytotoxic assay and flow cytometry analysis were performed to compare the rates of cell survival and apoptosis among different cell groups after cisplatin treatment.The protein expression levels of autophagy-related proteins,Mn-SOD and acetylated Mn-SOD?Ac-K68?,and the activity of Mn-SOD were analyzed by Western blot assay and Superoxide Dismutase typed assay kit,respectively.The interaction between Mn-SOD and SIRT1 was studied by co-immunoprecipitation assay.4.The expression of SIRT1 in tumor tissues from 453 cases of patients with ovarian carcinoma was detected by immunohistochemical staining.The weighted scores were obteined by a semi-quantitative scoring of SIRT1 cytoplasmic or nuclear expression.The associations between SIRT1 subcellular localization and the clinicopathological data,and overall survival duration of patients were studied by statistical analyses.The precise subcellular localizations of SIRT1 in ovarian carcinoma cells were confirmed by post-embedding immunogold staining for electron microscopy.5.The effects of subcellular localizations of SIRT1 on both migration and invasion in ovarian carcinoma cells were detected by Transwell assay.To explore the mechanism by which SIRT1 subcellular localization affects the migration and invasion of ovarian cancer cells,the differential levels of protein expression and acetylation between IGROV1 SIRT1cells and SIRT1^NLSmt cells were systematically studied by quantitative proteomic and acetylomic analyses,and the expression levels of specific proteins were confirmed by Western blot and/or real-time PCR assay.Results:1.Exogenous SIRT1 located in the cytoplasm and nucleus of SIRT1 cells.SIRT1^NESmt mainly located in the nucleus,while SIRT1^NLSmt predominantly located in the cytoplasm.The resluts of Western blot and real-time PCR confirmed the overexpression of exogenous SIRT1 in the SIRT1,SIRT1^NESmt and SIRT1^NLSmt cells.The mutation of NESs increased the distribution of SIRT1^NESmt in the nucleus,while the mutation of NLSs led to the enrichment of SIRT1^NLSmt in the cytoplasm.2.The protein expression levels of p53 and acetylated p53?Ac-K382?,toghter with the results of quantitative proteomic and acetylomic analyses suggested that wild-type SIRT1 and SIRT1^NLSmt had deacetylase activity,whereas the mutations of NESs resulted in deacetylase inactivation of SIRT1^NESmt.3.Compared with empty vector control cells,the SIRT1 cells and SIRT1^NLSmt cells showed enhanced and declined cisplatin sensitivity,respectively.Overexpression of both SIRT1 and SIRT1^NLSmt led to an accumulation of p62 and down-regulation of Beclin1 in tumor cells,suggesting an inhibition in autophagy flux.Overexpression of SIRT1upregulated the expression level of Mn-SOD,while SIRT1^NLSmt lacked of this effect.Although SIRT1 could bind to Mn-SOD,there was no significant effect on the acetylation level of Mn-SOD K68.4.The mean age of the 453 patients in our study was 57 years?range,15-92 years?.Higher cytoplasmic expression of SIRT1 in ovarian carcinoma tissues was associated with earlier FIGO stage?P<0.001?or more prolonged overall survival duration?P=0.047?.The SIRT1 expression level was not an independent predictor of overall survival.Moreover,there was no correlation between nuclear expression of SIRT1 and overall survival of the patients.The results of immunoelectron microscopy exhibited that SIRT1was located in the nucleus,cytosol,rough endoplasmic reticulum and mitochondria of ovarian carcinoma cells.5.Compared with empty vector control cells,IGROV1 SIRT1^NLSmt cells showed significantly decrease in migration and invasion,whereas the abilities of migration and invasion in the SIRT1 cells were markedly increased.The proteomic and acetylomic analyses revealed a down-regulation of mesenchymal biomarkers in the SIRT1^NLSmt cells compared with the SIRT1 cells,including vimentin and fibronectin,and an up-regulation of epithelial phenotype which were characterized by the increase in cytokeratins?CK-18and CK-19?,desmosome-associated proteins?desmoplakin,plakophilin-2,plakophilin-3,periplakin and epiplakin?and tight junction-associated proteins?claudin-1,JAM1 and nectin-1?,as well as acetylation level alterations in vimentin,CK-18 and desmoplakin.Among these differentially expressed proteins,mRNA levels of vimentin,fibronectin,CK-18,CK-19,plakophilin-2,plakophilin-3,epiplakin and nectin-1 were verified by real-time PCR assay,and protein levels of CK-18,CK-19 and vimentin were further confirmed by Western blot.Conclusion:1.The NESs and NLSs are the key factors affecting the subcellular localization of SIRT1.By mutation of NESs or NLSs,we obtained SIRT1^NESmt with a higher enrichment in the nucleus and SIRT1^NLSmt with a predominant cytoplasmic localization.2.Overexpression of wild-type SIRT1 increased cisplatin sensitivity while overexpression of SIRT1^NLSmt led to decreased cisplatin sensitivity in ovarian carcinoma cells.However,the mechanisms of autophagy and Mn-SOD were not able to reasonably explain the relationship between subcellular localization of SIRT1 and cisplatin sensitivity of ovarian carcinoma cells.3.Higher cytoplasmic expression of SIRT1 was associated with a favorable prognosis in the patients with ovarian carcinoma.4.Compared with ovarian carcinoma cells with overexpression of wild-type SIRT1,the tumor cells with overexpression of SIRT1^NLSmt showed declined abilities of migration and invasion.The mechanism was related to the inhibition of epithelial-mesenchymal transition?EMT?.To summarize,for the first time,our study suggest that subcellular localization of SIRT1 affects cisplatin resistance and the abilities of migration and invasion in ovarian carcinoma cells.Moreover,the cytoplasmic expression level of SIRT1 is associated with the prognosis of the patients with ovarian carcinoma.These results suggest that the role of SIRT1 as a tumor suppressor or promoter may depend on its subcellular localization,thus providing new insights into the role of SIRT1 in durg resistance and tumor progression.